The sonographic features of DAs were carefully observed and other associated cardiovascular anomalies were also evaluated during the screening. The architectures of DAs and the whole cardiovascular system were observed and analyzed, and then the cast findings were compared with prenatal ultrasonic results. In fourteen cases, 18 ductal abnormities were indicated by prenatal echocardiography in fourteen cases, while 25 were revealed by casting. Cast findings included 4 cases of ductal stenosis, 1 case of ductal dilation, 6 cases of ductal circuity, 3 cases of right-sided ductus, 5 cases of anomalous ductal connection, 1 case of bilateral ductus and 5 cases of absent ductus. Cast findings consisted with ultrasound in 10 ductal abnormalities, revealed additional 15 ductal abnormalities miss-diagnosed by sonography, and corrected 6 abnormalities misdiagnosed prenatally. Meanwhile, 3 ductal abnormalities (reversed flow) could not be demonstrated by casts but only by ultrasound. Cast models can visually display the anatomical characteristics of ductus arteriosus, and could be successfully used in the demonstration of ductus abnormalities in fetuses with severe CHDs. Comparing with ultrasound, casting technique has its own superiority in exhibiting ductus abnormalities, especially in certain types such as course, origin and absence abnormalities of ductus.The anatomy of ductus arteriosus (DA) can be varied in different congenital heart defects (CHDs), and it is difficult to fully discover the DA and other associated cardiac anomalies by prenatal ultrasound. This study was aimed to use the modified vascular corrosion casting technique to prepare fetal cardiovascular casts with DA anomalies, assess the casting effectiveness in evaluating the great vessels of the fetal heart and investigate the utility of cardiovascular casting for the demonstration of fetal DA abnormalities. This retrospective study enrolled fourteen fetuses (23 to 28 As an important physiological channel for fetal circulation, ductus arteriosus, a normal structure connecting pulmonary trunk (PT) with descending aorta (DAo), is playing an indispensable role in fetal cardiovascular system3. Fetal congenital heart defects (CHDs) are often accompanied by ductal abnormalities. In these cases, the connection, course and dimension of the arterial duct can be varied4. Sometimes it is difficult to fully discover the ductal and other associated cardiac anomalies by prenatal ultrasound. Vascular corrosion casting technique is capable of accurately exhibiting the three-dimensional (3D) architecture of cardiovascular system. Our modified cast models can visually display the origin, course, dimension and relative spatial position of the great vessels, and may become an important supplement to prenatal echocardiography6. This study was aimed to introduce the modified method of preparation of fetal cardiovascular casts and assess casting effectiveness in evaluating the great vessels of the fetal heart, Meanwhile, utility of modified cardiovascular casting for the demonstration of fetal ductal abnormalities.Nowadays, fetal cardiac screening has been paid more and more attention+2 (mean 24.8 ± 1.6) weeks, and the maternal age was between 22 and 36 (mean 28.5 ± 4.0) years. This study was approved by the Medical Ethics Committee of Xiangyang No.1 People's Hospital, Hubei University of Medicine. All families provided informed consents. All methods were performed in accordance with the relevant guidelines and regulations of the Medical Ethics Committee of Xiangyang No.1 People's Hospital, Hubei University of Medicine.From January 2013 to July 2019, this retrospective study screened all fetuses suspected with severe CHDs induced labor in our hospital. Fourteen cases with abnormal ductus arteriosus demonstrated by casting technique were enrolled as subjects. The gestational age of fourteen fetuses ranged from 23 to 287 and American Society of Echocardiography (ASE)8. The following standard views were included in the examination: transverse view of upper abdomen, four-chamber view, left and right ventricular outflow tract view, three-vessel view, three-vessel and trachea view, caval long-axis view, ductal and aortic arch view. The diameter of the great vessels of the heart of all cast specimens including  was measured with fetal echocardiography three times.Fetal echocardiographic examination was performed using an Voluson E8 or 730  ultrasound machine equipped with C4-8, C4-8D and RM6C transducers (probe frequency was 4.0 ~ 8.0 MHz), according to the guidelines recommended by the International Society of Ultrasound in Obstetrics and Gynecology (ISUOG)When the fetus was found to have severe CHD, the sonographic features of ductus were carefully observed to determine the presence of ductal abnormality or not. Other associated cardiovascular anomalies were also evaluated during the examination.Fourteen pregnancies underwent induced labor with informed consents. In fourteen cases, nine fetuses died in utero , and the other five fetuses were terminated because of complicated intracardiac and/or extracardiac malformations. Fetal specimens were donated to our hospital for further autopsy and cast model fabrication, according to the steps as follows: (1) Thorax was cut open along the median line and bilateral costal margins with an inverted Y-shaped incision, to expose the heart, thymus, lungs and diaphragm. Peeled off the thymus, opened the pericardium, and carefully observed the structure and course of the great vessels in the chest. Midline abdominal incision was made to observe the morphology of the organs and great vessels in abdominal cavity. (2) The umbilical vessels were separated, with umbilical vein intubated with a plastic catheter and the left side umbilical artery cut-off. Acetone was injected into the umbilical vein to expel the blood clots in the cardiovascular system. (3) Casting material, a mixture composed of 10 g acrylonitrile butadiene-styrene plastic (ABS), 100 ML acetone and 2 g dyestuff, was perfused slowly into the specimen at appropriate pressure. The total amount of casting fluid was controlled between 30 to 60 ML according to the specimen’s weight, and perfusion time was controlled in approximately 30 min. (4) After solidification of the casting material, the specimen was soaked in strong acid for about one week. (5) After corrosion, the specimen was immersed in clean water and carefully rinsed. Every cast specimen was preserved in a specimen box for further observation and evaluation.The diameters of the major vessels of the heart of all cast specimens was measured with vernier caliper three times.This study was approved by the Medical Ethics Committee of Xiangyang No.1 People's Hospital, Hubei University of Medicine.All families enrolled consented this study and provided informed consents.In fourteen cases, the diameters of the great vessels were measured by casting and compared with that measured by fetal echocardiography. The results showed that there were no significant differences in the diameter of the major blood vessels between casting groups and fetal echocardiography groups Table . 18 ductDA abnormalities included the following different types: 1) Ductal dimensional abnormality : five cases of ductal dimensional abnormality were found by casting, including 4 cases of ductal stenosis and 1 case of ductal dilation. One case of ductal stenosis was demonstrated both by ultrasound and cast, 2 cases with narrowed DA indicated by ultrasound were not confirmed by casts, while additional 3 cases revealed slim ductus in casts. One case demonstrated dilated ductus in cast, and another case of ductal dilation suspected by echocardiography was found to be bilateral ductus by cast. (2) Ductal course abnormality (including tortuous ductus and right-sided ductus): six cases of ductal circuity were exhibited in casts, in which only 2 cases were indicated by ultrasound. Three cases of right-sided ductus arteriosus were found by echocardiography, one of them diagnosis both in ultrasound and cast , even in fetuses with severe CHDs3. In recent years, several studies showed a variety of fetal CHDs were always associated with different ductal abnormalities4. Moreover, certain types of CHDs could even influence the ductal morphology and hemodynamics4. In these cases, sonographic features of ductus arteriosus could be diverse, which makes it hard to diagnose accurately in utero. Furthermore, prenatal echocardiography may often be affected by a series of factors, such as fetal position and gestational weeks, amniotic fluid volume, maternal abdominal wall thickness, ultrasonic resolution, etc. Hence, there are still some limitations in prenatal diagnosis of fetal CHDs by using ultrasound10.Ductus arteriosus is one of the main physiological shunts in fetal circulation, and it normally maintains open before birth. The vast majority of right ventricular output is carried by arterial duct from pulmonary trunk (PT) to descending aorta (DAo), leaving only a small amount of blood flowing to the lungs11. Cardiovascular casting is a method of injecting casting material into the vessels, corroding the soft tissues by strong acid, and leaving the real architectures of cardiovascular system12. This method can display the three-dimensional (3D) morphology of fetal severe CHDs. Its unique advantages will help to improve the understanding of fetal cardiac anatomy and the accuracy of prenatal diagnosis6. This study indicated cast models could vividly reveal the origin, course, dimension and spatial relationship of ductus arteriosus and other great vessels.In this study, we investigated the utility of vascular corrosion casting technique to assess casting effectiveness in evaluating the major blood vessels of the fetal heart and reveal the real anatomy of ductus arteriosus in different CHDs13. For instance, hemodynamic changes in ductus can only be displayed by color Doppler. As we know, blood in ductus arteriosus flows from the main pulmonary trunk to the descending aorta in normal fetus. However, some CHDs may change the hemodynamics. In our study, case 2, 3, 5 all demonstrated reversed flow across DA (from descending aorta to pulmonary artery), and were all associated with pulmonary atresia or pulmonary stenosis.Our study showed various ductal abnormalities were presented in severe CHDs fetuses. By comparing cast with ultrasound, we found more ductal abnormalities could be detected in cast models 25 vs. 18). Casts were not only helpful to confirm prenatal ultrasound results, but also beneficial to correct the missed diagnosis and misdiagnosis by echocardiography , hypoplastic left heart syndrome (HLHS), double-outlet right ventricle (DORV), aortic stenosis (AS), interrupted aortic arch (IAA)and double superior vena cava (DSVC).By using ultrasound combined with cast models, we found several different types of ductal abnormalities in these fourteen cases, which could be divided into dimension abnormality (DA stenosis and dilation), course abnormality , origin abnormality , absence abnormality (absent DA) and flow abnormality (reversed flow in DA). Eight cases had more than one abnormality. These abnormalities are always associated with complicated CHDs. And some of these abnormalities  are rarely seen. Moreover, the existence or absence of ductus arteriosus may change fetal prognosis and therapeutic strategy. Therefore, accurate prenatal diagnosis of ductus arteriosus abnormalities could help perinatal management. In our study, casting technique has its own superiority in revealing these ductal abnormalities, especially in the demonstration of abnormal course, origin and absence of ductus arteriosus. It will help the sonographers gain more insight into the real anatomy of complicated CHDs. Ductal circuity was found in six cases by casts, while only two cases were detected prenatally. Absent duct was suspected in three cases by ultrasound, while casts finally confirmed five cases with absent DA. Anomalous ductus connection was relatively rare, and we found five cases in cast models, with only three cases noticed by ultrasound. Moreover, as an unusual anomaly, one case of bilateral ductus arteriosus was detected by casting technique, which was missed in utero. In this case, the left-sided ductus was connected between left pulmonary artery (LPA) and left subclavian artery (LSA), while the right-sided ductus connected right pulmonary artery (RPA) and descending aorta (DAo). As we know, ductus arteriosus embryologically originates from the distal part of the left sixth arch, and the right sixth arch degenerates eventuallyCasting and ultrasound both have their own superiorities. Ultrasound is able to display hemodynamic changes by color Doppler, while casting could not. So, the flow direction changes in ductus arteriosus will certainly be missed on cast specimens. On the other hand, casting can demonstrate the real 3D architecture of cardiovascular system, while ultrasound usually display sonographic anatomy in 2D planes. Some abnormalities, such as anomalous ductal connection and bilateral ductus, the course and origin of ductus arteriosus is hardly to demonstrate in single plane by 2D ultrasound. 3D ultrasound may help to reveal these ductal abnormalities, but in fact, these vessels are always too tiny or too curved to be clearly observed or demonstrated.19. Comparing with ultrasound, casting technique has its own superiority in exhibiting ductus abnormalities, especially in certain types such as course, origin and absence abnormalities of ductus. Casting is of great value for sonographers and clinicians to better understanding the spatially anatomy of fetal ductus and even the whole cardiovascular system, and is helpful to improve the diagnostic capability of prenatal echocardiography20.We successfully applied the modified vascular corrosion technique to prepare fetal cardiovascular cast models. Cardiovascular cast models can not only effectively show the diameter of the major blood vessels of the fetal heart, but also visually display the anatomical characteristics of ductus arteriosus, and could be successfully used in the demonstration of ductus abnormalities in fetuses with severe CHDs


Histology is used to identify Kaposi’s sarcoma (KS) in countries with low resources to fund healthcare costs. Approximately 95% of KS cases can be detected using a polymerase chain reaction.To determine the presence of the open reading frame 75 (ORF75) gene associated with Kaposi’s sarcoma herpes virus among HIV-1/AIDS patients and to describe morphological presentations of KS.This was a retrospective, descriptive study of archived tissue blocks collected from 2013 to 2016. Haematoxylin and eosin staining was used to identify KS. Deoxyribonucleic acid from archived tissue blocks was extracted and a nested polymerase chain reaction was used to detect the ORF75 gene.p = 0.002). The ORF75 gene was detected in 60.5% (49/81) of tissue blocks; 27.2% were men (22/81) and 33.3% were women (27/81). The ORF75 gene was observed to be present in up to 15.4% (2/13) of the cases reported to have KS-like features.All 81 cases in this study had been diagnosed as HIV-1 positive, of which 68 had hallmark features of KS in the histology report and 13 had features suggestive of KS (‘KS-like’). Microscopic identification of KS by haematoxylin and eosin staining was considered a significant indicator of KS herpes virus ORF75 gene positivity (Following the initial diagnosis of KS by histology, the ORF75 gene was fur-ther detected from both cases that had hallmark features of KS as well as among cases with KS-like fea-tures. During the lytic cycle, the ORF75 genes are expressed resulting in the manifestation of KS.7 The ORF75 gene product has been proven to aid in lytic replication and enhancement of virus pathogenesis in host cells.8Kaposi sarcoma (KS) is a tumor formed from blood vessels; it later shows lesions on the skin or organs of HIV-positive people.11 The dis-tinct feature of HIV-associated KS is that it might affect the lymph nodes, gastrointestinal tract, lungs or liv-er.13 Despite the fact that saliva is the main route by which KSHV is transmitted,14 HHV-8 has been isolated from other body fluids.16 The main route of HHV-8 transmission to the opposite sex is through sexual relations.17 The pathogenesis of KS presents as an abnormal neoangiogenesis, proliferation of cancer cells and inflammation of endothelial cells.18 A classic KS lesion manifests various features ranging from maculopapular to nodular or plaque-like and, in most cases, is pain-less.21Kaposi’s sarcoma is listed among the defining malignancies of HIV/AIDS.22 Since KS is common among HIV/AIDS pa-tients,13 early detection of KSHV is essential in disease monitoring.22 The sensitivity of diagnostic tests for detection of KSHV depends on the sample selected for analysis.23 For example, biopsies obtained from patients with HIV/AIDS-KS were found to yield better results compared to using peripheral blood mononuclear cells from the same patient.23 Identification of KS in tissue biopsies by use of histological staining techniques should not be underestimated.24 In tissue biopsies, microscopic examination involves identification of proliferated spindle cells and oedema.25 Clinical diagnoses of KS have been shown to have limited predictive value.26 The use of molecular techniques such as polymerase chain reaction (PCR) permits the detection of the HHV-8 gene even for patients who present with early vascular lesions that histological techniques might miss.27 The use of PCR in the diagnosis of KS can detect approximately 95% of cases.28 The HHV-8 DNA has been successfully amplified using nested PCR previously.30 This study was aimed at determining the presence of the ORF75 gene linked to KSHV among HIV-1/AIDS patients. In addition, the objective of this current study was to describe the morphological presentations of KS among the studied cases.In sub-Saharan Africa, KSHV is endemic and approximately 84% of worldwide cases of KS occur in this region.The present study only included patients aged 18 years and older. Data on clinical information that was useful for this study were extracted from the registry records with the help of the data clerk. The following data were obtained from the registry records: sex, age, HIV-1 status, if patient was on antiretroviral or Highly Active Antiretroviral Therapy treatment, anatomic location of KS lesions, number of KS lesions, distribution of KS lesions, cluster of differentiation 4 cell count and histology diagnosis.Study approval number P682/11/2014 was assigned by Kenyatta National Hospital/University of Nairobi Ethics and Research Committee.A cross-sectional, descriptive, hospital-based study was used. Formalin-fixed, paraffin-embedded tissue blocks were retrieved from archives following histological reports of the patients who were diagnosed with KS or KS-like disease between 2013 and 2016. A consecutive sampling technique was used to select the archived tissue blocks from Thematic Unit of Anatomic Pathology, Department of Human Pathology, College of Health Sciences, University of Nairobi, and Department of Laboratory Medicine, Cytology Section, Kenyatta National Hospital.TM PCR DNA degradation solution . Each tissue section was cut to 10 µm thick. The tissue sections were processed for haematoxylin and eosin staining and a qualified pathologist reported on the results.For this study, a total of 81 tissue blocks were selected and analysed. A rotary microtome was used to section the formalin-fixed, paraffin-embedded blocks. A different blade was used for every formalin-fixed, paraffin-embedded block so as to avoid carry-over of genetic material. Once a block was cut, the microtomes surface was decontaminated using DNAZap31 ORF75 product size 804 bp Forward KS 2000 5′GGAAACAGGGTGCTGTG3′; Reverse KS 2034 5′CATGGCCTACGACGTCAC3′.32 The cycling conditions of the PCR for the targeted KS regions were similar and consisted of 30 cycles of: initial denaturation at 94 °C for 3 minutes, denaturation at 94 °C for 1 min, annealing at 63 °C for 1 min, extension at 72 °C for 1 min and final extension at 72 °C for 10 min. Amplified PCR products were analysed by electrophoresis on a 1% agarose gel containing ethidium bromide (1 µL/mL of agarose solution) and were visualised under ultraviolet light alongside a 1 Kilobase (Kb) deoxyribonucleic acid (DNA) ladder. For a positive control, a known case of KS was used. The ribonuclease-free water was used as a negative control.Isolation of DNA from tissue sections was done using a GeneRead DNA FFPE kit . The extraction kit removes paraffin and reverses formalin cross-links from tissue before DNA is bound to the QIAampMinElute column . The eluted DNA is then ready to be used for nested PCR to detect the ORF75 gene in HHV-8. A Taq PCR Core Kit  was used to detect the ORF75 gene. The set of primers used were; ORF75 product size 895 bp Forward KS 1000 5′CGGTTCGGTGGCATACAGGC3′; Reverse KS 1034 5′CTGACTACAGAGGGTGTCCCCG3′.t-tests. A p-value of less than 0.05 was considered to be statistically significant. Odds ratios in a cross-sectional study are known as prevalence odds ratios and were used as a measure of association.33The data were analysed using Statistical Package for Social Sciences version 21 ; the relationship between the ORF75 gene and clinical characteristics were tested by using chi-square and 3. Among the 81 cases, the ORF75 gene was detected in 49 cases (60.5%); 27.2% (22/81) were women and 33.3% (27/81) were men. Among cases positive for the ORF75 gene, 4.1% (2/49) were never on any form of antiretroviral therapy and 95.9% (47/49) were on antiretroviral therapy. No statistically significant association was found between the presence of the ORF75 gene and sex, antiretroviral treatment status, number of KS lesions or distribution of the KS lesions .Of the 81 tissue samples included in the study, 43.2% (35/81) were from women and 56.8% (46/81) were from men . All of n = 21; 42.9%). Age had a statistically significant association with ORF75 gene positivity .The mean age of patients with tissue blocks positive for the ORF75 gene was 41 years . Detection of the ORF75 gene was most common in the 30–39 years age group . The types of KS morphology identified included patchy, nodular, plaque and KS-like . The morP = 0.002)  and 13 cases (16%) had KS-like features . Among t= 0.002) .The amplified ORF75 genes of HHV-8 were identified by 1% agarose gel electrophoresis .36Retrieved clinical data revealed that all of the tissue blocks retrieved in the present study were collected from patients who had been diagnosed with HIV-1. These patients might have developed KS lesions due to immunosup-pression or because they were immunocomprised due to increased viral load that impaired their immune system. Other studies have also associated KS as an HIV/AIDS-defining illness.40 There is a lack of consensus as to why all forms of KS are more common among men than women.42 We hypothesise that gender-related factors such as hormones might influence the development of KS lesions. The results of this study showing KS pre-ponderance among men was consistent with the country’s published data on the distribution of malignancy cases as captured in the National Cancer Control strategy, 2017.43The findings of this study revealed that men were more prone to development of KS: 56.8% (46/81) compared with women 43.2% (35/81). This observation is concordant with others who also noted more frequent development of KS among men.46 In this study, KS lesions manifested among patients despite the fact that 77 (95.1%) were on antiretroviral treat-ment. Contrary to other findings that antiretroviral therapy alone can result in the resolution of KS,48 in our study, being on antiretroviral treatment did not have a statistically significant association with the presence of KS (P = 0.66). This finding is in agreement with another study that stated that there has been continued diagnosis of KS in HIV-positive patients, despite the availability of highly active antiretroviral therapy.49 Other studies have stated that patients infected with AIDS-associated KS respond to combined antiretroviral therapy by 50% depending on geographical location and severity of the presentation, thereby resulting in immune reconstitution and HIV suppression.52 In the current study, these manifestations of KS could be attributed to the weakening of the immune system by HIV-1.Kaposi’s sarcoma immune reconstitution occurs when a portion of AIDS-KS cases responds to the introduction of combined antiretroviral therapy with disease advancement.1 Kaposi sarcoma lesions are known to progress from asymptomatic to macule, papule, plaque and nodule forms.53 The findings of this study revealed that the KS lesions were disseminated in different body regions, including the lower limbs, upper limbs, genitalia, eyelids, palate, oral cavity and trunk (chest and back). In another study, fatality was witnessed in HIV-positive patients who had KS lesions manifested in the gastrointestinal tract, lungs and lymph nodes.54This study found patchy, plaque and nodular morphological presentations of KS. The morphological appearance of KS shows progression from plaques to nodular form and fungiform.30 Further to that, nested PCR has been used successfully to assess the prevalence of HHV-8 among HIV-positive patients in Brazil.27 A tissue biopsy excised from a KS lesion has been shown to have high viral load; hence, biopsies are the ideal sample for the detection of KSHV DNA.55 The detection of the ORF75 gene implies that this gene was present in 49 (60.5%) of the studied cases.The decision in this study to use tissue biopsy for detection of the ORF75 gene of HHV-8 is in agreement with another study that supported the use of tumor biopsies as suitable for viral DNA identification due to high viral load as opposed to the use of blood.56 Cases in the present study had a dark skin pigmentation. In another study of dark-skinned patients, KS had been confirmed to mimic a number of non-KS-like dermatological conditions.56 In another study, it was observed that it is difficult to identify KS in dark-skinned individuals, who presented with violaceous skin lesions.56Our study used the haematoxylin and eosin staining technique and the nested PCR method for detection of the ORF75 genes of the KSHV. However, HHV-8 immunohistochemical biopsy has been demonstrated to be the ‘gold standard’ for KS diagnosis.28 In addition, the PCR technique can detect approximately 95% of all KS cases.28 However, the cost associated with the use of PCR is quite high, which would limit the clinical application of HHV-8 DNA detection in resource-limited facili-ties.28The use of the PCR technique in the detection of KSHV has been shown to give the utmost specificity compared to the use of tests that determine exposure to infection.The present study considered microscopic detection of KS by haematoxylin and eosin as a significant indicator of KSHV ORF75 gene positivity. It therefore recommends the use of both clinical diagnosis and routine microscopy in the diagnosis of KS in resource-limited facilities. However, among individuals with dark skin pigmentation, there is the need to employ the use of a robust diagnostic technique to ascertain the true causative agent.The presence of the ORF75 gene of KSHV among immunosuppressed patients due to HIV-1 was successfully detected. Following the initial diagnosis of KS by histology, the ORF75 gene was further detected from both cases that had the hallmark features KS and those that had KS-like features. Microscopic detection of KS by haematoxylin and eosin should be considered a significant indicator of KSHV ORF75 gene positivity.


In general, composite materials are difficult to recycle. Tires belong to this class of materials. On top, one of their main constitutents, vulcanized rubber, is as elastomer, which cannot be remolten and hence is particularly challenging to put to a new use. Today, the main end-of-life routes of tires and other rubber products are landfilling, incineration in e.g., cement plants, and grinding to a fine powder, generating huge quantities and indicating a lack of sustainable recycling of this valuable material. True feedstock recycling is not feasible for complex mixtures such as tires, but devulcanization can be done to reactivate the cross-linked polymer for material recycling in novel rubber products. Devulcanization, i.e., the breaking up of sulfur bonds by chemical, thermophysical, or biological means, is a promising route that has been investigated for more than 50 years. This review article presents an update on the state-of-the art in rubber devulcanization. The article addresses established devulcanization technologies and novel processes described in the scientific and patent literatures. On the one hand, tires have become high-tech products, where the simultaneous improvement of wet traction, rolling resistance, and abrasion resistance  is hard to achieve. On the other hand, recycling and sustainable end-of-life uses are becoming more and more important. It is expected that the public discussion of environmental impacts of thermoplastics will soon spill over to thermosets and elastomers. Therefore, the industry needs to develop and market solutions proactively. Every year, approximately 40 million tons of tires are discarded. Through the devulcanization of end-of-life tires (ELT), it is possible to produce new raw materials with good mechanical properties and a superior environmental footprint over virgin products. The devulcanization process has become an interesting technology that is able to support the circular economy concept. With increasing global populations and welfare, consumption has been surging. Polymers—thermoplastics, thermosets, and elastomers—have shown significant growth over more than six decades from the 1950s onwards, with thermoplastics being by far the largest group. In 2018, the production volume has approached 350 million tons , one canIn the case of tires, which are a complex product made from completely different raw materials such as steel, cord, natural and synthetic rubber, additives, etc., full feedstock recycling will not be feasible, i.e., obtaining the original constituents or monomers. “Feedstock recycling” and “devulcanization” are two terms that are rather not to be used interchangeably, since the ambition is different. The expressions “depolymerization” or “molecular rearrangement” hit the meaning of devulcanization better.True feedstock recycling can be considered the “holy grail” of plastics recycling in that the monomers are obtained from collected scrap, and then, they are captured and reused. However, this route has not yet been developed sufficiently, and many approaches are still at a low technology readiness level. Another project in this direction is the Austrian mineral oil company OMV’s ReOil project, where 100 kg/h of plastics waste can be converted into a type of synthetic crude .For thermosets, recycling as for thermoplastics is not feasible, because the polymer chains have been converted into a rigid network that cannot be dissolved or molten anymore. There are some attempts to e.g., burn off the polymer matrix to recycle fibers from composite materials, which in an energy-efficient process can make sense for high-value materials such as carbon fibers. For elastomers, recycling options are strongly limited, too, because the polymer is also a network. Elastomers cannot be molten nor be dissolved. One of the huge volume applications of elastomers is tires, in which natural rubber is used next to a mix of synthetic rubbers. By vulcanization or curing, the properties of the natural rubber compounds are finalized . However, the biodegradability of the raw materials (mostly latex) is thereby lost. Tires are produced (and discarded) on the order of 40 million tons per year on a global basis, and they have become a huge environmental concern.Whereas waste tire dumps are visible to the public and are of general concern, end-of-life options for tires include incineration in cement plants and grinding them to a fine powder for addition into asphalt or concrete, which are rarely discussed in the general public. The attrition of tires on the roads which leads to microplastics formation is studied and discussed even less , althougIn the case of tires and rubber in general, feedstock recycling would be a very beneficial approach. For more than five decades, the devulcanization of rubber has been studied. Different technologies have been developed, and some of them have already made it to the market. This review article provides an update on the state-of-the-art in rubber devulcanization with an outlook on potential future developments.By vulcanization, as invented by Goodyear, sulfur can form bonds between unsaturated polymer chains found in latex to yield natural rubber. The process is also used for synthetic rubber. Accelerators can be added in the process, which is carried out at elevated temperatures. Accelerated sulfur vulcanizations are classified into three different types such as conventional (CV), semi-efficient (semi-EV), and efficient vulcanization (EV) depending on accelerator/sulfur ratio (A/S) between 0.1 and 12 .Common vulcanization accelerators are MBT, TBBS, TMTD, DPG, and CBS; for definitions, see Vulcanization gives the properties to natural or synthetic rubber; it renders the material into an elastomer.Tires are used on all sorts of vehicles. After several years, they need to be replaced, because their profiles have become worn out, and/or they have become brittle.Retreading is done for truck tires, while passenger car tires are single-use items. End-of-life-tires (ELTs) can be mainly recovered through two routes: the recovery of material and the recovery of energy . The calElastomers such as rubber are cross-linked, which prevents simple recycling, as it can be applied to thermoplastics ,16. The The higher the selectivity of the devulcanization process, the better will be the mechanical properties of the material. Horikx  has deveIt is estimated that 70% of global rubber production goes into tires, which consist of up to 60% of natural and synthetic rubber . TherefoTruck tires contain more natural rubber than tires for passenger cars, because they are subjected to more mechanical stress, which natural rubber sustains better .Truck tires typically contain natural rubber (NR) and synthetic rubber  ,21.The basic composition of tire rubber is shown in The tire rubber composition as shown in Regarding the useful life, there are two types of tires: the reusable tires and the non-reusable tires. The reusable tires are sent to tire retreading companies, providing them a new tread and run into service again. The non-reusable are tires that cannot be retreaded due to an advanced damage, structural deformation, or high degradation. These tires are the starting materials for recycling, according to Asaro et al. .For devulanization, waste rubber tire (WRT) material is typically first processed into ground tire rubber (GTR). While waste tires are often just landfilled or burnt in an ill-controlled manner, GTR can be processed into rubberized asphalt ,24, bituTo improve these, devulcanization has been researched for more than five decades . x-) bonds in the rubber matrix are cleaved.In the process, monosulfidic, (C-S), di-sulfidic (S-S), and polysulfidic  can be replaced by rubber recycled with devulcanized material . In gene2 (scCO₂) has been suggested [2 is chemically inactive, non-toxic, non-flammable, and inexpensive. Its critical point can be reached easily , and residual scCO₂ in the devulcanized rubber is removed easily.For the thermochemical approach in an extruder, the use of supercritical COuggested ,38. CO2 3) and benzoyl-peroxide (C14H10O4) was studied [As a chemical method, the oxidation of sulfur bonds using nitric acid , were also studied for thermochemical devulcanization by Zhang et al. . In that2O in organic solvents.Ghorai et al. ,51 propoSabzekar et al.  deployed2 (with DD) and ultrasonic methods as bulk treatments involve a high amount of energy and chemicals; however, they are quite selective processes. On the other hand, the biological process  is limited to the surface and is highly selective toward sulfur; it requires a low amount of energy and chemicals [Mangili et al. have compared different devulcanization methods . The scChemicals . Howeverhemicals .To study devulcanization, researchers have used ground tires, or they have prepared fresh ground natural rubber (GNR). For instance, in , Ghorai To analyze the quality of devulcanization, energy-dispersive X-ray (EDX), Fourier transform infrared spectroscopy (FTIR), field emission scanning electron microscope (FESEM) and thermogravimetric analysis (TGA) were used by Saputra et al. .In addition, solvent extraction and swelling, as well as attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, were applied by de Sousa at al. , alongsiIn order to reduce the processing costs of “full devulcanization”, the dynamic vulcanization of GTR/plastic blends was proposed. This is a cross-linking process between GTR and a plastic matrix initiated by sulfur ,57 or peAnother approach is to limit devulcanization to the surface of ground rubber tire powder. Thereby, particles can be reactivated to incorporate them into a new polymer matrix. This was investigated for PE by Jiang et al.  to prepaSurface devulcanization was achieved using intense shear and tetraethylenepentamine (TEPA), and then amine groups were grafted to the surface of devulcanized GTR by Jiang et al. . In thatAn innovative devulcanization/rubber reclaiming method is presented in  by DobroFor revulcanizing the devulcanized rubber, the following recipe has been suggested.On 100 parts of devulcanized rubber, 2 parts of stearic acid, 4 parts of ZnO, 1.5 parts of CBS (N-cyclohexyl-2-benzothiazole sulfonamide), and 1.5 parts of S were used by De et al. .In , 100 par*Conservation of natural resources *Conservation of energy *Avoidance of uncontrolled or high-emission end-of-life scenarios such as dumping or burning.*Cost savings for goods producers, since the devulcanized material is cheaper than its replacement, natural rubber.Reclaiming rubber from end-of-life products such as tires bears several advantages, as elaborated on by the pertinent literature : *ConserSabzekar et al. succeeded in adding 40% of reclaimed (devulcanized) rubber to natural rubber without a significant decrease in the mechanical properties .It was found by Seghar et al. that up to 65 wt % of virgin natural rubber (NR) can be replaced by rubber recycled with devulcanized material .Several studies confirm that devulcanized rubber can be reprocessed into rubber products such as tires without adverse effects.Some of the studies have used laboratory equipment, such as roller mixers, while others have utilized industrial equipment such as co-rotating twin-screw extruders.There are commercial offerings available, e.g., by Tyromer , Phenix The literature bears a wealth of information on rubber devulcanization, which can be achieved by thermal, thermochemical, mechanical, and biological means. The process as such has a good environmental performance, since virgin materials and energy are conserved. In addition, it can bring about significant cost savings. The general recycling hierarchy, which also applied to tires, is summarized in the following Reuse is better than recycling, and a material recycling path is to be preferred over feedstock recycling due to the lower energy requirements. Energy recovery should be the last step of a cascaded use model. Landfilling in general should be avoided. Although carbon is being sequestered, the burying of organic, reactive materials bears risks, and waste tire dump fires have been reported previously, see e.g., Escobar-Arnanz et al. ,64.The same properties for rubber that has been devulcanized and revulcanized as for virgin material were reported by Ghosh et al. .(a)make raw material manufacturing  more sustainable(b)make attrition to microplastics particles from tires less harmful, i.e., biodegradable. This might be achieved through suitable bioplastics materials.Apart from addressing the recycling of large volume rubber product streams such as tires, solutions need to be found to:Natural rubber today is mainly produced from the latex of the rubber tree or others. The rubber tree is grown in tropical areas, where plantations have often been established on previous rainforest land. Due to its nature to partially crystalize, natural rubber is harder than synthetic rubber, and it will give a longer lifetime to tires. This is also the reason while truck tires, which can run for well over 100,000 km , containOther approaches to make tires more sustainable can be found in the use of alternative raw materials. For instance, Midhun et al. have suggested replacing carbon black (CB) by rice husk derived nanocellulose (RHNC) ; see alsFeedstock recycling can be considered the ultimate goal for polymers in that the original monomers are recovered. It seems feasible for some pure polymers. For complex product mixtures such as tires, feedstock recycling back to isoprene and the other constituents seems not feasible today. However, a process to reverse vulcanization, and hence make the elastomeric material meltable and processable again, is devulcanization. Devulcanization offers a route to recycling end-of-life tires back into high value-added products, so that virgin natural and synthetic rubber can be partly replaced and saved with economical and environmental benefits. This review article has provided an update on the state-of-the-art in rubber devulcanization, as a promising alternative to tire landfilling, grinding to powder and incineration in cement plants. Previous reviews are e.g.,  by Manza2 as the sole carbon source. This offers the potential for biodegradable tires, where attrition would be significantly less harmful due to is shortened lifetime. In addition, microbial production would not require arable land and avoid competition with feed—and food—production, which is an issue often raised against biofuels and bioplastics. It is expected that the world fleet of cars will continue to grow, and that tires will be needed in future in large quantities. Therefore, sustainable end-of-life options are necessary, and more sustainable raw materials need to be sought. The circular economy concept needs to be extended to elastomers such as rubber and products made out of rubber. The devulcanization technology is a promising route with a realistic potential for large-scale implementation in the near future. Therefore, governments and the EU must introduce new laws for the circular economy and support companies to develop even more efficient recycling technologies.Another area in need of more technology development is the natural rubber feedstock base. Today, it is dominated by latex from rubber trees. Alternative isoprene sources such as fig tree milk offer the potential to be more sustainable in terms of land usage, transportation, and cultivation requirements. Medium chain-length polyhydroxyalkanoates (mcl PHA) are biodegradable, and they can either be made from carbohydrates or through photoautotrophic microorganisms using CO


Metagenomics can unveil the genetic content of the total microbiota in different environments, such as food products and the guts of humans and livestock. It is therefore considered of great potential to investigate the transmission of foodborne hazards as part of source-attribution studies. Source-attribution of antimicrobial resistance (AMR) has traditionally relied on pathogen isolation, while metagenomics allows investigating the full span of AMR determinants. In this study, we hypothesized that the relative abundance of fecal resistome components can be associated with specific reservoirs, and that resistomes can be used for AMR source-attribution. We used shotgun-sequences from fecal samples of pigs, broilers, turkeys- and veal calves collected across Europe, and fecal samples from humans occupationally exposed to livestock in one country . We applied both hierarchical and flat forms of the supervised classification ensemble algorithm Random Forests to classify resistomes into corresponding reservoir classes. We identified country-specific and -independent AMR determinants, and assessed the impact of country-specific determinants when attributing AMR resistance in humans. Additionally, we performed a similarity percentage analysis with the full spectrum of AMR determinants to identify resistome signatures for the different reservoirs. We showed that the number of AMR determinants necessary to attribute a resistome into the correct reservoir increases with a larger reservoir heterogeneity, and that the impact of country-specific resistome signatures on prediction varies between countries. We predicted a higher occupational exposure to AMR determinants among workers exposed to pigs than among those exposed to broilers. Additionally, results suggested that AMR exposure on pig farms was higher than in pig slaughterhouses. Human resistomes were more similar to pig and veal calves’ resistomes than to those of broilers and turkeys, and the majority of these resistome dissimilarities can be explained by a small set of AMR determinants. We identified resistome signatures for each individual reservoir, which include AMR determinants significantly associated with on-farm antimicrobial use. We attributed human resistomes to different livestock reservoirs using Random Forests, which allowed identifying pigs as a potential source of AMR in humans. This study thus demonstrates that it is possible to apply metagenomics in AMR source-attribution. Salmonella Typhimurium infections in humans using core-genome multilocus sequencing  bacteria and AMR determinants. The magnitude of such transmission has, however, been only limitedly estimated to date, usually targeting a single combination of pathogen-resistance type, e.g., ESBL-AmpC-producing E. coli , or by aE. coli . TraditiE. coli , which sDue to the genetic nature of antimicrobial resistance, and the possibility of horizontal transfer of resistance genes between bacteria of different species, we propose here to assess transmission of AMR by considering the abundance of genetic determinants of resistance derived from metagenomic sequencing . Metagenomics has indeed been previously demonstrated to be a valuable asset in trace-back studies to determine the origin of AMR contamination in water bodies . We usedThis study allowed to predict the relative attribution to different animal reservoirs of AMR determinants present in human resistomes, and thus sets the scene for future source-attribution studies of AMR transmission using metagenomics.1. Sampling protocol, number of samples collected, DNA extraction, sequencing method and diversity analysis of the resistomes have been described in detail elsewhere , pig farm workers (n = 54) and pig slaughterhouse workers (n = 70) have been described elsewhere , and turkey and veal farms in three among those nine countries . Individlsewhere . Here weThe procedures for DNA extraction and shotgun sequencing were described in detail elsewhere for the animal samples  and for 2. Adaptor sequences and low-quality nucleotides were also removed using BBduk23. The resulting trimmed read pairs were aligned to the ResFinder database4 described by The bioinformatic analysis of the metagenomic raw reads was similar to the one described by ResFinder-mapped counts were normalized to the length of each reference sequence as well as to the number of reads in each sample, thus converting counts into values of Fragments Per Kilobase of reference and Million reads (FPKM) for each ResFinder reference sequence. Genes with many alleles in ResFinder result in unspecific mapping and randomly assigned read pairs. To avoid sensitivity loss and wrong assignments, we kept ambiguous hits, but aggregated their abundances to higher levels, corresponding to 90% gene identity clusters (herein referred to as AMR determinants). To determine these clusters, we used CD-HIT-EST (v4.6.6)  at a 90%We applied the supervised classification ensemble algorithm Random Forests (RF)  in orderHieRanFor v.1.05 and Random Forests with the package caret v.6.0–84  as the square root of the number of predictor features (AMR determinants), and we initially split the data into a training and a testing subset, corresponding to random 70% and 30% of the total number of observations, respectively. Model performance was assessed on the basis of balanced accuracy  and 4 reservoir classes (pig (181), broiler (177), veal calves (61), turkey (60)). Two hierarchy levels were defined, L1 = ‘country’ and L2 = ‘reservoir’ and the data was considered balanced at the level of the reservoir class within each country , with approximately 20 farms sampled per reservoir, per country . The modVennDiagram v.1.6.20  5 countries with pig and broiler observations only, ii) 4 countries with observations of pig, broiler, turkey and/or veal calves and iii) 2 countries with observations of all four reservoirs. The overlaps were determined and visualized in Venn diagrams with the R package v.1.6.20 , and werThe aim with HRF1 was to identify country-specific and -independent AMR determinants with a positive impact on the classification of animal resistomes into corresponding reservoirs. The results of HRF1 informed the dimensionality reduction (reduction of the number of features in the model) performed in subsequent steps .We selected a set of 119 AMR determinants with MDA > 0 determined in HRF1 for the two countries where all four reservoirs were sampled. This country-independent subset of AMR determinants defined the feature space in HRF2. These AMR determinants allow distinguishing between the resistomes of animal reservoirs across all included countries. HRF2 was trained and assessed equally to HRF1. The aim of HRF2 was to assess the change in model performance after a targeted dimensionality reduction step. In order to compare the performance of HRF1 and HRF2, we investigated the percentage of concordance between the crisp class and the true class, the list of AMR determinants with positive MDA and the OOB errors at each country node, and the balanced accuracy at each terminal node (country-reservoir).upSample from the R package caret v.6.0–84  originating from country F, where the three groups of humans were sampled. RF3 considered thus an initial dataset with a total of 230 observations, distributed among the reservoirs ‘pig’ (20), ‘broiler’ (20), ‘veal’ (20), ‘turkey’ (21) and ‘human’ (149). Since turkey farms have not been sampled in country F, we assumed that consumers in country F are exposed to turkeys with resistomes similar to those that were sampled in country B, which represents the major turkey meat provider to country Fry F see . The algsimper from the R package vegan v.2.5–5   in order v.2.5–5 . The AMR v.2.5–5 . In each v.2.5–5 , applied v.2.5–5  and the . 1.0.12 . An idenWith the first hierarchical Random Forests model, HRF1, we identified a subset of AMR determinants that allows an accurate classification of a resistome into its animal reservoir, independently of the country of origin. This was achieved by identifying those AMR determinants with a positive mean decrease in accuracy concurrently in i) two countries where all four reservoirs were sampled (119 determinants), ii) four countries where more than two reservoirs were sampled (77 determinants), iii) five countries were only broilers and pigs were sampled (35 determinants) . We obseThe performance of the HRF2 model with dimensionally reduced data (119 AMR determinants) was compared to the performance of HRF1 with 389 features. The overall model accuracy was lower in HRF2 (mean accuracy = 0.826) compared to HRF1 (mean accuracy = 0.833), however, the distribution of the OOB errors in HRF2 were comparable to those of HRF1 for the classification of reservoirs within countries, for most countries . CountriOverall, HRF2 represented neither an overall improvement nor an overall decrease in performance compared to HRF1. Nonetheless, HRF2 resulted in a clear decrease in the proportion of AMR determinants important for country-specific classification and an increase in the proportion of those important for classification in a larger number of countries . This reThe models RF1, RF2 and RF3 were used to probabilistically attribute human resistomes to reservoirs, under different hypotheses see . RF1 wasRF3 was a country-specific model, trained on animal and human samples collected in country F . The predictor AMR determinants were those identified as specific to country F in model HRF1 see . RF3 hadWe assessed the pairwise dissimilarity between reservoirs across all countries and observed that among the four food-producing animals, the highest resistome dissimilarities were between broiler-veal calves and broiler-pig . Opposingly, the two reservoirs with most similar resistome compositions were pig and veal calves (38.9%). Compared to human resistomes, dissimilarity was highest for broiler and turkey  and lowest for veal calves and pig . The mean of all pairwise resistome dissimilarities was 66.9%. The top 10 AMR determinant contributors to each reservoir pairwise dissimilarity are indicated in aph(3’)-II, aph(3’)-IIIa and sul2 as markers of veal calves resistomes, tet(A), tet(L) and tet(S/M) as markers of turkey resistomes, ant(6)-Ia as marker of pig resistomes, lnu(A) as marker of broiler resistomes and cfxA6 as marker of human resistomes. Additionally, erm(F), mef(A), tet(40) and tet(Q) were identified as resistome signatures primarily in veal calves, and secondly in pigs in comparison with other reservoirs, while blaTEM-126 and erm(B) were signatures primarily in broilers or turkeys. The determinant cfxA2 was identified as a marker of veal calves resistomes primarily and secondarily of human resistomes. Nineteen unique AMR determinants were identified among the top contributors in all one-to-one reservoir comparisons . For eacant(6)-Ia, mef(A) and tet(40) were identified as ‘pig’ signatures, blaTEM-126, lnu(A) and erm(B) as ‘broiler’ signatures and cfxA2, cfxA6 and tet(Q) as ‘human’ resistome signatures. Three determinants – lnu(C), tet(O/W) and tet(W/32/O) – were always more abundant in the animal resistomes than in the human resistomes. The dissimilarity analysis performed separately for each human group against pigs and broilers  showed tIn order to assess the significance of the contribution of individual AMR determinants to the pairwise dissimilarities here investigated, multiple regression studies are needed including the abundance of selected determinants as predictors of reservoir for each pairwise combination. Additionally, the significance of the reservoir signatures here identified can be further determined via discriminant analysis.Supervised classification algorithms have been increasingly applied to trace back particular microbial contamination  or antimOne of the main challenges in the application of classification algorithms to highly dimensional data, such as metagenomics, is the identification of the set of features that results in the ideal discrimination level for future predictions . This chApplying a hierarchical model, with ‘country’ as one of the hierarchy levels, allowed, however, not only to reduce the number of features in subsequent steps, but also to identify country-specific and country-independent AMR determinants with a positive impact on the classification of animal resistomes into corresponding reservoirs. This step was important to subsequently define and assess a general source-attribution model, independent of the country of origin of the reservoirs. When assessing the performance of the first hierarchical Random Forests model, we observed that the number of AMR determinants necessary to classify a resistome across countries, i.e., in a country-independent manner, increases with a larger heterogeneity of reservoirs among the samples. While 35 AMR determinants could be coincidently used to attribute a resistome to the sources ‘pig’ or ‘broiler’ across five countries, our method selected 119 determinants to differentiate between the resistomes of pigs, broilers, turkeys and veal calves across two countries only. The reduction of predictor features to those most country-independent in a second hierarchical Random Forests model did not result in an overall improvement in model performance. However, improvements in classification were observed individually for resistomes originating in countries A, E and I. For these countries, there was a decrease in OOB error and/or an increase in percentage concordance between predicted- and true- country class and improved accuracy in the prediction of two reservoir classes. Contrarily, classification accuracy overall decreased for countries G, D and F, suggesting that the impact of country-specific resistome signatures in prediction may be higher in these countries. The performance of the second hierarchical Random Forests also showed an improvement in the classification of resistomes of broilers and turkeys, opposite to a decrease in prediction accuracy for resistomes of veal calves and pigs.We built three flat Random Forests models under different hypotheses and assessed their differences in prediction performance. The first model, trained exclusively on animal resistomes, proved obviously inadequate to correctly attribute the source of human resistomes, illustrating the need to account for an ‘unknown,’ ‘other,’ or ‘human’ source while building a source attribution of antimicrobial resistance. However, the predictions of this model were in accordance with those of the second Random Forests, in the fact that they both indicate that among the four livestock species considered, pigs seem to have the resistome composition closest to the composition of the human resistome. The third model, trained on observations of a single country and on a set of country-specific resistome signatures, showed a slight increase in accuracy during training compared to the previous (country-independent model) and a decrease in the proportion of votes attributed to ‘pig’ among samples collected from broiler farm workers. This finding supports the indication that occupational exposure to AMR determinants was higher among workers exposed to pigs than workers of broiler farms. Despite that difference between the models, both showed that workers on pig farms had resistomes more often attributed to the source ‘pig’ than workers in pig slaughterhouses. Furthermore, the country-specific model showed that, selecting the appropriate resistome signatures of the reservoirs sampled in that country, may be needed in order to improve attribution results and detect or strengthen the evidence for particular transmission links. This may not, however, be necessarily true for every country. For example, an overall improvement in prediction was observed for countries A, E and I when the feature space was reduced to a set of country-independent resistome signatures. The fact that country F was more difficult to classify with the same feature space may indicate that the country-independent set of features did not include important resistome signatures for source differentiation within this country.Lactobacillus isolated from chickens (erm(B) was highly prevalent in both cases, the gene lnu(A) was more commonly found among chicken-derived isolates.The results of the dissimilarity analysis between reservoirs clearly showed that human resistomes have a composition closer to resistomes of pigs and veal calves than to those of broilers and turkeys. Surprisingly, resistomes of the two poultry species were less similar than the resistomes of pigs and veal calves. Further studies that investigate differences in the microbiome of and account for antimicrobial use practices in different reservoirs may help to elucidate the dissimilarities here estimated. However, two recent studies have shown differences in the susceptibility to antibiotics of chickens  and fromchickens  in the slnu(A)) or predominant (blaTEM-126) markers for the reservoir broiler were also identified as exclusive of this reservoir’s core resistome in that study. In another study, the determinant lnu(A) was also one of the most prevalent (39%) AMR genes identified in Lactobacillus isolated from chicken (ant(6)-Ia) or predominant (erm(F), mef(A), tet(40), tet(Q)) markers of pig resistomes, were previously identified as part of both broiler’s and pig’s core resistomes. This difference between studies in the exclusive association of AMR determinants to a single reservoir’s resistome could be due to the fact that our study additionally included comparison to the resistomes of turkeys and veal calves.At least 50% of the resistome dissimilarity between every two reservoirs included in our study can be explained by a set of 10 AMR determinants. Among these top contributors to dissimilarity, we identified resistome signatures for each individual reservoir. These signatures are to be considered generally country-independent, since the analysis was based on the full set of 389 AMR determinants. We also assessed the similarity of the resistome of each human group  to the resistomes of broilers and pigs. The results clearly showed that the overall dissimilarity between pig resistomes and the resistomes of pig farm workers was lower when compared to the dissimilarity to the other two human groups, while dissimilarity between broiler resistomes and the resistomes of the three groups was similar. This finding suggests the occurrence of transmission of AMR determinants between workers and pigs within the farm environment. This is also supported by the clustering of samples based on the relative abundance of AMR determinants with a top contribution to dissimilarity, since samples from pig farm workers are those occasionally overlapping with samples from pigs. Resistome signatures found for pigs and broilers in this study included resistance genes previously also identified as elements of the ‘core resistome’ of those species, using the same set of samples . AMR det chicken . Genes herm(F), tet(40), or mef(A), respectively. Here, we identified these three AMR determinants as resistome markers predominantly in pigs. erm(B) or blaTEM clusters, respectively. We identified both AMR determinants as resistome markers predominantly in broilers. The interpretation of our findings in the context of those association studies suggests that the continued and generalized use of certain antimicrobials in specific production settings eventually will lead to the definition of the resistome composition, not only within individual farms or individual countries, but ultimately at a reservoir level, and may ultimately influence the dynamics of the transmission of antimicrobial resistance from livestock animals to humans.The same pig and broiler resistomes have also been recently assessed for associations with lifetime antimicrobial use at farm level. cfxA2 and cfxA6, that we identified here as resistome markers of the humans included in the study, have been identified in β-lactamase-producing oral anaerobic bacteria such as Prevotella spp. and Bacteroides spp. , tet(Q), erm(F) and tet(40), which were here identified as predominantly originating from pigs.In accordance with our findings, a study analyzing the same human occupational populations  previousIn this study, we showed that it is possible to use metagenomics data to attribute the occurrence of fecal antimicrobial resistance in humans to different livestock reservoirs. We used Random Forests algorithms and dissimilarity analysis (SIMPER) to identify country-specific and reservoir-specific resistome markers that can be used to design targeted source-attribution studies. The need for country-specific models needs to be assessed on a case-by-case basis, as countries may present different heterogeneity between reservoirs’ resistomes. In one of the countries, the results suggested a link between occupational exposure to pigs and the presence in human stools of antimicrobial resistance determinants mainly associated with pigs. This finding must be interpreted with caution, since gut resistome is expected to be influenced to some degree by gut microbiome composition, and among the reservoirs included in the study, pigs may have the gut microbiota closest to humans. Further studies are needed considering the relationship between microbiome and resistome in the different species and assessing the microbiome similarity between humans and livestock reservoirs.Additionally, some of the limitations of the present study need to be further addressed, including expanding the sampling of human resistomes to more than a single country and to the general human population , and collecting resistome samples of all livestock species considered for each country in the analysis. Furthermore, the number of samples collected for each population  and subgroup within a population  should be balanced.Despite the need for future improvements, this study sets the scene for the implementation of metagenomics in source-attribution in the food chain, and thus for an alternative, resistome-based approach to assess transmission of antimicrobial resistance.Members of the EFFORT consortium: H. Graveland (2), D.J.J. Heederik (2), L.A.M. Smit (2), H. Schmitt (2), R.E.C. Luiken (2), D.J. Mevius , A. van Essen (4), D. Ceccarelli (4), A. Hesp (4), J. van der Goot (4), B. Gonzalez- Zorn (6), G. Moyano (6), P. Sanders (7), C. Chauvin (7), J. David (7), A. Battisti (8), A. Caprioli (8), T. Blaha (9), K. Wadepohl (9), M. Brandt (9), P. Munk (1), D. Wasyl (10), M. Skarżyńska (10), M. Zajaç (10), H. Daskalov (11), H.W. Saatkamp (12), K.D.C. Stärk (13), P. Joosten (14), S. Sarrazin (14), J. Dewulf (14).(6) Complutense University of Madrid, Madrid, Spain; (7) French Agency for Food, Environmental and Occupational Health and Safety, Fougères, France; (8) Istituto Zooprofilattico Sperimentale del Lazio e della Toscana, Rome, Italy; (9) University of Veterinary Medicine Hannover, Bakum, Germany; (10) National Veterinary Research Institute, Pulawy, Poland; (11) National Diagnostic Research Veterinary Institute, Sofia, Bulgaria; (12) Wageningen University and Research, Wageningen, the Netherlands; (13) SAFOSO AG, Liebefeld, Switzerland; (14) Ghent University, Merelbeke, Belgium.https://www.ebi.ac.uk/ena, PRJEB22062, https://www.ebi.ac.uk/ena, PRJEB39685, and https://ega-archive.org/studies, ID EGAS00001003944.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: The studies involving human participants were reviewed and approved by the Medical Ethical Committee of the University Medical Centre Utrecht (NL) which confirmed that the Dutch ‘Medical Research Involving Human Subjects Act’ did not apply for the study of the EFFORT human populations  The patients/participants provided their written informed consent to participate in this study.AD: conceptualization, methodology, formal analysis, investigation, visualization, project administration, and writing of original draft. TR, LV, and AB: resources, data curation, and writing – review and editing. TP, RH, and IH: resources and writing – review and editing. FA: supervision and writing – review and editing. JW: funding acquisition, project administration, and writing – review and editing. TH: conceptualization, supervision, project administration, and writing – review and editing. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The thread–link–cut (TLC) approach has previously shown promise as a novel method to synthesize molecular knots. The modular second-generation approach to small trefoil knots described herein involves electrostatic interactions between an electron-rich bis-macrocyclic host compound and electron-deficient guests in the threading step. The bis-macrocyclic host was synthesized in eight steps and 6.6% overall yield. Ammonium and pyridinium guests were synthesized in 4–5 steps. The TLC knot-forming sequence was carried out and produced a product with the expected molecular weight, but, unfortunately, further characterization did not produce conclusive results regarding the topology of the product. Macrocycles have played a central role in the development of molecular recognition, self-assembled molecular devices, and molecular topology –6. For e1 is described (1 was designed to be a second-generation building block in the thread–link–cut (TLC) approach to molecular knots PF6 and ascorbic acid (see Scheme S2 in 1H NMR spectrum in DMSO-d6 solution at room temperature (≈24 °C) were broad, whereas the spectrum at elevated temperature  had sharper peaks. This effect of temperature on the spectrum is consistent with restricted dynamics of the backbone such as reputation +, 28; 1601.9 Da, [M + K]+, 52; 1626.8 Da, [M + Cu]+, 42. It is not surprising that the product would sequester metal cations given its electron-rich macrocyclic nature. Of course, the mass spectral data do not address whether the unknot, trefoil knot, or both were formed , rather than unimolecular threading involving solvophobic forces. The flexible and modular approach involving several guests was designed to test the lower size limits on trefoil molecular knots, as the proposed TLC sequence can produce trefoil knots with 73 or 75 backbone atoms. Several synthetic routes to bis-macrocyclic host 1 were explored and the optimized route required eight steps and proceeded with a 6.6% overall yield. Bis(ammonium) guest 2 was synthesized in five steps with a 1.9% overall yield and bis(pyridinium) guest 3 was prepared in four steps and 17% overall yield. The TLC sequence using host 1 and guest 2 produced a product with the expected molecular weight, but the data were inconclusive on whether this was the unknot, trefoil knot, or a mixture of both.The design of a second-generation thread–link–cut (TLC) approach to molecular knots was described. This differed from the first-generation TLC approach  in that File 11, TLC knot-forming scheme, experimental procedures and copies of 1H NMR spectra.Conformations of host


Using the speed vs. lactate concentration curve, the speed at lactate threshold (sLT) and parameters corresponding to sLT were calculated . In the following day, a T30 corresponding to sLT was performed and BL-T30, VO2-T30, HR-T30, SR-T30, and SL-T30 were measured after the 10th and 30th minute, and average values were used for comparison. VO2-sLT was no different compared to VO2-T30 (p > 0.05). BL-T30, HR-T30, and SR-T30 were higher, while SL-T30 was lower compared to BL-sLT, HR-sLT, SR-sLT, and SL-sLT (p < 0.05). Continuous swimming at speed corresponding to lactate threshold may not show the same physiological and biomechanical responses as those calculated by a progressively increasing speed test of 7 × 200 m.The purpose of this study was to verify the physiological responses and biomechanical parameters measured during 30 min of continuous swimming (T30) at intensity corresponding to lactate threshold previously calculated by an intermittent progressively increasing speed test (7 × 200 m). Fourteen competitive swimmers (18.0 (2.5) years, 67.5 (8.8) kg, 174.5 (7.7) cm) performed a 7 × 200 m front crawl test. Blood lactate concentration (BL) and oxygen uptake (VO Progressive discontinuous swim protocols, such as a 7 × 200 m progressively increasing speed test, are commonly used to evaluate both physiological ,2 and biIn swimming, the most frequently used method for sLT calculation is x-axis projection of the intersection of two lines connecting the three higher and four lower points of the speed lactate curve . SubsequThus, the purpose of the current study was to verify the physiological responses and biomechanical parameters during continuous swimming at intensity corresponding to the lactate threshold previously calculated by an intermittent progressively increasing speed test (7 × 200 m). We hypothesized that the calculated parameters would be verified during the continuous swimming effort.Fourteen regional and national level male and female swimmers (ten sprinters and four middle-distance swimmers) that specialized in competitive distances of 100, 200, and 400 m volunteered to participate in the study . Partici−1) low volume endurance training (~3000 to 4000 m). The study was conducted during the specific preparation mesocycle of training. All swimming tests were performed using front crawl in a 25 m indoor swimming pool with a constant water temperature of 25 °C to 26 °C and 60% ambient humidity.Physiological and biomechanical parameters calculated during a progressively increasing speed swimming test  were compared to those measured during 30 min of continuous swimming. Swimmers were tested in two sessions 48 h apart , and all+, SensLab GmbH, Leipzig, Germany). Immediately after the completion of each 200 m repetition, a mouthpiece and a nose clip were attached to swimmers during recovery. Expired air collected during the first 20 s of recovery was analyzed for oxygen uptake (VO2) using a portable gas analyzer , mean r = 0.99 (0.01); 1). VO2-sLT and HR-sLT were calculated by interpolation using linear regression of swimming speed versus VO2 (mean R2 = 0.99 (0.02), mean r = 0.99 (0.01)) or HR (mean R2 = 0.99 (0.01), mean r = 0.99 (0.01). During the 7 × 200 m test, SR was calculated by the time (T) used to complete three arm-stroke cycles (180·T−1) and SL was calculated by dividing swimming speed every 50 m (V) by SR. The time for three arm-stroke cycles was recorded using a handheld chronometer. SR-sLT and SL-sLT were calculated by interpolation using the best fit regression line of SR and SL versus swimming speed during the 7 × 200 m test (SR: mean R2 = 0.98 (0.02), mean r = 0.99 (0.01); SL: mean R2 = 0.96 (0.04), mean r = 0.98 (0.02)).All swimmers participated in a standardized swimming warm-up, and 10 min later performed a 7 × 200 m front crawl test at intensities calculated using the most recent 200 m race time and corresponding to 60%, 70%, 75%, 80%, 85%, 90%, and maximum effort. Participants were familiarized with the pace of the first two repetitions during a previous training session. During testing, one of the experimenters walked alongside the swimming pool providing guidance during each 200 m repetition. During the 7 × 200 m test, each repetition started every five minutes and 30 s with a push-off start from within the water [N, USA.; ). HR was2-T30 were measured after the 10th and 30th minute of T30, while HR-T30 was recorded continuously. SR-T30 and SL-T30 were measured every 50 m during the T30 continuous swimming session. The 10th min was used to identify variations in physiological adjustments compared to the 30th min. Nevertheless, the mean values of BL, HR, SR, SL, and speed (s-T30) measured during the T30 were used for the statistical analysis. The swimmers participated in a continuous swimming session 48 h after the completion of the 7 × 200 m test. Continuous swimming speed session was completed after a standardized warm-up that consisted of 1000 m of swimming . Ten minutes after warm-up, the swimmers started a T30 at a constant speed corresponding to sLT. During T30, the swimmers kept the individual sLT speed constant while guided by a sound signal emitted by a transmitter placed next to the ear and under the swimming cap . The swimmers were instructed to adjust their speed in order to touch the wall at each sound signal. Additionally, one of the experimenters recorded the time for each 50 m split . BL-T30 and VOt-test for dependent samples was used to examine the differences in physiological and biomechanical parameters calculated during the 7 × 200 m progressively increasing speed test and those measured in T30. Specifically, comparisons between sLT, BL-sLT, VO2-sLT, HR-sLT, SR-sLT, SL-sLT vs. s-T30, BL-T30, VO2-T30, HR-T30, SR-T30, SL-T30, respectively, were applied. Pearson r correlation coefficient was used to examine the relationship between the calculated values from a 7 × 200 m test with that measured during T30. The effect size for paired comparisons using the pooled standard deviation as denominator was calculated with Cohen’s d [d was less than 0.20, small if it was between 0.20 and 0.50, medium if it was between 0.50 and 0.80, and large if it was greater than 0.80. The 95% confidence limits (95% CL) were also calculated for the mean differences between parameters. G-Power 3.1.9.4 software [N = 14), an ES of 0.80 was required to get a statistical power value greater than 0.80. For the estimation of agreement between parameters, Bland and Altman plots were used [p < 0.05.The student ohen’s d . The effsoftware  was usedere used . SPSS so−1, mean difference (SD): −0.002 (0.010) m·s−1, 95% CL: −0.008, 0.005·m·s−1, d = −0.02, p = 0.634). A significant correlation was observed between sLT and s-T30 .sLT calculated after the 7 × 200 m test was similar with s-T30 (sLT: 1.317 (0.078) vs. s-T30: 1.316 (0.082) m·s−1, mean difference (SD): 1.3 (2.4) mmol·L−1, 95% CL: 0.00, 2.35 mmol·L−1, d = 0.83, p = 0.05; R2 = 0.001, r = 0.03, p = 0.92). A Bland and Altman plot indicated agreement between calculated and measured values : 11 (11) b·min−1, 95% CL: 6, 18 b·min−1, d = 1.24, p = 0.02; R2 = 0.06, r = 0.25, p = 0.39). Agreement between calculated HR-sLT and measured HR-T30 values was observed (2-T30 was not different compared to VO2-sLT (VO2-T30: 41.7 (6.8) vs. VO2-sLT: 42.8 (6.2) ml·kg−1·min−1, mean difference (SD): −0.88 (4.5) ml·kg−1·min−1, 95% CL: −3.22, 1.45 ml·kg−1·min−1, d = −0.16, p = 0.43) and these parameters were correlated . A Bland and Altman plot showed agreement between calculated VO2-sLT and measured VO2-T30 values (Measured BL-T30 was higher compared to BL-sLT (BL-T30: 4.7 (2.3) vs. BL-sLT: 3.4 (0.8) mmol·Ld values . During observed . Moreove0 values .−1, mean difference (SD): 4.0 (3.9) cycles·min−1, 95% CL: 1.97, 6.07 cycles·min−1, d = 1.15, p = 0.002; R2 = 0.16, r = 0.41, p = 0.15). A Bland and Altman plot indicated agreement between calculated SR-sLT and measured SR-T30 values : −0.3 (0.2) m·cycles−1, 95% CL: −0.40, −0.14 m·cycles−1, d = −0.88, p = 0.001; R2 = 0.55, r = 0.74, p = 0.003). A Bland and Altman plot showed agreement between calculated SL-sLT and measured SL-T30 values  vs. SR-sLT: 29.7 (4.1) cycles·min0 values . Measure0 values .2 was similar to measured VO2, while a higher BL, HR, and SR and lower SL were recorded during T30. Bland and Altman plots indicated agreement, although a great bias was observed for all the physiological and biomechanical parameters.The purpose of the current study was to verify the physiological and biomechanical parameters measured during continuous constant speed swimming corresponding to lactate threshold with those calculated after a 7 × 200 m test. The calculated sLT was successfully maintained during a T30 session. Calculated VOA similar speed compared to sLT was expected in T30 on account of our experimental design. This is because swimmers were guided to follow a constant speed using audio signals. While maintaining the required speed during T30, some physiological adjustments were made by the swimmers. Increased BL and HR were observed during T30, and several factors may have contributed to this increment. First, we should examine the validity of sLT calculation with the method used in the present study. Previous studies report that using the intersection of two lines provides a speed corresponding to lactate threshold similar to MLSS . However−1 [A second factor that needs to be mentioned is that lactate threshold as well as MLSS may present lactate concentration at a range of 2 to 8 mmol∙L−1 . In such−1 . Support−1  found a −1 . Nonethe−1 . It seem−1 .−1) higher HR corresponding to lactate threshold using 400 m compared to 200 m stages [−1) was observed between HR-sLT and HR-T30, possibly attributed to the training status and specialty of the swimmers. Additionally, a likely HR drift towards the last minutes of exercise attributed to cardiovascular adjustments during the long exercise duration in T30 cannot be excluded [2-T30 was no different compared to VO2-sLT. It has been indicated that oxygen uptake reaches values between ~80–100% of VO2peak at the end of an endurance training set with a duration of 15 to 30 min [2 values that were ~85% of VO2max, similar to the current study (85.5%). A combination of steady VO2 response and increased BL during a continuous exercise may be observed during continuous efforts or in very heavy exercise intensity domains [A higher HR was observed during T30, indicating a higher effort during continuous swimming. A higher HR was measured in continuous exercise compared to that calculated corresponding to lactate threshold after a 7 × 200 m test in a previous study [excluded . In conto 30 min ,27. Spec domains .SR-T30 was increased whereas SL-T30 was decreased compared to SR-sLT and SL-sLT, respectively. Such changes are associated with an increased energy cost . It is p2-sLT, HR-sLT, SR-sLT, and SL-sLT and BL-T30, VO2-T30, HR-T30, SR-T30, and SL-T30, respectively, as indicated by Bland and Altman plots and has also been confirmed previously in a homogenous group of female middle- and long-distance swimmers [2-sLT, and HR-sLT as well as SR-sLT and SL-sLT were calculated by equations obtained by the best fit of these parameters versus swimming speed, thus reducing the error of calculation. However, this was not reflected in the measured values, indicating that the observed differences represent real physiological and biomechanical gaps between predicted and measured variables. Further research may examine various mathematical models for lactate threshold calculation in swimmers.The abovementioned differences in physiological and biomechanical parameters indicate that the calculated parameters may not always correspond to the measured values during continuous swimming. However, this is in contrast to the observed agreement between BL-sLT, VOswimmers . DespiteThe physiological and biomechanical parameters calculated by a progressively increasing swimming speed test corresponding to sLT may not be verified during continuous 30 min swimming in sprint and middle-distance swimmers. Swimmers maintaining constant speed corresponding to the second lactate threshold in a long duration 30 min swimming present individualized biomechanical and physiological adjustments that may not reflect the expected responses. In this case, an additional test for verification or a different mathematical model of lactate threshold calculation may be required to provide a valid guidance of training pace. Coaches should be aware that the individual data obtained by a progressively increasing speed test should be examined thoroughly and tested in training practice before planning a training set.


Dopamine is a key neurotransmitter that regulates attention through dopamine D1 and D2-receptors in the prefrontal cortex (PFC). We previously developed an object-based attention test (OBAT) to evaluate attention in mice. Disruption of the dopaminergic neuronal system in the PFC induced attentional impairment in the OBAT. However, previous studies have not systematically examined which specific brain regions are associated with the blockade of PFC dopamine D1 and D2-receptors in the OBAT. In this study, we investigated the association of dopamine D1 and D2-receptors in the PFC with attention and neuronal activity in diverse brain regions. We found that both dopamine D1 and D2-receptor antagonists induced attentional impairment in the OBAT by bilateral microinjection into the PFC of mice, suggesting that both dopamine D1 and D2-receptors were associated with attention in the OBAT. Our analysis of the neuronal activity as indicated by c-Fos expression in 11 different brain regions showed that based on the antagonist types, there was selective activation of several brain regions. Overall, this study suggests that both dopamine D1 and D2-receptors play a role in attention through different neuronal circuits in the PFC of mice. Attention is critical to high-level cognition, while attention deficit is a hallmark of neuropsychiatric disorders . The dopIn an earlier study, we established an object-based attention test (OBAT) to evaluate attention in mice . This te2, 42) = 0.0686, p = 0.9337; objects,  = 3.7780, p = 0.0586; groups × objects interaction,  = 0.1640, p = 0.8493; Fig. 2, 22) = 0.2772, p = 0.0032, Fig. Six-week-old mice were habituated for a 1-week period in an animal facility (Fig. Figure Our study is limited because the exact role of the brain area being manipulated can be obscured by pharmacological suppression . DopaminAdditional file 1. Additional materials.


This is the first time that nanofiber containing more than one polymer having a core–sheath configuration has been generated in this way. Water-soluble polymers polyethylene oxide (PEO) and polyvinyl pyrrolidone (PVP) are used as the core and sheath layers, respectively. Core–sheath nanofibers with a diameter in the range of 331 to 998 nm were spun using 15 wt % PEO and 15 wt % PVP polymer solutions. The forming parameters, working pressure and rotating speed, had a significant influence on the size, size distribution and the surface morphology of the nanofibers generated. Overall, fibre size decreased with increasing working pressure and rotating speed. The fibre size was normally distributed in all cases, with 0.2 MPa working pressure in particular showing narrower distribution. The fibre size distributions for 0.1 and 0.3 MPa working pressure were broader and a mean fibre size of 331 nm was obtained in the latter case. The fibre size was evenly distributed and narrower for rotating speeds of 2000 and 4000 RPMs. The distribution was broader for rotating speed of 6000 RPM with a mean value obtained at 430 nm. Continuous, smooth and bead-free fibre morphologies were obtained in each case. The fibre cross-section analysis using a focused ion beam machine showed a solid core surrounded by a sheath layer. Our findings demonstrate that the pressurised gyration could be used to produce core–sheath polymer nanofibers reliably and cost-effectively with scale-up possibilities (~4 kg h The applications of polymer nanofibers are boundless and have made a significant breakthrough in regenerative medicine, drug delivery, and electronic devices over two decades ,7,8,9,10Modifying the nanofibers by incorporation of additional layer or layers can enhance the performances of functional scaffolds and architectures that are used in regenerative medicine to repair damaged tissues and develop artificial organs ,14. In aElectrospinning is a favourite and arguably the best technique to produce nanofibers. Many researchers have taken advantage of its versatility and easy to use aspects over many years despite its dependence on high-voltage . It is wPressurised gyration is an alternative electric field free production technique to process polymer nanofibers on a large scale in a single step by combining pressure and spinning of solute contained in a perforated aluminium vessel ,29,30,31In this work, we report the generation of core–sheath polymer nanofibers with a combination of two water-soluble polymers, polyethylene oxide (PEO) and polyvinyl pyrrolidone (PVP) by twin-reservoir pressurised gyration. The individual polymers (PEO and PVP) have been spun in single material pressirised gyration investigations and shown to generate nanofibers in our previous work ,31. TherMw = 200,000 g mol−1) and polyvinyl pyrrolidone (PVP)  were obtained from Sigma Aldrich . Deionised water was used as a solvent for PEO and PVP.Polyethylene oxide (PEO) . Similarly, PVP powder was dissolved in deionised water in the weight ratio of 15:85 in an airtight bottle and magnetically stirred for 24 h at ambient temperature. The surface tension and viscosity of the polymer solutions were characterised using calibrated a Kruss tensiometer  and a Brookfield viscosity meter , respectively.2). The rotational speed of the vessel can go up to 6000 RPM and the pressure can be elevated to 0.3 MPa. Before commencing the gyration experiments, the equipment was calibrated for its rotational speed. The generated fibres were collected on a stationary aluminium foil attached in the device mount 150mm away from the centre of spinning  and a fixed working pressure of 0.1 MPa. To evaluate the influence of working pressure on generation of core–sheath fibres, 15 wt % PVP and 15 wt % PEO polymer solutions were spun at different working pressure  and a fixed rotating speed of 6000 RPM.Scanning electron microscopy  on the samples was carried out at 5 kV applied voltage. Before imaging, the samples were coated with gold using an Edwards Sputter S1 50B coater  for 75 s. Fibre diameter was quantified for various forming conditions in the planar microstructure of the core–sheath nanofibers using Image J analysis. For this, more than 100 fibres were chosen at random points in the scanning electron microscopy images. Focused ion beam (FIB) milling and imaging were carried out on the samples to verify the core–sheath structure using a Zeiss NV40 FIB/SEM machine . The milling and polishing of the samples were achieved with a current of 150 pA and 80 pA, respectively. The images were obtained at 5 kV applied voltage.The polymer solution’s physical properties are important governing parameters of pressurised gyration in order to generate quality products. Thus, the viscosity and the surface tension are key processing parameters in pressurised gyration to generate defect-free fibres . A higheThe yield of the core–sheath nanofibers obtained here has been compared with other manufacturing techniques, as shown in Here core–sheath nanofibers were obtained by choosing two water-soluble polymers, PEO and PVP, as the core and the sheath fluids, respectively. The water-soluble polymer solutions had higher viscosities compared to non-aqueous polymers used in our earlier work . The visIn addition, PEO and PVP could be swapped as the sheath and core, respectively, to form the core–sheath nanofibers. The structures of PEO and PVP are different: PEO is an ether and PVP is a ketone. The molecular weight of these polymers are also different, but both are water-soluble polymers. The obtained viscosities for 15 wt % of PVP are higher than the 15 wt % PEO, even though they have the same number of solutes in a given amount of solvent. However, PVP will evaporate more slowly than PEO since it has a higher viscosity. The rate of evaporation also depends on other factors, such as temperature, humidity and blowing of gas. The temperature and humidity in this work are fixed  so the only factor that will affect the rate of evaporation is the blowing of gas. It should also be noted that the temperature of the vessel can go up during gyration, but the rise in temperature in water is not overly significant (1–2 °C) to causes a dramatic change in viscosity.If PEO is the sheath fluid, it will evaporate more quickly than the PVP core fluid and will cause the encapsulation of the inner core. Moreover, the selection of water-soluble polymers must be based on molecular weight and viscosity. The molecular weight of the polymer affects the concentration of the polymer solution, and by extension the viscosity of the solution. The greater the molecular weight, the higher the viscosity for a given polymer concentration. The polymer chain entanglement increases with an increasing molecular weight and viscosity. The other factor we must consider is the formability of the polymer. The polymer chains must deform/stretch during spinning to make fibres. For example, gelatine is not soluble in cold water, but it is soluble in hot water. If it is cooled, it will become a gel. Gels have higher viscosity, but their excessive viscosity make formability poor. Therefore, the selection of polymers must take into account the viscosity and formability of polymers.Water-soluble polymers are used in drug delivery and tissue engineering due to their cost-effectiveness, biocompatibility and non-toxicity. However, the hydrophilic and hygroscopic properties make them weaker in handling pressure. Other water-soluble polymers like hydroxyethyl cellulose (HEC) are used in the biomedical arena, paint industry, soil amendments (they improve permeability and water retention) in agriculture, coal dewatering, cosmetics, wastewater treatment, absorbent pads and gel electrolyte membranes . Moreover, water-soluble polymers could be made as a compound and grafted with other polymers to modify their physical/chemical/biological properties, e.g., combining hydrophilic and hydrophobic polymers for controlled drug release, for example grafting HEC with polylactic acid to functionalise the main polymer chain to alter the properties ,36,37.2) and will shield the core fluid. Thus, the sheath fluid evaporates more quickly than the core fluid, but both have the same boiling point and encapsulate the core fluid to form nanofibers. The interaction between the core and sheath fluid jets during spinning is negligible, and as it is a rapid process, it does not provide sufficient time for two fluids to diffuse into each other. Moreover, miscible solvents (water) will reduce the interfacial surface tension during spinning and produce thinner fibres.To further examine the fibre morphology and to verify core–sheath bicomponent formation, the nanofibers produced at a constant rotating speed 6000 RPM and different working pressures were analysed using focused ion beam milling and imaging. In this study, core–sheath nanofibers containing a polyethylene oxide core and polyvinyl pyrrolidone sheath layers were fabricated using novel pressurised gyration manufacturing technology. The effect of the processing conditions such as rotating speed and working pressure on fibre size and fibre size distribution were analysed. Overall, the fibre size decreased with increasing working pressure at a constant rotating speed. The fibre size was normally distributed for no pressure (spinning only), 0.1 and 0.3 MPa. The fibre size distribution obtained at a working pressure of 0.2 MPa showed a skewed distribution. The rotating speed had a significant influence on the fibre diameter. The fibre size decreased with increasing rotating speed. The fibre size was evenly distributed for all the rotating speeds used in this study. Overall, the surface morphology of the nanofibers revealed continuous, smooth and bead-free structures. Focused ion beam analysis confirmed core–sheath bicomponent structure formation.