The linear economy paradigm in place to date has to be seriously challenged to give way to a new school of thought known as the circular economy. In this research work, precast kerbs and paving blocks made with recycled concrete (RACC-mixture) bearing 50 wt% mixed recycled aggregate (masonry content of 33%) and an eco-efficient cementitious material as 25 wt% conventional binder replacement were evaluated to assess their intrinsic potential to replace traditional raw materials, in keeping with circular economy criteria. Therefore, precast products were subjected to mechanical strength, durability and microstructure tests and were compared to conventional concrete units . Although a class demotion was observed for water absorption and some decreases in flexural strength (26%), splitting tensile strength (12.8%) and electrical resistivity (45%) and a lower class water absorption were registered, and the recycled mixture also exhibited a greater performance in terms of compressive strength (6%), a better abrasion resistance classification and a comparable porosity and microstructure, which ensures a good concrete durability. In any case, the results showed that precast pieces were European standard-compliant, thus supporting the viability of the mixed recycled aggregates and eco-efficient cementitious replacement in footways. Millions of kerbs and pavement blocks are made every year to repair or to build new footways. They are bulk-concrete precast, made usually with natural aggregates. There is a good chance to use recycled concrete in this kind of industrial products. However, so far, there are not many studies that deal with this matter. All of them investigate the effect of using recycled aggregates in its properties ,5,6,7,8.At the European Council’s 1998 meeting at Cardiff [Against that backdrop, for a number of decades the international scientific community has produced hosts of studies attesting to the potential of secondary aggregates and pozzolanic additions sourced from construction and demolition waste, CDW (Construcition and demolition waste) as replacements for natural aggregate and primary binders, primarily clinker.Of the various types of recycled coarse aggregates in place, the one where output is highest in many EU countries is so-called mixed recycled aggregate, MRA, in whose composition masonry waste—brick, roof and indoor tiles and similar—accounts for over one-third of the total . MasonryWhereas the use of mixed recycled coarse aggregate and CDW masonry—primarily brick—powder has been amply studied, their joint application in recycled concrete manufacturing has been seldom explored by the scientific community ,13,14,15ex professo preparation of the secondary aggregates took place); this mixed recycled aggregate could be regarded as a selling point in the future acceptance of its use and could demonstrate a competitive advantage towards the conventional products, as it would surely affect the costs: in other words, as it was not explicitly conditioned or prepared for this study, but the findings revealed its intrinsic potential.In the non-structural bulk concrete formulated and tested here—RACC concrete mixture—50 wt% of the natural aggregate was replaced with mixed recycled aggregate as supplied by a waste treatment plant  were compared to recycled concrete units made with mixed recycled aggregate and eco-efficient cement containing masonry powder sourced from CDW, since those are the products that eventually the recycled precast pieces would have to substitute.The purpose of the study was to ascertain the potential mechanical strength and durability of recycled kerbs and paving blocks bearing secondary raw materials drawn from construction and demolition waste as an ideal replacement for traditional raw materials, with a view to envisaging a more responsible and environmentally sustainable future for the construction industry.The natural aggregates applied here  are ordinarily used to manufacture non-structural precast concrete paving blocks, kerbs, pan forms and similar. The aim was to prepare a control concrete with those materials as comparable as possible to the recycled concrete formulated in this study.The concrete was made with fine and coarse aggregate, both siliceous, conforming to the specifications on aggregates apt for use in concretes set out in the Spanish Structural Code  and EuroThe particle size distribution for those materials, determined further to European standard EN 933-1 , is plotThe chemical composition of the natural aggregates found with X-ray fluorescence (XRF) is given in The mixed recycled aggregates used in this study were obtained by mechanically separating, crushing and sieving construction and demolition waste at the TEC-REC: Tecnología y Reciclado S.L. CDW management plant located in the province of Madrid, Spain.Their EN 12620:2003+A1 -determinThe non-floating components in the recycled aggregate listed in The chemical composition of the European and Spanish standard , followed by aluminium (Al2O3) and iron (Fe2O3) oxides.The macroscopic composition of the masonry waste deployed, sourced from a management plant in the Spanish province of León, was 100% fired clay. Prior to use it was pre-conditioned by drying at 105 C to a constant weight, crushed in a jaw crusher, ground in a ring grinder and sieved to <63 μm. That powder was subsequently blended with ordinary portland CEM I 42.5 N cement in a shaker-mixer. The X-ray fluorescence (XRF)-determined chemical composition of the masonry powder is given in The chemical composition of the resulting recycled eco-efficient cement, a blend of 75% CEM I and 25% clay brick powder, is listed in 3 and Cl− content, minimum initial and final setting times and soundness. Further to the values for those parameters  clay, added during cement manufacture, does not have to be burnt in the cement kiln and therefore consumes no energy for desiccation, dehydration or calcination.Further to Spain’s Structural Code , the cleThe concretes in this study were mixed with potable, Structural Code -complianConcrete batching calls for a detailed study of the characteristics of the components, given the highly significant effect of their inter-relationships on mix behaviour. Design requirements for the specific project where the concrete is to be used must also be scrupulously honoured. Here the concrete was intended for non-structural precast elements conforming to the mechanical and durability requirements set out in the European (EN) standards governing paving block and kerb production.Bearing in mind the basic batching for commercial precast concrete and earlier findings ,19,20, tPrecast monoblock kerbs and pavers were manufactured in the laboratory as specified in Spanish standard UNE 127340  and EuroIn keeping with the dimensions routinely found in Spanish class A2 urban footways (standard UNE 127340 ), the keThe paving blocks were likewise dimensioned to routine practice in Spanish urban settings, i.e., to a cross-section of 100 mm × 80 mm cross-section and a length of 200 mm.The geometric/dimensional tolerance, surface finishes and visual  characteristics of the units were verified against the specifications laid down in standards EN 1340 ,47 and UBoth recycled and conventional concrete mixtures were assessed on the laboratory hydraulic press. Standard 150 mm × 300 mm cylindrical specimens conforming to  were tesEight recycled precast kerb units (200 mm × 100 mm × 1000 mm) were tested for 28 d flexural strength in keeping with standards EN 1340 ,47 and UEight 28 d recycled paving blocks (100 mm × 80 mm × 200 mm) were tested for splitting tensile strength as laid down in standard EN 1338 ,43. RACCAbrasion resistance was evaluated as per standards EN 1340 ,47 and UThree paving blocks (100 mm × 80 mm × 200 mm) and kerbs (200 mm × 100 mm × 1000 mm) RACC concrete were tested for water absorption according to standard EN 1338 ,43. MoreCC and RACC concrete electrical resistivity was found at 20 °C on 100 mm × 200 mm saturated surface-dry cylindrical specimens in keeping with the protocols set out in standard UNE 83988-1 . To ensuConcrete microstructure was analysed on a Hitachi S-4800  scanning electron microscope (SEM) fitted with a tungsten X-ray emitter and Si/Li detector and coupled to a Brucker XFlash 5030 EDS analyser. All samples were mounted on a metal sample holder for readier placement in the microscope and carbon-coated to ensure conductivity and avoid signal masking.This technique, which yields information on material pore structure, is based on the capillarity generated in liquids that do not soak adjacent solids. A liquid such as mercury with a 141° contact angle against a solid surface can only be inserted into the material pores if the pressure applied is greater than that exerted by the capillarity. Porosity tests on 28-day cured RACC concrete samples  were conducted on a Micromeritics  9500 mercury porosimeter featuring a pressure range for mercury intrusion of 0.00345 MPa to 227.53 MPa. To ensure moisture removal, the samples were previously dried to a constant weight at 40 °C and degasses with a vacuum pump (30 min).Both concretes, CC and RACC, met the dimensional requirements for paving blocks and kerbs laid down in the respective European standards ,43,46,47The visual appearance, texture and colour of the two materials were also compared. No surface cracking or flaking was observed in any of the units, irrespective of the material used in their preparation. No significant textural differences were perceived between the CC and RACC specimens. Colour differences were readily detected, however , for theDespite that obvious visual difference between the two concretes, the use of RACC should not be ruled out on those grounds only. Rather, it should be valued for its potential as a sustainable alternative to dying precast elements, in keeping with routine practice in the commercial manufacture of urban footway paving blocks.The strength values for CC and RACC are given in Compressive strength in CC and RACC is plotted against time in The filler effect and pozzolanicity of recycled brick powder-based cement additions. The findings reported by Olofinnade et al.  endorse The presence of unhydrated cement particles in the recycled aggregate, which may induce secondary hydration.The effect of internal curing prompted by non-compensation for the water absorbed by recycled materials such as aggregates  or cemenAt the earlier ages, CC exhibited higher compressive strength than RACC , with va2/g) was higher than the 3593 cm2/g, characterising the CEM III/A found in the reference concrete, whilst the replacement ratio defined lay within the range recommended in the literature.the pozzolanicity of the recycled materials used, which has even been shown to effectively prevent strength loss induced by the use of recycled aggregates . MoreoveThe absence of any technique to offset water absorption, lowering the effective w/c ratio and favouring higher mechanical performance in the RACC.The strength of the interfacial transition zone (ITZ) between the recycled aggregate and the cement paste, as per the microstructural analysis discussed below.The CC exhibited 28 d compressive strength of 35 MPa and the RACC 37.1 MPa , substanThe present findings contrast with those of other research published in the existing literature for the same replacement ratios (50% RA and 25% masonry addition), where compressive strength was reported to decline. In an initial study, Cantero et al.  recordedAggregate shape is known to have a significant impact on concrete flexural strength. The jaggedness and surface roughness typical of recycled aggregate may be expected to have a beneficial effect on recycled concretes. Whereas some authors have reported comparable strength for recycled and conventional aggregate ,65,66 orThe authors of a number of studies found flexural strength to be similar in concrete manufactured with conventional and blended (brick powder-additioned) cement, especially where low replacement ratios were applied: 10% , 15% 1111,13 andIn a subsequent study, Ge et al.  indicateFurther to the data in To date, very few studies have been published on the effect of including both recycled materials to replace gravel and conventional cement on concrete flexural strength. Cantero et al. , howeverFurther to a very thorough review of the literature conducted by Silva et al. , the repGe et al. , explorithe pozzolanicity of the recycled materials used , along w2/g) than of CEM III/A (3593 cm2/g), which affects the strength of the bonds that can be developed [the greater specific surface of the brick powder applied  met the class 4 (product marking ‘I’) requirement, the most demanding was defined in standard EN 1340 ,47. StanAuthors such as Mas et al.  also tesThe 21 d and 28 d electrical resistivity observed for the control and recycled specimens  show thaFurther to the pattern visible in the values observed for the samples , electriWater absorption is one of the most difficult parameters to control when working with recycled aggregate due to its higher sorptivity than found in the natural material. As the bars in 2) weathering resistance requirements. Sadek and Nouhy [The adverse effect of recycled aggregate on water absorption in precast concrete units has been observed by earlier authors. In a study jointly replacing different proportions of natural fine and coarse aggregate with brick masonry waste in paving blocks and flags, Jankovic et al.  detectednd Nouhy , who alsnd Nouhy , in turnnd Nouhy , howeverThe structures characteristic of cement hydration products such as portlandite and CSH gels  are readily recognisable in the SEM micrographs of the RACC concrete reproduced in Similar results were published by other researchers such as Bignozzi and Saccani , who expAccording to the log differential intrusion-measured pore diameter (mL/g)/pore size (µm) and ore size distribution (mL/g)/cumulative intrusion (µm) curves for CC and RACC in At pore sizes of 0.2 µm and smaller, the voids volume was considerably greater in the RACC than in the CC , due priThe experimental investigation carried out in this paper showed that the use 50 wt% mixed recycled aggregate (masonry content of 33%) and an eco-efficient cementitious material as 25 wt% conventional binder replacement is a technically viable option, as well as and European standard-compliant, for the manufacture of recycled precast elements (kerbs and paving blocks) used in footways.Although no significant textural differences were detected between the CC and RACC specimens, highly visible variations in colour were perceivable, as the inclusion of masonry CDW lent a reddish tone to the RACC units. In any case, no cracking or surface flaking was observed in any of the specimens prepared.The strong recycled aggregate-cement paste ITZ, the non-compensation of water absorption by recycled materials and the pozzolanicity and greater specific surface of those materials relative to ordinary cement were identified as positive factors contributing to the mechanical strength of the RACC-mixture. Although early age mechanical strength was lower in the RACC than in the CC, the 28 d compressive strength was 37.5 MPa in RACC, values comfortably higher than the batching target, and 6% higher in RACC than in the CC. Nonetheless, RACC precast units exhibited a 26% lower flexural strength and a 12.8% lower splitting tensile strength compared to the conventional kerbs and paving blocks (CC-concrete), although the variation was much narrower than that reported in the literature.The product marking classification of the recycled kerbs and paving blocks according to the European standards was also used in the comparison to the conventional precast products. On one hand, as a result of the strong recycled aggregate-cement paste bond and the higher abrasion resistance of the recycled coarse aggregate, the recycled precast elements were compliant with the strictest European criterion  whereas those of CC-concrete qualified for class 3 (product marking ‘H’). On the other hand, recycled kerbs and paving blocks were classified under class 1/product marking ‘A’ as the 6% water absorption limit was exceeded; thus, it was a less favourable category than that obtained by the conventional precast elements.Regarding the electrical resistivity, an overall lower performance (51% at 21 days and 45% at 28 days) was noticed for RACC concrete. Nonetheless, as the literature suggests, the use of different replacement ratios and/or types of recycled materials might mitigate the adverse effect of CDW on this property.In terms of microstructure, the use of recycled aggregate and masonry additions did not alter the formation of portlandite, CSH gels or ettringite and no risk of hydration reactions was identified. Moreover, the higher volume of pores with diameters of under 0.2 µm noticed in the RACC compared to CC due to the presence of recycled coarse aggregate does not compromise recycled concrete durability but reduces the number of pores larger than 0.2 µm, potentially improving concrete unit durability by refining pore structure.The conclusions drawn include the following:


Several outbreaks of highly pathogenic avian influenza (HPAI) caused by influenza A virus of subtype H5N8 have been reported in wild birds and poultry in Europe during autumn 2020. Norway is one of the few countries in Europe that had not previously detected HPAI virus, despite widespread active monitoring of both domestic and wild birds since 2005.Anser brachyrhynchus), and several other geese, ducks and a gull, from south-western Norway in November and December 2020. Despite previous reports of low pathogenic avian influenza (LPAI), this constitutes the first detections of HPAI in Norway.We report detection of HPAI virus subtype H5N8 in a wild pink-footed goose . Anseriformes  and Charadriiformes  .Norway is one of the few countries in Europe that had not previously detected HPAI virus, despite widespread active monitoring of both domestic and wild birds since 2005 [Anser brachyrhynchus) from Sandnes municipality, in Rogaland County  notified the Norwegian Food Safety Authority (NFSA) of a confirmed case of HPAI virus subtype H5N8 in a wild pink-footed goose  2018/1136 of August 10th 2018 on risk mitigation and reinforced biosecurity measures and early detection systems in relation to the risks posed by wild birds for the transmission of highly pathogenic avian influenza viruses to poultry. This included a housing order for poultry and birds kept in captivity in the coastal municipalities from Rogaland county in the southwest to the Swedish border in the southeast. As a result of multiple detections of HPAI in two counties in the two weeks following the initial detection  with wild bird subtypes H5N8 and H5N5 sequences from neighbouring countries including the United Kingdom, the Netherlands, Germany, Denmark, Belgium and Russia with the most recent common ancestor being A/chicken/Iraq/1/2020 (H5N8).As of the 22nd of December 2020, HPAI virus of subtype H5N8 has been detected in ten wild and four captive birds, all in Southern Norway Table .Larus marinus) was observed sick for a couple of days in Bergen municipality, Vestland county, and died on the 29th of November 2020. Bergen is around 170 km north of Sandnes  and three roosters  were found dead in late November and early December, respectively, in a publicly operated bird park in Hå municipality, about 15 km south of Sandnes  between 2016 and 2018 . Of the From the analyses of data deriving from counts of geese in Jæren, as well as neck band observations and ordinary recoveries of ringed birds , some poWild birds migrating from Russia to the south-west during autumn have been proposed as a potential pathway for the introduction of HPAI virus to Europe , but it Similarly, we cannot rule out that geese from Iceland brought the virus to Norway during autumn migration. However, HPAI virus has not been detected in Iceland and this route of introduction is considered less likely.A northward return migration of geese from their wintering sites in areas extending from Denmark to Belgium, to sites in Southern Norway, has recently been documented . Thus geThe Barnacle goose, like the pink-footed goose, also migrates through Norway and breeds on Svalbard. It was the species with the highest number of reported cases of HPAI virus of subtype H5N8 in Europe during the autumn of 2020 . TherefoHPAI virus has been detected for the first time in Norway, but the mode of introduction remains unclear. However, a northward migration of infected geese or gulls from Denmark or the Netherlands during the autumn of 2020, is currently our main hypothesis for the introduction of HPAI to Norway. More detailed studies of the migratory routes of wild birds species, bird genetics and molecular studies of H5N8 viruses, are needed to answer this question. The presence of HPAI in wild birds constitutes a new, and ongoing, threat to the Norwegian poultry industry, and compliance with the improved biosecurity measures on poultry farms should therefore be ensured. Finally, although HPAI of subtype H5N8 has been reported to have very low zoonotic potential, this is a reminder that HPAI with greater zoonotic potential in wild birds may pose a threat in the future. This underpins the importance of implementing a One Health strategy in handling avian influenza in Norway.The NFSA is responsible for implementing the active surveillance programme for avian influenza (AI) in wild birds in Norway. The programme started in 2005 and is based on screening of cloacal and oropharyngeal swabs from healthy birds shot during the hunting season .Preparation of DNA libraries were performed using the NexteraXT kit , indexed libraries were quantified using Quantifluor dsDNA System , pooled and sequenced on a NextSeq550 sequencing platform using a High Output Kit v2.5 with 2 × 150 base- paired end reads. These analyses were performed at APHA, Surrey, United Kingdom.https://github.com/AMPByrne/WGS/blob/master/RefGuidedAlignment_Public.sh). Final consensus sequences were obtained by mapping to the genome of A/chicken/Iraq/1/2020 (EPI_ISL_623074). Closest genetic relatives on the GISAID EpiFlu database (https://platform.gisaid.org/ accessed on 25th February 2021) were identified using the BLAST function. The top 50 BLAST hits were downloaded and added to a manually curated alignment with related avian influenza virus HA sequences, the resultant H5Nx dataset was aligned using MAFFT v7.450 [https://github.com/rambaut/figtree), rooted by A/Goose/Guangdong/1/96 and nodes were placed in ascending order (Fig. Raw sequencing reads were assembled using a custom in-house pipeline, available on github (T v7.450 , inspectT v7.450 . We infeT v7.450  and obta


Male infertility is a major health issue with an estimated prevalence of 4.2% of male infertility worldwide. Oxidative stress (OS) is one of the main causes of male infertility, which is characterized by excessive reactive oxygen species (ROS) or lack of antioxidants. Meanwhile, it is reported that oxidative stress plays an important role in the spermatogenic impairment in Inner mitochondrial membrane peptidase 2-like (Immp2l) mutant mice. In this study, we focused on the potential mechanism of Guilingji in protecting the spermatogenic functions in Immp2l mutant mice. The results revealed that Immp2l mutant mice exhibit impaired spermatogenesis and histology shows seminiferous tubules with reduced spermatogenic cells. After administration of Guilingji [150 mg/kg per day intragastric gavage], however, alleviated spermatogenesis impairment and reversed testis histopathological damage and reduced apoptosis. What’s more, western blotting and the levels of redox classic markers revealed that Guilingji can markedly reduce reactive oxygen species. Moreover, Guilingji treatment led to inhibition of the phosphorylation of mitogen-activated protein kinase (MAPK), regulated apoptosis in the cells. In summary, Guilingji can improve spermatogenesis in Immp2l mutant mice by regulating oxidation-antioxidant balance and MAPK pathway. Our data suggests that Guilingji may be a promising and effective antioxidant candidate for the treatment of male infertility. Infertility is defined as having regular and unprotected sex with the same partner and not getting pregnant after 1 year . Half ofGuilingji is a TCM from the Ming Dynasty. According to the 2015 edition of the Chinese Pharmacopoeia, one of the confidential products of national brands of TCM with Red ginseng as the main raw material . It has In order to test this hypothesis, we selected Immp2l mutant mice as our animal model. Inner mitochondrial membrane peptidase 2-like (IMMP2L) is a functional domain of mitochondrial intima proteina that plays a crucial role in cleaving space-sorting signal peptide sequences of cytochrome c1 (CYC1) and mitochondrial glycerol phosphate dehydrogenase 2 (GPD2) . TherefoIn this study, Immp2l mutant mice were used as the animal model of oligosthenospermia, and Guilingji was selected as the treatment drug by using the unique characteristics of traditional Chinese medicine to explore the influence on these specific antioxidative pathway on oligosthenospermia in mice. This research will provide valuable insights for understanding the pathological changes and treatment mechanisms of spermatogenic dysfunction caused by oxidative stress.The main components of Guilingji  are presented in Immp2l mutant mice have been described previously . Mice we+/+ mice and 20 Immp2l-/- mice. There were 4 groups in this experiment: Immp2l+/+ group; Immp2l+/+-Guilingji group; Immp2l-/- group; Immp2l-/--Guilingji group. And there were 10 mice in each group. Immp2l-/- and Immp2l+/+ mice were administered intragastrically with Guilingji for 30 days, respectively. The appropriate therapeutic doses of Guilingji is 150 mg/kg per day . a, b, c, d grade respectively represents different motility states of sperm. Sperm motility is quantitatively evaluated by motile sperm (grade a + b)/all sperm*100.After the mice were euthanized, fresh testicular tissue was taken and cleaned with cold PBS (pH 7.4). Then, fixed in formalin for 48 h and embedded in paraffin after conventional treatment. The wax block was cut into 5 μm thin slices with a microtome, and the slices were stained with hematoxylin and eosin, which were scanned by a light microscope.Fresh testicular tissue was taken, Radio Immunoprecipitation Assay (RIPA) lysis buffer containing phosphatase inhibitors and protease was added, and total protein was extracted from the tissue after centrifugation. Western blotting analysis was based on previous reports . ConciseFor immunofluorescence analysis, the paraffin sections were dewaxed, dehydrated, and incubated overnight at 4°C with primary antibodies. Then, the tissues were incubated with secondary antibodies for 1 h in the dark. Finally, the nuclei were counterstained with DAPI for 10 min. Immunofluorescence signals were visualized and recorded using a laser scanning confocal microscope . The primary and secondary antibodies used in this study are listed in After the mice were euthanized, the tissues were homogenized, then placed in cold sterile phosphate buffered saline (PBS). The homogenate was centrifuged at 2,500 rpm/min at 4°C for 10 min, and the supernatant was extracted for further analysis of oxidative stress level. Assays of Malondialdehyde (MDA), Superoxide dismutase (SOD) and Glutathione (GSH) levels in testicular tissues were performed using commercial kits . We further analyzed the test data according to the instructions.The TUNEL assay was executed by using a One‐Step TUNEL Apoptosis Assay Kit. First, paraffin-embedded sections were placed in 65°C drying oven, followed by deparaffinized in xylene, then treated with a range of different concentrations of alcohol  and rinsed with PBS at pH 7.5. The tissue was then treated with protease K solution and refixed with 4% paraformaldehyde solution. The prepared TUNEL reaction mixture was dropped onto the tissue and incubated at 37°C for 1 h in a dark environment. The nuclei were stained with DAPI. TUNEL positive cells were counted under fluorescence microscope.http://herb.ac.cn) (http://herb.ac.cn). Then, using Oncomine (https://www.oncomine.org) for Go and KEGG analysis. Go analysis can reveal the main enrichment terms in Biological Process (BP), Cellular Component (CC) and Molecular Function (MF). KEGG analysis can reveal the major enriched signaling pathways. Pathway with enrichment p < 0.01 was considered as significant.Traditional Chinese medicine (TCM) prescriptions can effectively delay the progression of disease through the coordination of various compounds and targets . Therefob.ac.cn) . Differep < 0.05.All experiments were performed at least three times and the data are expressed as the mean ± SEM. When the data were normally distributed, one-way ANOVA followed by Tukey test or two-way ANOVA followed by Bonferroni’s multiple comparison test. If these data were not normal distributed, Kruskal-Wallis test was used. GraphPad Prism 7.0 Software  was used for Statistical analysis chart. The differences between groups were considered statistically significant when -/- mice weighed less than Immp2l+/+ mice (+/+ and Immp2l-/- mice) were not different than in saline-treated mice of the same genotype  in sperm motility percentage and sperm concentration, when compared to the Immp2l-/- group, as presented in The detection of sperm motility by Computer-assisted sperm analysis (CASA) was used to evaluate whether Guilingji affected sperm motility and concentration. Results in -/--Guilingji group mice showed thicker seminiferous epithelium, and more tubules with the higher Johnsen scores  activity were decreased. On the contrary, Guilingji administration can significantly reduce the above effects. The results showed that Guilingji alleviated oxidative stress by balancing oxidation and anti-oxidation.To assess testicular antioxidant status, we measured the levels of classic markers of redox. As shown in -/- mice was dramatically increased. After Guilingji treatment, the level of apoptosis of germ cells were considerably strangled in Immp2l-/- mice. Meanwhile, Guilingji treatment had no vital effect on the germ cell apoptosis in Immp2l+/+ mice. To further confirm this result, we also detected apoptosis-associated proteins active-Casp3, Bcl-2, and Bax expression by western blotting, and found that Guilingji inhibited cleaved Casp3, Bax expression and promote Bcl-2 expression  is an assay for the detection of apoptosis. As shown in Functional Enrichment Analysis of Ginsenoside Re, the Main Ingredient of Guilingji The structural formula of ginsenoside Re is shown in -/- mice. However, compared with the untreated group, the levels of phosphorylated proteins in MAPK pathway were decreased in the treatment group (p < 0.05). In conclusion, these results suggested that Guilingji may play a protective role in spermatogenesis by inhibiting the above signal pathway.Previous studies have shown that activation of MAPK pathways is closely related to oxidative stress-related apoptosis . PathwayIn this study, we showed that the level of oxidative stress in Immp2l mutant mice testis was higher compared with wild-type mice, leading to spermatogenic impairment. We revealed that the Guilingji administration reduced ROS level, inhibited germ cell apoptosis and improved spermatogenic function. Importantly, we found that the therapeutic effects of Guilingji was depended on MAPK pathways. In summary, we elaborated the therapeutic effects of Guilingji administration in oligospermia animal models, which was depended on the ant-oxidative capacities.-/- mice act as one of the ideal animal models of infertility, alleviating oxidative stress is conducive to ameliorating spermatogenesis of Immp2l mutant mice.As we know, oxidative stress refers to the imbalance between the oxidative system and the antioxidant system, which is the main factor in the pathology of infertility in 30–80% of infertile men . ComparePrevious studies have shown that red ginseng and ginsenosides can decrease and/or eliminate the production of free radicals by modulating the activity of antioxidase . While rIntracellular antioxidants such as glutathione (GSH), superoxide dismutase (SOD) protect cells against oxidative damage by eliminating cytoplasmic ROS . GuilingThe apoptotic cascade is caused by excessive production of ROS and oxidative stress, which hinder the basic functions of sperm, thereby making sperm inactive and immotile. Therefore, the development of therapeutic methods to improve or inhibit sperm cell apoptosis induced by oxidative stress is an urgent requirement . In this-/- mice, demonstrate that Guilingji strongly inhibit the MAPK signal transduction pathways. All in all, our experiment revealed that Guilingji could attenuated oxidative stress and apoptosis in Immp2l mutant mice via restrain activation of the MAPK signaling pathways.The separation of active ingredients from TMC and further analysis of their mechanism of action are of great significance for discovering drug targets and evaluating drug efficacy . TherefoIn short, the results of this study systematically proved that Guilingji can attenuate ROS, oxidative stress and apoptosis by inhibiting MAPK-mediated signal pathways in the immp2l mutant mouse model. This study provides evidence that Guilingji may be effective in the clinical treatment of male infertility. However, the use of a single dose of Guilingji is deficiency of our experiment, which will be improved in future studies. In addition, the regulation mechanism of Guilingji on metabolites needs further experimental verification.


Crops assemble and rely on rhizosphere-associated microbiomes for plant nutrition, which is crucial to their productivity. Historically, excessive nitrogen fertilization did not result in continuously increasing yields but rather caused environmental issues. A comprehensive understanding should be developed regarding the ways in which crops shape rhizosphere-associated microbiomes under conditions of increased nitrogen fertilization. In this study, we applied 16S and 18S ribosomal RNA gene profiling to characterize bacterial and fungal communities in bulk and rhizosphere soil of rice subjected to three levels of nitrogen fertilization for 5 years. Soil biochemical properties were characterized, and carbon-, nitrogen-, and phosphorus-related soil enzyme activities were investigated, by assays. Increasing nitrogen fertilization led to a decreasing trend in the variation of microbial community structures and demonstrated a more definite influence on fungal rather than bacterial community compositions and functions. Changes in the level of nitrogen fertilization significantly affected chemical properties such as soil pH, nutrient content, and microbial biomass levels in both rhizosphere and bulk soil. Soil enzyme activity levels varied substantially across nitrogen fertilization intensities and correlated more with the fungal than with the bacterial community. Our results indicated that increased nitrogen input drives alterations in the structures and functions of microbial communities, properties of soil carbon, nitrogen, and phosphorus, as well as enzyme activities. These results provide novel insights into the associations among increased nitrogen input, changes in biochemical properties, and shifts in microbial communities in the rhizosphere of agriculturally intensive ecosystems. Arabidopsis  and leucine aminopeptidase (LAP) play a role in the N-acquisition process by decomposing N-acetyl-β-D-glucosamine or catalyzing the cleavage of amino acids or proteins, respectively. Finally, acid phosphatase (ACP) represents P-acquiring enzymes that hydrolyze phosphate monoesters and organic molecules  is a staple food for more than half of the global population, and its perpetual production is crucial for food security  what is the response pattern of soil microbial taxonomy, microbial functional composition and soil biochemical properties, including nutrients, microbial biomass and carbon (C), nitrogen (N), and phosphorus (P) related enzymes to excessive N fertilizer application? (b) what are the differences and similarities between bacteria and fungi in response to the increase of N fertilizer application rate? To comprehensively understand these questions, a 5-year field experiment was conducted from 2016 to 2020. Rhizosphere and bulk soil samples were collected from rice plants grown in soil subjected to three levels of N fertilization, 8 weeks after transplantation, once the root microbiota had been well established and stabilized . The eff−2 – per rice-growing season. According to the rice production management practice, the recommended nitrogen application rate of Liaoxing 1 was 150 kg hm–2 during the growing season, and the N application treatments selected in this study were 100% and 200% of the recommended N application rate, respectively. Each treatment was replicated three times; hence, nine experimental plots were completely randomized and surveyed during the experimental period. Urea (N: 46%), as the only type of nitrogen fertilizer was applied in three sessions, i.e., 50% before transplanting, 30% at tillering stage, and 20% at panicle initiation stage. The application rates of P  and K  fertilizers were the same in all experimental plots, and 100% P and K fertilizers were applied before the transplanting. Rice seeds were disinfected – soaked in 0.01% potassium permanganate solution for 24 h – and then seeded were sown on the substrate soil of rice seedlings in the greenhouse. The substrate can meet the requirements of nutrients for seeding growth. The environment of the greenhouse was very suitable for the growth of rice seedlings with the temperature maintained at 25–27°C. It was ventilated twice a day to keep sufficient oxygen in the greenhouse. After 1 month of growth in the greenhouse, the rice seedlings were transplanted into the experimental field. This experimental design was consistently applied from 2016 to 2020. To prevent fertilizer leakage, different N fertilizer treatment plots were separated by PVC partitions and artificial ridges. The distance between plots was 30 cm, rice on the boundary of plots were designed for protection and not sampled. In order to promote rooting and prevent ineffective tillers, a week of water control management was carried out at tillering stage (early July). Irrigation in paddy field was carried out after water control, and the remaining growth period of rice was completed under flooded condition. During this period, field management shall be carried out according to local field management practice. Rhizosphere and bulk soil samples were collected at the full heading stage, 8 weeks after transplantation of the rice seedlings (2020). At this stage, the rhizosphere associated microbiota had been well established and stabilized. Another reason why we chose to take samples in this period is that for the growth period of Liaoxing 1, the plant was in the heading and filling period at this time. This period is important for rice growth and development, which directly affects the grain yield at the harvest stage. Therefore, it is of great significance to explore the associated microorganisms and soil biochemical properties in this period. Three replicate samples of rhizosphere and bulk soil were collected from plants (randomly select 12–15 rice plants in each plot) subjected to each of the three N fertilization levels. Each complete rice plant was removed with its immediate block of soil included. The soil loosely attached to the roots was collected as bulk soil samples, whereas remaining soil on the root surfaces was brushed off gently with a sterile brush and gathered as rhizosphere soil samples . Average annual temperature and precipitation in the area are 8.4°C and 715.5 mm, respectively. We tested typical paddy soil, in which rice had been planted for more than 30 years. The experimental land was demarcated into plots, 8.6 m × 9 m in size. A Japonica rice variety – Liaoxing 1 (CNA20040621.3) – was provided by the Rice Research Institute of Shenyang Agricultural University and used in experimental planting; this cultivar, with its stable yield, moderate growth cycle, and strong resistance, is widely planted in Northeast China. The experiment evaluated three levels of N fertilization – 0, 150, and 300 kg/hm samples .2O. The PCR conditions were as follows: 95°C for 5 min; 25/30 cycles of 30 s at 95°C, 30 s at 50°C, and 1 min at 72°C, with a final extension at 72°C for 7 min. The products were examined using agarose gel electrophoresis and purified using AMPure XP beads , as per the manufacturer’s protocol. The purified PCR products were quantified and homogenized to form a sequencing library, which was then subjected to library quality inspection; after passing the quality inspection, the library was sequenced using the PacBio Sequel System .We used the NucleoSpin 96 Soil kit , as per the manufacturer’s protocol, to extract total genomic DNA from rhizosphere and bulk soil samples. Furthermore, we tested the integrity, concentration, and purity of DNA using electrophoresis on a 1% agarose gel. The 16S and 18S rRNA genes were amplified with primers 27F:1492R (AGRGTTTGATYNTGGCTCAG/TASGGHTACCTTGTTASG ACTT) and ITS1F/ITS2 (5′-CTTGGTCATTTAGAGGAA GTAA-3′/5′-GCTGCGTTCTTCATCGATGC-3′), respectively. PCR amplification was performed in a 20 μl mixture containing 1 μl VnF (10 μM) and 1 μl VnR (10 μM), 1 μl DNA template (approximately 5–50 ng of DNA), 0.4 μl KOD FX Neo (TOYOBO), 10 μl KOD FX Neo Buf, and 4 μl 2 mM dNTP and then supplemented with 20 μl ddH1 was used to identify circular consensus sequences (CCSs) through barcodes, and obtain barcode-CCS sequence data. Second, the barcode-CCSs were filtered to obtain valid sequences, and third, UCHIME v4.2 software2 was used to identify and remove the chimera sequences in to obtain the optimization-CCS sequence. Sequence data were clustered into operational taxonomic units (OTUs) at a 97% similarity level using the USEARCH3 method. Species annotation and abundance analyses were performed using bacterial and fungal 16S and 18S rRNA datasets from the SILVA database to reveal the species compositions of samples. The raw FASTQ files obtained in the study for the sequencing libraries have been deposited into NCBI Sequence Read Archive (SRA) under BioProject accession numbers PRJNA725973  and PRJNA725981 .Sequencing data analysis was divided into three steps. First, Lima v1.7.0 software2. After 30 min, the pH was measured using a pH meter . We further used the potassium dichromate external heating method to measure total organic carbon (TOC), whereas soil total nitrogen (TN) was measured using the semi-micro Kelvin method , as per the manufacturer’s instructions. The weight of soil used to determine the activity of enzyme was 1 g. Then the soil was evenly divided into two parts, one part was used to measure the enzyme activity and the other part was for the determination of water content (drying method). Considering LAP as an example, the microplate was coated with purified LAP antibody  to prepare a solid-phase antibody; LAP was then added to the microwells of the coated monoclonal antibody, before being combined with horseradish peroxidase (HRP)-labeled LAP antibody, to form an antibody–antigen–enzyme-labeled antibody complex. After thorough washing with deionized water, tetramethylbenzidine  was added for color development. TMB turned blue in response to the catalytic activity of HRP  and then yellow after the addition of acid . Color change was measured spectrophotometrically at a wavelength of 450 nm. The color intensity was positively correlated with the activity of LAP in the sample, and then the activity level of LAP in the sample was determined by comparing the OD  value of the samples to the stand curve. Activity levels of NAG, BG, and ACP  were measured in a similar manner.4, principle coordinate analysis (PCoA) and analysis of similarities were performed to determine the differences in microbial community structure, and alpha diversity analysis was performed to study microbial community richness and diversity. We annotated bacterial functions using the FAPROTAX database . Analysis of variance (ANOVA) and least significant difference analyses were used to test the significance of the effect of increased N fertilization on soil chemical properties and enzyme activity. Bioinformatics analyses were performed using BMKClouddatabase , based odatabase  was usedp < 0.05) higher than that without N application treatment (N0), but the rice yield under nitrogen application treatment N150 and N300 had no significant difference (p > 0.05). In 2018 and 2020, the rice yield showed a downward trend with the doubling of N fertilizer application.We recorded the grain yield data of Liaoxing 1 from 2017 to 2020, and the results are shown in Principle coordinate analysis based on the Bray-Curtis distance algorithm was performed at the OTU level . The OTUp > 0.05) difference in bacterial species diversity between rhizosphere and bulk soil samples, whereas fungal species diversity in rhizosphere soil samples was significantly (p < 0.05) higher than that in bulk soil samples. Notably, the species richness of both bacteria and fungi in rhizosphere soil samples was significantly (p < 0.05) higher than that in bulk soil samples.We evaluated the alpha diversity of each sample using the Shannon and Chao1 indices to measure species diversity and richness, respectively . We founp < 0.05) decrease, particularly in soil subjected to the N300 treatment, it was significantly lower than that in N0- and N150-treated soil. With increasing N application, fungal species diversity in rhizosphere soil samples increased at first and then decreased, whereas an opposite pattern was observed in the bulk soil samples. Furthermore, the species richness of fungal communities in both rhizosphere and bulk soil samples showed a significantly (p < 0.05) decreasing trend with increasing N application.With increased N application, bacterial species diversity and richness in both the rhizosphere and bulk soil samples showed a significant  in rhizosphere and bulk soil samples subjected to increasing levels of N fertilization application are shown in Increased N fertilizer input affected the functions of bacterial  and fungCharacteristics of soil pH, TOC, TN, AN, and AP are shown in Next, we measured microbial biomass carbon (MBC), nitrogen (MBN), and phosphorus (MBP), the results of which are presented in The activities of four key enzymes related to C, N, and P are shown in We conducted RDA on microbial communities and soil enzyme activities  at the fThe excessive use of N fertilization is currently one of the main problems affecting agricultural production in China , particuIn this study, we explored the characteristics of the microbial community in the rhizosphere and bulk soil of rice grown in soil that was subjected to three N treatment levels. Increased N fertilizer input altered the bacterial and fungal community structures, which highlighted apparent differences in these structures, in both the rhizosphere and bulk soil. As a result, changes in bacterial community structures followed a generally increasing trend, whereas fungal community structures adhered to a generally decreasing trend, which might be related to the differences in N-acquisition strategies between bacterial and fungal communities . Wang etA previous study  has showNitrosomonas – a chemoautotrophic bacterium – which oxidizes ammonia to nitrous acid  can be found below: DM, HD, and SF conceived the study. HD wrote the manuscript. HD, HS, CC, and AW performed the experiments. HD, HS, and SF performed the statistical analyses. HD, CC, and LJ involved in the field management and soil sampling. All authors discussed the results and commented on the manuscript.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Every year, over 65,000 Australians experience an acute coronary syndrome (ACS) and around one-third occur in people with prior coronary heart disease. Cardiac rehabilitation (CR) aims to prevent a repeat ACS by supporting patients’ return to an active and fulfilling lifestyle. CR programs are efficacious, but audits of clinical practice show variability of program delivery, which may compromise patient outcomes. Core components, quality indicators and accreditation of programs have been introduced internationally to increase program standardisation. With Australian quality indicators (QIs) for cardiac rehabilitation recently introduced, we aimed to conduct a survey in one state of Australia to assess the extent to which programs adhere to the measurement of QIs comparing country, metropolitan, telephone and face to face programs.A cross- sectional survey design with face validity testing was used to formulate questions to evaluate cardiac rehabilitation program and personnel characteristics and QI adherence. Between October 2020- December 2021, 23 cardiac rehabilitation programs across country and metropolitan areas were invited to participate. Quality improvement was defined as adherence to the Australian Quality Indicators, and we developed an objective score to calculate program performance categorised by quartiles. Significance of CR completion and time to enrolment between program type (telephone versus face to face) and location .Among the 23 CR programs, 15 were country and 8 metropolitan-based and 22 were face to face and 1 telephone-based. Median wait time from discharge was 27.0 days, (interquartile range 19.3–46.0) across all programs and country completions of enrolled were 76.9% versus metropolitan 56.5%, A survey of 23 cardiac rehabilitation programs, showed variability in adherence to measurement of the Australian Cardiovascular and Rehabilitation Association and Australian Heart Foundation Cardiac Rehabilitation Quality Indicators.ACTRN12621000222842, registered 03/03/2021.Australia New Zealand Clinical Trials Registry (ANZCTR), The online version contains supplementary material available at 10.1186/s12913-022-07667-2. Every year, over 65,000 Australians experience an acute coronary syndrome (ACS) (heart attack or unstable angina) and around one-third occur in people with prior coronary heart disease (CHD)–3. In AuGlobally and in Australia, core components –24 and q2 (5 times the size of the United Kingdom) with a population of 1.7 million in 2020, with 22.4% living in country areas  19.3–46.0) across all programs, with country having a median of 33.0 days (IQR 21–45) and metropolitan a median of 23.0 days (IQR 18–37), Program length and sessions.Mean total program length was 7.0 weeks SD ± 1.11) with country slightly longer than metropolitan programs  2004 CR framework,  NHF PathAll the primary coordinators were registered nurses with 78.2% completing a postgraduate qualification relating to cardiovascular care (Masters or Graduate Diploma). Eighty seven percent belonged to the Australian Cardiovascular and Rehabilitation Association (ACRA) peak body. All participated in some type of professional development in 2019 with the majority choosing cardiovascular-based conferences, webinars, workshops, and seminars.Australian Quality indicator (QI) adherence.All QIs except QI 1 were measured by some programs and program adherence is presented in Table Seventy four percent of programs were ranked at a medium level of performance with a mean performance score of 11.4/16, (SD ± 0.79). No programs were ranked as poor, 13% ranked low and 13% high Table . Comparip < 0.001) and the telephone program had higher completions than face to face programs . We were unable to measure total patients eligible in this dataset and this is something that our quality improvement initiatives hope to address in the future. However, an audit conducted between 2013- 2015 amongst the same programs showed that approximately 16,600 patients per year are eligible and of these 5,000 are referred. [The aim of this study was to assess the extent to which programs met the national QIs. This study surveyed 23 cardiac rehabilitation services in South Australia, Australia for program and personnel characteristics and quality performance adherence, comparing country and metropolitan, telephone and face to face programs. In 2019, of the 69.5% of programs that could provide data, there were 1,972 patients enrolled with 66.7% of these patients completing. Country and metropolitan enrolments were similar with 42.8% of country patients attending the telephone program. Country completions were higher than metropolitan  across country and metropolitan, telephone and face to face. Program length and sessions were similar amongst country and metropolitan, telephone and face to face programs (mean 7.0 weeks or 10.5 sessions) and indeed 6–8 weeks (12–16 sessions) is the norm across Australian programs. However, if we look at international benchmarks, program duration is far less in Australia than our international counterparts in the UK and Europe, who have a standard of ≥ 24 -36 sessions or around 12 -18 weeks, raising the question of what ‘dose’ of CR is most effective to improve outcomes? , 36 TotaThe ideal cardiac rehabilitation program consists of a multidisciplinary team. In South Australia where programs do not have all types of professionals as part of their team, this is enabled by referral pathways. The multidisciplinary team amongst country, metropolitan, telephone and face to face programs consisted of a nurse and either a physiotherapist or exercise physiologist and to a slightly lesser degree a dietician or pharmacist. Where they were not part of the team, they were accessible via a referral pathway, with the exception of the telephone program Table , which cThe most measured QI was comprehensive assessment across country, metropolitan, telephone and face to face programs, followed by depression screening, smoking assessment and counselling thereof Table . There iA key finding of this survey was the higher completion rates in country compared to metropolitan, and telephone compared to face to face programs. This was likely driven by 42.8% of patients accessing country programs attending the telephone option. While this measure reflects a process indicator, we also know from the survey that the telephone program delivers only one exercise session, where efficacy evidence tells us that exercise is a significant driver of improved CR outcomes , 14, 23.Quality improvement measurement and comparisons often require a multi-component approach of standardisation, measurement, reporting and change management. We were not able to conduct a validation process because of the variability of data entry into the CATCH database. This identifies the need for universal data entry to accurately measure program quality and will require a change management strategy to achieve. Accreditation can be a tool to promote change management among clinicians to drive standardisation of care and deliver evidence-based scope of practice, qualifications, and program content delivery .This survey was self-reported for each program and thus is subject to reporting bias. We were not able to objectively validate the questionnaire responses nor correlate these with clinical outcomes, thus we don’t know whether QI adherence is associated with better quality program content. Further, as the performance score is derived from program adherence to the QIs, the discriminatory capacity of the score may be limited. Since data entry into the CATCH database is not mandatory across the 23 programs, not all survey items measured have 23 programs as the denominator. Further enrolment and completion numbers are underestimated. We were not able to measure enrolment against eligible or referred patients for 2019, though we know from our previous audit work that approximately 5,000 patients are referred to cardiac rehabilitation each year in SA. (16) The sample size of 23 programs is small to measure any meaningful differences between country and metropolitan, telephone and face to face programs. Further the Australian Quality Indicators were only published in 2020 and therefore programs have not had time to implement improvements.(31) Despite this we have surveyed 23 CR programs across SA, evaluating program and personnel characteristics and determining adherence with the Australian Quality indicators, giving us an indication of the level of program quality to inform development of an accreditation system.A survey of 23 cardiac rehabilitation programs in one state of Australia, showed variability in adherence to measurement of the Australian Cardiovascular and Rehabilitation Association and National Heart Foundation Cardiac Rehabilitation Quality Indicators as measured by a survey. These data indicate that there are gaps in the delivery of evidence-based content in South Australian cardiac rehabilitation programs, which could be addressed by quality improvement initiatives such as an accreditation system. A state-wide engagement and education program will be required as an essential preliminary step towards an accreditation program.Additional file 1. Cardiac Rehabilitation Program Performance Scoring Criteria.Additional file 2. The South Australian Cardiac Rehabilitation Program Accreditation Survey.


A. mellifera workers with different treatments. These results suggest that though the residue of antibiotic treatment may not have direct effect on honey bee gut community, the gut microbiota dysbiosis caused by high dosage of antibiotic treatment has a cascade effect on the gut community of the nestmates in honeybee colonies.Gut microbial community plays an important role in the regulation of insect health. Antibiotic treatment is powerful to fight bacterial infections, while it also causes collateral damage to gut microbiome, which may have long-lasting consequences for host health. However, current studies on honey bees mainly focus on the impact of direct exposure to antibiotics on individual bees, and little is known about the impact of social transmission of antibiotic-induced gut community disorder in honey bee colonies. In order to provide insight into the potential pass-on effect of antibiotic-induced dysbiosis, we colonized newly emerged germ-free workers with either normal or tetracycline-treated gut community and analyzed the gut bacteria composition. We also treated workers with low dosage of tetracycline to evaluate its impact on honey bee gut microbiota. Our results showed that the tetracycline-treated gut community caused disruption of gut community in their receivers, while the direct exposure to the low dosage of tetracycline had no significant effect. In addition, no significant difference was observed on the mortality rate of  Apis spp.) is one of the most important insect pollinators, contributing an estimated 153 billion euros to the world’s agriculture in 2005 , of which fecal-oral transmission is the most important route for the establishment of a typical gut microbiota community in the receiver . In thisreatment  may alsoApis mellifera) were kept in the experimental apiary of the Honey Bee Research Laboratory, College of Animal Sciences, Zhejiang University. Four different colonies were used as four experimental replicates (referred as E1–E4). The laboratory worker bees were raised in cages  and incubated at 31°C ± 1°C and 75% ± 5% relative humidity (RH). Dead bees were counted and removed daily. All pollen and syrup supplied to workers were irradiated and sterilized.Honey bees . The gut homogenate was centrifuged at 10,000 × ad libitum tetracycline solution at 450 μg/mL (450 μg/mL Tetracycline in filter-sterilized 0.5 M sucrose solution) for another 5 days, whereas CV workers were only fed with 0.5 M sucrose solution for 5 days. Afterward, bees were immobilized at 4°C and marked with red and green paint pens for the AT and CV workers, respectively, and then returned to their original colony. Two days later, 15 CV and 15 AT workers were sampled again, of which 5 were used for 16S rRNA sequencing, 5 for quantification of bacterial loads, and the remaining 5 workers were sampled to prepare normal or dysbiotic gut bacteria for downstream experiments.Antibiotic treated (AT) workers and conventional gut community (CV) workers were obtained using a protocol described by Germ-free workers were obtained using the protocol described by 1 Sequence Read Archive with a BioProject accession number PRJNA225925.Bacterial genomic DNA was extracted using the TIANamp Stool DNA Kit  according to the manufacturer’s protocol. Polymerase chain reactions (PCR) targeting the V4 regions of the 16S rDNA genes were carried out using the specific primer with the barcode. Sequencing libraries were generated using the NEBNext Ultra DNA Library Prep Kit  following manufacturer’s recommendations and index codes were added. The library quality was assessed on the Qubit@ 2.0 Fluorometer  and Agilent Bioanalyzer 2100 system . The library was sequenced on an Illumina NovaSeq platform and 250 bp paired-end reads were generated. The 16S rRNA gene sequences were deposited in NCBIPaired-end reads from the original DNA fragments were merged using FLASH software . SequencBacterial loads of workers were determined by quantitative PCR (qPCR) using universal bacterial 16S rRNA primers . Sampled guts of workers were obtained according to the protocol described by t-test or Mann-Whitney test was used to calculate the statistical difference of bacterial load and the alpha diversity among groups. Survival analysis was performed using Kaplan-Meier survival curve and log-rank test. All graphs were generated in GraphPad Prism 8.SPSS software version 22.0  and GraphPad Prism 8  were used for statistical analysis. After verifying variance homogeneity and data normality, independent sample t-test: for E1: t = 3.14 df = 8, p = 0.0138; for E2: t = 2.151, df = 8, p = 0.0079; for E3: t = 2.894, df = 8, p = 0.0201; for E4: t = 3.158, df = 8, p = 0.0134). 16s rDNA amplicon sequencing demonstrated that Lactobacillus, Frischella, Commensalibacter, Bartonella, Snodgrassella and Gilliamella are the core bacterial species dominating in CV workers and AT workers . The absolute abundance of Lactobacillus was significantly decreased in AT workers in E1 and E3, and showed a decreasing trend in E2 and E4 . However, no significant difference was found on the alpha diversity (Shannon index and Chao 1 index) of microbiota species between CV workers and AT workers in any of the replicates (p = 0.006), but not in other 3 replicates.The absolute abundance of gut bacteria was significantly reduced in antibiotic treated (AT) workers compared to conventional gut community (CV) workers in all the four replicates . There were no significant differences in Shannon index and Chao 1 index between the NorA and the DysA in the four experiments. PCoA analysis revealed that the overall microbiota structure was significantly different according to the type of gut bacteria they received, as samples of Nor and Dys formed two distinctive cluster in three of the four replicates  and samples of NorA and DysA formed two distinctive cluster in two of the four replicates .To reveal the possibility of pass-on effect to their nestmates of tetracycline-treated gut microbiota disequilibrium, we determined the gut microbiota composition and size of workers that were exposed to either normal or dysbiotic gut community. Quantification with 16s rDNA copy numbers revealed that the total bacterial abundances of these workers were not influenced by the type of gut microbiota they received , but 16sischella . The speischella  in any oTo evaluate the possible negative impact of low dosage antibiotic residue on the establishment of honey bee gut microbiota, 10 μg/mL tetracycline was used to treat workers during the development of their gut communities. However, no significant impact was observed, as neither the community size nor the makeup was significantly different between Nor and NorA, or between Dys and DysA , 4A.In addition, we evaluated and compared the longevity of workers from different group, and the survival rates of bees of Dys, NorA or DysA were not significantly different from that of control group .Gilliamella relative abundance. In E2, Dys workers harbored lower genus_Snodgrassella and genus_Gilliamella relative abundance. In E3, Dys workers harbored higher class_Gammaproteobacteria and phylum_Proteobacteria relative abundance. And in E4, Dys workers harbored higher phylum_Firmicutes, genus_Lactobacillus and genus_Frischella relative abundance but lower phylum_Proteobacteria, genus_Snodgrassella and genus_Bombella.The linear discriminant analysis effect size (LEfSe) algorithm was then applied to determine which microbiota taxa caused the significant difference between Nor workers and Dys workers. Significant differences on the relative abundances of some taxa were found, but the taxa showing differences were not consistent in the four replicates . For exavia certain pathways due to the sociality of honey bees. Therefore, we investigated the effect of honeybee gut microbiota dysbiosis caused by tetracycline treatment on the establishment of gut community of their nestmates.Antibiotics have been widely used in animals for disease control and have significantly contributed to the development of animal husbandry industries. However, the collateral damage caused by antibiotic treatment has also been a great concern . The appA. mellifera gut microbiota and reduced genetic diversity of core species in A. mellifera gut community  can be found in the article/SJ, YW, FH, and HZ conceived and designed the study. SJ, YW, GC, and SW performed experiments. SJ and YW analyzed data and drafted the manuscript. All authors edited and approved the final version of the manuscript.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


We explored the potential impact of Crohn’s disease on the intragastric environment of fasted adults with a view to potential effects on intragastric performance of orally administered drugs in the fasted state. Data were collected from 15 healthy individuals and 15 patients with Crohn’s disease. All subjects remained fasted for at least 12h prior to gastroscopy. Intragastric resting volume and pH were measured upon aspiration. Osmolality, surface tension, pepsin activity, and content of six bile acids were measured within 4 months upon sample collection. Unlike intragastric volumes, intragastric osmolality was significantly increased by Crohn’s disease. However, mean osmolality value in patients was only slightly higher than in healthy individuals , therefore, unlikely to affect intragastric drug product performance. Primarily due to the high variability of data in healthy individuals, the potential effects on intragastric pH and surface activity could not be evaluated on a statistical basis. However, based on average (mean and median) values, even if they are statistically significant, it seems unlikely to be of clinical significance. Inter-subject variability of pepsin activity, and total bile acids content was high in both the healthy and the patients’ groups. Statistical investigation of the potential impact of Crohn’s disease on these parameters requires prior designation of the minimum differences to be detected; such differences will determine the minimum sample size required of relevant investigations. During the last two decades, substantial progress has been made in characterizing physicochemically the gastrointestinal (GI) contents of healthy adults  resultinCrohn’s disease is a heterogeneous disorder with a multifactorial etiology, including genetic factors, environmental insults and intestinal microbiota, characterized by chronic, segmental and transmural inflammation that affects the gastrointestinal tract and may involve any segment of the oral cavity up to the anus. The involvement of the upper GI lumen is less known, as a routine upper endoscopy is not commonly indicated .To date, apart from few data on the pH along the GI lumen of patients with inflammatory bowel disease , data onThe purpose of the present exploratory study was to evaluate whether the fasting gastric environment of patients could be affected by the Crohn’s disease.Human aspirates were collected at the Red Cross Hospital of Athens, after receiving approval by the Scientific and the Executive Committee of the Hospital (AP 152/160307).Fifteen healthy adults  and fifteen adults with Crohn’s disease  participated in the study during a routine gastroscopic procedure for diagnostic purposes at the Gastroenterology Department of Red Cross Hospital of Athens .Diagnosis of Crohn’s disease was based on histologic examination. Fourteen had the disease in the distal and one in the proximal region of the digestive tract. All subjects gave their informed consent for performing gastroscopy and collecting gastric contents.Subjects remained fasted for at least 12 h prior to gastroscopy. Following the standard protocol, gastrointestinal fluids were aspirated during the procedure to facilitate the diagnostic process i.e., improve visibility of the gastric wall. Normally, these fluids are directed to the waste upon aspiration. Complete aspiration of gastric contents was performed to determine volume of resting gastric fluid. Upon aspiration, each sample was kept for 2 h in the freezer, its pH was measured, and it was then transferred to -70 °C. In all cases, to avoid multiple thaw-freezing cycles, prior to freezing the material to be stored was distributed to five smaller vials . OsmolalPepsin from porcine gastric mucosa was from Sigma Chemical Co., St. Louis, MO, USA. All other chemicals used were of analytical grade and all solvents were of LC-MS grade and were purchased from Sigma Aldrich Chemie GmbH.pH values were measured by a pH electrode . Surface tension was measured at 37 °C using the DeNouy ring method . Osmolality was measured by using the freezing point depression technique . Pepsin activity was measured by a modification of the method described by Anson, and quantification was based on pepsin from porcine gastric as a standard .th, 25th, 75th, and 90th percentiles and the individual outlying data points. Within each box, horizontal solid lines indicate median values and horizontal dotted lines indicate mean values.Raw data are presented as Box-Whisker plots showing the median, 10Where possible, i.e. the power of the test allowed and normality and equal variance tests passed, the difference between the two groups was evaluated with the unpaired t-test. Type I error was set to 0.05. All statistical comparisons were performed using SigmaStat 3.5 .Mean (SD) values for the resting gastric volumes were 5.8 (3.6) mL and 6.1 (4.5) mL for Crohn’s disease patients and healthy subjects, respectively . These vIntragastric pH values of patients with Crohn’s disease were less variable than in healthy adults. Median (range) pH values of gastric contents in Crohn’s patients and healthy adults were 2.0 (range: 1.2-8.1) and 1.7 (range: 1.0-8.1), respectively . SimilarSurface tension of gastric contents was less variable in Crohn’s disease patients . Median Mean(SD) osmolality values of gastric contents were 293 (35) mOsmol/kg and 257 (39) mOsmol/kg in the Crohn’s disease and healthy adults, respectively . IntragaHigh inter-subject pepsin activity was observed in both the Crohn’s disease patients and the healthy individuals groups. Coefficients of variation were 101% and 94%, respectively. Median (range) pepsin activity values of Crohn’s disease gastric contents were 131,300  USP units/mL and 137,000  USP units/mL for the Crohn’s disease and healthy adults, respectively. Generally, these data are in agreement with a study in which the effect of malnutrition and subsequent re-feeding on digestive function in Crohn’s disease patients was investigated [Individual bile acid concentrations were highly variable in both study groups; concentrations were below the quantification limit (6 μM) in 11 out of 15 patients and in 7 out of 15 healthy individuals. Coefficients of variation of individual bile salt concentrations in Crohn’s patients and in healthy individuals ranged between 290% and 387% and between 206% and 304%, respectively. The predominant bile salts were glycoconjugates and the relative mean luminal concentrations were GC > GCDC > GDC ∼TC ∼ TCDC > UDC .Mean (median) values for total bile salts concentration in the gastric contents of patients with Crohn’s disease and healthy individuals were 141(12) μΜ and 99(10) μΜ, respectively .Based on pepsin activity data and bile acids data, investigation of the impact of potential differences between Crohn’s patients and healthy individuals requires prior designation of the minimum differences to be detected, as these will determine the minimum number of samples sizes required for reaching statistically relevant conclusions.Compared to healthy individuals, intragastric osmolality was significantly different but only slightly higher in Crohn’s patients; this difference is unlikely to affect the performance of drugs administered orally in the fasted state.Based on this exploratory study, average values of all other characteristics also suggest that differences on the physicochemical characteristics of gastric contents between adults with Crohn’s disease and healthy adults if any, are unlikely to affect the performance of orally administered drugs in the fasted state.However, especially for the most highly variable parameters, i.e. pepsin activity and bile acids concentrations, designation of the minimum differences to be detected is required in order to reach statistically relevant conclusions.


Berry cell death assessment can become one of the most objective parameters to assess important berry quality traits, such as aroma profiles that can be passed to the wine in the winemaking process. At the moment, the only practical tool to assess berry cell death in the field is using portable near-infrared spectroscopy (NIR) and machine learning (ML) models. This research tested the NIR and ML approach and developed supervised regression ML models using Shiraz and Chardonnay berries and wines from a vineyard located in Yarra Valley, Victoria, Australia. An ML model was developed using NIR measurements from intact berries as inputs to estimate berry cell death (BCD), living tissue (LT) (Model 1). Furthermore, canopy architecture parameters obtained from cover photography of grapevine canopies and computer vision analysis were also tested as inputs to develop ML models to assess BCD and LT (Model 2) and the intensity of sensory descriptors based on visual and aroma profiles of wines for Chardonnay (Model 3) and Shiraz (Model 4). The results showed high accuracy and performance of models developed based on correlation coefficient (R) and slope (b) . Models developed based on canopy architecture, and computer vision can be used to automatically estimate the vigor and berry and wine quality traits using proximal remote sensing and with visible cameras as the payload of unmanned aerial vehicles (UAV). In grapevines, berry cell death (BCD) occurs from 90–100 days after anthesis within the mesocarp tissue of berries  . Sensor 20–1650; ), which 20–1650; .This paper aimed to develop machine learning (ML) models based on NIR spectroscopy and canopy architecture parameters as inputs to predict the BCD, LT of berries, and potential wine acceptability by consumers for two of the most important cultivars in Australia: Shiraz and Chardonnay. Specifically, ML Model 1 was developed using the NIR absorbance values of berry samples  and canopy architecture parameters (Model 2) as inputs to predict living (LT) and dead tissue (BCD). On the other hand, Models 3 and 4 were constructed using canopy architecture parameters, obtained from upward pictures of canopies and computer vision algorithms as inputs to predict the intensity of sensory descriptors for Chardonnay (Model 3) and Shiraz (Model 4).Implementing these models close to harvest to predict the BCD, LT, and final wine quality traits may offer a more comprehensive berry maturity assessment to target the specific wine quality or style that characterizes a particular region. The assessment of canopy architecture parameters required for these models can be obtained from smartphone applications such as VitiCanopy  or aerian = 9 plants per cultivar; 18 in total)  at an elevation of 83 m. The commercial vineyard is located in the Yarra Valley with a total extension of 49 Has. The region is characterized by an oceanic climate with an average maximum temperature of 20.5 °C and a mean January temperature (MJT) of 28.1 °C with an average annual rainfall of 732.3 mm . Samples were obtained from two different cultivars: Chardonnay, clone Schwarzmann from Block B and Shiraz clone R99 in Block F , which un total) .n = 18 per cultivar; 36 total per date). Images were analyzed using a customized Matlab® R2020b code [e), crown cover (ff), canopy cover (cf), crown porosity (Φ), and Clumping Index (Ω) as described in previous publications [For canopy vigor analysis based on imaging, an iPhone 11  was mounted on a selfie stick. Two upward-looking images  at groun20b code ,16 , rotating the berry a third after each measurement. For data analysis, the first derivative based on Savitzky–Golay was calculated using Unscrambler X ver. 10.3  software to enhance the peaks in the hydrogen peroxide (H2O2) range . This apparatus can measure within a spectral range between 1596 and 2396 nm. For each measurement, the whole berry was placed on the nose of the device, covering the entire reading area. Three NIR readings were taken per berry .n = 216). All berries collected were halved using a sharp blade longitudinally through the pericarp at the highest diameter. One half was used to measure total soluble solids (°Brix) using an Alla France REFBX010 optical refractometer . The other half was used for staining with fluorescein diacetate (FDA) to acquire fluorescent microscopy images. The staining and fluorescence imaging followed the method described by Fuentes et al. [From each of the nine plants marked of each cultivar, two bunches were marked from the east and west sides. From each bunch, six berries were collected from the middle of the bunches n = 21. All ber® R2020b  [Images were obtained using the Leica Application Suite (LAS) software  color cooled digital camera with the same gain and exposure settings for all images A. Berry MA, USA) ,6, which−1; Enartis Pacific, Malvern East, VIC, Australia) was added to undergo fermentation, whilst for Shiraz, Saccharomyces cerevisiae EnartisFerm Vintage Red yeast was used (0.25 g L−1) for alcoholic fermentation, all batches were stored at 20 °C for 2 weeks.Bunches of berries from each plant and cultivar were collected at harvest for micro-vinification. Three batches (referred as groups in results) of wine from each cultivar were made using berries from each group of plants, which were the same used for BCD and canopy architecture analysis. Wines were made in the servery area from the sensory laboratory at the Faculty of Veterinary and Agricultural Sciences (FVAS) from the University of Melbourne (UoM). For both cultivars, bunches were destemmed (~3–3.5 kg) and crushed, followed by their transfer to 5 L carboys. For Chardonnay, Saccharomyces cerevisiae EnartisFerm Vintage White yeast (0.25 g L®) sensory session. Due to the lockdown in 2020 as a result of the COVID-19 pandemic, the sensory session was conducted remotely in the panelists’ residences. For this purpose, the wine samples were poured into 30 mL plastic test tubes, labelled with 3-digit random codes and mailed to the participants as express delivery in boxes with foam insulating material so that they received them within 1 day to avoid the wines from developing any off-aromas. For safety issues, panelists were asked to assess the visual and aroma characteristics of the wines only. Prior to the sessions, participants were asked to read and sign a consent form (Ethics ID: 1953926.4). Participants were asked to join a Zoom meeting  and open a link to RedJade software  with the questionnaire. Participants were also asked to place the samples in front of them. The questionnaire consisted of assessing the intensity of visual parameters, aromas, and perceived quality  and the Tukey test (α = 0.05) in XLSTAT 2020.3.1  to assess significant differences between samples. The mean and standard error (SE) values were also calculated for all parameters.Canopy architecture and sensory data were analyzed using ANOVA  as inputs to predict (i) living tissue (LT) and (ii) dead tissue (DT). Data were randomly divided, with 75% of the samples used for the training stage and 25% for testing using the mean squared error (MSE) as performance algorithm. Models 2–4 were developed using the Levenberg–Marquardt training algorithm. Model 2 was constructed using the canopy architecture parameters (i) LAI, (ii) LAIe, (iii) fc, (iv) ff, (v) φ, (vi) Ω as inputs to predict (i) LT and (ii) DT. On the other hand, Models 3 and 4 were developed using the canopy architecture variables as inputs to predict the intensity of sensory descriptors  were developed using a code written in Matlabcriptors  for Charcriptors . A diagrp > 0.05) between the three groups of samples for LT in both cultivars. For LAIe, there were non-significant differences (p > 0.05) between the groups of samples for Chardonnay, but there were significant differences (p < 0.05) for Shiraz. For the latter, Group 3 (0.91) presented significant differences from Group 1 (1.44).For LT and LAIe at harvest, it was found that Chardonnay had lower LT and higher LAIe than Shiraz . Accordip > 0.05) between samples. For Shiraz, there were significant differences (p < 0.05) for all parameters except for Ω. Group 3 of Shiraz presented significantly higher LAI (2.00), fc (0.68), and ff (0.84) than Group 1 . On the other hand, Group 3 was significantly lower in φ (0.19) than Group 1 (0.28) and 2 (0.21).p > 0.05) differences between the groups of samples for any of the sensory attributes in wines from both cultivars  available from a specific wine region could be used to apply the model and obtain wine quality trait targets from available wine libraries . Many wiThe implementation of AI in viticulture and winemaking offers powerful tools to assess berry quality traits, such as potential aroma profiles of wines based on berry cell death. Canopy architecture parameters used as ML inputs can be easily acquired through cover imagery or unmanned aerial vehicles (UAV), making the applications and models presented in this paper very practical to assess the spatial and temporal variability of quality traits. Furthermore, the canopy architecture model (Model 2) showed higher accuracy and better performance to Model 1 produced using NIR spectroscopy to predict LT and BCD. However, NIR instrumentation within the range required for the models can be economically prohibitive for small and medium vineyards, requiring know-how for the use, download and management of data. In the case of canopy architecture inputs, they have been already automated and can be easily incorporated as inputs to ML models developed. These models can be used as objective maturity assessments of berries in the field using precision viticulture tools and potentially assess wine quality traits if specific winemaking processes are incorporated within the ML procedure. Further research is required to model other cultivars and winemaking techniques. The results from this research can be incorporated into the models developed here by using the learning capabilities of ANN modelling techniques to acquire a universal model applicable to different regions and cultivars. Specific deployments of machine learning models developed should be determined according to the wine style from different wine producers and feed new relevant data into the models for retraining purposes. The latter procedure will not require an entirely new experiment but enough site-specific data for models to learn the variations required.