Enterobacterales. In contrast, only a few of the root samples inoculated with a single-bacterial strain (Group B) harbored a rich bacterial flora with relatively high bacterial diversity. However, wheat roots inoculated with a mixed bacterial complex were colonized by a more diverse and abundant bacterial flora, which was mainly composed of Enterobacterales, Actinomycetales, Bacillales, Pseudomonadales, and Rhizobiales. The results demonstrated that inoculation with bacterial complexes could help plants establish more balanced and beneficial endophytic communities. In most cases, bacterial inoculation does not result in successful colonization by the target bacterium in wheat roots. However, bacterial inoculation consistently had a significant effect on the root microbiome in plants. CAP analysis demonstrated that the variation in wheat root endophytic communities was significantly related to the taxonomic status and lignocellulose decomposition ability of the inoculated bacterial strain (p < 0.05). To reveal the role of lignocellulose degradation in shaping the root endophytic microbiome in wheat, four bacterial strains with different colonization abilities were selected for further transcriptome sequencing analysis. The results showed that, compared with that in the dominant bacterial species Ent_181 and Ent_189 of Group A, the expression of lignocellulose-decomposing enzymes was significantly downregulated in Bac_133 and Bac_71 (p < 0.05). In addition, we found that the dominant bacterial species of the tomato endophytic microbiome were more likely to become dominant populations in the wheat root microbiome. In general, our results demonstrated that lignocellulose-decomposing enzymes played a vital role in the formation of endophytes and their successful colonization of root tissues. This finding establishes a theoretical foundation for the development of broad-spectrum probiotics.The introduction and inoculation of beneficial bacteria in plants have consistently been considered as one of the most important ways to improve plant health and production. However, the effects of bacterial inoculation on the community assembly and composition of the root endophytic microbiome remain largely unknown. In this study, 55 strains were randomly isolated from tomato roots and then inoculated into wheat seeds singly or in combination. Most of the isolated bacterial strains showed an ability to produce lignocellulose-decomposing enzymes and promote plant growth. The results demonstrated that bacterial inoculation had a significant effect on the wheat root endophytic microbiome. The wheat root samples inoculated with single-bacterial species were significantly separated into two groups (A and B) that had different community structures and compositions. Among these, root endophytic communities for most wheat samples inoculated with a single-bacterial strain (Group A) were predominated by one or several bacterial species, mainly belonging to  Plants harbor a set of taxonomically and functionally diverse microbial communities on their surfaces and within their tissues. Similar to the human microbiome, the plant microbiome contains an enormous number of genes, and the genetic content far exceeds that of the host plant itself . These mPseudomonas as an example, when these strains come in contact with the plant root surface, they first form a colony and then form biofilms. In this way, Pseudomonas strains can colonize host plants as well as the fungi in the rhizosphere (Pseudomonas aeruginosa (P. chlororaphis) strain PCL1391, P. fluorescens in different parts of wheat roots and the number of spots of wheat take-all disease was studied, and the results confirmed that the colonization level of the biocontrol strains was inversely proportional to the number of disease spots on the host plants. The higher the level of colonization was, the lower the number of disease spots. When the root colonization level reached 107–108 CFU/cm, almost no disease spots were produced  examine the effect of bacterial inoculation with a single bacterium or a mixed bacterial complex on the community assembly and composition of the root endophytic microbiome of plants; (2) identify the factors that determine whether a microorganism becomes a root endophyte or successfully colonizes the inner tissue of plant roots; and (3) identify the reasons for the differences in colonization ability, which is why some strains exhibit colonization or even become excellent endophytes. A better understanding of the colonization ability of bacterial endophytes will contribute to the study of plant–endophyte interactions in agroecosystems and natural ecosystems.-7, inoculated on different media, and incubated in an incubator at 28° or 37°C. The experimental medium included Luria-Bertani agar (LB), nutrient agar (NA), tryptic soy agar (TSA), Gauze No. 1 medium, and Hoagland nitrogen-free medium. The bacterial colonies on the plate were selected according to size, color, and shape. The isolated endophytic bacteria were tested for growth on LB at 28° or 37°C and transferred to a new plate two times for purification. Then, the purified strains were divided into two portions for seed preservation and stored at 20% glycerol in a -20°C freezer for further study.The tomato cultivar Xinzhongshu No. 4, which was grown in a greenhouse under natural light conditions, was used for the isolation of endophytic bacteria. After 55 days of growth with nutrients, the tomato plants were pulled from the soil, and their roots were shaken to remove large soil particles. The harvested roots were carefully rinsed with tap water to remove the tightly attached soil, separately placed in 75% ethanol solution for 1 min and 5% NaClO solution for 3 min for sterilization, and then rinsed again with sterile water three times. The sterilized tomato roots were placed on LB agar plates at 37°C overnight to assess sterility to ensure that the isolated bacteria were from endophytic bacteria within the roots. The surface-sterilized root tissues were ground, serially diluted to 10The genomes of the isolated bacterial strains were extracted according to instructions for the Ezup Column Bacteria Genomic deoxyribonucleic acid (DNA) Purification Kit. A pair of universal primers, 27F and 1492R, was used for PCR amplification of bacterial 16S rDNA, using the extracted DNA as a template. The basic conditions for amplification were as follows: denaturation at 94°C for 30 s, followed by 30 cycles of annealing at 60°C for 30 s, and extension at 72°C for 90 s. The reaction volume was 50 μL, and 2 μL of DNA template was added for each reaction. The concentration of the PCR products was determined by 1% agarose electrophoresis. The purified PCR products of the 16S rRNA gene for each strain were sent for sequencing and molecular identification. After removing the repetitive strains, 55 isolated endophytic strains were selected for the subsequent bacterial colonization experiments, and the results of the molecular identification are displayed in 3, 0.1% K2HPO4, 0.05% MgSO4, 0.05% KCl, 0.2% carboxymethyl cellulose (CMC), 0.02% peptone, and 1.7% agar in 1,000 mL of H2O) with a sterile tip. The CMC agar plates were incubated at 28°C for 48 h and stained with Gram’s iodine solution (2.0 g of KI and 1.0 g of iodine in 300 ml of distilled water) for 3–5 min. The cellulase activity was determined by observing the presence or absence of hydrolytic circles. Standard cellulose solution was used as a positive control, and sterile water was used as a negative control. To screen for xylanase activity, similar agar plates were prepared using xylan as a substrate rather than CMC, and then, the plates were incubated at 28°C for 48 h and stained with Gram’s iodine solution for 3–5 min.In this experiment, the cellulase and xylanase activities of 55 isolated bacterial strains were detected. To screen for cellulase activity, the overnight cultured bacterial solutions were spotted on CMC agar plates (0.2% NaNO-1) for 8 h (some strains were shaken for 10–24 h). The concentrations of the bacterial cultures were determined at an OD600 of 0.5, and 100 μL of bacterial suspension was collected for each strain. Wheat seeds were surface-sterilized with NaClO (5%) for 10 min and thoroughly rinsed with sterile water three times. The sterilized seeds were sown in sterile plastic chambers with 2–3 layers of filter paper and incubated overnight at 28°C under dark conditions. Ten healthy wheat seeds were then placed in sterile chambers with 2–3 layers of filter paper and were separately treated with 50 μL of bacterial culture suspension. All the treated samples were transferred into a light incubator at 28°C for 3–5 days and then incubated under natural light at room temperature for 10–12 days. Sterile water was added promptly to keep the filter paper moist. The control plants were treated with a sterile medium solution. The experiment for each bacterial strain was carried out in triplicate.For the single-bacterial inoculation experiment, the 55 endophytic strains were separately inoculated in liquid LB medium and cultured at 28°C , Ent_189 (absolute dominance), Bac_71 (relative dominance), and Bac_133 (non-dominance), according to their abundance in the inoculated root samples. Furthermore, we constructed 3 mixed bacterial complexes using the 4 selected bacterial strains and their taxonomic relatives . Based o® DNA Isolation Kit  according to the instructions. The extracted total genomic DNA was stored at –80°C until being subjected to high-throughput sequencing.The healthy wheat plants were separately collected for each treatment, and roots were removed with sterile scissors. Wheat roots were disinfected in 5% NaClO solution for 30 s with sterilized forceps and then rinsed with sterile water three times. Subsequently, they were disinfected with 75% ethanol for 5–10 s and rinsed with sterile water three times again. The sterilized roots were used for the extraction of total genomic DNA by using the PowerSoilDNA fragments of the V5–V7 region of the bacterial 16S rRNA gene were amplified using the primer pair 799F (AACMGGATTAGATACCCKG) and 1193R (ACGTCATCCCCACCTTCC) and fused with Illumina MiSeq adapters and a 6-bp barcode sequence unique to each sample. PCR amplifications were carried out in triplicate in a 50-μL reaction system using the extracted genomic DNA as templates. The PCR amplification products were subsequently purified, combined in equimolar ratios, and subjected to high-throughput sequencing with the Illumina MiSeq sequencing platform, which produced 250 paired-end nucleotide reads, at Sangon Biotech . Two rounds of sequencing were conducted to ensure that an adequate amount of bacterial DNA  was available for subsequent analysis after the removal of all the chloroplast sequences.de novo method using USEARCH (version 8.1.1861)  were spliced with FLASH software (version 1.2.3). Barcodes and primer sequences were removed using Cutadapt (version 1.9.1). Low-quality sequences with ambiguous bases, average quality scores < 25, or sequences shorter than 200 bp were removed to control sequence quality. Then, chimeric sequences were identified and removed with a .1.1861) . After s.1.1861) . Accordip-value less than 0.05 . Through statistical analysis of the degree of influence of species on the samples, the value of the influence of each significantly different species on the sample was obtained, and significantly different microorganisms with a value of influence greater than two could be visualized.The taxonomic units and their relative abundances in each sample were visualized by drawing bar charts and heatmaps based on the number of reads using the R package gplots (version 2.17.0). The diversity indexes (Shannon and Simpson index) and species richness estimators (Chao1 diversity and ACE index) for each sample with respect to a sequence depth of 3% were calculated using QIIME (version 1.8.0). Rarefaction and rank abundance curves were generated at a 97% OTU similarity level. Principal co-ordinates analysis (PCoA) and hierarchical cluster analysis in QIIME were used to evaluate the beta diversity of samples and the similarities and differences in community composition of different samples. Statistical analysis was performed based on the unweighted UniFrac distances and Bray–Curtis matrix to determine the significant differences between samples. The differences in bacterial taxa among the groups of samples were identified by LEfSe (LDA effect size) analysis . First, To reveal the potential functional traits corresponding to the bacterial localization in the wheat root microbiome, four endophytic strains, Ent_181, Ent_189, Bac_71, and Bac_133, with colonization ability were selected for inoculation into wheat roots in sterile Petri dishes. The inoculated plants were grown, and the roots were harvested as described above. After surface disinfection, the sterilized plant roots were cutoff and placed into sterile bowls for grinding. Then, Phosphate Buffer Saline (PBS) was added to dilute the grinding solution to prepare the bacterial suspension. The bacterial suspension was obtained by serially filtering through 100, 20, and 11 μm membranes to remove plant tissues. Finally, the obtained endophytic bacterial precipitates for each sample were collected and sent to a commercial service for RNA extraction and transcriptional sequencing by using Illumina HiSeq™.de novo  were removed to obtain clean data. The clean data were assembled into transcripts de novo  and thende novo . The colAccording to the annotation results for the transcripts, the annotated GeneOntology (GO) function information was obtained, and the top GO functions were used for the analysis of GO enrichment to draw the network diagram. KAAS was used to obtain the annotated Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway information of the transcripts, and clusterProfiler was used for the analysis of the KEGG pathways and COG enrichment . Salmon The total genomic DNA of wheat root samples infected with 55 single-bacterial strains, parallel control samples, and mixed bacteria was amplified and sequenced in the V5–V7 region of bacterial 16S rDNA using Illumina MiSeq with 2 × 250 bp reads. The obtained raw sequence reads were separately merged and filtered for quality control . Finally, a total of 23,36,079 high-quality sequences for 58 root samples were obtained. On average, 41,713 high-quality sequences were obtained per sample . For the inoculation experiment with the mixed bacterial complex, 806,643 pairs of raw sequences (reads) were obtained for 21 root samples from 7 groups after high-throughput sequencing. These sequences were also filtered for quality control, and 690,046 high-quality sequences were obtained. On average, 32,859 high-quality sequences were obtained per sample .Achromobacter, Bacillaceae, Chryseobacterium, Lysinibacillus, Pantoea, Pseudomonadaceae, Acidovorax, Rhizobiaceae, Serratia, Staphylococcus, Stenotrophomonas, and Burkholderia, covering a broad range of bacterial phyla. The wheat root samples with single-bacterial inoculation were divided into Group A, Group B, and the control group for further bioinformatic analysis according to the Shannon index and community diversity of the endophytic microbiome. Compared with those of the control wheat group, the community structure and the composition of the root endophytic root microbiome of the inoculated groups changed greatly, suggesting that single-bacterial inoculation greatly affected the structure and proportion of the flora in the wheat roots (p < 0.05), suggesting that single-bacterial inoculation had a significant effect on the bacterial diversity  . The result was further verified by a Venn diagram, in which only a few OTUs were shared among the three groups or between pairs of samples  . Simulta samples . In summBefore identifying the endophytic strains that colonized wheat roots, bacterial strains with more than 99% sequence identity were first merged and regarded as one type of representative strain by pairwise alignment and the construction of a phylogenetic tree using the full-length 16S rDNA sequences of the 55 bacterial strains. Finally, 22 representative bacterial species with unique sequences were obtained . To idenp = 0.005). Among these, cellulase activity was significantly related to the root samples that harbored the dominant bacterial population in the endophytic microbiome , indicating that lignocellulose-decomposing enzymes play an important role in the mechanism of bacterial infection in plants. Among the 55 endophytic bacterial strains used in this study, most strains showed clear cellulase and xylanase activities  with the environmental variables was performed . The restivities , indicatBacillus strains (BacM), and mixed Enterobacter strains (EntM), resulted in an Enterobacteriales-dominant microbiome, with a proportion of 45–50% , Ent_189 (absolute dominance), Bac_71 (relative dominance), and Bac_133 (non-dominance), according to their abundance in the inoculated root samples. Furthermore, we constructed 3 mixed bacterial complexes using the selected 4 bacterial strains and their taxonomic relatives . Based of 45–50% . In contoportion . Compareoportion . However samples .Enterobacter strains could easily infect and colonize root systems via complex interactions. The Enterobacter strain corresponding to OTU101 had a relatively high abundance in Ent_181. Although the inoculated Bacillus strains were also found to be localized in wheat roots, such as the Bacillus strain corresponding to OTU5 in Bac_71 and that, corresponding to OTU4 in BacM, the abundance of the corresponding Bacillus OTUs was relatively low, and the infection was less effective than that with the Enterobacter strains , and the same result was observed when comparing Group A with Group B , which indicated that the assembly process was mainly influenced by external factors; that is, inoculation had an effect on the assembly of the bacterial flora in wheat roots . Among tPlant roots can release a large number of chemicals or organic compounds, such as amino acids, proteins, and organic acids, into soils to affect the microbial community of the rhizosphere and further affect the recruitment of bacterial endophytes in plant roots . Howeverp-value in the combined model with cellulase activity was less than 0.05.With regard to microbial ecology, it has been hypothesized that microbial assembly is mainly dominated by deterministic and stochastic processes . The stoIn the single-bacterial inoculation experiment for 55 strains, we found that infection by single bacteria was clearly dominated by 1–3 bacterial species with high abundance, and the dominant bacteria were most likely to be pathogenic bacteria, although these bacteria were also found to be the earliest colonizers among root endophytes in previous studies. In contrast, the community composition of root samples inoculated with the mixed bacterial strains was highly diverse and varied, with a relatively high abundance of Actinomycetales. These results were consistent with the ecological process analysis for the root endophytic microbiome; that is, bacterial inoculation significantly affected the community assembly of the root microbiome in plants. In addition, CAP analysis demonstrated that the lignocellulose-decomposing enzyme activities of the inoculated bacterial strains were among the main factors that affected the community assembly and composition. The further transcriptomic analysis also showed the significant differences in the expression of functional pathways and genes involved in lignocellulose degradation. It was then speculated that the formation of the flora did not simply follow the neutral theory but that there was a certain selective deterministic process (screened by the lignocellulose barrier) within the neutral theory. SRX1489 1103–SRX14891181 and SRX14938537–SRX14938540 for community and transcriptomic analysis of the wheat root microbiome and the GenBank database, accession numbers ON242109–ON242163 for the full-length 16S rDNA of the isolated bacterial strains.The data presented in the study are deposited in the NCBI Short Read Archive, accession numbers BT and YZ designated and supervised the project. TZ, JX, and RT performed the experiments. TZ performed all the bioinformatic analyses and prepared figures and tables. BT, TZ, KL, and XX contributed to the manuscript’s writing and revision. YL, QZ, KL, XX, and LL suggested protocols, data analyses, and interpretation of results. All authors have read and approved the submitted version of the manuscript.


Background and objectivePituitary apoplexy (PA) is a possible life-threatening disorder due to spontaneous hemorrhage or impaired blood supply in the pituitary gland. It may present as an acute or subclinical form, and treatment options include either surgery or a conservative approach. The purpose of this study was to retrospectively analyze the clinical, imaging, and hormonal features, as well as the therapeutic outcomes, in a relatively short period of time in a series of consecutive patients with pituitary apoplexy (PA).ResultsThirty-six patients were included, 50% presenting typical symptoms of PA. The presenting symptoms were headache (44.4%), visual abnormalities (44.4%), and digestive symptoms (22.2%). At diagnosis, hormonal deficiency was observed in 22 (61.1%) patients. Of the evaluated patients, 78.2% of the 23 operated cases and all unoperated cases presented tumor remnants. Vision improved in 81.8% of the operated and 100% of conservatively managed cases. Of all cases, 69.4% remained with long-term hypopituitarism.ConclusionComplex management of PA frequently leads to visual improvement but long-standing hypopituitarism. First described by Bailey in 1898, pituitary apoplexy (PA) represents a rare condition characterized by abrupt hemorrhage and/or ischemia of the constituents of sella turcica, mainly concerning a pituitary adenoma . The claThe objective of our study was to analyze predisposing or precipitating factors, clinical status, imaging and hormonal features, and therapeutic management and its outcome for 36 cases with PA evaluated in the course of one year in a single tertiary care endocrinological center. The rationale of our study came from the lack of an existing guideline for the treatment of PA based on previous studies, so adding our experience to the literature may help elaborate specific recommendations regarding the management of these cases.We performed a retrospective study that included 36 patients (sample size determination) diagnosed with PA and examined for one year  in the Department of Pituitary and Neuroendocrine Pathology at the “C.I. Parhon” National Institute of Endocrinology, Bucharest, Romania. The inclusion criteria regarding PA diagnosis divided our group of patients into two categories: the ones with classical clinical presentation, i.e., patients exhibiting signs of acute typical manifestations  accompanied by imagistic proof of hemorrhage or tumor necrosis, and the ones with subclinical presentation defined as brain imaging evidence of pituitary adenoma hemorrhage in asymptomatic/oligosymptomatic patients. The exclusion criteria included patients with a lack of conclusive investigations. Tumor hemorrhage was histopathologically confirmed in all operated cases. In all cases, a full pituitary function evaluation , testosterone/estradiol, cortisol, corticotropin stimulation test when needed, insulin-like growth factor 1 (IGF-1), growth hormone (GH) suppression test when needed, urine osmolality, and water deprivation test when needed), formal ophthalmological evaluation, and pituitary imaging (magnetic resonance imaging (MRI) or computed tomography (CT)) were performed at diagnosis and during follow-up. Close endocrinological and ophthalmological follow-up was performed. Hypopituitarism was established according to the current criteria of diagnosis . PituitaClinical presentation and predisposing events Half of the patients (18) presented a classical PA episode (eight of these patients have at least one precipitating factor before the apoplectic event), while the other half had pituitary adenoma hemorrhage described on sellar imaging accompanied by minimal, atypical symptoms (seven of them presented at least one precipitating factor prior to the apoplexy diagnosis). Overall, 41.6% of our patients had at least one possible predisposing factor . Nine (25%) patients were previously known to have a pituitary adenoma . These nine cases include five nonfunctioning pituitary adenomas, three prolactinomas, and one adrenocorticotropic hormone (ACTH)-secreting pituitary adenoma. Two of the nine cases underwent surgery before the apoplectic event, and all patients with prolactinoma were under treatment with dopamine agonists (DAs) at the time of PA. Regarding the type of pituitary adenoma in the patients who were not previously known to have a pituitary tumor (27), one newly discovered secreting pituitary tumor was found after the PA diagnosis was established (GH-secreting pituitary adenoma).Eight patients retrospectively recognized various signs or symptoms possibly suggestive of pituitary pathology present before the apoplectic event Table .The most common symptoms of apoplexy in all of our patients  were headache (44.4%), visual abnormalities (44.4%), and digestive manifestations (22.2%). Regarding the most common manifestations of apoplexy in the 18 patients presenting with a sudden and severe onset of symptoms, headache and ocular disorders were the most common (72.2% each), followed by digestive phenomena 38.8%). Oculomotor palsies were found in nine patients with classical PA. Mild similar symptoms were described in the subclinical group (Table 8.8%. OcuPituitary Apoplexy Score (PAS)PAS was determined retrospectively for 30 patients : 17 patients who underwent surgery (73.9%) and all 13 patients who were managed conservatively. An important number of patients (11) were attributed zero points, and the majority presented with PAS ≤ 4 points (82.3% of the operated patients and 92.3% of the patients who had a conservative management approach)  cases were previously known to have a pituitary adenoma: five nonfunctioning pituitary adenoma, three prolactinomas, and one ACTH-secreting pituitary adenoma. Eighteen patients were evaluated using MRI alone, 10 patients underwent CT scans alone, and both investigations were performed for eight patients. Pituitary macroadenomas were diagnosed in 25 (69.4%) cases, four patients presented pituitary adenoma under 10 mm maximum diameter, and no available data regarding tumor dimension was mentioned for seven patients.Hormonal assessmentWhen PA diagnosis was established, 22 patients were diagnosed with hypopituitarism . Corticotropic deficiency was noted in seven cases and was the most common deficit in patients with classical PA , while gonadotropic deficiency had the greatest prevalence in subclinical cases .Management and outcomesTwenty-three (63.8%) patients underwent neurosurgical intervention; 11 of them have typical clinical presentations. Twelve patients with a subclinical form of PA underwent delayed surgery, mostly within one month from diagnosis. Neurosurgical decompression was performed . The decision was made by a multidisciplinary team .Tumor OutcomeAfter the apoplectic event, imaging follow-up results showed that the majority of the evaluated patients by CT/MRI presented remnant intrasellar mass (85.3%), irrespective of the management type (78.2% of those operated and 100% of those conservatively managed)  of the operated patients and all cases with conservative treatment had improvement in vision Figure .Hormonal OutcomeNewly developed pituitary insufficiency was more important in patients undergoing surgery versus patients treated conservatively Figure . A totalWe have retrospectively analyzed the clinical course and therapeutic outcome for 36 patients with PA referred to our center during 2019. The mean age at diagnosis in our patients is similar to previous studies ,9,12-15.Inconsistent symptoms of pituitary disorder were retrospectively found in a minority of our patients  a few months before the diagnosis of PA, mostly of the subclinical type. Similar clinical features suggestive of endocrine dysfunction were also described as being rare in other cohorts . Other sAlthough the Pituitary Apoplexy Score (PAS) suggested by the UK Pituitary Apoplexy Guidelines Development Group was not calculated and taken into consideration in our patients before the therapeutic decision, it could represent a solution for better division of cases when defining a major neuro-ophthalmic insult and therefore making the best choice between management options. In the future, the use of this type of tool may improve the follow-up and perhaps help elaborate guidelines for the optimal management of PA ,6,9.In our series, both CT and MRI were used to diagnose pituitary lesions. For some of our patients, an MRI scan was used even after a CT examination was made, which supports what other authors claim about the fact that CT is not sensitive enough for PA diagnosis and the accurate assessment of the contents of the pituitary sella ,13,18. MPatients affected by PA were diagnosed with a nonfunctioning pituitary adenoma in most of our cases as in other studies ; perhapsIn the classical symptomatic form of PA, corticotropic deficiency is considered the most common acute endocrine dysfunction ,13,18,20The main limits of our study are represented by the retrospective nature and relatively small sample of patients. Also, the retrospective calculation of the PAS might not prove to be accurate in all patients. The design of our study did not allow long-term follow-up of the tumor remnant.Pituitary apoplexy is a rare and challenging endocrine condition that can be difficult to diagnose and treat. We consider achieving the objective of our study, and our verdict is in large part similar to preexisting literature. The visual recovery in these patients is notable, but the outcome of pituitary function is less encouraging. As no consensus guidelines of optimal management are available, our study offers a global image and raises awareness about the management of this disorder in our country and underlines the need for multidisciplinary evaluation of these patients and future practice guidelines to optimize specialized care for these rare patients.


B. indicus × Bos taurus bulls were randomly assigned in pens with or without shade from a galvanized steel-roof structure. Performance data  and heat stress indicators  were assessed along the study period. The economic outcomes derived from shade implementation were determined using the net present value. Meteorological variables were also monitored every 1 min, and grouped in a thermal comfort index for feedlot cattle, the InComfort Index (InCI). The shade structure efficiently reduced radiant heat load on cattle in pens with shade. According to the classification of the InCI, during very hot days , greater proportion (80%) of animals in shaded pens were using shade. Under such circumstances, cattle in shade had water intake reduced by 3.4 L per animal, body temperature was lower by 5°C, subcutaneous temperature was lower by 1°C and respiration rate was lower by 10 breaths min−1 compared to animals in pens without shade (P = 0.0001). Although dry matter intake was similar (P = 0.6805), cattle in pens with shade had higher average daily gain reflected in a heavier hot carcass weight . Considering an initial investment of $90 per animal to build a structure that lasts 15 years, the expected payback time is four finishing cycles (~110 days per cycle). In conclusion, this study confirms that the proposed novel shade design is economically profitable, improves performance, and enhances animal welfare.Provision of shade reduces radiant heat load on feedlot cattle, thus reducing demand of water and energy for thermoregulation. While the positive effects of shade on animal welfare are widely known, the literature lacks data on the magnitude of its economic impacts. In this study, we propose the concept of novel shade design to prove that a correctly oriented and dimensioned roof structure, which optimizes shade to be displaced within the pens, motivates cattle to seek shade, protect them from short-wave solar radiation, and is resilient to counteract weather adverse conditions. The beneficial outcome is improvement in animal welfare and productive performance, as well as increments on financial return and sustainability. To attest these benefits, eight hundred  Artificial shade is in the forefront of environmental modification to mitigate the negative impacts of heat stress and to improve welfare of beef cattle, particularly in tropical environments where animals face high levels of short-wave solar radiation , 2. The Although the positive wellbeing and behavioral effects of providing shade for animals are unquestionable, their economic benefits are still inconclusive. Some studies have found positive outcomes on animal performance and economic gains , 14, othIn this study, we propose a novel concept of shade that could be used in tropical areas. It is a design that combines rectangular pens with a shade structure mounted in a north-south orientation. This design makes shade to be projected within the pens, motivates shading-seeking behavior of cattle, and efficiently protects them against short-wave solar radiation, while improves animal welfare, increases productivity performance, and offers an interesting 1–2 year payback time. To attest these benefits, 800 steers were randomly assigned in shaded and unshaded pens. The animals and the environment were monitored to (1) describe the thermal environment experienced by the animals, (2) determine the impact of shading on heat stress indicators, (3) assess the impacts of shading on dry matter intake, water intake, average daily gain, and hot carcass weight, and 4) provide analyses concerning economic outcomes of the shading structure. To the best of our knowledge, this is the first robust study to show the economic benefits of shade in feedlot cattle under commercial conditions of Brazil, where close to seven million cattle are kept in feedlots without shade . This is provide 2, with precipitation rate above 1200 mm year−1 occurring at summer months (From November to February), and dry winters (From May to July). The novel concept of shade design used in this study comes from a shade structure that optimizes maximum availability and displacement of shade within pens, blockage against solar radiation, resilience to weather adverse conditions, and ease of daily management operations. We proposed rectangular pens with 15 × 50 m (width × length) instead of square pens, and the shade structure installed on the north-south fence line. This design favors a moving shade from east to west within the pen, and motivates shade-seeking cattle to follow the shade, which results in reduced wet areas because of congregation of animals  . Overall animals . AdditioThe shade structure was made from galvanized coated aluminum/zinc/silicon (55% × 43.5% × 1.5%) steel-roof structure . The anchors were made of steel instead of wood because the cost of steel is the same as that of wood in Brazil. The frame assembly was made of tubular beams which is easier to assemble . PillarsB. indicus × Bos taurus, predominantly Nellore bulls were used in the study. Approximately 2 weeks before the beginning of the study, the animals were treated with anthelmintic medication , immunized against bovine respiratory disease , and clostridia .All animal procedures were approved by the Animal Ethics Committee of the São Paulo State University (Protocol number 016339/19). A total of 800 2 of shade floor area/animal. The 800 bulls were equally divided between pens with shade, and pens without shade . Each bull was provided with 15 m2 space, 0.30 cm of concrete bunk space, and 3.0 × 0.8 × 0.25 m (length × width × height) water trough. The bulls were assigned in blocks by their fasted initial body weight (BWi ± SEM) , and weekly harvested (one block per week) in the order of heavier to lighter block. Furthermore, bulls were phenotypically characterized by a trained observer on the basis of muscularity, structure, body length, hip and wither height. They were then grouped into five different phenotypes: Nellore, Black Angus x Nellore, crossbred , crossbred (European dairy breeds), and crossbred (European beef breeds).Sixteen soil-surfaced pens were used, eight of them had the novel shade design that provided 3.0 mCynodon ssp., equipped with feeding bunk. During this phase, all animals received pre-experimental (maintenance) diet (−1) of 1.5 kg (−1 day−1) and for chemical analyses. All the samples from diets were dried at 55°C in a forced-air oven for 72 h for DM determination. Dried samples were grounded with a Wiley-type mill  and analyzed for ash (method 924.05)  and allocated in three 16 ha pastures of ce) diet , for at f 1.5 kg  24). Bu. BuCynodf 1.5 kg . Diet dr 924.05) , NDF (27 924.05)  and EE ( 924.05) .3 animal−1 day−1). Hydrometer readings were taken every morning. The average daily water intake , dry matter intake , average daily gain , and conversion  were determined for each pen. The DWI was determined by measuring water flow to the water troughs as follows:Water was freely available. Each water trough was equipped with hydrometers to determine daily water intake  and N is the number of animals in the ith ordinal day of the experiment in the jth pen. The DMI was determined based on the difference of the offered and refused feed, as follows:Where, Hfo is the amount of dry matter offered (kg day−1), while fr is the amount of dry matter refused collected daily at the feed bunk. The ADG were calculated based on fasted initial body weight  and fasted final body weight  as follows,Where, n is the total number of days on feeding. The BWi and BWf were obtained using a scale that was regularly calibrated with 450 kg standard-weight. The feed conversion (FC) was calculated as follows,Where, f, the animals were transported (330 km) to a commercial packing plant, and hot carcass weight  was obtained after complete evisceration and remotion of kidney, pelvic and heart fat of the carcass.After reaching their expected BWS, W m−2; CMP-22, Kipp and Zonen, Delft, Netherlands; spectral range = 0.3–3.6 μm), ultraviolet solar radiation , air temperature , black-globe temperature in the sun , relative humidity , wind speed  and daily precipitation , were all continuously recorded every minute with a portable weather station  placed near (~50 m) the pens with shade and without shade. Temperature sensors were also placed inside the pens and water troughs to better characterize the microclimate experienced by the bullocks in the shaded and unshaded pens. These measurements were recorded every 5 min.Solar irradiance  and exposed to clear sky . Three temperature sensors (i-buttom) were previously waxed  and placed inside the water troughs to obtain water temperature (°C). The ground surface temperature underneath the shade and in the full sun were periodically measured with an infrared thermal camera .A set of six black-globe devices were placed in two shaded pens , and thrSC,°C), respiratory rate , and body surface temperature  were measured both in bulls housed in shaded and unshaded pens. Behavior of bulls in the shaded pens were also observed. Ten days before the experimental period, miniature implantable bio-loggers  were surgically implanted in forty animals, twenty in the shaded pens, and the other twenty in the pens without shade. Before the implantation, all the loggers were calibrated at 2°C increments between 30 and 42°C in a thermally insulated box against a highly-accurate thermocouple . Logging and storage of TSC data was set to measure every 2 min during the experimental period.Subcutaneous temperature  during 20 days. Respiration rate was observed by five observers positioned outside the pens  by visually counting the flank movements of the bulls. Body surface temperature was measured using an infrared thermal camera  scanning the dorsal right side of the animals at a distance of 3.0 m. For each bull, the TS was considered as a mean of the scanned dorsal right side, from the scapular to rump region. Images were analyzed using FLIR Thermal Studio. Number of bulls using shade or at feed bunk was periodically monitored by using direct, focal and instantaneous samplings every 15 min, from 08:00 to 17:00 h. A total of 1500 observations from 250 animals were scanned along 20 days during adaptation, growing and finishing phase. Shade use was recorded whenever the head or one of the hooves of an animal was underneath the shade area. If not, it was considered as exposed to solar radiation. Feed bunk use was recorded whenever an animal was at standing position close (~0.5 m) to the feed bunk.The RTo determine the economic outcomes and the payback time of the proposed shade design, we calculated the net present value (NPV) as0 is the initial investment of the shade structure ($), FCn). We estimated the initial investment of the artificial shade structure in USD$ 90.00 animal−1 .Where, ΠA, HR, RS, U, WS and TG) experienced by the animals. Principal components were obtained by computing eigenvalues (λi) and its respective eigenvectors PCAj1 = e11TA + e12HR + e13RS + e14U + e15WS + e16TG), and second principal component (PCAj2 = e21TA + e22HR + e23RS + e24U + e25WS + e26TG). All principal components were used in order to develop an environmental index, the InComfort Index (InCI) based on the Membership Function Value Analysis:Principal component analyses –34 were R(λi) is given byWhere, n is the number of principal components and InCI is the weighted membership value calculated with principal components for each day linked with its meteorological condition and level of heat stress experienced by the animals. The i is the value of ith principal component, λi(min) and λi(max) are the maximum and minimum values of ith principal component, respectively. The W(ei) is given byWhere, λW(ei) is the weight of the ith principal component among all the principal components selected for evaluating level of heat stress experienced by animals on ith day, and ei is the contribution rate of the ith principal component. Based on daily water intake and respiratory rate, the InCI were grouped into four classes: rainy days, when the mean is 0 ≤ InCI ≤ 1, and precipitation rate above 20 mm day−1; cloudy days, when the mean is 0 ≤ InCI ≤ 0.4; hot days, when 0.4 < InCI ≤ 0.6; and very hot days, when the mean is 0.6 < InCI ≤ 1.Where, In this study, we investigated the effects of the following independent variables on cattle performance and heat stress responses: shade vs. no shade, weight block, coat color (black vs. light-colored cattle), time of day, heat load experienced during the days of feeding and association between them. Confirmatory models were then fitted by applying conventional statistical techniques through mixed model based on Generalized Least Squares (GLS) using the Statistical Analysis System . BecausYijkl is the nth measurement of the dependent variable; S is the fixed term of the ith shade treatment (S = shade and unshaded pens); B is the fixed effect of the jth class of initial fasted body weight ; SB is the interaction between the ith shade treatment and jth class of body weight; H is the fixed effect of the kth class of the InComfort index ; SH is the interaction between the jth shade treatment and kth class of the InComfort index; SBH is the interaction between the ith shade treatment, jth class of body weight and kth class of the InComfort index; and D is the fixed effect of the lth days on feeding  within the interaction between ith shade treatment, jth class of body weight and kth class of the InComfort index. Models c and d had SP as interaction between ith shade treatment and jth phenotypic trait of cattle, and C is the fixed effect of the lth corral pen within the interaction between ith shade treatment and kth class of body weight shade treatment. The μ is the parametric mean and εijklm is the residual term. For subcutaneous temperature (e), body surface (f), and respiratory rate (g) the best fitted models were:Where, ith shade treatment; C is the fixed effect of the jth coat color (C = Dark and light colored cattle); SC is the interaction between the ith shade treatment and jth coat color; H is the fixed effect of the kth class of the InComfort index; D is the fixed effect of the lth days on feeding within kth class of the thermal comfort; T is the fixed effect of the mth time of the day ; and SCHT is the interaction between ith shade treatment, jth coat color, kth class of the InComfort index and mth time of the day. For models f, g, and h, SCT is the interaction between ith shade treatment, jth coat color and kth classes of solar irradiance. εijklm and εijkl are residual terms. Behavioral data were analyzed using nonparametric regression analyses through the Generalized Additive Models (GAM Procedure), by fitting air temperature, black-globe temperature, solar irradiance, and wind speed as independent variables.Where, S is the fixed term of the Figure 7). During these days heat stress indicators were significantly reduced compared to bulls without access to shade. Overall, the values were 5°C lower in body-surface temperature, 1°C lower in subcutaneous temperature, 10 breaths min−1 lower for respiration rate, and 3.4 L animal−1 lower for water intake . Even though dry matter intake was similar (P = 0.6805) for the bulls in shade and no shade (Table 4), those in pen with shade presented better feed conversion (P = 0.0004) and heavier hot carcass weight . The increment on hot carcass weight of the bulls in pens with shade was sufficient reason to make the proposed shade design economically viable. The payback time is expected to be within four feeding cycles .We confirmed the hypothesis that the proposed novel shade design not only benefits cattle comfort, but is also economically sustainable. Three main findings support this hypothesis: First, based on the radiant temperatures taken within the shade and full sun, the structure with shade efficiently reduced radiant heat load on the animals, especially on very hot days . The variable accounted for the combined effects of solar irradiance, black-globe and air temperature, humidity, wind speed, and precipitation rate. Feeding days were grouped into four classes , 5. Durivery hot , 37–40. very hot .−2 . Animals in shade also received less long-wave radiation emitted from the shaded surface. During the hottest time of the very hot days, the radiant temperature of the shaded ground surface was 15°C lower than the unshaded areas, a difference that represents 100 W m−2 less long-wave radiation being emitted from the ground surface  and B. indicus light hair coat cattle (Brahman) sought shade as the solar load increased, and panted less when they were in the shade. Considering that increments on respiratory rate and peripheral vasodilatation are positively correlated with mass and heat transfer through respiratory and cutaneous surface . The increase was greater during the very hot days , particularly when exposed to hot days. In this study, using mean difference of 3.43 L animal−1 d−1 for cattle in pens without shade vs. cattle in pens with shade during 23 very hot days, and extrapolating to 100 days in a year with similar meteorological conditions and for 20 000 animals, the use of shade would potentially save 6860 m3 of clean water. The saving emphasizes the effectiveness of using shade to alleviate heat stress of cattle and reduce water consumption.When cattle are exposed to hot conditions and are unable to access shade, they can sustain high rates of evaporative water loss to maintain their thermal equilibrium, provided that body fluids can be replaced timeously , a physi−1 d−1, for shaded and unshaded cattle, respectively. However, cattle in pens with shade presented better feed conversion (P = 0.0389), which was nearly improved by 4.5 % (6.83 vs. 7.15 ± 0.10) as well as increased average daily gain (P = 0.0004) by 5%  compared to cattle housed in pens without shade (Use of the proposed novel shade design increased efficiency in dry matter intake. Both groups of cattle in shade and no shade ate less during hotter and rainy days. Bulls may decrease intake during hotter days as a tentative mechanism to reduce heat generation by metabolism . During ut shade . This reP = 0.0002) hot carcass weight than those kept in pens without shade  and wind (80 km hr−1).The economic feasibility of this study was done on the basis of net present value. The increment in hot carcass weight resulted in a payback time within the first four feedlot cycles . Considen et al.  reportedife-span , especiaife-span . The shaThe concept of this novel shade design proposed herein challenges the conventional east-west orientation design normally used for shade structures in tropical environments. The later design projects a “static” shade under the roof structure, and consequently becomes causes for cattle congregation, soil compression and mud formation. To alleviate these problems, we propose a combination of rectangular pens with a roof structure-oriented north-south that makes the shade to displace along the east-west direction along the pen . The outThe following conclusions can be drawn from this study which involved 800 feedlot cattle randomly assigned in pens with and without shade access to determine the effectiveness and economic viability of shade use:The proposed novel shade design combines a roof structure with a north-south orientation, rectangular instead of square pens (length at least three times higher than width), and roof height of at least five meters. This design allows almost 100% of the pen area to be in shade throughout the day.Cattle in pens with shade presented higher average daily gain, reflecting in heavier hot carcass weight  compared to cattle with no access to shade.−1 precipitation combined with >80 km hr−1 wind speed.The novel shade design effectively buffers the negative impacts of high radiant heat load on cattle and resists adverse weather impacts as high as 50 mm hrThe expected payback period for the novel shade design was within four finishing cycles (~110 days per cycle), assuming an initial investment of USD$90 per animal to build the structure that lasts 15 years.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.All protocols performed in the study were approved by the IACUC of the São Paulo State University, Brazil.Conceptualization: AM, RP, GM, and VC. Methodology, validation, and data collection: AM, RP, and GM. Formal analysis: AM and GM. Writing original draft–manuscript: VF. Original draft-figures: AM, GM, and VF. Writing–review and editing: AM, HM, KG, and RP. Visualization: AM, KG, RP, GM, HM, MC, and VF. Supervision: AM and RP. Funding acquisition: AM, VC, and RP. All authors contributed to the article and approved the submitted version.


Astronauts suffer skeletal muscle atrophy in microgravity and/or zero-gravity environments. Artificial muscle-actuated exoskeletons can aid astronauts in physically strenuous situations to mitigate risk during spaceflight missions. Current artificial muscle fabrication methods are technically challenging to be performed during spaceflight. The objective of this research is to unveil the effects of critical operating conditions on artificial muscle formation and geometry in a newly developed helical fiber extrusion method. It is found that the fiber outer diameter decreases and pitch increases when the printhead temperature increases, inlet pressure increases, or cooling fan speed decreases. Similarly, fiber thickness increases when the cooling fan speed decreases or printhead temperature increases. Extrusion conditions also affect surface morphology and mechanical properties. Particularly, extrusion conditions leading to an increased polymer temperature during extrusion can result in lower surface roughness and increased tensile strength and elastic modulus. The shape memory properties of an extruded fiber are demonstrated in this study to validate the ability of the fiber from shape memory polymer to act as an artificial muscle. The effects of the operating conditions are summarized into a phase diagram for selecting suitable parameters for fabricating helical artificial muscles with controllable geometries and excellent performance in the future. The zero-gravity and/or microgravity environments astronauts must endure are made incredibly physically demanding through skeletal muscle atrophy, which is caused by leaving Earth’s gravitational field. Muscle strength and articulation are imperative for astronauts’ effectiveness and safety when aboard the International Space Station (ISS), traveling through deep space, or adjusting to the gravity of terrestrial bodies beyond Earth . To helpExoskeletons can be used as muscular assistive devices for astronauts during human space exploration in strenuous situations to mitigate risk. Using actuators, linkages, and pivots, exoskeletons aid the wearer’s muscles, bones, and joints ,4,5. In Spinning, twisting, and three-dimensional (3D) printing are the most widely used manufacturing techniques for producing SMP artificial muscles. Three subcategories of spinning are used to fabricate artificial muscles: melt spinning, wet spinning, and electrospinning. In melt spinning, viscous polymer melt is drawn from a spinneret, cooled, and spooled onto a rotating drum, resulting in a long, single polymer fiber ,11,12. TRecently, a material extrusion system  has been3, glass transition temperature of 55 °C, melting temperature of 155 °C, and thermal conductivity of 0.132 W/m·K [Luminy LX175 polylactic acid (PLA) pellets  were selected as the SMP build material for all helical artificial muscles in this study. This particular PLA has a density of 1240 kg/m32 W/m·K .The representative helical artificial muscles in this study were printed using the same extrusion system and process described by our previous research . PrintheThe average outer diameter, fiber thickness, and pitch of each muscle group were measured using a high-precision measurement system  and metrology software . The averaged geometries across all muscle groups were then evaluated against each other to determine how extrusion conditions affect the helical geometry of the muscles. The average geometries are also compared to the corresponding flute diameter, depth, and pitch of the mandrel to determine which extrusion conditions result in geometries that closely resemble the mandrel.−1 and 10 s−1 and fit to the Carreau model to determine the zero-shear rate viscosity using an Anton Paar rheometer . The rheology tests were conducted using a 25 mm diameter parallel plate tool at a tool gap of 1 mm.Shear rate sweeps were performed at 160 °C to investigate the zero-shear rate viscosity just above the melting temperature of the PLA build material. The viscosity was measured at shear rates between 0.01 sThe mechanical properties of the artificial muscles were characterized through tensile tests using a mechanical property tester . Muscle groups 2 and 6 were used as representative artificial muscles to evaluate the effects of extrusion conditions on mechanical properties. The artificial muscles were tensioned along the helical axis at a strain rate of 25 mm/min. The average cross-sectional area was calculated for each muscle group tested using the measured geometries of the artificial muscles. Load and displacement data from testing, along with the calculated average cross-sectional area, were used to construct respective stress–strain curves and calculate elastic moduli for the respective muscle groups.The previously described high-precision measurement system and inspection software were used to inspect the surface morphology and measure the surface roughness of the helical artificial muscles. Muscle groups 2 and 6 were selected as the representative artificial muscles to compare how extrusion conditions affect surface morphology and roughness. The arithmetic mean surface roughness was measured on the linear portions of the artificial muscles using the InSpec metrology software. In total, 100 equally spaced points were fitted across 2 mm of the artificial muscle edge at linear sections using the measurement software. The locations of the fitted points were used to calculate a mean line and the arithmetic mean of all 100 points from the mean line, resulting in a surface roughness value for a section of the muscle.Muscle group 2 was also selected as the representative artificial muscles for contraction testing. To perform contraction tests, each artificial muscle was programmed at the glass transition temperature (55 °C) of the SMP material, cooled to harbor the necessary internal stress that causes contractions, and reheated above the glass transition temperature to generate contractions. To begin programming, an artificial muscle is heated to 55 °C for five minutes using a 150-watt heat lamp . Then the muscle is elongated to a strain of 10% using the previously described material property tester while still under heat. The artificial muscle was removed from heat and allowed to cool back down to room temperature at the elongated state. Finally, the muscle was reheated to 60 °C to induce contractions. During reheating, the mechanical property tester was used as a load cell to measure the contraction stresses of the artificial muscle over time. From the contraction stress data, an average max contraction stress and stress rate were calculated for the representative artificial muscles.n = 3 samples per group.All artificial muscles were trimmed to a target length of 75 mm for all measurements and tests. During trimming, the tail end of the muscle was removed to reach the target length. All quantitative values of artificial muscle measurements in the text and figures were reported as means ± standard deviation (SD) with The extrusion system, shown in Not only does the helical mandrel support the muscle as it extrudes, but it also aids in the shape memory effects of some SMP materials. As the polymer melt flows through the mandrel, the polymer chains within the melt align to the longitudinal direction of the helical flute of the mandrel. SMPs with anisotropic coefficients of thermal expansion benefit greatly from this polymer alignment and helical geometry, allowing for larger contraction strains and forces, as well as fewer muscles within an artificial muscle array to produce the desired muscle contractions ,18,34. Consistent and predictable muscle geometry is paramount for artificial muscle production. Varying the key extrusion conditions will either generate condition combinations that successfully extrude muscles of various geometries or conditions that cannot produce proper helical structures. Furthermore, specific combinations of key extrusion conditions may result in different helical geometries using the same internal mandrel. The ability to produce muscles with different geometries from the same mandrel increases the usefulness of the extrusion system by allowing the operator to produce a wide variety of muscle geometries without altering the printhead’s internal mandrel. Therefore, it is critical to discover what ranges of each key extrusion condition produce suitable helical artificial muscles, what combinations produce geometries close to the geometry of the mandrel, and how the conditions affect the overall geometry.To determine the effects of extrusion conditions on muscle geometry, the single-factor experiments described in The geometries for muscle groups 1, 2, 3, and 4 are illustrated in For muscle groups 5, 6, 2, and 8, helical geometry measurements are shown in Lastly, the geometries for muscle groups 9, 2, 11, 12, and 13 are shown in The measured geometry variance caused by single-factor experimentation can be attributed to either the die swell phenomenon or self-gravity. When the printhead temperature increases or the cooling fan speed decreases, the exiting polymer has not solidified and therefore is in a more molten state; allowing the velocity profile to relax and causing the fiber thickness to enlarge or swell ,35,36. ALastly, muscle group 2 was found to have an average muscle diameter (3.58 ± 0.16 mm), fiber thickness (1.16 ± 0.09 mm), and pitch (11.59 ± 2.44 mm) closest to that of the ideal geometries of the mandrel flute diameter, depth, and pitch , making the extrusion conditions for this muscle group best suited for printing geometries close to the mandrel geometry. Herein, muscle group 2 will be a representative muscle for all further testing.To better evaluate the printability and artificial muscle formation, a 3D phase diagram was constructed using the key extrusion conditions and geometry measurement results. As seen in Furthermore, To simplify printing condition selections, the Brinkman number Nua=3), viscosity (184.6 × 10−7 N·s/m2), thermal conductivity (26.3 × 10−3 W/m·K), and Prandtl number (0.71) of air at room temperature, are all known constants and shown in At room temperature, the Prandtl number is assumed to be 0.71 , and the−1 by fitting the data to the Carreau model. The Carreau model is defined as:−19 Pa·s, 0.343 s, and 0.0333.The measured viscosity of molten PLA at 160 °C is shown in −8 and 5.96 × 10−8 and Nusselt numbers between 1.9143 and 2.3318 will result in adequately formed helical artificial muscles. On the other hand, using extrusion conditions that result in Brinkman and Nusselt numbers outside of these ranges will either produce a molten filament without a helical profile or a clogged printhead.Using the zero-shear rate viscosity calculated from the rheological testing, the Brinkman and Nusselt numbers of the polymer during the extrusion process can be calculated. 2 and 1.07 mm2, respectively. Muscle group 6 was fabricated with a lower fan speed than those of muscle group 2, therefore, the muscle group 6 muscles exited the printhead at a higher temperature. The difference in surface morphology and surface roughness is shown in Helical geometry is only one critical aspect of artificial muscle production and cannot drive all extrusion conditions. The mechanical properties of the artificial muscles are also affected by the extrusion process. 2, the artificial muscles were able to induce a contraction stress of 49.52 ± 6.01 kPa and an average stress rate of 0.308 kPa/s, validating their ability to contract as artificial muscles.Lastly, the helical artificial muscles must contract when stimulated, and the contraction must be quantifiable to determine its viability. PLA is a thermally actuated, one-way SMP; therefore, the muscles must be programmed at or above their glass transition temperature before they can contract ,46. The The formation of PLA helical artificial muscles through a specifically designed extrusion system was evaluated at various extrusion conditions. The geometric measurements and general observation of muscle shape fidelity were used to determine successful printing condition combinations. Depending on the combinations of the key extrusion conditions, the polymer build material cooled too rapidly and clogged the printhead, lacked proper solidification, or fell into a suitable temperature and polymer melt flow range that successfully extrudes artificial muscles. Three-dimensional and two-dimensional phase diagrams were constructed using the results of the 13 printing experiments. After measuring the geometry of all muscles, it was found that increasing the printhead temperature or decreasing the cooling fan speed increases fiber thickness. Furthermore, increasing printhead temperature, reducing the cooling fan speed, or increasing inlet pressure increases muscle diameter and pitch. Additionally, shape morphology and mechanical properties were shown to be affected by the extrusion conditions. Lastly, representative artificial muscles printed with a printhead temperature, cooling fan speed, and inlet pressure of 125 °C, 75%, and 12 psi were programmed and contracted using thermal stimulus. These artificial muscles had a contraction stress and stress rate of 49.52 kPa and 0.308 kPa/s. Future work may focus on other extrusion conditions and extrusion through mandrels of differing geometries. Helical artificial muscle extrusion using different SMP build materials will also be tested and compared to previous works. PLA was chosen in this study as a proof-of-concept material to determine whether helical geometry, mechanical properties, and surface morphology could be controlled for a shape memory material. However, the contraction stress of PLA artificial muscles measured in this study is lesser than the two-way SMPs commonly used in artificial muscle fabrication, such as nylon and ethylene-vinyl acetate copolymer. Therefore, artificial muscles will be extruded using nylon and ethylene-vinyl acetate copolymer, and the contraction stresses and strains will be compared to the results published by Haines et al.  and Qi e


Polypropylene (PP) has become the most promising and candidate material for fabricating lightweight products. Microcellular injection molding (MIM) is a cost-effective technology for manufacturing porous plastic products. However, it is still challenging to fabricate high-performance PP microcellular components. Herein, we reported an efficient strategy to produce lightweight and high impact toughness foamed PP/polyethylene terephthalate (PET)/polyolefin-based elastomer (POE) components by combining in situ fibrillation (INF) and MIM technologies. First, the INF composite was prepared by integrating twin-screw compounding with melt spinning. SEM analysis showed PET nanofibrils with a diameter of 258 nm were achieved and distributed uniformly in the PP due to the POE’s inducing elaboration effect. Rheological and DSC analysis demonstrated PET nanofibrils pronouncedly improved PP’s viscoelasticity and crystal nucleation rate, respectively. Compared with PP foam, INF composite foam showed more stretched cells in the skin layer and refined spherical cells in the core layer. Due to the synergistic toughening effect of PET nanofibrils and POE elastic particles, the impact strength of INF composite foams was 295.3% higher than that of PP foam and 191.2% higher than that of melt-blended PP/PET foam. The results gathered in this study reveal potential applications for PP based INF composite foams in the manufacturing of lightweight automotive products with enhanced impact properties. Polypropylene (PP) is one of the most widely used thermoplastic all over the global plastic market. Owing to its advantageous performances, including low cost and density, good processability, excellent chemical and temperature resistance and high mechanical properties, PP has been widely used in many fields such as automotive, packing, construction and consumer electronics . Using aMaterial lightweighting, structural optimization design and manufacturing process innovation are three effective ways to achieve lightweight products . PolymerTherefore, promoting the foaming ability of PP during MIM process, and further achieving uniform micro/nano-scale cells, plays an important role in enhancing mechanical properties. In situ fibrillation-based technology is increasingly recognized as an environmentally sustainable, inexpensive and high-throughput process for the fabrication of in situ nanofiber (INF) reinforced composites. INF composites are potential candidates to be substituted by carbon/glass fiber reinforced composites ,17. DuriThe incompatibility between the matrix and the dispersed phase is beneficial for the formation of physical fibrillar network. While on the other hand, the weak interfacial interaction originating from the incompatibility makes the micro/nanoscale fibrils strip out of the matrix at the interface due to the stress concentration . TherefoIn this study, INF technology associated with the MIM process was applied to fabricate lightweight and excellent toughness INF composite foams. First, the PP/PET/POE composites with PET nanofibrils were prepared by coupling twin-screw compounding with melt spinning. Afterwards, the phase morphology, rheological properties, and isothermal crystallization behaviors of prepared the PP-based composites were analyzed. Then, MIM experiments were carried out to fabricate PP foams, PP/nanofibrous PET foams, and PP/nanofibrous PET/POE foams. PP/spherical PET and PP/spherical PET/POE foams were also prepared for comparison with the same process setting. The cellular morphologies of all foams, including parallel and perpendicular to flow direction, were investigated to identify the role of PET nanofibrils and POE particles in improving PP’s foaming ability. Finally, impact testing was performed to evaluate the mechanical performance of PP and PP-based composite foams. The synergistic toughening mechanism of MIM products by PET nanofibrils and POE were further explored.3 and a melt flow rate (MFR) of 25 g/10 min (230 °C/ 2.16 kg). The dispersed phase polymer employed in this work was PET (FC510) by Yizheng Chemical Co., Ltd. . The melt point of PP and PET was determined by DSC as 167°C and 253°C, respectively. Polyolefin elastomer  was provided by Dow Chemical Investment Co. , with an MFI of 5 g/10 min at 190 °C/2.16 kg. The physical blowing agent, CO2 (99% purity), was obtained from Ningbo Wanli Gas Corporation . An isotactic PP  was supplied as matrix polymer by Maoming Petrochemical Co., Ltd., with a density of 0.9 g/cmThe in situ nanofibril (INF) process combining twin-screw compounding with high-speed hot drawing is presented in this study, and is shown in The processing temperature of extruder barrel was set to 7 segments from feeding zone to die, specifically 160 °C, 190 °C, 230 °C, 265 °C, 265 °C, 265 °C and 255 °C. A screw speed of 80 rpm was used and feeding speed was kept at about 7.5 kg per hour. The extrudate from the die was connected with a custom-built fiber spinning system, comprised of a variable speed motor and a high-speed roller with diameter of 25 mm. The rotational motion of the roller drew the extruder, and the spherical PET domains was deformed into nanofibrils uniformly dispersed in PP matrix. By maintaining the rotational rate of the roller, the extrudate draw ratio was kept at 12.5:1. For comparison, an undrawn sample which only experienced traditional melt blending was also studied. The neat PP went through the same process to ensure the same thermal history. It should be mentioned that many trials prior to the formal experiments have been performed to acquire the above processing parameters.The pelletized INF composites were used as raw materials for fabricating fibrillary PET reinforced PP composite foams by a 120-ton injection molding machine , equipped with a Mucell SCF delivery system . MIM experiments were conducted to prepare PP based composite foams. The main processing parameters, including melt temperature, mold temperature, injection speed and gas dosage were set to be 190 °C, 35 °C, 75 mm/s and 0.7 wt.%, respectively. The shot size adjusted to maintain the resulting weight reduction was 20% compared with the solid sample. For comparison purposes, neat PP and PP based melt-blended composite foams were also prepared under the same conditions.A scanning electron microscope  was used to observe the sample’s micromorphology. All pelletized materials were first compressed into a rectangular plate at 190 °C under 10 MPa by a vacuum hot compression machine (K14L20VHE), and then were cryogenically fractured in liquid nitrogen. The fracture surface of the sample was coated with a thin layer of platinum to avoid electron charging during SEM observation. In order to observe the variation of PET domains, all composite samples were exposed to xylene vapors for 1.5 h at 140 °C, followed by ultrasonic cleaning in deionized water twice to remove the PP matrix. With the obtained micrographs, Image J software (version 1.8.0.) was used to define the spherical and fibril PET domain size.In order to observe the cellular morphology inside the foamed sample, the impact spline was cooled in liquid nitrogen for 30 min and immediately fractured along melt flow direction (FD) and thickness direction (TD) to obtain the real cellular morphology. The geometry of the impact sample and SEM characterization regions are illustrated in n is the number of cells.The average diameter of the cells can be calculated by the following formula:M is the magnification of the SEM microscope, and A is the area of the image.The population density of the cells can be calculated using the following equation:d) was calculated by the following equation [As in conventional injection molding, a typical three-layer structure is obtained with the increase in distance from the mold wall during the MIM process . In ordeequation :(3)d=baω) ranging from 0.01 to 100 rad/s at 190 °C to obtain storage modulus (G’), loss factor (tan δ) and complex viscosity (η*). All tests were conducted under the protection nitrogen atmosphere to avoid oxidative degradation of melt.A rotational rheometer  was used to investigate the rheology behavior of the PP and PP based composites. A dynamic strain sweep was conducted first to identify the linear viscoelastic region. Afterwards, the dynamic frequency sweep tests were applied with the frequency  was used to investigate the isothermal crystallization behavior. The samples were heated from 40 °C to 200 °C at a 10 °C/min heating rate and held for 5 min to eliminate thermal history. Subsequently, the samples were cooled at 10 °C/min to 130 °C and held for 20 min. All measurements were performed under nitrogen atmosphere protection.Izod impact testing was conducted by using an impact tester  according to ISO 180. Before testing, all samples were dried at 60 °C in a vacuum for at least 3 days to remove any moisture. All specimens were notched in the middle with a depth of 2 mm using a standard cutter, and then placed horizontally with the notch oriented away from the pendulum and broken by a hammer with an energy of 2 J. At least five specimens were tested for each test to evaluate the reliability and stability of results.For polymer composites, the phase morphology and distribution of dispersed domains play an important part in determining their foaming behavior and mechanical performances. G’), complex viscosity (η*) and loss tangent (tan δ) as a function of frequency. It is observed from G’ curve for neat PP, PP/PET(S) and PP/PET(S)/POE was about 2, indicating the rheological behavior of a melt with liquid-like properties [G’ curve than that of neat PP, especially in the region of low frequencies. We attributed this shift in G’ to the presence of the solid spherical PET domains that behave as rigid and influence the stress relaxation behavior of the PP matrix [G’ and a plateau occurred at low frequency, owing to the constraint formed by the PET fibril network as well as the increased entanglements between PET fibrils and PP molecular chains [G’ of PP/PET(F)/POE was bigger than that of PP/PET(F), particularly at low frequencies, mainly attributing to the higher fibril aspect ratio and increased molecular entanglements caused by the spatially linked structure of PET nanofibrils [As rheological behavior has a significant influence on the foaming behavior of polymers, dynamic frequency sweep tests were carried out to characterize the effect of PET fibrils and POE particles on the linear viscoelastic responses of the PP matrix. operties . PP/PET as a function of time for all samples. As seen, all curves have a similar sigmoidal shape. PP crystallization characteristic was mostly enhanced for INF composites. Compared with melt-blended composites, PET nanofibrils in INF composite have much bigger specific surface areas than that of spherical PET domains, which thereby can promote the crystallization rate as function of nucleation agents. Besides, previous studies have indicated that PET nanofibrils incorporation could also facilitate the PP molecular mobility effectively [Aside with rheological behavior, crystallization is another important factor in determining a composite’s foamability. The heat flow traces during isothermal crystallization at 130 °C for both neat PP and PP-based composites are displayed in ectively , and henn exponent of all samples varied between 2–3. Although the heterogeneous crystal nucleating effect of the additive could generally promote spherulitic crystal growth resulting in the increase of n value beyond 3 [n values between 2–3. It is also of note that PP/PET(F)/POE shows a significant three-magnitude and one-magnitude higher Zt value than that of neat PP and PP/PET(F), respectively. It is well known that polymer crystallization contains crystal nucleation and growth. It is necessary to note that the existence of PET nanofibrils can promote the crystal nucleation rate as explained above. However, the crystal nuclei are normally subject to high resistance during the growth owing to the big viscoelastic property. It is difficult to compare the overall crystallization rate directly from the values of tZ because the unit of tZ is s-n and n is not constant for the samples. Therefore, the half-time of crystallization  is computed for the purpose of discussing the kinetics of crystallization [1/2t as a function of materials. The experimentally determined t1/2 from the relative crystallinity data was plotted alongside the t1/2 values derived from Avarima equation. It can be clearly distinguished from 1/2t of INF composites took dozens of seconds and was much shorter than that of neat PP. Intriguingly, compared with PP/PET(F), PP/PET(F)/POE had a shorter 1/2t. We attributed this result to the smaller diameter and larger aspect ratio of PET nanofibrils caused by adding 3 wt.% POE, which offered a larger surface-to-volume ratio for crystal nucleation. Therefore, the gathered results give rise to the suggestion that the synergistic effect of PET nanofibrils and POE particles substantially enhance the PP matrix crystallization kinetics thermodynamically.A thermodynamic analysis of isothermal crystallization based on the Avrami model was conducted at 130 °C to further evaluate the influence of PET nanofibrils on crystallization. The Avrami plots are shown in beyond 3 , the tralization . Figure It is known that the differences of shearing stress and temperature along TD and FD usually generate a nonuniform cell structure, which deteriorates the mechanical properties of the foamed parts. An RMIM experiment was performed to fabricate neat PP and PP-based composite foams. To investigate the effect of PET dispersed phase morphology evolution on the foaming behavior of PP, To quantitatively characterize the cell distribution, the cell size distribution of foamed samples was plotted in 7 cells/cm3, respectively, which was greatly improved compared with that of neat PP. This indicates that the PET nanofibrils can homogenize the cell distribution, which was in accordance with the crystallization and cell morphology results discussed above.The cell morphologies parallel to FD are shown in d) is affected by the cell deformation, and normally higher cell deformation corresponds to a larger d value. The offset angle (θ) is affected by the melt temperature and local melt viscosity, and higher temperature and lower viscosities means a larger θ. It is clear that when compared with neat PP, the d of INF composite foams is larger, and the θ become smaller. This demonstrated that a lot of cells have occurred larger deformation. The main reason maybe that the presence of PET nanofibrils increase the resistance to polymer molecular chain motion, resulting in an increase in local viscosity and enhanced shearing stress. At the same time, the cell wall thickness decreased, and the ability of the cells to undergo deformation was reduced, leading to an increase in cell deformation. In particular, the biggest d and smallest θ were obtained for PP/PET(F)/POE foams. Besides the effect of PET nanofibrils, the POE with long chain branching can also improve the resistance of molecular chain motion [n motion .2, increasing by 295.3% compared with the neat PP foams, and followed by PP/PET(S), PP/PET(S)/POE and PP/PET(F), which were increased by 191.2%, 71.2% and 27.1%, respectively. The sharply improved impact strength should definitely be correlated with the synergistic effect of cell structure and filler system (PET fibrils and POE additives). For one thing, the refined crystals caused by PET fibrils can promote the flexibility and toughness of PP matrix. Due to the weak cohesive forces between PET molecular chains, the PET fibrils can enhance the sliding ability of PP crystals [Izod impact testing was conducted to evaluate the ductility of the injection molded foams at a relatively high deformation rate. crystals . Previoucrystals . Figure crystals  during tIt is noteworthy that a large number of PET fibrils and POE additives dispersed uniformly in the frozen layer and cell wall, which is efficient for ductile fracture behavior. In summary, a cost-effective and environmentally friendly strategy based on in situ fibrillation and microcellular injection molding process was developed to fabricate lightweight and high toughness PP/PET/POE composite foams. Nanofibrillar PP/PET/POE composite was prepared by combing twin-screw compounding mixed with melt spinning process. Incorporation of POE particles in PP/PET blend played a role of compatibilizer and plasticizer. The average diameter of PET nanofibrils were decreased after introducing POE due to the particle-induced PET domains elaboration. A long and an average diameter of 258 nm were achieved and distributed uniformly in the PP matrix. The rheological analysis data demonstrated that the PET nanofibrils dramatically increased melt viscoelasticity and strength. The isothermal crystallization results showed that PP’s crystal nuclei rate as well as the chain mobility were effectively improved because of the dual heterogeneous nucleation effects of PET nanofibrils and POE particles, and eventually promoted the PP’s crystallization kinetics.The INF composite foams prepared by MIM achieved a sandwich-like structure, including a solid frozen layer, a shear layer contained stretched cells, and a core layer that consisted of numerous spherical cells. Owing to the improved crystallization and rheological properties, the obvious refined foam structure in the core layer of the PP matrix, with decreased cell size and increased cell density, was obtained by introducing the PET nanofibrils and POE particles. Furthermore, compared with neat PP foams and other melt-blended composites, the PP/PET(F)/POE composite foams exhibited the largest cell deformation in skin layer because the presence of PET nanofibrils increases the resistance to polymer molecular chain motion. Notably, the Izod impact results showed that the PP/PET(F)/POE composite foams exhibited the highest impact strength, increasing by 295.3% compared to PP foams. The existence of oriented PET nanofibrils may induce a crack deflection perpendicular to the impact direction and hinder the crack propagation, thus increasing the absorbed energy. The toughening effect of POE’s uniform dispersion in the PP matrix is another reason for the outstanding impact strength.The results obtained in this study reveal potential applications for PP based INF composite foams in the manufacturing of lightweight automotive products with enhanced impact properties. Future work would focus on investigating various mechanical and functional properties of INF composite foams, including tensile, flexural and thermal insulation properties.


Cotton fibers with ultra-high purity cellulose are ideal raw materials for producing nanocellulose. However, the strong hydrogen bond and high crystallinity of cotton fibers affect the dissociation of cotton fibers to prepare nanocellulose. The structures of two kinds of cotton fibers (CM and XM) in different growth stages from 10 to 50 days post-anthesis (dpa) were studied by Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD). In the process of cotton fiber growth, the deposition rate of cellulose macromolecules firstly increased and then stabilized. Then, the surface morphology, the chemical composition, and the crystal structure of the nanocellulose prepared from cotton fibers with different growth stages by deep eutectic solvent, a green solvent, were characterized by Transmission Electron Microscope (TEM), scanning electron microscopy (SEM) analysis, XRD, and Thermo Gravimetry (TG). The growth time of cotton fibers affected the properties of prepared nanocellulose, and nanocellulose obtained from cotton fibers at about 30 dpa had less energy consumption, higher yield, and milder reaction conditions. Cotton is an important economic crop in China, and cotton fiber is one of the natural resources with the highest fiber purity. Cotton fiber is a single-celled fiber formed by the development of ovules epidermis cells ,2. AfterIn 2003, the research group of Abbott  first prIn this paper, the cellulose deposition and organization during different phases of cotton fiber development from 10 dpa to 40 dpa are studied by Fourier transform infrared (FTIR), X-ray diffraction (XRD), and SEM spectroscopy. Then, the cotton fibers at different growth stages were dissolved in deep eutectic solvent, and nanocellulose from cotton fibers at different growth stages was prepared by ultrasound. The structural properties of nanocellulose were analyzed by TG, XRD, SEM, and TEM. This study will provide theoretical support for the high-value utilization of cotton fiber.Oxalic acid dihydrate (purity: >99%) and choline chloride (purity: >98%) were obtained from Sinopharm Chemical Reagent Co., Ltd. . Sodium chloride (purity: >99%) was also obtained from Sinopharm Chemical Reagent Co., Ltd. . Two kinds of cotton, CM and XM, were applied in the experiment ), as shown in Two kinds of cotton were used as materials, and single flowers were marked on flowering day (0 dPa) and 10 growth bolls were harvested at different growth stages. These stages represent the major developmental stages of cotton fibers. The pericardium was immediately removed, and the isolated ovules were transferred to a cryogenic flask and stored in a cryogenic biological storage system filled with liquid nitrogen until analysis.DES with a molar ratio of 1:2 was prepared with 63.0 g oxalate dihydrate and 69.8 g choline chloride, and stirred continuously for 60 min in a glass reactor at 95 °C. About 0.1 g of treated cotton fiber was added to the reactor. After the reaction, 200 mL of deionized water was added to the reactor to reduce the viscosity of the mixture. The treated cotton cellulose was cleaned and filtered. The pretreated fibers were first mixed with water at a concentration of 0.5 wt.%. The suspension was then treated with a JY99-IIDN ultrasonic generator  equipped with a 25 mm cylindrical titanium alloy probe. The ultrasonic frequency was 20 kHz, the output power was 1000 W, and the ultrasonic time was 30 min.The FTIR spectra were recorded using a Nicolet Magna 560 FTIR instrument  with a diamond attenuated total reflectance.X-ray diffraction analysis was performed on a Bruker D8 ADVANCE instrument with copper target radiation. The acceleration voltage was 50 kV, the scanning range 2θ was from 10° to 50°, the scanning speed was 5°/min, and the tube current was 40 mA. Crystallinity was calculated according to the following formula.200 is the peak intensity of the diffraction of the main crystal plane at 22.7°; Iam is the diffraction intensity of the amorphous region at Angle 2θ close to 18°.In the formula, IThe morphology of the pretreated cotton fibers in different growth stages was observed using a JSM-7500F scanning electron microscope (SEM)  at 15 kV.TEM observation on the CNFs was performed using Thermo Fisher FEI Quanta FEG 450  at 120 KV acceleration voltage. A drop (5 µL) of diluted CNFs slurry was dropped on a carbon-coated electron microscope grid, and then negatively stained with 1 wt.% phosphotungstic acid solution to enhance the contrast of the image. The size of the nanofibrilated cellulose was measured by transmission electron microscope.−1, respectively, and the absorption peaks were consistent with the structure of cotton cellulose  method was used to study the crystal structure and relative crystallinity of nanocellulose. As shown in onset) of cotton cellulose at different growth stages were slightly different. The initial degradation temperature of the original cotton fiber was approximately 300 °C, while the initial degradation temperature of the prepared nanofibrillated cellulose was raised to approximately 305 °C. Compared with original cotton cellulose, nano-granulated cellulose has a smaller size, larger specific surface area, and lower molecular weight, thus its thermal stability is greatly affectedThe TG curves of nanocellulose obtained from different stages of cotton fibers can be seen in The SEM of nanocellulose obtained from different stages of cotton fibers can be seen in The size of NFC at different growth stages prepared by deep eutectic solvent pretreatment was analyzed by TEM as shown in In order to better clarify the accumulation and structure change of cellulose during cotton growth, FTIR and XRD were used to analyze the structure and crystallinity of cotton fiber. Both CM and XM cotton fibers showed characteristic absorbance peaks of the cellulose from the analysis of FTIR. The major change in cellulose organization occurs between 16 and 30 dpa. This phase represents the active cellulose deposition phase during the secondary cell wall synthesis. The crystalline size of cotton fibers increased fast in less than 10 dpa, while after 30 dpa, the cotton fibers of CM and XM had a consistent crystalline size.The properties of the nanocellulose prepared from cotton at different growth stages showed that the relatively optimal treatment condition is the reaction temperature of cotton fibers at 90 °C in eutectic solvent. Considering the yield and the process energy consumption, cotton fibers at about 30 days after flowering were suitable for the preparation of nanocellulose. The maximum yield of nanocellulose can be as high as 74.2%.