Lactobacillus acidophilus, Lactobacillus plantarum, Bacillus subtilis and Enterococcus faecalis for 14 days was assessed used 16S amplificon sequencing. The results showed that the four probiotics caused changes in the composition and structure of the gut microbiota in mice, but they did not cause changes in the diversity of the gut microbiota. These results provide a strong basis for the preparation of probiotics and theory regarding their targets.The overuse of antibiotics has led to an increase in resistant bacteria and unnecessary culture contamination. As one of the best green pollution-free antibiotics, probiotics and their preparations have become research hotspots. The different effects of different probiotics on gut microbiota are still unclear. In this study, the gut microbiota of mice treated with Lactobacillus acidophilus (La), Lactiplantibacillus plantarum (Lp), Bacillus subtilis (Bs), Enterococcus faecalis (Ef), LB broth medium, and MRS broth medium. We sequenced 16S rRNA from fecal samples from each group 14 d after gavaging. According to the results, there were significant differences among the six groups of samples in Firmicutes, Bacteroidetes, Proteobacteria, Bacteroidetes, Actinobacteria, and Desferribacter (p < 0.01) at the phylum level. Lactobacillus, Erysipelaceae Clostridium, Bacteroides, Brautella, Trichospiraceae Clostridium, Verummicroaceae Ruminococcus, Ruminococcus, Prevotella, Shigella, and Clostridium Clostridium differed significantly at the genus level (p < 0.01). Four kinds of probiotic changes in the composition and structure of the gut microbiota in mice were observed, but they did not cause changes in the diversity of the gut microbiota. In conclusion, the use of different probiotics resulted in different changes in the gut microbiota of the mice, including genera that some probiotics decreased and genera that some pathogens increased. According to the results of this study, different probiotic strains have different effects on the gut microbiota of mice, which may provide new ideas for the mechanism of action and application of microecological agents.Probiotics, also referred to as “living microorganisms,” are mostly present in the genitals and the guts of animals. They can increase an animal’s immunity, aid in digestion and absorption, control gut microbiota, protect against sickness, and even fight cancer. However, the differences in the effects of different types of probiotics on host gut microbiota composition are still unclear. In this study, 21-day-old specific pathogen-free (SPF) mice were gavaged with  The gut microbiota and the host complement each other and are indispensable in the intestinal tract of animals . The gutEscherichia and Klebsiella [There are many factors that can influence gut microbiota. Diet plays a key role in the regulation of gut microbiota composition. Different diets can cause different changes in the composition of gut microbiota . Antibioebsiella . With thebsiella . Becauseebsiella . Some prebsiella .Lactobacillus and its subspecies are widely used in food processing, preservation, and fermentation. Because most Lactobacillus have the biological characteristics of acid and bile salt tolerance, they can utilize their good characteristics in the intestine [L. acidophilus and L. plantarum are currently widely used in food as safe probiotics [B. subtilis [E. faecalis can tolerate relatively severe environments, such as pH 9.6 and high concentrations of salt, and is often used in fermented products [As commonly used probiotics, ntestine ,16,17. Lobiotics ,19. B. ssubtilis  can prodproducts . Therefoproducts ,23,24.L. acidophilus, L. plantarum, B. subtilis, and E. Faecalis, which act as probiotics on the gut in the body, the differences in the effects on gut microbiota are still unclear. In this study, mice were orally gavaged with a fresh bacterial culture of four probiotics with specific concentrations, and changes in the gut microbiota of the mice were analyzed.As for L. plantarum, L. acidophilus, B. subtilis, and E. faecalis; the Kunming mice  in this study were fifteen days old and pre-fed for one week (21 d old) for the test. The animal experiments performed in this study strictly followed the national guidelines for experimental animal welfare announced by the Ministry of Science and Technology of the People’s Republic of China in 2006 ) and were approved by the Animal Welfare and Research Ethics Committee at Qingdao Agricultural University, Shandong, China . An LB broth medium and an MRS broth medium  were used in the study.L. plantarum, L. acidophilus, B. subtilis, and E. faecalis were incubated for 6 h, 8 h, 10 h, 12 h, and 14 h. L. plantarum and L. acidophilus were incubated in a warm oven with an MRS broth, while B. subtilis and E. faecalis were incubated in a shaker with an LB broth at 180 rpm/min. The turbidity of each bacterium was measured at different incubation times .Thirty-six mice of approximately the same size and weight, eighteen males and eighteen females, were selected and divided equally into six groups.L. plantarum (Lp) and L. acidophilus (La) were selected as the test groups where the MRS broth medium was selected as the control group (MRS). B. subtilis (Bs) and E. faecalis (Ef) were selected as the test groups where the LB broth medium (LB) was selected as the control group. Bacterial liquid and broths were administered in quantities of 0.1 mL/animal and gavage took place at 17:00 BST daily.Gavage was administered to each group of mice separately and continued for 14 d. The experiment was conducted for 14 d, and at the end of the experiment, fresh feces from each group of mice were collected separately and transferred to a −80 °C freezer for storage.Total microbial genomic DNA samples were extracted using an OMEGA Soil DNA Kit (D5625-01)  following the manufacturer’s instructions, and they were stored at −20 °C prior to further assessment. The extracted DNA was determined in terms of quantity and quality using a NanoDrop ND-1000 spectrophotometer  and agarose gel electrophoresis, respectively.The quality DNA was sent to Personal Biotechnology Co., Ltd.  for sequencing of the bacterial 16S V3V4 region.Sequence quality control and splicing were performed using the DADA2 method. QIIME2(2019.4) and R software were used to analyze the taxonomic composition, α-diversity, and β-diversity of the samples.L. plantarum and L. acidophilus still grew rapidly after 6 h of incubation. The proliferation of bacteria started to slow down after 8 h of incubation. The growth of B. subtilis and E. faecalis slowed down after 6 h of incubation. The proliferation of bacteria almost stopped after 8 h of incubation. In this study, the selected bacterial liquid was cultured for 8 h, and 0.1 mL was used, with the concentration of bacterial liquid being ≥108 CFU/mL.The results in The results show that the sequence lengths were mainly distributed as follows: 405 bp; 406 bp; 424 bp; 425 bp; 429 bp; 430 bp. The numbers were 41,115; 11,065; 16,081; 50,169; 9203; 128,783.As shown in p < 0.01). Firmicutes and Bacteroidetes were dominant in the Bs group; Firmicutes, Bacteroidetes, and Proteobacteria were dominant in the Ef group. Compared with the control LB group, Firmicutes, Bacteroidetes, and Proteobacteria in the Bs and Ef groups did not change significantly (p > 0.05). A few Bacteroidetes, Actinobacteria, and Deferribacteria were detected in the LB group, where Actinobacteria and Deferribacteres were significantly higher than those in the Bs and Ef groups (p < 0.01).To study the effects caused by the four probiotics on the gut microbiota in mice, the top ten species with relative proportions at the phylum level and the genus level were selected, and a small number of species were classified as other species to evaluate the distribution of gut microbiota composition in mice. The species composition at the phylum level is shown in Lactobacillus was present in all six groups. Lactobacillus, Bacteroides, Blautia, Lachnospiraceae Clostridium, and Erysipelotrichaceae Clostridium were dominant in the La group. Lactobacillus, Shigella, Blautia, Ruminococcus, Erysipelotrichaceae Clostridium, and Clostridiaceae Clostridium were dominant in the Lp group. Compared with the control MRS group, Lactobacillus and Lachnospiraceae Clostridium in the La group were significantly higher (p < 0.01) and Bacteroides was significantly lower (p < 0.01). Lactobacillus, Shigella, Blautia, and Clostridiaceae Clostridium in the Lp group were significantly higher (p < 0.01), and Ruminococcus was significantly lower (p < 0.01). Lactobacillus and Ruminococcaceae Ruminococcus were dominant in the Bs group. Lactobacillus, Prevotella, Shigella, Bacteroides, Blautia, Ruminococcus, and Erysipelotrichaceae Clostridium were dominant in the Ef group. Compared with the control LB group, Ruminococcaceae Ruminococcus in the Bs group was significantly higher (p < 0.01), and Prevotella, Bacteroides, Ruminococcus, Erysipelotrichaceae Clostridium, and Lachnospiraceae Clostridium were significantly lower (p < 0.01). Prevotella, Shigella, Blautia, and Ruminococcaceae Ruminococcus in the Ef group were significantly higher (p < 0.01) and Lactobacillus, Ruminococcus, Erysipelotrichaceae Clostridium, and Lachnospiraceae Clostridium were significantly lower (p < 0.01).The species composition at the genus level is shown in Lactobacillus was significantly higher in the La and Bs groups than in the other two groups (p < 0.01). Blautia and Erysipelotrichaceae Clostridium in the La, Lp, and Ef groups were significantly higher than those in the Bs group (p < 0.01). Bacteroides and Prevotella had the highest proportion in the Ef group. Blautia had the highest proportion in the Lp group. Shigella in the Lp and Ef groups were significantly higher than those in the other two groups (p < 0.01), and Shigella was higher in the Lp group. Clostridiaceae Clostridium in the Lp group was significantly higher than in the other three groups (p < 0.01). Lactobacillus accounted for the highest proportion in the Bs group.Compared with the four experimental groups, The rarefaction curves indicate the magnitude of the effect of sequencing depth on the diversity of the observed samples. The results are shown in In order to assess the effect of four probiotics on the diversity and richness of the intestinal microbial community in mice, this study characterized the richness by the Chao1 index and the diversity by the Shannon and Simpson indices, and the individual indices of the samples are shown in p < 0.01). The diversity was not significantly different from the MRS group (p > 0.05). The abundance of Bs and Ef groups was significantly higher than the control LB group (p < 0.01). The diversity was not significantly different from the LB group (p > 0.05).Relatively high Chao1, Shannon, and Simpson indices indicated high bacterial richness and diversity. In this study, the abundance of the La and Lp groups was significantly higher than in the control MRS group (p > 0.05). The richness of the Bs group was significantly higher than that of the Ef group (p < 0.01). Both the La group and the Lp group were significantly higher in richness and diversity than the Bs and Ef groups (p < 0.01).Compared with the four test groups, there was no significant difference between the La and Lp groups (p < 0.05) −0.108); LB group . There were different clusters in all six groups of samples, and the distance between the six groups of samples was large. NMDS uses rank ordering, and the farther the distance between two points, the greater the difference between the microbial communities in the two samples, and vice versa, as shown in p < 0.05). −1.178); MRS group ; Bs group ; Ef group ; LB group . The greater distance between the six groups of samples in the NMDS analysis indicates that the difference in community composition is more significant (p < 0.01).To show the variability and similarity of microbial communities among the six groups of samples, this study used the principal co-ordinates analysis (PCoA) method and NMDS analysis. PCoA was used to expand the sample distance matrix in the low dimensional space after projection and to retain the distance relationship of the original samples to the maximum, as shown in L. acidophilus, L. plantarum, B. subtilis, and E. faecalis on the structure and diversity of the gut microbiota in mice.According to the available studies, animal gut microbiota mainly includes bacteria, fungi, archaea, protozoa and viruses, which maintain the gut microecological balance in the host and interact with the host, thus influencing the host’s physiology and health . The balL. acidophilus can increase the proportion of Firmicutes, Lactobacillus, Erysipelotrichaceae Clostridium, and Lachnospiraceae Clostridium in the gut and reduce the proportion of Bacteroides. L. plantarum can increase the proportion of Firmicutes, Proteobacteria, Lactobacillus, Shigella, Blautia, and Clostridiaceae Clostridium in the gut and reduce the proportion of Ruminococcus. B. subtilis can increase the proportion of Lactobacillus and Ruminococcaceae Ruminococcus, and reduce the proportion of Prevotella, Bacteroides, Ruminococcus, Erysipelotrichaceae Clostridium, and Lachnospiraceae Clostridium. E. faecalis can increase the proportion of Prevotella, Shigella, Blautia, and Ruminococcaceae Ruminococcus and reduce the proportion of Lactobacillus and Lachnospirace Clostridium.The results of this study show that. L. acidophilus, L. plantarum, B. subtilis, and E. faecalis could significantly increase the richness of the gut microbiota in mice. The effects of L. acidophilus and L. plantarum were more obvious. In addition, the four strains did not significantly affect the diversity of the gut microbiota in mice. The colony structure was greatly affected by the four species.Lactobacillus and decreased the abundance of Bacteroidetes, Vibrio spp., and Ruminococcus [L. plantarum increased the abundance of Firmicutes, Proteobacteria, and Lactobacillus and decreased the abundance of Bacteroidetes, [B. subtilis can increase the abundance of Firmicutes, Bacteroidetes, and Lactobacillus [E. faecalis can increase the abundance of Bacteroidetes and Ruminococcus and decrease the abundance of Proteobacteria [p < 0.01). In this study, although the four probiotics added did not have a significant effect on flora diversity, it was found that the four experimental groups differed significantly in their flora structure when analyzed between the groups.In previous studies, probiotic strains have been shown to be able to maintain the stability of the gut microbiota and interact with the intestinal microbiota by competing for nutrients, such as oxygen, through antagonism and cross-feeding in the gut . The usenococcus ; in otheoidetes, ,31; B. sbacillus ; E. faecbacteria . This isB. subtilis after colonization and the formation of an anaerobic environment, resulting in an increase in the abundance of anaerobionts such as Lactobacillus. In general, Firmicutes are associated with the ratio of Bacteroidetes and the susceptibility to disease states [E. faecalis may reduce the abundance of these two clades.In the present study, the increase in the abundance of Firmicutes may be related to the increase in beneficial bacterial species, such as Lactobacillus . Notablye states , but in Shigella [E. faecalis could inhibit the growth of Shigella and alleviate the symptoms caused by Shigella, which differed significantly from the results of the present study, probably due to the use of Blautia, which is a newly discovered potential probiotic, the abundance of which is influenced by some prebiotics [Blautia may be influenced by L. acidophilus, L. plantarum, and E. faecalis; B. subtilis did not increase its abundance, and whether its growth is influenced by the growth of B. subtilis needs to be studied. Prevotella is abundant in the body as a key player in the balance between health and disease [L. acidophilus can increase the abundance of Prevotella [L. plantarum can decrease the abundance of Prevotella and Bacteroides [B. subtilis and E. faecalis can increase the abundance of Prevotella [In addition, the present study found different variations at the genus level, with increased abundance of Shigella  in the LShigella ,38 and iShigella ,40 testsebiotics . In this disease . It has evotella , which iteroides ,44, and evotella ,46. All L. acidophilus, L. plantarum, B. subtilis, and E. faecalis, were gavaged into mice. The study showed that the four probiotics exerted different effects on the structure and richness of the gut microbiota in the intestines of the mice, and the study elucidated the mechanism of probiotic interactions in the intestine, which further provided a strong basis for the preparation of probiotics and theory regarding their targets.In this study, four probiotics:


Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae), is an important pest of stored products and at the same time a species with great potential as food and feed that has recently attracted increasing interest as nutrient source. Future predictions show a massive increase of insect meals’ production in the near future, therefore, as expected in the case of other durable stored products, insect meals may get infested by insects during their storage. In this context and as a continuance of our previous study on the susceptibility of meal of the yellow mealworm, Tenebrio molitor L. (Coleoptera: Tenebrionidae), to storage insect infestations, the objective of this study was to test the susceptibility of A. diaperinus meals to infestations of three stored products pests, namely A. diaperinus itself, T. molitor, and the red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae). The population growth of the three species was evaluated on pure A. diaperinus meal, as well as on A. diaperinus meal-based substrates with different percentages of wheat bran . Our results showed that all three insect species tested were able to grow and develop on the A. diaperinus meal-based substrates evaluated, giving in a short time high population densities. This study confirms again our initial theory for insect infestations during storage of insect-based products.The lesser mealworm,  Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae), is an important pest of stored products that has been found infesting dozens of agri-industrial commodities (A. diaperinus is among the first list of species that had been authorized for aquafeed production in 2017 in the European Union (EU) [Hermetia illucens (L.) (Diptera: Stratiomyidae), and the yellow mealworm, Tenebrio molitor L. (Coleoptera: Tenebrionidae)  , but theproducts .T. molitor meals to infestations caused by different stored product beetle species, including T. molitor itself. The rationale behind this study was simple: if there is an infestation in insect rearing units, then control can be a very complicated procedure, and any control methods should be extremely selective  (Coleoptera: Tenebrionidae), which is a major pest of stored products with global distribution  was used. T. molitor and A. diaperinus were weekly supplied with fresh potatoes and apples, respectively, as a moisture source. T. castaneum and T. molitor were kept at 26°C, 55% relative humidity (RH), whereas A. diaperinus was kept at 30°C and 55% RH. All insect species were kept at continuous darkness. The adults used in the tests were <1 month-old.All insect species used in the bioassay were reared at the Laboratory of Entomology and Agricultural Zoology, Department of Agriculture, Crop Production and Rural Environment, University of Thessaly. Specifically, A. diaperinus meal preparation late-stage larvae were used, after harvesting them by separating them from the feeding substrate with sieving. Larvae were frozen at −20°C, chopped using a stainless-steel mill , dried for 72 h at 60°C, and finally sieved with an 1-mm opening sieve. The insect meal produced was stored at −20°C until the initiation of the bioassays.For the T. castaneum, T. molitor, and A. diaperinus was evaluated in a series of laboratory bioassays, in which pure A. diaperinus meal was used, as well as mixtures of it with wheat bran in different percentages. Specifically, five substrates based on insect meal were evaluated: 100% A. diaperinus meal (0% wheat bran), a mixture of 75% A. diaperinus meal and 25% wheat bran, a mixture of 50% A. diaperinus meal and 50% wheat bran, a mixture of 10% A. diaperinus meal and 90% wheat bran and 100% wheat bran . Wheat bran was selected as substrate ingredient, as it is a major feed commodity, commonly used for animal livestock feeding  were placed into the vials, using different vials for each species. All vials were then placed at the proper conditions for each species mentioned above and were kept there for 65 d. In the case of T. molitor and A. diaperinus, a slice of potato and carrot, respectively, was placed inside the vials twice a week to cover the insect moisture needs. After 65 d the vials were opened for the evaluation of the progeny production and the produced adults , larvae, and pupae were counted. As a direct measure of population growth, for all species, the instantaneous rate of increase was calculated using the following equation: ri = ln(Nf/No)/ΔT, where Nf was the final number of individuals, No was the initial number of individuals, and ΔT was the change in time, i.e., the duration of the experiment. Positive values of ri suggest a growing population, ri = 0 shows a stable population, and negative ri values specify a population in decline  was counted in this treatment by the end of the bioassay , as the total number of the produced individuals ranged from 154 to 358 at the termination of the bioassay, corresponding to instantaneous rates of increase ranging between 0.031 and 0.044. Significantly higher instantaneous rates of increase were recorded on the mixtures of the insect meal with wheat bran, i.e., on the substrates with 10, 50, and 75% A. diaperinus meal, than on wheat bran alone. The population growth of T. castaneum, expressed as total number of individuals at the end of the bioassay, as a function of the percentage of A. diaperinus meal in the substrates was best described by a quadratic equation   . Additionificant . A quadrT. molitor among the substrates tested for all life stages examined, with the exception of dead adults  showing the ability of T. molitor to grow and develop on this substrate. However, the total number of individuals was lower on this substrate at the termination of the trial than on the rest of the substrates tested, differences though were significant only with the 50% A. diaperinus meal. For the latter substrate, at least 24-fold increase of the T. molitor population was recorded at the termination of the bioassay. As far as the instantaneous rates of increase of all substrates are concerned, those ranged between 0.035 and 0.048, with the value of the substrate with 50% insect meal being the highest one (0.048). The population growth of T. molitor, as a function of the A. diaperinus meal percentage in the tested substrates, was best described by a quadratic equation , were not able to develop on 100% T. molitor insect meal , in comparison with whole grain kernels (T. granarium (While we are unaware of the possible source of infestation, we hypothesize that care should be taken in the raw materials, as these may contain various stored product insects, that can subsequently expand to the standard insect rearings. From the three species tested here, modities , constit kernels . Even if kernels , and thu kernels . Monitor kernels , but theranarium .A. diaperinus meals can be easily infested by A. diaperinus itself, but also by T. castaneum and T. molitor, that can easily build high population densities in a very short period of time. The actual impact of such a damage in the final product is poorly understood, and should be examined more thoroughly, chiefly in terms of certain quality characteristics. The increase in production of insect-based products for food and feed necessitates the need for improved storage, under the same principles that storage happens in other durable commodities, like wheat, rice, and maize. Apart from minimizing insect infestations, improved storage technologies can be adopted to be used for insect-based commodities, in conjunction with improved food security attributes.The present work provides data that show that


Smartphone interventions effect in pediatric subjects on the day of surgery: a meta-analysis Li L, Ma J, Ma D and Zhou X. (2021) Front. Surg. 8:759958. doi: 10.3389/fsurg.2021.759958A Retraction of the Systematic Review Article The journal and Chief Editors retract the 16 December 2021 article cited above.Following publication, concerns were raised regarding abnormal similarities with the contents of other articles published by unrelated research groups. A subsequent investigation, which was conducted in accordance with Frontiers' policies, raised strong concerns over the authorship of the articles, resulting in a loss of confidence in the findings presented in the article.The authors have not responded to this retraction.This retraction was approved by the Chief Editors of Frontiers in Surgery and the Chief Executive Editor of Frontiers.


Aspleniumtrilobum Cav. was identified. The plants were found growing on the bark of a Myrceugeniaparvifolia (DC.) Kausel tree in a small swamp next to specimens of Drimyswinteri J.R.Forst. & G.Forst. . Several previously unrecorded species were found, including Carexcf.excelsa Poepp. ex Kunth, Chusquea cf. quila Kunth, Ercillacf.spicata (Bertero) Moq., and Boquilatrifoliolata (DC.) Decne., highlighting the importance of exploring and documenting this biodiversity hotspot. The discovery in this wilderness area extends the distribution 86 km north on the continent, which was previously limited to the east of the municipality of Penco in the Biobío region . This paper presents an observed specimen, its locality, and associated species.The biodiversity hotspot of central Chile is home to a high proportion of endemic species, but some of these species are inconspicuous and not easily observed. During a botanical exploration in the Los Queules National Reserve (Chile), a population of  Asplenium L. is one of the fern genera with the greatest species diversity and geographical distribution. This genus and Hymenasplenium (at least 66 species) comprise the family Aspleniaceae, with Asplenium known for its remarkable species richness , while the southern limit is in Laguna San Rafael, Aysén Region   Baill.). The forest on the hilltops is mainly dominated by Nothofagusglauca (Phil.) Krasser and Nothofagusobliqua (Mirb.) Oerst. In the wetter areas, species such as Aextoxiconpunctatum Ruiz & Pav., Cryptocaryaalba (Molina) Looser and Persealingue (Ruiz & Pav.) Nees make up the dominant canopy layer.The Los Queules National Reserve is administratively located in the municipality of Pelluhue, in the Maule region Fig. . It coveA.trilobum  Kausel individual with a remarkably large diameter of about 40 cm at breast height, which is an unusual attribute for this species, located at 404 m a.s.l.  Gasper & Salino, Luzuriagapolyphylla (Hook.) J.F. Macbr., Carexcf.excelsa Poepp. ex Kunth, Chusqueacf.quila Kunth, Myrceugeniaexsucca (DC.) O. Berg, Myrceugeniaparvifolia (DC.) Kausel, Drimyswinteri J.R. Forst. & G. Forst., Hydrangeaserratifolia (Hook. & Arn.) F. Phil., Ercillacf.spicata (Bertero) Moq., Rhamnusdiffusus Clos, Boquilatrifoliolata (DC.) Decne., and Ugnicandollei (Barnéoud) O. Berg.The site is located on the eastern edge of the wilderness area, about 60 metres from forest plantations. The accompanying species were Carexcf.excelsa  are new records for this wilderness area (Ercillaspicata in the study area is highly plausible, as it is one of only two species within its genus. Chusqueaquila is also highly possible, as its morphology is clearly different from the only Chusquea species previously recorded at the site: C.cumingii. However, the identification of these species still needs to be confirmed. In a similar case, although Carexaphylla Kunth and Carexexcelsa L. have been reported from nearby wild areas (C.excelsa. If confirmed, this would be the first record of this genus at the site.It is important to note that lsa Fig. a, Chusquila Fig. a, Ercillata Fig.  and Boquess area . The speld areas , the plaThe importance of botanical exploration in advancing our taxonomic knowledge is inestimable. However, many biodiversity studies have shown a marked accessibility bias, with sampling favouring areas close to major roads and other access routes, leaving interior and remote regions largely undersampled . This saNew species and distribution records are frequently detected in previously explored areas e.g. . This hiA.trilobum in Chile may be more extensive than we know. The discovery of A.trilobum in the Los Queules National Reserve, and the detection of other previously unrecorded species in the area, underlines the existence of a diversity and distribution of species yet to be explored and understood in these ecosystems. Consequently, the development of inventories and basic research in under-explored areas, such as ravines and forest remnants, should be promoted. Finally, it is crucial to encourage the training of advanced human capital in botany, taxonomy and genetics, areas that have received little attention from the Chilean State.In the context of this study, it is possible to suggest that the distribution of


Balangia and Duyunaspis represent related Cambrian oryctocephalid trilobites from South China, are comprehensively discussed over the ontogeny, and the results show that, from B. balangensis via D. duyunensis to D. jianheensis, their exoskeletal morphology shows a directional evolution. Based on the direction of evolutionary changes in the development of Balangia and Duyunaspis, we speculate that Duyunaspis likely evolved from Balangia instead of Balangia evolved from Duyunaspis, as was previously assumed. This inference is also supported by the phylogenetic tree. This research provides not only a better understanding of the mechanisms of evolution in trilobites, but also new insights for the relationship between developmental evolutionary changes and phylogeny in trilobites.The evolution process can be reconstructed by tracking the changes in the dynamic characters of life cycles. A number of related trilobites from the Cambrian of South China provide additional information for the study of trilobite evolutionary patterns, which has been hampered by previous incomplete fossil record though. Here,  This ime.g., e.g., Phylogenetic tree provides a possible solution for the determination of ancestries . The conThe effaced trilobites provide phylogenetic information, and certain morphological characters of trilobites appeared repeatedly throughout their history . Thus, tDuyunaspis  in this study are housed at the Guizhou Research Center for Palaeontology (GRCP). The phylogenetic position of D. paiwuensis is not discussed in this article due to its small number of specimens, unclear stratigraphic range . The number of pygidial segments is present in two or three types at the same degree  are almost identical to those of the other morph in the same meraspid degree. Thus, the average ontogeny pathways for D. jianheensis can reflect the changes in morphological characters during ontogeny. The case of B. balangensis and D. duyunensis are similar to that of D. jianheensis. Based on that, possible ontogenetic pathways of B. balangensis and D. duyunensis are shown in The ontogenetic pathways of pathways . Referripathways  was derinheensis . Two segDuyunaspis jianheensis, which can approximately be regarded as isopygous. It could be inferred that it might be isopygous in the M1 , D. duyunensis had a pit-like glabellar furrows. The fourth pair of glabellar furrows (S4) is indistinguishable throughout the ontogeny. Though unconspicuous in the early stage, the inter-ring furrow in the pygidium developed at the M1. The type of facial suture changes from proparian (M0–M6) to gonatoparian (M7–M8) and finally to opisthoparian (M9–H)  . The antn (M9–H) .Balangia balangensis was nearly the same size as the cephalon, being isopygous  to reconciled meraspid periods with different numbers of pygidial segments , for these morphologic changes are progressive (with the thoracic segment increasing) rather than sudden. Here by using the ontogenetic pathway of The postembryonic developmental stages of trilobites are delineated based on the development of articulations between segments . A seriee sample . The oryenomenon . So, theenomenon . Degreesenomenon  and ellienomenon  trilobite.g., glabellar furrow, occipital furrow, and axial furrow) are usually augmented during the ontogeny or clade of a group  trend  in the shape of the glabellar and the anterior border are both representation of ontogenetic sequences and possibly evidence of phylogeny. In addition, D. duyunensis and D. jianheensis both changed progressively from isopygous to micropygous during the ontogeny , while the isopygous condition  is more likely to occur in the early stratigraphic range   to D. duyunensis (proparian–gonatoparian–opisthoparian). In fact, this change is the result of the intersection between the end of the posterior branch of the facial suture and the cephalon from the lateral margin of the head, which progressively approaches to the posterior margin of the cephalon. Generally, the facial suture type remained the same in ontogeny. Considering that D. duyunensis is largely variable in the facial suture type during the ontogeny  trend . The graontogeny . Balangieries 2) . The numxpressed , it chanontogeny , the varnheensis , the promoulting . FurtherBalangia might have derived from Duyunaspis based on heterochrony. As a potential pattern for an evolutionary change, the key issue in heterochrony is to determine the ancestral-descendant relationships and the timing of sexual maturation , the co-occurrence of B. balangensis and D. duyunensis in the same strata in Banglang Formation is clearly incompatible with the second element proposed by B. balangensis and D. duyunensis  from D. duyunensis. The developmental evolutionary changes of phenotypic traits appear to be directional in B. balangensis, D. duyunensis, and D. jianheensis, therefore, we believe that the clade B. balangensis–D. duyunensis–D. jianheensis (yunensis . Nearly yunensis  trilobit species , and thuyunensis  implies nheensis  is reasoBalangia balangensis–Duyunaspis duyunensis–Duyunaspis jianheensis in terms of the cephalon morphology, size of the pygidium relative to the cephalon and the trunk segments number in developmental evolutionary change. Furthermore, according to the four common elements in the speciation process, the lowest datum of B. balangensis is higher than that of D. duyunensis, which is probably caused by the incompleteness of the fossil record. We speculate that Balangia is not necessarily the descendant of Duyunaspis, on the contrary, Balangia may be the ancestor of Duyunaspis. This inference is also supported by the phylogenetic tree.There is a directional gradual evolution of 10.7717/peerj.15068/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.15068/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.15068/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.15068/supp-4Supplemental Information 4Click here for additional data file.


Notobitus meleagris (Hemiptera: Heteroptera: Coreidae). The results show that there were eight subtypes of sensilla on the antennae of the nymphs and 11 subtypes of sensilla on the antennae of the adults. With the growth of instars, the type, quantity, and size of the sensilla gradually increased. Sexual dimorphism did not appear in the type of sensilla but existed in the length and diameter of some sensilla. In addition, we also discussed the functions of different types of sensilla through previous published studies, which will be helpful for further research on the behavioral and biological control of N. meleagris.In this paper, we used scanning electron microscopy to describe the morphological types, number of sensilla, and their distributions on the antennae of each nymphal instar and adult of Notobitus meleagris  is a serious pest of bamboo shoots in China, India, Myanmar, Vietnam, and Singapore. The antennae of the nymphal instars and adults of N.meleagris are involved in communication among individuals and finding the host plants. In order to understand the morphology of antennal sensilla, their types, and the distribution of sensilla on the antennae of nymphal instars and adults in N. meleagris, we studied the morphology of antennal sensilla with a scanning electron microscope. The antennae of the nymphs and adults comprised the scape, pedicel, and two flagellomeres. Four types and eight subtypes of sensilla were identified in the nymphal instars , whereas those of the adults had five types and eleven subtypes of sensilla . There are significant differences in the number, type, and size of the sensilla in different nymphal instars, which increases with the increase in nymphal instars. There was no sexual dimorphism observed in the adult sensilla; however, the length and diameter of St.3, Sb.2, and Sb.3 were sexually dimorphic. The potential functions of each sensillum were discussed based on the morphology and distribution of the antennal sensilla and were compared with similar published studies. Our results provide primary data for further research on the behavioral mechanism, green prevention, and control of N. meleagris.The bamboo bug  Notobitus meleagris (Fabricius) (Hemiptera: Coreidae) is an important pest of bamboo shoots and is distributed in China , India, Myanmar, Vietnam, and Singapore [N. meleagris in a year, and they overwinter in weeds or bark as adults. The occurrence period is in line with the bamboo shoot growth period. In April, the adults of the overwintering generation begin to move [N. meleagris are gradually increasing. In severe cases, >20 individuals are simultaneously present on each bamboo shoot . Our findings provide basic information regarding the sensory-communicative system of N. meleagris nymphs and adults, their behavioral mechanisms associated with biological control, and the host selection and management of N. meleagris.During the investigation, we found that N. meleagris , Duyun City, Guizhou Province, on 29 August 2021.Ten individual adults with each nymphal instar of eleagris  were colThe photographs of the morphological characteristics of the antennae in the adults and nymphs were acquired using a Canon camera  equipped with a Canon EF 100 mm F/2.8L IS USM Macro Lens, with Godox MF12 Twin Flash used as a light source. Antennae were individually photographed under an ultra-depth-of-field 3D microscope , and the pest parameters of each instar were determined.N. meleagris adults and nymphs  at 4 °C for 24 h. The antennae were carefully detached from the head using tiny-tipped forceps and were fixed in 2.5% glutaraldehyde for 24 h. After fixing, ultrasonic cleaning was performed for 15 min. The samples were rinsed thrice with phosphate buffer . Serial dehydration was performed with 30%, 50%, 70%, 80%, 90%, and 100% ethanol; the samples were dehydrated at each concentration for 20 min and then at 100% for 1 day. After dehydration, the samples were dried in an electric blast dryer at 40 °C for 6 h and placed on the sample holder with conductive adhesive based on different observation surfaces. Finally, ion sputtering gold plating was performed for 2 min. The shape and number of sensilla on the entire antennae and segments of the male and female adults, as well as each nymphal instar, were photographed using SEM , and the length of each sensillum was measured.We anesthetized t-tests in SPSS 22.0 software  and expressed as mean ± standard error. Kolmogorov–Smirnov tests were used to assess the normality of the data, and Levene tests were used to determine the homogeneity of variance. Data conforming to the normal distribution and showing homogeneity of variance were analyzed using one-way analysis of variance, and Tukey’s method was used for multiple comparisons.Scaler software , associated with the SEM system, was used to measure the length of each sensillum and identify and enumerate the antennae of the adults and nymphs. The identification and naming of the antennal sensilla were performed as previously described by Schneider and Zacharuk ,43. The N. meleagris comprised the scape, pedicel, and two flagellomeres. The total antennal length of the nymph and adult was measured between 3422.86–16587.89 μm , sensilla basiconica (Sb), sensilla chaetica (Sc), and sensilla coeloconica (Sco). Based on the differences in size and external morphological characteristics, St was divided into three subtypes , Sb was divided into two subtypes (Sb.1 and Sb.2), and Sc was divided into two subtypes (Sc.1 and Sc.2). Each sensillum showed various distributions among the nymphal instars.Among the three St subtypes, St.1 was the most abundant on the antennal surface in each nymphal instar and was distributed in the second flagellomere. The number of St.1 on the antennae of the first to fifth nymphal instars was 231.0–1226.3 . They arAmong the two Sb subtypes, Sb.1 was distributed vertically in the second flagellomere of all the nymphal instars. The number of Sb.1 on the first to fifth nymphal instar antennae was 8.6–43.1 . They arAmong the two Sc subtypes, Sc.1 was strictly distributed in the scape, pedicel, and first flagellomere of all the nymphal instars. The number of Sc.1 on the antennae of the first to fifth nymphal instars was 90.9–552.8 . They arSco.1 was distributed vertically in the second flagellomere of all the nymphal instars. The number of Sco.1 on the antennae of the first to fifth nymphal instars was 1.2–19.8 . They arThe antennae of the adults were observed via SEM, which revealed seven types of sensilla: St, Sb, Sc, Sco, and sensilla campaniformia (Sca). Based on the differences in size and external morphological characteristics, St was divided into three subtypes , Sb was divided into three subtypes , Sc was divided into two subtypes (Sc.1 and Sc.2), and Sco was divided into two subtypes (Sco.1 and Sco.2). These 13 subtypes of sensilla were distributed in the antennae of both the male and female adults; however, the size of these sensilla varied between the sexes.Among the three St subtypes, St.1 was distributed in the second flagellomere of the adults. The number of St.1 on the antennae of the males and females was 1683.7–2058.8 . The shaAmong the three Sb subtypes, Sb.1 was distributed in the second flagellomere of the adults. The number of Sb.1 on the antennae of the males and females was 101.5–120.1 . The shaAmong the two Sc subtypes, Sc.1 was distributed in the scape, pedicel, and first flagellomere of the adults. The number of Sc.1 on the antennae of the males and females was 737.6–960.1 . The shaAmong the two Sco subtypes, Sco.1 was distributed in the second flagellomere of the adults. The number of Sco.1 on the antennae of the males and females was 30.3–40.5 . The shaSca was oval-shaped and clustered at the base of the scape in adults. They consist of an oval plate with an ecdysial pore, situated inside a tight columnar socket. . The lenThere were four types and eight subtypes of sensilla on the antennae of the nymphs; St.2 and St.3 were unique to the fourth and fifth nymphal instars, whereas Sb.2 was unique to the third, fourth, and fifth nymphal instars. Except for the abovementioned four sensilla, the other sensilla were shared among all the nymphal instars. Additionally, five types and 11 subtypes of sensilla were identified on the antennae of the adults. There was no sexual dimorphism among these types, and Sb.3, Sco.2, and Sca were unique to the adults.N. meleagris nymphs to adults gradually increased from 5 to 11. The structure and type of these sensilla were similar to those of other Hemiptera insects [N. meleagris, particularly in the nymphs, can be divided according to antennal segments. The types of sensilla in the first, second, and third antennal segments were identical, whereas the types of sensilla in the fourth antennal segment differed remarkably from those in the other three segments (N. meleagris had the highest number and density of sensilla, indicating that it plays an essential role in sensing in N. meleagris. The length of the antennae increased with the increase in the instar stage, with altered morphology and a gradual increase in the types and number of sensilla. However, N. meleagris nymphs lacked Sb.3, Sco.2, and Sca, suggesting that these three sensilla are related to the sexual maturity of N. meleagris. There was no sexual dimorphism in the types and numbers of sensilla in N. meleagris, except for the length and thickness of St.3, Sb.1, Sb.2, Sb.3, Sc.2, Sco.1, and Sca. It is speculated that these sensilla play an essential role in the mating or oviposition behavior of N. meleagris.We identified St.1, Sb.1, Sc.1, Sc.2, and Sco.1 on the antennae of the first and second nymphal instars. The antennae of the third nymphal instar had Sb.2 in addition to the sensilla present on the antennae of the first and second nymphal instars. The antennae of the fourth and fifth nymphal instars had St.2 and St.3 in addition to the sensilla present on the antennae of the third nymphal instar. The antennae of the adults had Sb.3, Sco.2, and Sca, in addition to the sensilla present on the antennae of the fourth and fifth nymphal instars. The subtypes of sensilla in the  insects ,29, withsegments . The fouN. meleagris is similar in appearance to those described for Riptortus pedestris [N. meleagris; therefore, the ability to perceive pheromones is stronger in higher instar stages. Additionally, Sb.1 is similar in appearance to those described in Pyrrhocoris sibiricus [N. meleagris showed sexual dimorphism, which may be related to the oviposition behaviors of the females. The sensilla chaetica of N. meleagris is similar in appearance to those described for Apolygus lucorum [N. meleagris, and usually protects other receptors [N. meleagris is similar in appearance to those described for Eocanthecona furcellata [N. meleagris nymphal instars and is speculated to be the main sensilla through which they sense external temperature and humidity. The sensilla campaniformia of N. meleagris is similar in appearance to those described for Gerromorpha [N. meleagris, that is to say, the mechanical reception of the adult is stronger than that of the nymphs [N. meleagris adults have a stronger ability to perceive signals than nymphs. The specific functions of different sensillum subtypes should be further verified by conducting detailed electrophysiological studies on single sensilla.The antennae are the principal organs of the insect sensory system. Various sensilla form essential functional elements to screen and select environmental factors and perceive chemical, mechanical, and physical signals ,45. Sensedestris , which iedestris ,47,48 anedestris , with paedestris ,51. Howeibiricus , and senibiricus , which oibiricus . The len lucorum , which ieceptors ,55,56. Frcellata , which ircellata ,58. Sco.romorpha , found iromorpha ,35,59. Te nymphs . This alN. meleagris and a basis for blocking the function of sensilla and controlling N. meleagris through synthetic pheromones. This could lay a theoretical foundation for the green prevention and control of insects and aid in forestry plant protection.Receptors are the primary means of communication between individual insects and the environment; moreover, insects use sensilla to locate their partners and host plants . In addi


Author Contributions, author Xiaotong Liu was erroneously excluded.In the published article, there was an error. In the Author Contributions, paragraph 1. This sentence previously stated:A correction has been made to “CH, RZ, and XiyL contributed to conception and design of the study. CH organized the database. RZ and XiyL performed the statistical analysis. HL and GH wrote the first draft of the manuscript. SL and XiaL wrote sections of the manuscript. CH, XiyL, and XW contributed to manuscript revision, read, and approved the submitted version.”The corrected sentence appears below:“CH, RZ, and XiyL contributed to the conception and design of the study. CH organized the database. RZ and XiyL performed the statistical analysis. XiaL wrote sections of the manuscript. CH, XiaL, XiyL, and XW contributed to manuscript revision, read, and approved the submitted version. All authors contributed to the article and approved the submitted version.”The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.


Despite the high incidence of appendicitis, accurate diagnosis of suspected appendicitis has long been challenging, with a high rate of negative (unnecessary) appendectomies in clinical practice. Mohamed and colleagues have tried to identify laboratory biomarkers for diagnosing suspected appendicitis, which is a very important topic in a clinical setting. However, there was no sample size calculation of the minimum number of samples required in this study. The authors applied a limited number of participants in the normal appendix group n=2, compliBesides, they reported relatively high diagnostic accuracy of circulating fibrocyte percentage  in the diagnosis of uncomplicated appendicitis, and similarly high diagnostic accuracy of neutrophil–lymphocyte ratio in the diagnosis of uncomplicated appendicitis et al.Multiple types of computed tomography (CT), have shown similarly high diagnostic accuracy in the diagnosis of appendicitis, with a pooled high sensitivity of 89–97% and specificity of 93–95%Overall, there is still no enough evidence that laboratory biomarkers could act as an alternative to CT imaging. Lose-dose CT should still be preferred for the diagnosis of suspected appendicitis in current clinical practiceNot applicable.C.-D.Z. was partly supported by the China Scholarship Council (201908050148). R.Z. was partly supported by the National Natural Science Foundation of China (61976249). These funding sources had no role in the study design, in the collection, analysis, and interpretation of data, in the writing, and in the decision to submit the article.R.Z.: writing and editing. Z.-M.Z.: editing and revision. C.-D.Z.: conceptualization, writing, editing, revision, and submission.The authors declare that they have no financial conflict of interest with regard to the content of this report.Not applicable.Chun-Dong Zhang.The data supporting the findings of this study are available from the corresponding author upon reasonable request.Not applicable.


As the critical component of automotive engine coolant, ethylene glycol (E.G.) significantly matters in heat dissipation. In this study, the key aim is to investigate the heat transfer behaviour of E.G. as nano-additives base fluid. The heat transfer capability of E.G./SiC nanofluid (N.F.) was experimentally and theoretically evaluated via transient hot wire methods and equilibrium molecular dynamics (EMD) simulation, respectively. M.D. simulation exhibited a great ability to accurately forecast the thermal conductivity of N.F. compared with the experiment results. The results confirmed that the thermal stability of N.F. is relatively greater than that of E.G. base fluids. An improvement mechanism of thermal conductivity and thermal stability under an atomic scale via the analysis of mean square displacement (MSD) and radial distribution function (RDF) calculation was elaborately presented. Ultimately, the results indicated that the diffusion effect and the increasing transition rate of liquid atoms are responsible for thermal conductivity enhancement. Fluids with excellent heat transfer performance are urgently required due to the continuous development in industry and academia fields . In the 2/E.G. hybrid N.F.s were fabricated, and the heat transfer of the hybrid N.F.s was studied by Toghraieeret al. [Temperature and nanoparticle concentrations are critical physical quantities affecting fluid’s thermal conductivity because they are closely related to Brownian motion and other important thermal factors ,18,19,20eret al. , who inderet al.  presenteIn addition to experimental investigation of thermal conductivity, regression fit analysis was utilized to predict thermal conductivity, including the most famous Maxwell and Einstein models. However, such simulation analysis based on experimental data can only speculate or estimate the underlying mechanism without firm evidence. Fundamentally, the key reason for the increase in thermal conductivity cannot be well analysed from the microscopic level. Conversely, molecular dynamic simulation can reasonably predict the thermal conductivity and analyse the microscopic atomic motion to achieve convincing mechanisms. Non-equilibrium molecular dynamics (NEMD) and EMD methods were the widely used two simulations for evaluating the heat transfer characteristics of N.F. NEMD simulation was adopted by Zhou et al.  to invesE.G., as the main component of automobile coolant, is critical to the cooling system of automobile engines because the heat dissipation performance of the engine was related to the E.G. fluids ,30. TherThis paper aims to reveal several thermal conductivity improvement mechanisms for N.F.s with diverse concentrations at varying temperatures. Thus, the EMD means was employed to model and simulate the glycol/SiC N.F. system and the heat transfer performance was discussed. It was profoundly revealed the diffusion behaviour of SiC nanoparticles in E.G. from the micro scale. Eventually, the active adsorption layer between SiC and E.G. base liquids promoted the improvement of thermal conductivity.−1 [−1 may be assigned to the C-Si vibration mode of SiC. In addition, the stretching vibration peak at approximately 3414 cm−1 may be attributed to the O-H formation from the water molecule in the air.For the exploration of the morphology and size distribution of SiC nanoparticles used, TEM as well as SAED characterisation under different locations, were conducted in −1 . The traThe thermal conductivity of pure E.G. and 0.1–1% N.F. at different temperatures were derived by the EMD simulation approach and compared with the experiment results using transient hot wire methods. t is the time, The MSD is used to measure the bias between the location of particles’ motion over time. This phenomenon is a standard method to analyse the dynamic characteristics of the N.F. system. The formula for calculating MSD is as follows:The diffusion coefficient can be calculated to account for the aggregation and adhesion of the nanosystem. The MSD curves of the total atoms in the whole E.G./SiC N.F.s system with various molar fractions under diverse temperatures are depicted in The RDF reflects the fluid microstructure and reveals the aggregation behaviour of different atoms in the fluid. Molecular dynamics, a multidiscipline technology combining mathematics, physics and chemistry, is a simulation method using Newtonian mechanics to simulate and calculate the motion state of microscopic molecule for obtaining a series of macroscopic physical quantities, such as thermal conductivity, friction coefficient, dynamic viscosity, velocity, temperature, shear stress and other parameters. With the development of simulation, M.D. simulation has been gradually adopted by multiple researchers to remedy the shortcomings of experimental methods. The movement law of atoms was accurately detected by M.D. analysis, and it elucidated the reasons for the improvement of dispersion behaviour, rheological behaviour, thermal performance, and friction performance from the atomic scale. This paper uses the open-source software LAMMPS 64-bit 29 October 2020 . A large-scale Atomic/Molecular Massively Parallel Simulator was considered for the simulations to investigate the heat transfer characteristics of pure E.G. and E.G./SiC N.F.s with different concentrations.Prior to the model construction, the SiC and E.G. structure cells can be seen, as shown in r is the equilibrium bond distance, E1 and E2 are the stretching and bending energy, θ is the equilibrium value of the angle, and To achieve the reliability, creditability, and precision of the simulation results, several force fields between atoms are carefully chosen. Lennard-Jones (LJ) Long range Coulomb force potential for C-C, O-H, Si-Si interactions with a cut-off distance of 10 Å was used. This force field is suitably extended on the basic of LJ potential, which increases the Coulomb pair interaction between atoms, making the output results more accurate. The formula of this force field can be expressed as follows:V denotes the volume, Bk denotes the Boltzmann constant, T is the temperature, <xJ(0)xJ(t)> is the heat flow autocorrelation function.First, the entire simulation system needs to be balanced for several ns to obtain a constant temperature of 300–350 K in the NVT ensemble using the Nose Hoover thermostat. Then, EMD operation is conducted in the NVE ensemble to calculate the thermal conductivity and heat flux. In this EMD method, the thermal conductivity is calculated by heat flux. The method for calculating the heat flux is based on the contribution of atoms in the designated group. It can be used to measure the heat flow through a group of atoms , and the thermal conductivity can be calculated by Green Kubo formula as follows:−4 Å) in Lammps. Ovito software  was used to post process the simulation results.For the detection of M.D. model effectiveness, the equilibrium relaxation of E.G./SiC N.F.s with different molar fractions at different temperatures under initial conditions was calculated between the time steps of 0–250 ps. In this investigation, the thermal conductivity of E.G. was simulated by the EMD method. The CVFF force field is used in the simulation research system. The molecular optimisation function determines the bond length and angle of the E.G. molecule. Finally, SiC cells are introduced to obtain E.G./SiC models with molar fractions of 0%, 0.1%, 0.5%, and 1%. The conjugate gradient method eliminates unreasonable interactions in the system and minimises the energy. Then, the Nosé Hoover hot bath method is used to relax for 1 ns under the constant pressure and temperature (NPT) ensemble. The temperature gradient is accordingly set . The pressure is set as standard atmospheric pressure. Then, after withdrawing the NPT ensemble, the simulation was relaxed for 1 ns under the constant volume and temperature (NVT) ensemble. When the whole system reached equilibrium, the NVT ensemble was removed. The system finally runs 200 ps under the micro-canonical ensemble (NVE) to obtain the kinetic energy, potential energy, and potential force of atoms. The heat flow density through the energy contribution of atoms in the group is calculated and sampled every 1000 steps. Finally, we connect the heat flow density J with the autocorrelation set average of the thermal conductivity k through the Green-Kubo formula, and the thermal conductivity is accordingly calculated. The time step is set as 1 fs. Periodic boundary condition (PBC) is chosen in the simulation system, the Leap Frog algorithm is used for the equation of motion integration, and the long-distance electrostatic interaction calculation needs to be matched with PPPM , and E.G. is obtained from Shanghai Aladdin Bio-Chem Technology Co., Ltd., Shanghai, China. The detailed physical parameters of SiC nanoparticles were displayed in In the preparation of N.F.s, a conventional two-step method was applied. First, SiC nanoparticles were added into E.G. base fluid, and the mechanical dispersion, including high-pressure homogeniser and magnetic stirrer was used. Under the premise of mechanical stability, ultrasonic dispersion is carried out to further improve its dispersion stability, including ultrasonic probe and ultrasonic bath dispersion. In this way, stable N.F.s are well prepared. The detailed preparation process is shown in In nanoparticle characterisations, we use TEM  to detect the particles’ average diameter, overall distribution, and aggregation conditions in E.G. fluids. XRD  and FTIR  were used for the crystal construction, chemical bonds, and micro-valence change. In the manufacturing process of N.F.s, a high-pressure homogeniser and magnetic stirrer are used to mix nanoparticles and base liquid physically. The ultrasonic energy generated by the ultrasonic disperser due to the cavitation role makes the N.F. uniform and stable. Finally, the prepared N.F. is put into the ultrasonic bath for re-stablisation. Thermal stability of N.F. and E.G. was assessed by TGA analysis conducted by Thermogravimetry meter . Thermal conductivity meter  is experimentally used to measure thermal conductivities of N.F. and base fluid via a transient hot wire method. Each experiment was performed by 3 times and the average with standard deviations were taken.This study explores the heat transfer capability of E.G. and reveals the enhanced mechanism of E.G. thermal conductivity by adding SiC nanoparticles via an EMD simulation under varying temperatures and concentrations. Based on the results and discussion, the primary conclusion can be presented as follows:Thermogravimetric analysis confirmed that the SiC/E.G. N.F.s appear thermally stable compared with the E.G. alone, which is mainly attributed to the small size effect caused by SiC nanoparticles addition.The E.G./SiC N.F. thermal conductivity was predicted and simulated using M.D. simulation. Most of simulation values of the thermal conductivity increased with the increasing temperature except the several contrary results. In addition, compared with the E.G. solution alone, thermal conductivity of SiC N.F. was improved, by up to approximately 5.4% and 7.3%, for experiment value and simulation value, respectively, which showed a credibility for simulation results.The MSD curve proves that the addition of nanoparticles greatly accelerates the movement velocity of liquid atoms in N.F.s, widens the diffusion layer, and improves the diffusion efficiency. The interface effect and the soaring motion of liquid atoms are the fundamental reason, rather than the Brownian motion improvement, for improving thermal conductivity.RDF analysis confirmed that the microscopic structure of E.G. base fluid changed from the liquid phase into a solid–liquid state due to the introduction of SiC nanomaterials, which may also be the reason responsible for the enhancement of thermal conductivity.


Correlation analysis between required surgical indexes and complications in patients with coronary heart disease by Tao M, Yao X, Sun S, Qin Y, Li D, Wu J, Xiong Y, Teng Z, Zeng Y and Luo Z (2022) Front. Surg. 9:948666. doi: 10.3389/fsurg.2022.948666A Retraction of the Original Research Article Following publication, the publisher uncovered evidence that several false identities were used in the peer-review process. The assignment of false reviewers was confirmed by an investigation conducted in accordance with Frontiers' policies and the Committee on Publication Ethics (COPE) guidelines.The investigation also uncovered concerns about the presentation and validity of the data in the article. The authors did not respond to contact regarding the data presented in the article.This retraction was approved by the Chief Editors of Frontiers in Surgery and the Chief Executive Editor of Frontiers.The authors did not respond to contact regarding this retraction.


Based on data from three imported mpox cases in South Korea, the overall attack rate was determined to be 1%, while a secondary attack rate of 14% was estimated in the high-exposure group. In East Asia, mpox was initially detected in June 2022 (South Korea) among returning travellers who had engaged in sexual activity with men from Europe. As of May 2023, several European countries had reported a single-digit incidence of mpox infections, seemingly indicating the end of the epidemic.,A recent study demonstrated the importance of prioritizing public health and social measures, including mpox vaccination of high-risk subpopulation, to reduce mpox transmission across the community.We collected publicly available data on mpox cases from Korean public health authorities . Based oWe measured the overall attack rate, which is the probability that an infection occurs among susceptible individuals, and the secondary attack rate in the high-exposure group and estimated the 95% confidence intervals (CIs) using the bootstrap method .The overall attack rate based on the first three imported cases was found to be 0.9% . After stratification by degree of exposure, the secondary attack rate was estimated at 14.2% among contacts in the high-exposure group . However, no transmission cases were identified in the intermediate- and low-exposure groups .The secondary attack rate provides useful information on how the interactions in a specific setting relate to the transmission risk. Our findings suggest that mpox transmissibility from the first three imported cases to the South Korean population was low and that mpox transmission was potentially driven by the high-exposure group. This finding is consistent with those of previous studies in that the major risk for mpox transmission was associated with a high degree of exposure, including sexual contact.Notwithstanding, this study has certain limitations. First, behavioural heterogeneity might have influenced the estimation of the attack rate. Thus, further research involving a greater number of cases with contact-tracing data is warranted to avoid possible bias and to reduce CI width. Second, a secondary mpox infection from the imported case occurred during medical practice.The continual estimation of mpox transmissibility is important for evaluating control measures against mpox. A comparison of transmissibility estimates according to different mpox-exposure levels and modes of transmission  is warranted. Furthermore, with the easing of SARS-CoV-2-related international travel measures in Asian countries,This work was supported by the Basic Science Research Program of the National Research Foundation of Korea of the Ministry of Education (NRF-2020R1I1A3066471).S.R. conceived the study. D.L., H.D., A.C., M.-K.K. and B.C. were involved in data collection and assimilation. D.L., S.C., H.D., A.C. and S.R. performed data analysis, discussed the results and drafted the manuscript. All authors have critically read and approved the final manuscript.Dayeong Lee , Sangbum Choi , Hyunkyung Do , Achangwa Chiara , Min-Kyung Kim , BumSik Chin , and Sukhyun Ryu Conflict of interest: S.R. was supported by the Korea Disease Control and Prevention Agency (contract numbers: 2023020748C-00 and 2023040522C-00). All other authors report no potential conflicts of interest.This study did not require institutional review board approval or informed consent because all data used are anonymous and are publicly available on websites.The data underlying this article will be shared on reasonable request to the corresponding author.Supplementary_Materials_taad080Click here for additional data file.


China. The correct affiliation is College of Horticulture Science and Engineering, Shandong Agricultural University, Taian, Shandong, P.R. China.These details have been corrected only in this correction notice to preserve the published version of record.


Brachystele Schltr.  comprises 20 species distributed from Mexico to Argentina, with 10 species found in Brazil. Anatomical studies of Orchidoideae Lindl. have been scarce, and the anatomy and histochemistry of Brachystele are still largely unknown. In this study, we conducted a characterization of the vegetative organs of B. guayanensis (Lindl.) Schltr. using standard anatomical and histochemical microtechniques. In this study, we provide the first information about the anatomy and histochemistry of Brachystele. The studied species was observed to display anatomical characters commonly found in the vegetative organs of representatives of the Cranichideae tribe . Others can be interpreted as adaptive strategies conditioned by the environment and their terrestrial life form ; and raphides). In this study, cataphylls, and the presence of spiranthosomes in leaves, including stomatal guard cells, as well as alkaloids in these structures, are anatomically described for the first time in Orchidaceae. The presence of hyphae and pelotons in the stem of B. guayanensis is described for the first time in Cranichideae. Histochemical tests confirmed the presence of lignin, proteins, and alkaloids, the lipidic nature of the cuticle, starch grains stored in spiranthosomes, and the composition of the raphides. Alkaloids were observed in abundance, particularly in the roots, suggesting a potential role in defense against pathogens and herbivores, as well as potential medicinal activities, as seen in phylogenetically related groups to Brachystele.The orchid genus  Orchidoideae Lindl. (Orchidaceae) is monophyletic and comprises approximately 3650 species and 208 genera, distributed throughout the temperate and tropical regions of the Old and New World ,2. In BrIn anatomical terms, Orchidoideae is poorly studied since most of the species sampled in previous studies ,10 occurBrachystele Schltr.  comprises 20 species distributed from southern Mexico to northern Argentina [Among the genus of Orchidoideae, rgentina ; 10 specrgentina . The taxrgentina ,17.Brachystele are currently limited to the work of Bernal et al. [B. widgrenii (Rchb.f.) Schltr. However, there have been no histochemical studies of any Brachystele species, although some medicinal properties of B. dilatata (Lindl.) Schltr. and B. unilateralis (Poir.) Schltr. were mentioned  in ethnobotanical studies [Anatomical descriptions of the vegetative organs of l et al. , who exa studies ,19.B. guayanensis (Lindl.) Schltr.  Ormerod and Zeuxine strateumatica (L.) Schltr. by Andreota et al. [Aa paleacea (Kunth) Rchb. f. and Pterichis multiflora (Lindl.) Schltr. by Corredor and Arias [B. guayanensis  Rolfe and Gomesa crispa (Lindl.) Klotzsch ex Rchb. f. According to these authors, the strategic positions of these structures contribute to the maintenance and (re)colonization of internal tissues, serving as important sources of inoculum for adventitious roots extending from the rhizome, corroborating the findings of Pridgeon [Eulophia epidendraea C.E.C. Fisch., Malaxis acuminata D. Don, Oreorchis indica (Lindl.) Hook. f. and Rhizanthella gardneri R.S. Rogers). Such structures were also found in the rhizomes of epiphytic and rupicolous orchids of the Epidendroideae (Promenaea rollisonii (Rchb. f.) Lindl. and P. xanthina Lindl.) by Pedroso-de-Moraes et al. [B. guayanensis is first reported for Cranichideae. According to some authors [Fungal hyphae and pelotons were observed to be concentrated mainly in the more peripheral portions of the cortical parenchyma g, which Pridgeon  for terrPridgeon , Uma et Pridgeon  and SuetPridgeon  for Epids et al. . In this authors ,47,48, t authors ,50,51. M authors ; increas authors , and conBetween 28 and 45 scattered collateral vascular bundles can be observed internally, surrounded by a parenchyma sheath i, which Brachystele guayanensis has transversely circular roots of variable calibers (Pelexia dolichorhiza Schltr. (=Pachygenium pteryganthum (Rchb. f. and Warm.) Szlach., R. González and Rutk.), Prescottia colorans Lindl. (=P. stachyoides (Sw.) Lindl.), and Sauroglossum elatum Lindl. Moreira and Isaias [calibers a, which calibers b,c. Our d Isaias  reportedd Isaias , Pridgeod Isaias , and Chod Isaias  noted thBrachystele widgrenii, Lankesterella caespitosa (Lindl.) Hoehne, L. ceracifolia (Barb. Rodr.) Mansf., Pelexia orthosepala (Rchb.f. and Warm.) Schltr, and Sacoila lanceolata (Aubl.) Garay. by Bernal et al. [Cranichis candida (Barb. Rodr.) Cogn. by Andreota et al. [Aspidogyne Garay and Microchilus C. Presl. by Bona et al. [Simple, unicellular trichomes, such as those observed here c, were rl et al. ; for Craa et al. ; and fora et al. . Accordia et al. , Andreota et al. , and Bera et al. , simple The root cortex consists of 18–20 layers of rounded parenchyma cells of varying sizes with innumerable small, triangular intercellular spaces d. In addThese characters are frequently observed in Orchidoideae ,21,56. IB. guayanensis are uniseriate and composed of rounded, elliptical cells with thin walls. The cells of the exodermis are slightly thickened , are responsible for renewing their aerial portions and help prevent pathogens and herbivores from reaching the vascular system  and causing local and/or systemic damage.According to Sut et al.  and Bulpd Nakata  and Vizzd Nakata  for pland Nakata  for the B. guayanensis indicates its potential as a medicinal species. Further (bio)phytochemical investigations could be informative, including the subsequent isolation and toxicity testing of any bioactive substances and the characterization of their chemical nature.Among the bioactive substances found in Orchidaceae species are alkaloids  and nitrogenous heterocyclic organic molecules derived from the secondary metabolism of amino acids . More than 100 types of bioactive alkaloids have been identified in over 2000 orchid species ,81. ThesB. guayanensis (I.S. Santos 1160 and 1161).For the anatomical studies, samples of vegetative organs  from five adult individuals of yanensis  were colv/v) [® digital camera attached to a Leica DM500® microscope, using Motic 2.0 Image Plus Software.The collected samples for anatomical studies were fixed in 70% FAA  in hermetically sealed containers for 48 h. After this period, they were preserved in 70% ethyl alcohol . For thev/v) . They wev/v)  and mounv/v) . For theFor histochemical studies, in natura samples obtained at the time of collection were stained with the following reagents: Coomassie blue and xylidine ponceau ,90 for dBrachystele. The studied species exhibit foliar, cauline, and root characters shared with other Cranichideae species . Others can be interpreted as adaptive strategies that have evolved in response to the environment and their terrestrial life form, including cuticle thickness, the presence of amphistomatic leaves, fibers, and lignin for internal tissues support, fleshy roots with reduced velamen compared to the cortex, the presence of trichomes to increase water and nutrients absorption, and raphides for calcium reserve, osmotic and ionic regulation, detoxification, and defense. In this study, the cataphylls are received in anatomical terms for Orchidaceae, in addition to verifying that the tiny starch grains are aggregated in spiranthosomes throughout the vegetative systems of B. guayanensis, and such structures, as well as the alkaloids, are first referred to leaves, especially for the stomatal guard cells. The presence of hyphae and pelotons in the stem of B. guayanensis are first described for Cranichideae. We believe that such fungal structures, located in the peripheral portions of the rhizomes, are important strategies for the maintenance of internal tissues and the efficient inoculation of still young adventitious roots. In addition, the presence and abundance of alkaloids and raphides in the reserve organs (roots and rhizomes), as well as in the guard cells of the stomata, may be associated with defense against herbivores and against the entry and systematization of pathogens and diseases, since its reserve organs are more durable than the aerial ones and are responsible for the maintenance and renewal of its aerial parts by storing water and other energy reserves , and the stomata for configuring an entryway for harmful microorganisms. Additionally, B. guayanensis is likely to be a potentially medicinal species, mainly due to the abundance of alkaloids in its roots and rhizomes, similarly to phylogenetically related groups of the family. Furthermore, this study reveals the importance of anatomical and histochemical studies, in particular, directed to neotropical orchids that are poorly studied, guiding the development of studies with different aspects, especially biochemical and phytochemical studies.In this study, we have provided the first information regarding the anatomy and histochemistry of


P. harmala, T. ramosissima, and P. reptans. The suitability of the tDNAs for molecular studies was evaluated by polymerase chain reaction (PCR) of the fragments of the internal transcribed spacer (ITS) in nuclear DNA and the trnL-F region in chloroplast DNA. Some significant differences were found between the tDNAs extracted by five extraction methods. With the exception of P. harmala, where the PCR of both the ITS fragments and the trnL-F region worked successfully in all DNA samples, but only the ITS fragments, not the chloroplast trnL-F region, were amplified in the DNA samples of T. ramosissima and P. reptans. The chloroplast trnL-F region was amplified only in DNA samples extracted from fresh and dried leaves of the three studied herbs using the commercial kit. Gene All kit, the main CTAB method, and its modified protocols were the less time-consuming protocols that yielded DNA suitable for downstream PCR vis-a-vis the modified Murray and Thompson method.Extracting high-yield, high-quality DNA from plant samples is challenging due to the presence of the cell wall, pigments, and some secondary metabolites. The main CTAB method, two of its modified protocols (beta-mercaptoethanol or ammonium acetate were eliminated), the modified Murray and Thompson method, and the Gene All kit were statistically compared based on the quantity and quality of the total DNA (tDNA) extracted from fresh and dried leaves of three medicinal herbs  Medicinal herbs and their products are of health and economic value. Extraction of high-quality DNA is required to study the genetic, morphological, and ecological characteristics of medicinal herbs . The extPeganum harmala L. (Zygophyllaceae), Tamarix ramosissima Ledeb. (Tamaricaceae), and Potentilla reptans L. (Rosaceae) were investigated. P. harmala, known as espand in Persian, is a perennial herbaceous plant native to the arid regions of North Africa, the Mediterranean, the Middle East, Pakistan, and India [P. harmala has been traditionally and commonly used for medicinal and psychoactive purposes [P. harmala is traditionally used as an antiseptic by burning its seeds [P. harmala has many other pharmacological properties, including analgesic, anticonvulsant, antihistamine, anti-inflammatory, antioxidant, antitumor [T. ramosissima, called gaz in Persian, is a halophytic herb [T. ramosissima is used as a skewer [Tamarix species are useful in leucoderma, spleen disorders, eye diseases [T. ramosissima has been shown to possess antibacterial, antioxidant, astringent, appetizing, and invigorating properties [P. reptans is a perennial plant from Caspian and Iranian-Turanian regions and is common in Afghanistan, Europe, Iran, Iraq, the former Soviet Union, Turkey, and North Africa. This herb is common in Azerbaijan, Boyer-Ahmad, Gorgan, Gilan, Kohgiluyeh, Lorestan, Semnan, and Tehran provinces of Iran [Potentilla has been known for its therapeutic properties since ancient times. Extracts from the aerial or underground parts of P. reptans are used in traditional medicine against bacterial [In the current study, three medicinally important herbs, including nd India . The hernd India . Since apurposes . In Irants seeds . The herts seeds . P. harmntitumor , 16, antntitumor . T. ramotic herb  that pretic herb  and is wtic herb . Becausea skewer . The Tamdiseases , and woudiseases . T. ramooperties , 23. P.  of Iran , 24. Theacterial , fungal,acterial . P. harmala, T. ramosissima, and P. reptans using five extraction methods: the main CTAB method [trnL intron and trnL-F spacer in the chloroplast genome [trnL-F region is one of the most common chloroplast markers. trnL(c) and trnL(f) primers are used to amplify specific sequences of tRNA genes in the chloroplast [Against this background, the present study aims to compare the quality of the extracted total DNA (tDNA) from fresh and herbarium samples of B method , two modB method , 29, andB method  and the t genome  were amporoplast , 33.Herbs: Fresh and dried leaves of P. harmala, T. ramosissima, and P. reptans were studied. P. harmala was collected on July 17, 2018, a Tuesday, on our way from Shahrood to Azadshar . On the same day, T. ramossissima, known commonly as salt cedar, was collected on our way from Damghan to Sari above Cheshmeh Ali (Semnan province). Finally, P. reptans, commonly known as creeping baby's breath, was collected on August 3, 2018, a Friday, from field margins in Sari . Dr. Atefe Amirahmadi (Ph.D. in Plant Biosystematics) identified the plants. The specimens of P. harmala (Amirahmadi: 2048.), T. ramosissima (Amirahmadi: 2539.), and P. reptans (Amirahmadi: 2666.) were kept in the herbarium of Damghan University. The young leaves which were free of wounds and pests were isolated. Once they had been washed with distilled water, some of the leaves were frozen to -80°C, and the rest were dried at room temperature.DNA extraction: Using five methods, the total DNA was extracted from dried and fresh (stored in the freezer) leaves. The three main extraction methods included the CTAB method [B method , the modB method , 29 and Main and modified CTAB methods: In the main CTAB method [B method , 0.25 g The first modified CTAB method differed from the main CTAB method in that no BME was used. In the second modified CTAB, only 76% ethanol without ammonium acetate was used to wash the DNA pellets. The modified Murray and Thompson method: In this study, the modified method of Murray and Thompson published by Riahi et al. in 2010 [ in 2010  was usedGene All kit method: The total DNA was also extracted using the Gene All kit  as per the instructions of the kit. The quantity and purity of the extracted tDNA were determined using a UV-Vis spectrophotometer  at 230, 260, 280, and 320 nm. The absorbance ratios of 260/280 and 260/230 were calculated for the evaluation of DNA purity and the detection of protein and non-protein contamination, respectively ,35. The trnL-F region in the chloroplast genome. Of the different trnL-F regions in the chloroplast, trnL (UAA) (c) and trnF (GAA)(f) were analyzed [The quality of the tDNA extracted from the fresh and dried leaves of the studied herbs was examined by the PCR of the ITS fragments in the nrDNA and the analyzed . Table 1analyzed . trnL-F region was the same as the main PCR program used for the nuclear region, except for the annealing temperature, which was 57°C for the chloroplast sequences. For amplification of the target sequence, 500 ng of DNA was used per 20 µl reaction volume containing 10 µl of 2X Ampliqon Master Mix 2SO4, 3.0 µM MgCl2, 0.4 µM dNTPs, 0.05 units µL-1 Amplicon Taq DNA polymerase, inert red dye, and a stabilizer) and 10 picomoles of each forward and reverse primer. Five μL of the PCR products were electrophoresed on a 1.5% agarose gel. The size of the PCR products on the agarose gel was determined using a DNA molecular weight marker.P. harmala by the five extraction methods was significant in terms of the concentration of the tDNA and the A260/A230 ratio. For T. ramosissima, a significant difference between the extraction methods was found only in terms of the A260/A230 ratio for the dried leaves. In comparison, only the tDNA extracted from the dried leaves of P. reptans by the methods studied showed significant differences in terms of both the concentration of the tDNA and the A260/A230 ratio. For a more detailed investigation, the paired comparisons were made with the Mann-Whitney test which has been shown in There was no significant difference between the tDNAs extracted from both fresh and dried leaves of the studied herbs using each of the five extraction methods . When thP. harmala, the highest and lowest concentrations of the tDNA extracted from the fresh leaves were obtained using the modified Murray and Thompson method and the Gene All kit, respectively. In comparison, the first and second modified CTAB methods gave the highest and lowest concentrations of the tDNA extracted from dried leaves, respectively. As for the A260/A230 ratio of the tDNA extracted from the fresh leaves, the second modified CTAB and the main CTAB methods resulted in the lowest and highest non-protein contaminants. However, for the dried leaves of P. harmala, the modified Murray and Thompson method and the main CTAB method ranked first and last, respectively. In the next step, the results of the five extraction methods that showed a significant difference were compared to determine the best method for DNA extraction from fresh or dried leaves of each herb. As shown in T. ramosissima, the second modified CTAB method and the Gene All kit resulted in the highest and lowest A260/A230 ratio  and lowest (0.48±0.070) ratios with the second modified CTAB method and the Gene All kit, respectively. For the fresh leaves, the highest (1.21±0.300) and lowest (0.46±0.035) ratios of A260/A230 were obtained with the second modified CTAB method and the Gene All kit, respectively.The only parameter that changed significantly for T. ramosissima did not change significantly across the extraction methods  and dried (1.25±0.170) leaves, respectively. The ratio of A260/A280 of the DNA extracted from  methods . The higP. reptans, the five extraction methods differed significantly in the concentration and A260/A230 ratio of the DNA extracted from the dried leaves (both p=0.034), but not for the fresh leaves  from the tDNA extracted by all extraction methods studied. trnL-F region in the chloroplast genome of P. harmala, T. ramosissima, and P. reptans, respectively. As expected, the PCR products were amplified at 700 bp with the tDNA extracted by all extraction methods examined. However, the PCR of the trnL-F region gave no results using the tDNA extracted by the main CTAB method, the first CTAB method, and the modified Murray and Thompson method from fresh or dried leaves of T. ramosissima when the main PCR programs  Poir. In this method, which resulted in intact DNA successfully amplified in PCR, the concentrations of CTAB, NaCl, polyvinylpyrrolidone (PVP), and BME were increased, phenol was used to eliminate proteins, and the incubation time was decreased at lower temperatures [BME is a reducing agent that can remove tannins and polyphenols and denatures proteins by reducing disulfide bonds . In manyeratures .P. harmala, such as Zygophyllum coccineum L., Zygophyllum album L.F., and Zygophyllum aegyptium A. Hosny using a modified CTAB method similar to the main CTAB method used in this study. They successfully used the extracted DNA for random amplified polymorphic DNA (RAPD).Hammad and Qari  extracteT. ramosissima and P. reptans. Not all research teams, though, have access to centrifuges or ultracentrifuges necessary for the isolation of intact chloroplasts and direct extraction of cpDNA from them.The chloroplast genome (cpDNA) can provide a wealth of information on plant phylogeny, molecular ecology, population genetics, and evolution. The extraction of cpDNA and the nuclear genome usually reduces the quality of the cpDNA -48. TherIn the present study, plant samples were homogenized without liquid nitrogen. Despite its great influence on the quality of DNA extraction, researchers performing DNA extraction often try to exclude liquid nitrogen from their protocols to save money, as tight research budgets do not allow such extravagances. Extreme care must also be taken when using this substance in a laboratory. It is highly toxic and poses a hazard to the user -51. Ali P. harmala [T. ramosissima [P. reptans [T. ramosissima and P. reptans was not determined. The second shortcoming is that only the extraction protocols were performed in three replicates. Therefore, it was impossible to analyze the results' normality and apply parametric methods. Despite all these shortcomings, this study is important for two reasons: 1) we statistically compared the results from five different extraction methods, and 2) the results apply to laboratories that have the same limitations as we do.This study is one of the few studies that have been statistically analyzed. However, it has several major shortcomings due to a lack of equipment and adequate research funding. The most important deficiency was the lack of detailed phytochemical analyses. We did not examine phytochemicals in any of the fresh or dried leaves. However, in other similar studies on these herbs, researchers have found useful information on  harmala , 52, T. osissima , 21, and reptans , 53. It Damghan University for the financial support of this study.The authors would like to thank The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.