Research on the development of the dorsal neural tube is particularly challenging. In this highly dynamic domain, a temporal transition occurs between early neural crest progenitors that undergo an epithelial-to-mesenchymal transition and exit the neural primordium, and the subsequent roof plate, a resident epithelial group of cells that constitutes the dorsal midline of the central nervous system. Among other functions, the roof plate behaves as an organizing center for the generation of dorsal interneurons. Despite extensive knowledge of the formation, emigration and migration of neural crest progenitors, little is known about the mechanisms leading to the end of neural crest production and the transition into a roof plate stage. Are these two mutually dependent or autonomously regulated processes? Is the generation of roof plate and dorsal interneurons induced by neural tube-derived factors throughout both crest and roof plate stages, respectively, or are there differences in signaling properties and responsiveness as a function of time? In this review, we discuss distinctive characteristics of each population and possible mechanisms leading to the shift between the above cell types. Pattern formation during embryonic development relies on precursor cells adopting one of several alternative fates. These decisions are determined by a combination of extrinsic signals, such as morphogen gradients, intercellular interactions mediated by Notch-Delta activities, and cell-intrinsic factors that respond to the precedent signals and comprise a downstream transcriptional regulatory network. Together, these are believed to specify particular cell identities ,2,3.The generation of distinct cell identities along the dorsoventral axis of the neural tube (NT) is an excellent model for investigating cell decisions in time and space. It involves the integration of opposing concentration gradients of Sonic hedgehog ventrally and of Bone Morphogenetic Protein (BMP) and Wnt dorsally ,5,6,7. IHence, a major challenge is that the above transitions are intrinsically dynamic, an outcome of time and stimulation. At first, the dorsal domain of the NT is transiently populated by premigratory NC cells. These exit the NT to form a rich collection of cell types, such as sensory and autonomic neurons, satellite cells and Schwann cells of the peripheral nervous system as well as pigment cells, ectomesenchyme and endocrine derivatives whose combination varies along the neuraxis ,14. SubsThe sequential generation of the above cell types raises fundamental questions on the dynamics of dorsal NT behavior. When does fate segregation occur; prior to cell emigration due to cell-cell interactions or to a graded response to morphogens, and/or during migration when multipotent NC cells may be instructed by environmental cues to form specific derivatives? Evidence points to a heterogeneous nature of the NC at the population level, being a mixture of both fate restricted and multipotent progenitors at the various stages. As such, the measured proportion of cells with different degrees of commitment is likely to vary as a function of the methods used to follow cell lineages, on animal species, and on specific axial levels considered at progressive stages. Along this line, analysis at a given axial level over time showed a stereotypic pattern of migration of NC progenitors followed by ordered target colonization ,18. In aDespite this still active controversy, significant progress in other aspects of NC ontogeny, such a cell emigration, migration and differentiation, has been achieved over the years ,26,27,28The dorsal region of the NT that contains premigratory NC cells is a pseudostratified epithelium in which progenitor cells undergo interkinetic nuclear migration and exhibit typical patterns of cell proliferation ,30. ThisDiscrete labeling of the dorsal NT in several species revealed that, following EMT, NC cells migrate in a stereotypic manner and colonize their peripheral targets in a general ventral to dorsal sequence ,36,37,38FoxD3, Sox9 and Snai2, whereas late progenitors do not [Foxd3 reporter, confirmed that neural progenitors are Foxd3-positive but prospective melanoblasts downregulate Foxd3 and segregate from neural lineages already before emigration. Moreover, when the normal downregulation of Foxd3 is prevented by gene misexpression at a late stage corresponding to the end of neural lineage production, the late-emigrating precursors failed to upregulate the melanogenic markers Mitf and MC-1 and the guidance receptor Ednrb2, generating instead glial cells that expressed P0 and Fabp. In a reciprocal experiment, loss of Foxd3 function in mouse NC resulted in ectopic melanogenesis in the dorsal tube, in sensory ganglia and along ventral roots [Mitf and melanogenesis, albeit in this study Foxd3 was misexpressed at very early stages, thus comprising both neural as well as melanogenic precursors, rather than attaining exclusively the prospective pigment cell subset [Foxd3, Snai2 and Sox9 are also lost from the dorsal NT prior to melanoblast emigration, it is likely that the latter two, along with Foxd3, form part of a network that influences neural vs. melanocyte development. The differential expression of these three genes to prospective neural lineages but not to melanoblasts, highlights a temporal sequence of molecular differences between the above fates apparent already at the premigratory stage.The dynamic yet precise cellular behavior described above would indicate that the dorsal NT is molecularly heterogeneous, either at the spatial and/or temporal levels. Several lines of evidence lend support to this notion. Early premigratory NC progenitors have been shown to express s do not . Furtheral roots ,43. Thesl subset . Since, Oct4, which acts in this context, on specification and survival of the ectomesenchyme. Along this line, no re-expression of Oct4 was detected along more caudal regions of the axis corresponding to trunk levels, suggesting a different mechanistic scenario [Additional evidence for molecular heterogeneity in the dorsal NT stems from an unbiased hierarchical clustering of 35 genes that was performed at a cranial level of the avian axis. This analysis revealed five distinguishable clusters that mapped to different domains of the dorsal neural primordium. For example, a central portion expressed a combination of NC, pluripotency and differentiation markers of NC, whereas a more lateral subdomain exhibited “NC only” genes, together highlighting spatial differences within the premigratory region of the NT . In addiscenario .EdnrB enhancer, a comprehensive temporal map of the chromatin and transcriptional landscape of vagal-level NC cells revealed the existence of three clusters: neural, neurogenic and mesenchymal, each predetermined epigenetically prior to NC delamination [Similarly, using an mination . Recentlmination . In micemination . Availabmination  will enaIs there any relationship between the observed molecular heterogeneity of premigratory NC progenitors described above, the precise localization of a cell in the dorsal NT and the acquisition of distinct fates by the migrating cells? One extreme possibility is that there is no relationship between the localization of presumptive progenitors in the dorsal tube, the sequence of their emigration and their final fates. McKinney et al. suggested this possibility for most trunk derivatives except for sympathetic progenitors . A seconSnail genes in this domain [One of the hallmarks of NC development is the ability of premigratory precursors to exit the neuroepithelium via a process of epithelial-to-mesenchymal transition (EMT) followed by cell delamination and generation of cellular motility . Notablys domain . In the s domain  and refsIn contrast, no synchronization to the S-phase seems to be required for emigration of cranial NC cells , perhapsSubstantial evidence supports the notion that the process of EMT invokes the concerted action of signaling proteins with a network of transcription factors, affecting downstream cytoskeletal and cell adhesion properties ,57, as wWnt1 transcription that in turn promotes G1/S transition, a necessary step for delamination of trunk NC [A balance between BMP and its inhibitor noggin, in association with the developing somites, was found to underlie the emigration of trunk-level NC ,59. BMP trunk NC . N-cadhetrunk NC ,61,62. Strunk NC . A role trunk NC .YAP is expressed and is active in premigratory NC of avian embryos. Gain of YAP function stimulates NC EMT, and attenuation of YAP inhibits cell exit. This is associated with reduced G1/S transition and enhanced apoptosis. Using specific in vivo reporters, loss of YAP function in the dorsal NT was found to inhibit BMP and Wnt activities whereas gain of YAP function stimulates these pathways. Reciprocally, inhibition of BMP or Wnt signaling downregulates YAP activity. In addition, YAP-dependent stimulation of NC emigration was compromised upon inhibition of either BMP or Wnt activities. These data established for the first time a positive bidirectional crosstalk between these pathways and incorporated YAP signaling into a BMP/Wnt-dependent molecular network responsible for emigration of trunk-level NC [Another important player in this genetic network is Yes-associated-protein (YAP), an effector of the Hippo pathway, that controls various aspects of development including cell proliferation, migration, survival and differentiation ,66. YAP level NC . YAP siglevel NC . Open quSnai2, one of the earliest described genes in developmental EMT and metastasis [Snai2 does not appear to affect EMT of trunk NC [Snai2 plays a pivotal role in regulating EMT of NC precursors in the head, where Cad6B acts as a direct target of Snai2 repression [Snai2, forming a complex at the Cad6b promoter [Snai2/Slug was shown to cooperate with the Polycomb repressive complex 2 (PRC2) to regulate various aspects of NC development including specification and EMT/migration [Hmga1 was found to act both on NC specification at the neural plate border, and at a later stage, on NC emigration via canonical Wnt signaling [Snai2 [Transcription factors, regulated by the above signaling proteins, are an essential component of the molecular network leading to NC EMT and delamination ,33,69. Atastasis ,71. Notatrunk NC ,73, neittrunk NC . Howeverpression . Furtherpromoter . In Xenoigration . Recentlignaling . Along tg [Snai2 . The prec-Myb appears to regulate both the formation as well as EMT of NC cells downstream of BMP [Sox9, Snai2, and Foxd3, but not each factor separately, effectively induced ectopic EMT on the dorsoventral extent of electroporated NTs, along with other traits of NC cells [Sip1/Zeb2-defective mouse embryos revealed persistent E-cadherin expression in NC precursors that accumulate in the NT, indicating defects in EMT [Zeb1 and Zeb2 were found to overlap. By interfering with their expression using shRNAs, the authors showed that both factors share an equivalent stimulatory function on NC EMT [Less is known about transcription factors with effects on NC EMT at thoracic levels, and few examples will be provided here. m of BMP . FurtherNC cells , but losNC cells ,74,81. ZNC cells . Sip1/Zes in EMT . Involves in EMT . In avian NC EMT . Future n NC EMT .HES in a non-cyclic, persistent manner and do not undergo neurogenesis [The dorsoventral organization of the vertebrate central nervous system (CNS) is coordinated by two groups of cells known as organizers, the RP and the floor plate (FP). As such, both cell subsets are composed of post-mitotic cells with the nascent FP exiting the cell cycle earlier than RP ; furtherogenesis ,89. ThusMafB, Msx, Lmx1a/b, etc., which are considered as RP markers, are produced in the dorsal NT from closure of the neuroepithelium onward, including the early NC period [The RP has classically been considered the dorsal domain of the vertebrate NT along the entire rostro-caudal axis, where it produces morphogens responsible for dorsal cell fates, including BMPs ,93,94,95C period ,15,98,99C period , but theC period  and see Foxd3, as evidenced by lineage analysis with a Foxd3 reporter [BMPR1A (Alk3), and cease to express Foxd3 and the direct BMP target genes Id2/3 , LRP8, Znf536, and Zic4. This recent RNAseq analysis provided a set of many spatiotemporal-specific genes appropriate for cell type identification and for functional studies [Second, our understanding of RP formation is hindered by the lack of genes uniquely transcribed in either NC or RP populations. To overcome this limitation, a transcriptome analysis was performed at the trunk level of quail embryos comparing the dorsal NT at premigratory NC and at RP stages . In addiRspo1  and HES4 studies . Among t studies .A subject worth mentioning is the differing morphology of the RP along the neuraxis . WhereasGdf7-positive RP boundary was evidenced between the rhombic lip neuroepithelium and the RP proper. This boundary was shown to signal bidirectionally to maintain on the one hand expression of atonal1 in the rhombic lip and on the other hand, to specify the early expression of RP-derived choroid plexus markers such as Transthyretin [Gdf6a-positive cell type, which was termed ‘veil cell’. Notably, veil cells contribute to RP expansion; they are able to generate squamous RP cells by direct transformation that occurs predominantly in the lower rhombic lip region and is accompanied by the downregulation of Gdf6a. Veil cells undergo both symmetric divisions that account for self-renewal and also asymmetric divisions that generate both types of progeny [Gdf-expressing lineage [Gdf7 is also expressed in two lateral bands flanking a central domain [Gdf7-positive cells function as signaling boundaries to induce adjacent interneuron progenitors and/or act on the central RP, or whether they differentiate into derivatives distinct from those of the central domain.In the hindbrain of chick embryos, a thyretin . In the  progeny . Hence,  lineage ,105,106.l domain ,102, we BMP4, Wnt1 and Lmx can be induced up to stage 12HH, yet at later stages, dorsal progenitors lose their competence to generate these cell types and instead generate dorsal interneurons via a Msx3-dependent mechanism [Little is known about the timing of RP specification and differentiation vis-a-vis the period of NC production and emigration from the NT. Evidence suggests that BMP signaling is necessary for early development of both populations ,95,107. echanism . In favoechanism .Hes/Hairy in the nascent RP, which in turn, downregulates responsiveness to BMP and reduces G1/S transition of premigratory NC, a prerequisite for cell emigration [Hes gene expression in RP, which is associated with a lack of cell proliferation in this and other boundary cell types [Hes1 dynamics and the cell-cycle was found such that in most cells, division takes place at or near the peak of Hes1 expression. This peak in Hes1 protein expression is then followed by a dip, the onset of which is followed by the G1-S transition, leading to a second period of increase in Hes1 protein concentration before the next division. When Hes1 oscillations were dampened, the cell-cycle slowed down, indicating the functional significance of Hes1 oscillations for an efficient cell-cycle progression [Hes at all, or alternatively, whether they exhibit an oscillatory behavior of this gene, characteristic of cycling progenitors, which turns into a permanent mode of expression upon RP formation.In this context, we reported that BMP is necessary for initial expression of igration ,30. Thisll types . Along tgression . An intrLmx1a [Lmx1a expression is restricted to the dorsal NT of wild-type mouse embryos encompassing both NC and RP phases of development (E8.5–E11.5). In Dreher mutants, while expression of Lmx1a begins normally and persists through E9.5, the NC stage, expression is not maintained through the mature RP. The loss of Lmx1a was accompanied by a complete failure of Bmp6 and Gdf7 expression throughout NT development, yet Msx1 disappeared only by E11.5 and Wnt1 and Wnt3a were not altered [Lmx1a is not necessary for all aspects of RP development or that additional factors or sources of Lmx-independent BMPs compensate for the loss of Lmx1a. In addition, in the hindbrain, only certain rostro-caudal regions of the RP are lost in the absence of Lmx1a [Lmx1a is not critically involved in the early NC program.Important information on RP formation stems from the Dreher mutant mouse, a spontaneous neurological mutation defective in Lmx1a . Lmx1a e altered , suggestof Lmx1a ,113, hinAnother substantial signaling system is Notch-Delta, found to mediate the maintenance of the hindbrain RP epithelium . This leOtx2 was found to be a master regulator of choroid plexus development and maintenance. Whereas conditional deletion of Otx2 under the regulation of Gdf7 affected primarily the hindbrain choroid plexus, deletion of Otx2 by the Otx2-Cre-ERT2 driver resulted in lack of all the plexi in the brain [Msx1 and Id4 and of signaling factors such as BMP6 and Gdf10. A similar regionalization was reported for the ventral part of the ependymal zone  [As previously shown, fate mapping analysis revealed that the lateral part of the RP generates the choroid plexus at the hindbrain level of the axis ,102 Fig. The trahe brain . In addihe brain ,117,118,he brain . These rhe brain ,120,121 he brain ,121. NotZic6 and Rho-associated kinase. Interestingly, Mib mutants defective in Notch signaling showed a loss of rostral RP cells [see also ref. [BMP receptor type 1B expression was evident around the central canal lining with an associated reduction in Sonic hedgehog signaling [In zebrafish, the dorso-ventral stretching of RP cells on their way to generate a radial glial scaffold, was shown to associate with the conversion of the primitive lumen into a central canal. Stretching of the RP was evidenced along the whole spinal cord with RP cells extending over 2/3 of the NT diameter. This process involves extension of the RP cytoskeleton and depends on activity of lso ref.  for mouslso ref. . In addiignaling . Hence, In this context, it is worth elaborating on a poorly studied structure, termed the glycogen body, that is thought to derive from RP cells and is present across avian species . The glyMath1 in vitro was induced by and subsequently became dependent upon BMP signaling [BMP family members have been found to mediate major functions exerted by the RP . Manipulignaling ,130.To note is that signaling by BMPs is highly complex and time dependent. BMP7 was shown to be active at a post-patterning phase, when neural progenitors exit the cell cycle and enter the terminal differentiation pathway. In both avians and mice, BMP7 is required for the generation of dI1-dI3-dI5 interneurons, an activity mediated by Smad1 and Smad5, that operate through the canonical Smad pathway .With regard to the Smad family of transcriptional BMP effectors, the presence and activity of inhibitory Smads, Smad6 and Smad7, was shown to be a possible contributing factor to limit the dorso-ventral extent of BMP action. Both Smad 6 and 7 are expressed in the NT and restrict the action of BMP signaling to its dorsal domain. For instance, ectopic misexpression of Smad7 suppressed dI1 and dI3 neural fates and concomitantly increased the number of dI4–dI6 spinal neurons. In contrast, Smad6 mostly blocked dI1 axon outgrowth. Taken together, these experiments suggest that inhibitory Smads have distinct roles in spatially limiting the response of cells to BMP signaling .Wnt1 and Wnt3a caused a reduced number of dI1 and dI2 interneurons and a compensatory increase in dI3. This was mimicked by treatment of isolated neural plates with Wnt3a in a BMP-independent manner [Wnt factors are also produced in the dorsal NT at both NC and RP stages. Absence of both t manner , highligGdf7, an RP-specific gene. RP ablation had no effect on initial patterning of the dorsal NT resulting in normal NC development. Selective loss of the RP prevented the formation of both dI1 (Math1) and dI2 (Ngn1) interneurons and the dorsal midline was occupied instead by dI3 cells expressing Mash1 [A more direct and time-controlled way to address the functions of the RP as a structure was its ablation by targeting diphtheria toxin under the regulation of ng Mash1 . suggestLmx1a mutants (Dreher mice), however, Math1+ dI1 interneurons were generated in reduced numbers and no effect on dI2 was monitored [Math1-expressing neural progenitor cells, which comprise the rhombic lip, were lost [Gdf7, BMP6, Msx1 and Wnt1 in the Gdf7 mutants compared with the Dreher mice in which residual expression of Wnt1 and Msx1/2, factors shown to operate on interneuron development, persisted.In onitored . At the ere lost . This diIn contrast to the spinal cord, less is known about functions of the RP at telencephalic levels. Using the Gdf7-diphteria toxin-mediated ablation paradigm, RP ablation resulted in a failure of midline induction and holoprocencephaly in the dorsal telencephalon. This was accompanied by a reduced activity gradient of BMPs. In dissociated cells and mutant explants, exogenous Bmp4 was sufficient to rescue RP-dependent midline patterning. Hence, the telencephalic RP is required for normal dorsal cortical patterning at least partially through BMP signaling .Mib under the regulation of Wnt-Cre, also caused a specific loss of the RP with no apparent effect on the NC. In its absence, dI1 interneurons did not develop but dI2 were expanded up to the dorsal midline [Loss of Notch signaling in the dorsal NT, achieved by mutating the ubiquitin ligase  midline . Differe midline ,131,136, midline . In thisIn addition to its role in dorsal interneuron development, the RP was shown to act as a barrier to axon growth. An early study reported that keratan sulfate is specifically expressed in the rat definitive RP and hypothesized that this and other glycosaminoglycans might inhibit bilateral crossing of incoming sensory or of commissural nerve fibers . BMP7 anDuring neural development, NC cells that generate the peripheral nervous system, and definitive RP cells of the central nervous system, are sequentially formed in the same anlagen. Thus, it is the dorsal domain of the NT where the major decision of becoming peripheral or central nervous system takes place. How the dorsal neural primordium transits between these phases remains largely unknown. Evidence suggests that these peripheral and central neural branches segregate from a common progenitor in the dorsal NT already prior to the completion of NC emigration ,19. TranOur knowledge of the fate and functions of the RP is growing. Along this line, investigating the heterogeneity of RP properties at various levels of the neuraxis and its functional significance are also a subject of considerable interest. The development of the dorsal NT embodies basic processes in development: the regulation of cell proliferation, cellular movements and cell elongation, epithelial-mesenchymal transitions, lineage decisions, and relationships between them. Aberrant signaling during critical phases leads to disease, ranging from defects in NT closure ,143,144,


This study revealed that soil samples from Mamuju are moderately contaminated. There was a strong positive relationship between 226Ra, 232Th, ambient dose equivalent rate, and Pb. Ecological risk index (RI) and cumulative pollution index (IPI) values in Mamuju are 2.05 and 125, respectively, which are possible hazards to human health as a result. Pb concentration in the Mamuju soil samples ranged from 109 to 744 mg kg−1, exceeding the worldwide average of 27 mg kg−1.Mamuju, Indonesia, is an area with high natural background radiation. This study assesses heavy metal content in soil samples from this area to determine the level of public and environmental hazard it presents. This study analyzes natural radionuclide elements using high purity germanium (HPGe) gamma spectrometry and performs heavy metals analysis using a flame atomic absorption spectrometry (FAAS). Moreover, pollution indices and descriptive analyses were used to assess heavy metal contamination in the environment and the correlation between heavy metals and radionuclides. The results demonstrate that soil samples in several areas of Mamuju contain a high concentration of the natural radionuclides  Heavy metals are widely distributed in the environment and are often associated with pollution, contamination, and toxicity due to non-bio-degradable and persistence in nature ,2. In thRecently, heavy metals in the environment have increased beyond acceptable limits through development activities, including industry and agriculture. These heavy metals can be toxic and accumulate in the soft tissues of animals, plants, and even humans when they enter the body through food, water, air, or skin. Soil is often a repository of heavy metals because soil particles, such as clay and humus, have a charge that helps metal cations bind to the soil, thereby preventing their release, even temporarily . Heavy mPb is a harmful environmental pollutant with high toxic effects on many body organs. Pb is absorbed from the respiratory and digestive systems. Due to immune modulation, oxidative, and inflammatory mechanisms, Pb exposure can induce neurological, respiratory, urinary, and cardiovascular disorders. Cr is found in the Earth’s crust and industrial processes. The main route of Cr exposure is via ingestion and can cause various diseases, such as renal, neurological, and several cancers, including lungs, bladder, kidneys, thyroid, testicles, and bone. Cd, although rare, occurs naturally in soil. A high concentration of Cd in the soil can occur following industrial activities, with the main exposure route being ingestion. Health effects due to this heavy metal are degenerative bone diseases, kidney failure, and lungs diseases. Zn and Ni are essential heavy metals in the human body. However, exposure to extreme elements can be harmful to respiratory diseases. Cu, as well as Cr, is found in the Earth’s crust. The health effect of this heavy metal concerns abdominal disorders and metabolic activity abnormalities ,6,7,8.Several studies have also discussed heavy metal pollution in areas with high levels of natural radionuclide elements in the soil, such as areas around uranium mines with high background radiation ,10,11. A−1 with a range of 200 to 2300 nSv h−1 was found [Mamuju, Indonesia has a relatively high average radiation dose rate due to its high natural background radiation level ,14,15,16as found .238U from 539 to 128,699 Bq kg−1 and 232Th from 471 to 288,639 Bq kg−1 [A previous study has found that Mamuju has a unique geological condition, with 15 types of rock with different mineral contents, 11 types of class C minerals, 6 types of Class B minerals, and 5 types of class A minerals found in this area . Volcani Bq kg−1 . Accordi Bq kg−1 . Moreove Bq kg−1 .This study assesses heavy metal levels in soil samples taken in Mamuju in order to determine the possibility of environmental and public hazards resulting from heavy metals in this area. Heavy metal pollution in the soil can have harmful effects on groundwater, agricultural production, food safety, and human health because the soil is the most important ecosystem for human survival and development. Most people living in Mamuju are farmers, and their daily life in the area is, therefore, heavily dependent on nature. Most residents mainly consume locally grown food from traditional markets. Therefore, the determination of the heavy metal content in the local soil is fundamental to the identification, monitoring, and assessment of potential sources of pollution in the area ,9.This paper describes the first approach to assess heavy metal contamination in Mamuju. Previous studies on Mamuju examined specific areas rather than the region as a whole, and had limited scope, focusing purely on natural radioactivity, radiation dose assessment, and radioactive mineral exploration ,15,19,21Mamuju is located on the western edge of Sulawesi Island, between Mamuju Bay to the north and Labani Bay to the south. The topography of the Mamuju area consists of a western coastal area and an eastern mountainous area ,22. The Soil samples were obtained from the sub-districts of Mamaju city , Tapalang sub-district , and Northern Botteng Village. Soil samples were collected from 18 different locations in Mamuju. Each sample consisted of 1 kg of soil extracted at a depth of 5–20 cm from the soil surface using a trowel. Then, soil samples were stored in polymer bags. In the laboratory, the extraneous plant material, rocks, gravel, and roots were removed from the soil samples, then dried in an oven at 105 °C for 24 h to remove the moisture content. The soil samples were then crushed by hand, sieved using a 50–60 mesh sieve, and separated into two parts for natural radionuclide element analysis and heavy metal analysis.3–HCl) of 1 g of the soil sample for 2 h at 90 °C. Afterward, the sample was diluted to 100 mL with de-ionized water, left for 3 h, and then filtered [−1, respectively.The samples after preparation were heated in a furnace at 400 °C for 2 h to remove organic matters. An aqua-regia extraction method was then used to determine the total metal content of each sample. This method consists of the partial digestion  is used to calculate the radioactivity concentration from these measurements:E is counting efficiency, Y is energy yield, N is net count per second, W is sample weight (kg), and cF is the correction factor  [A high purity germanium (HPGe) detector gamma spectrometer  was used to measure the radioactivity of the sample. Each approximately 1 kg sample was weighed and sealed in a standard Marinelli container, and placed for approximately 30 days before further gamma spectrometer measurement to allow the decay products to reach secular equilibrium. The counting time was approximately 80,000 s. The peak energy values of 351 and 609 keV were used to calculate  factor) ,19,25.iE) are aimed to assess the level of contamination in soil based on the toxicity of heavy metals and environmental response.This study used three assessment tools to determine the level of contamination of a research area. Pollution indices also involve some heavy metal measurement and geological background data of an area. Geochemical index (I-Geo) aims to determine metal contamination in soil by comparing current concentrations with preindustrial levels. The pollution index (PI) aims to determine metal contamination in soil by comparing current concentration with the regional background. Moreover, ecological risk (RI) and the potential ecological risk of each heavy metal  is a quantitative indicator to measure the degree of soil pollution by heavy metals . I-Geo icontents , and 1.5contents ,30.Cn is the heavy metal content, Bn is the regional background heavy metal content, and PI is the pollution index. The baseline data of regional background defined by Reimann and Caritat [The pollution index (PI) of each metal is the ratio of the heavy metal content in the sample to the regional background and calculated according to the following equation: Caritat  are Pb = Caritat ,30.iE) and was used to evaluate the potential ecological risk factor of soil pollution by assessing the heavy metals [iT is the toxic response factor for heavy metal, and if is the ratio of the measured concentration (iC) to the background concentration (iB) of metal in the soil. The iT values for the heavy metals studied were 5 for Pb and Cu, 1 for Zn, and 30 for Cd. The potential ecological risk of each heavy metal (iE) was classified into five categories as either low (iE < 40), fairly (40 ≤ iE < 80), considerable (80 ≤ iE < 160), high (160 ≤ iE < 320), or very high (iE  > 320). Tang et al. (2013), categorized the RI into four classes: low (RI < 150), moderate (150 < RI ≤ 300), high (300 < RI ≤ 600), and very high (RI > 600) [The ecological risk index (RI) consisted of the sum of individual heavy metals . The equation below expresses the Pearson correlation coefficients:n is the sample size, ix and iy are the individual sample points indexed, and We used MAPINFO PROFESSIONAL  to create maps, and ORIGIN PRO 2021  to perform bivariate analysis in order to find significant relationships between the distribution coefficients of radionuclides and heavy metals ,33. We c226Ra, 232Th, and 40K in surface soil samples from Mamuju is presented in 226Ra varied from 232 to 2761 Bq kg−1, with an average of 784 Bq kg−1. The activity concentration of 232Th varied from 424 to 3310 Bq kg−1, with an average of 1008 Bq kg−1. The activity concentration of 40K ranged from 203 to 1655 Bq kg−1, with an average 770 Bq kg−1.The activity concentration of −1 (Cu), 11 to 293 mg kg−1 (Cr), 0.40 to 1.40 mg kg−1 (Cd), 109 to 744 mg kg−1 (Pb), 4 to 69 mg kg−1 (Ni), and 175 to 392 mg kg−1 (Zn).The concentrations of heavy metal in soil samples from the Mamuju area presented in The I-Geo, EI, RI, PI, and IPI pollution indices were used to identify heavy metal concentrations of environmental concern in the Mamuju soil samples. These indices numerically describe pollution levels in soils and can be used to calculate the exchangeable soil fraction by representing the real bio-available fraction. The pollution indices of the Mamuju samples are shown in The I-Geo values found for Cu, Cr, Cd, Ni, and Zn show that the samples are within the uncontaminated to fairly contaminated category with an I-geo value of <1, except for the Pb value which has an I-Geo average of 4.11 and a range of 1.48–10. The average I-Geo score for Pb in the Mamuju soil samples is within the heavily contaminated to extraordinary contaminated category (4 < I-Geo ≤ 5).The PI values, i.e., the ratio of heavy metal content in the samples to the regional background heavy metal content were <1 for Cu, Cr, and Ni, and were therefore within the low contamination category. The PI values of Cd, Pb, and Zn were >3, and were therefore in the high contamination category. The IPI value for the Mamuju soil samples was 2.05 with a range from 1.16 to 3.06, and was therefore within the moderate contamination category.iE < 160). The RI value is used to evaluate the combined ecological risk of heavy metal soil pollution. The RI values of the samples ranged from 53 to 277, with an average of 125, and were therefore mainly within the low contamination category with some areas included in the moderate contamination category.The EI value is used to evaluate the potential ecological risk of heavy metal soil pollution. EI values for Cu, Cr, Cd, Ni, and Zn in the samples were all <40, and were therefore within the low contamination category. The average EI value for Pb was 102 with a range of 37–251, which is within the medium contamination category  were 1387 Bq kg−1, 1468 Bq kg−1, and 301 Bq kg−1, respectively [−1 for 226Ra, 1756 Bq kg−1 for 232Th, and Bq kg−1 for 40K [In general, Mamuju soil samples were indicated to have a level of environmental radioactivity that is above the worldwide average. Worldwide average activity concentrations of ectively . The stu for 40K .226Ra is a decay product of uranium with a half-life of 1600 years. 226Ra contained in the soil can potentially accumulate into plants or animals which are consumed by people. Geological conditions strongly influence natural radionuclide activities, and rocks of the alkali basalt group  found in the Mamuju area (in sub-districts Botteng and Takandeang) are radioactive. Extrusive mafic stone basalt is the most extensive of all frozen rocks, and comprises more than 90% of all volcanic rocks [−1. A similar value was reported by Nugraha et al. (2021) with a geometric mean of 613 nSv h−1 and a range of 200 to 2300 nSv h−1, and Shilfa et al. (2021) reported an ambient dose equivalent rate in Northern Botteng which ranged from 420 to 1430 nSv h−1 [Uranium and thorium are terrestrial naturally occurring radioactive materials (NORMs) which form in the Earth’s crust. ic rocks . Moreove nSv h−1 .−1, exceeding the worldwide average of 27  mg kg−1 [−1, 100 mg kg−1, and 200 mg kg−1, respectively [The heavy metal concentrations in the soil decrease in the sequence Zn > Pb > Cr > Cu > Ni > Cd. The Pb concentration in the Mamuju soil samples ranged from 109 to 744  mg kg mg kg−1 . Moreoveectively ,38,39.iE <  160) and the combined ecological risk of heavy metal soil pollution (RI) values of the samples ranged from 53 to 277, with an average of 125.Similar results with heavy metal assessment using pollution indices indicate a possibility that humans may be exposed to these pollutants. The lead value has an I-Geo average of 4.11 and a range of 1.48–10. The average I-Geo value for Pb in the Mamuju soil samples is within the heavily contaminated to extremely contaminated category (4 < I-Geo ≤ 5). The highest values are in Botteng (I-Geo = 6), North Botteng (I-Geo = 7), and Takandeang (I-Geo = 10) areas. The PI values of Cd, Pb, and Zn were >3, and were therefore in the high contamination category. Although the PI values for Cd and Zn are high, these concentrations are still below the limits set by the Indonesian Ministry of Environment. The IPI value for the Mamuju soil samples of 2.05 was, therefore, within the medium contamination category. Moreover, the EI values for Cu, Cr, Cd, Ni, and Zn in the samples were within the low contamination category. However, the average EI value for Pb was 102 with a range of 37–251, which is within the medium contamination category , Cu, and Ni. Severodvinsk city is the largest industrial center for the navy in north-western Russia with concentrations of As (the average concentration 12.6 mg kg−1), Cd, and Pb, which are higher than others. Bayanwula and Tongliao cities in China are uranium mining areas. The Tongliao area has very high Cd, 7–8 times the average soil concentration in China, while the Bayanwula area has a higher concentration of Pb than other heavy metals. In Kerala, India, which is also a high natural background radiation area, the concentration of Pb was found to be higher than in other elements. Several studies of heavy metals in soils in several regions of the world include Toplica (Serbia), Severodvinsk (Russia), Bayanwula (China), Tong Liao (China), and Kerala (India). ,42,43,44p ≤ 0.001) between 226Ra, 232Th, ambient dose equivalent rate, Pb, and Cu with correlation coefficients of 0.92 (226Ra and 232Th), 0.90 (226Ra and Pb), 0.83 (232Th and Pb), 0.98 , 0.91 , and 0.91 (Cu and Pb). The strong correlation between 226Ra, 232Th, and Pb is due to the decay processes of the two radionuclides into Pb isotopes/elements, such as 214Pb, 212Pb, 210Pb, 208Pb, and 206Pb. The Pb isotope is a decay chain product of uranium (the parent of 226Ra) and thorium. 214Pb is a radioactive element and has a half-life of 26.8 min, 210Pb has a half-life of 22.3 years, and 206Pb is the stable isotope (the end product of the uranium decay chain). As for thorium decay, 212Pb is a radioactive element with a half-life of 10.6 h, and 208Pb is stable, which is the end product of the thorium decay chain [214Pb will decay to 214Bi, 212Pb will decay to 212Bi, and 210Pb will decay to 210Bi. Beta radiation will be hazardous if it enters the human body, either from breathing or digestion [The correlations between heavy metals and natural radionuclides in soil samples were performed using the ORIGIN PRO 2021 . The statistical significance of bivariate analysis is useful to find significant relationships between the distribution coefficients of radionuclides and heavy metals. The correlation coefficient results between radionuclides, ambient dose equivalent rates, and heavy metals in the soil samples are presented in ay chain ,34,45,4640K and the ambient dose equivalent rate was a negative correlation. This negative correlation occurs because the 226Ra and 232Th contents are high, so the K concentration is not significant.Meanwhile, moderate and even negative correlation coefficients were found between radionuclides and some heavy metals . The relationship between Lead occurs naturally in the environment. However, anthropogenic activities, such as mining and burning fossil fuels, including transportation, contribute to the release of high concentrations of Pb to the environment. However, there are no mining and industrial activities in Mamuju. The higher correlation coefficient for these elements is due to the fact that the main source of these metals in this area might be due to the parent rock or soil  and burning of fossil fuels. Regarding the geogenic sources, the previous study reported rock types in the Mamuju area are classified into alkaline basalt groups, such as gummite (>70% Th), davidite (15% Fe), thorianite (12% Pb), and autunite (>48% U) .226Ra, 232Th, and Pb. The strong correlation between 226Ra, 232Th, and Pb is due to the decay processes of the two radionuclides into Pb isotopes/elements, such as 214Pb, 212Pb, 210Pb, 208Pb, and 206Pb. The Pb isotope is a decay chain product of uranium (the parent of 226Ra) and thorium. The ecological risk index (RI) and cumulative pollution index (IPI) values in Mamuju are 2.05 and 125, respectively, and are therefore within the moderately contaminated category. Lead concentrations in the Mamuju soil samples exceeded the thresholds for heavy metals in soil set by the World Health Organization (WHO), the Indonesian Ministry of Environment, and the United States of America Environmental Protection Agency (US EPA). Moreover, Pb concentration in the Mamuju soil samples ranged from 109 to 744 mg kg−1, exceeding the worldwide average of 27 mg kg−1. It is possible that these factors might cause negative health effects among the residents of the area.Mamuju is a unique area because it is highly naturally radioactive and has a high concentration of heavy metals. The heavy metal concentrations in the soil decrease in the sequence Zn > Pb > Cr > Cu > Ni > Cd. A strong positive correlation was found between The determination of the heavy metal in a soil sample is a fundamental study to the identification, monitoring, and assessment of potential sources of pollution in the area. This study is expected to assist local governments, central government, and various stakeholders in making policies regarding environmental safety and population health. This study did not conduct heavy metal measurements in foodstuffs and drinking water. Therefore, we suggest that it is necessary to carry out continuous research, especially the measurement of heavy metals in food, water, and humans (bioassays), to obtain more comprehensive data. With these facts, Mamuju can be a prospective area for epidemiological studies to be carried out for both low-dose-rate radiation and heavy metal radiation.


Parasitic diseases represent a common issue in fish and, when histozoic forms are present, this elicits a chronic inflammatory reaction leading to granuloma formation. Despite the large knowledge of granuloma formation due to parasites in visceral organs, little is known about the development and the evolutive stages of granulomas in naturally infected fish. Mullets (Osteichthyes: Mugilidae) are a widespread euryhaline fish species that harbor different parasites, thus representing a suitable model for the study of parasite-induced granulomas. Combining histopathology and immunohistochemical tools, we identified three developmental granuloma stages , that ranged from an intact parasite with mild signs of tissue reaction to the formation of a structured granuloma. The identified histological patterns could be reliable tools in the staging of the granulomatous response associated with histozoic parasites and are an attempt to broaden the knowledge of the inflammatory response in different host–parasite systems.Histozoic parasite–fish host interaction is a dynamic process that leads to the formation of a granuloma, a specific chronic inflammatory response with discernible histological features. Mullets (Osteichthyes: Mugilidae) represent a suitable model concerning the development of such lesions in the host–parasite interface. The present work aimed to identify granuloma developmental stages from the early to the late phase of the infection and to characterize the immune cells and non-inflammatory components of the granuloma in different stages. For this purpose, 239 mullets were collected from 4 Sardinian lagoons, and several organs were examined by combining histopathological, bacteriological, and immunohistochemical methods. Granulomas associated with trematode metacercariae and myxozoan parasites were classified into three developmental stages: (1) pre-granuloma stage, characterized by intact encysted parasite and with no or mild tissue reaction; (2) intermediate stage, with partially degenerated parasites, necrosis, and a moderate number of epithelioid cells (ECs); and (3) late stage, with a necrotic core and no detectable parasite with a high number of ECs and fibroblasts. The three-tier staging and the proposed morphological diagnosis make it conceivable that histopathology could be an essential tool to evaluate the granulomas associated with histozoic parasitic infection in fish. The chronic inflammatory response is a non-specific reaction against a multitude of factors, such as harmful substances, bacteria, mycobacteria, fungi, and parasites, particularly common in fish ,12,13,14In natural and experimental infection due to bacterial and particularly mycobacterial and oomycetes species, granulomas have been described as a core of macrophages and bacteria associated with central necrosis, surrounded by a fibrous layer mostly evident in the later stages of infective granulomas ,20,21. UIt is well known that also some histozoic parasites  can elicit chronic granulomatous inflammation .More details of the cellular components of granuloma and their distribution have been provided by electron microscopy studies, where ECs have been typically found near or in contact with the parasite body or cyst wall . AlthougRodlet cells (RCs), although not a regular component in parasite granulomas, increase in the course of infectious diseases, parasitic infections, and environmental pollutants ,36.Granuloma formation is a dynamic process, and many times the structure of these specific chronic inflammatory responses can give useful information about the timing, evolution, and prognosis of the disease.Labratrema minimus  metacercariae has been accomplished [Nevertheless, detailed studies on granuloma progression against histozoic parasites have not been carried out, although numerous details of parasite granuloma cellular components have been reported both in saltwater and freshwater fish ,30,37. Amplished . The premplished  allows aThe present work aims to characterize the histological features of the granulomatous inflammatory response in visceral organs of mullets naturally infected with trematode and myxozoan parasites.For this purpose, different developmental stages of granuloma, from the early to the late phase of the histozoic parasite infection, were identified using discernible histological features, and afterward, the immune cells and other non-inflammatory components associated with the different stages of the granuloma were characterized by histochemical and immunohistochemical tools.From July to October 2014, 239 mullets were collected by fishing nets from 4 different coastal lagoons in Sardinia (western Mediterranean Sea): 60 from Calich lagoon , 60 from San Teodoro lagoon , 60 from Mistras lagoon , and 59 from Marceddì lagoon .Mugil cephalus (11), Chelon labrosus (35), Chelon auratus (83), and Chelon ramada (110), variably combined in the different lagoons. All the specimens were identified at species level following Ben-Tuvia (1986) and Bauchot (1987); fish names conform to FishBase [Identification of Mugilidae species revealed a heterogeneous group composed of FishBase ,45,46. SThe mullets were transferred to the Department of Veterinary Medicine, University of Sassari, and the internal organs  of a subsample of 12 fish were analyzed for the presence of tissue parasites to identify the main taxa present. Squashes and scrapings of organs were placed on slides, pressed under a coverslip, and observed under an optical microscope. The parasites found were trematode metacercariae (TM) and myxozoan plasmodia (MP), and they were identified to the lowest taxonomic level possible according to the observable characters, following the specialistic literature ,49,50,51A culture test was carried using the anterior kidney of the 239 subjects on a primary isolation medium (blood agar). Colonies present in the plates were cloned and identified by phenotypic , biochemical , mass spectrometry (MALDI-TOF), and molecular (using generic primers and subsequent sequencing) methods.Fresh tissues from sampled organs (liver and spleen) were homogenized and decontaminated and inoculated in Stonebrink medium tubes and 2 Löwenstein–Jensen medium tubes  for mycobacteria culture. Decontamination of the samples was carried out using 1.5% cetylpyridinium chloride, following the standard protocol aimed at eliminating contaminating bacteria, in order to allow mycobacteria growth on specially sown soils .Samples of the liver, spleen, heart, and kidney of the 239 mullets were collected and immediately fixed in 10% buffered formalin for 48 h, dehydrated with increasing alcohol concentrations and xylene in an automatic tissue processor, and paraffin embedded. Sections of 3 μm thickness were obtained with a microtome  and stained with hematoxylin and eosin in an automatic multistainer . Slides were then evaluated at light microscopy . Lesions found in visceral organs  were evaluated according to the parasite class involved ,54.A total of 75 well-preserved granulomatous lesions from 47 of the 239 fish were included in this study. All granulomas were selected considering different organs and different classes of parasites involved. Lesions were classified into three developmental stages based on the evaluation of selected histological features  and were investigated to evaluate different structural components  by immunohistochemistry (IHC). Serial sections were mounted on positively charged slides  for IHC according to a previously developed protocol ,56,57. BThe presence of collagen, mast cells, and rodlet cells was also evaluated with Masson’s trichrome and Giemsa staining, respectively . To inveGranulomas were further evaluated for their proximity to the blood vessel and assigned to one of the following three categories: close to the vessel , near the vessel , far from the vessel (granuloma distance from the vessel >100 microns or not present in the field of view at 200× magnification). Additionally, macrophage aggregates (MAs) associated with granulomas were also evaluated. The 75 granulomas were assigned to the “present” or “absent” categories based on the presence or absence of macrophage aggregates near the granuloma in a field of view at 200× magnification.https://imagej.net/ImageJ, accessed on 15 May 2020). The quantitative evaluation of the collagen area, associated with the lesions, was accomplished using ImageJ software on microphotographs taken at 200× magnification on slides stained with Masson’s trichrome. In detail, the total area of the granuloma was selected by the “freehand selection” tool, measured, and then isolated from the background with the “clear outside” option. The central area of the lesion, containing parasite or necrotic material, was selected by the freehand tool, measured, and deleted by the “cut” function to avoid erroneous measurements of color values similar to the collagen area. After that, “color threshold” tool settings were adjusted to identify color values corresponding to collagen, and the obtained area was selected and measured. The color threshold measurement was repeated three times, and the final collagen area was calculated as the mean of the three consecutive measurements and expressed as a percentage of the total area of the granuloma.Each lesion was microphotographed and images were analyzed using a Java-based open-source image processing program  was used to correlate the prevalence of parasites in examined organs. Furthermore, categorical and ordinal variables were compared using the Spearman rho (ρ) rank correlation coefficient. A value of ρ approximately equal to 1 indicates a good correlation, a value near 0 indicates a poor correlation, and a negative value indicates an inverse correlation. A p-value < 0.05 was considered significant.Microscopic features of the lesions were analyzed using Stata 11.2 software . Immunohistochemical results were compared using the non-parametric Kruskal–Wallis test with Dunn’s post hoc comparison. The chi-square test  (Myxobolus sp. (Myxobolidae)  A,B and tbolidae) C,D in thbolidae) .The microbiological examination of all samples was negative for the presence of pathogenic bacteria and mycobacteria.Among the 239 sampled fish, 172/239 (71.96%) presented at least 1 parasite, of which 78/239 (32.63%) were affected only by TM and 40/239 (16.73%) only by MP, whereas in 54/239 fish (22.59%), both classes of parasites were detected. Concerning organ distribution, TM were detected in all examined organs, with a higher prevalence in the kidney (19.6%) and heart (14.2%), whereas MP were more frequently detected in the kidney (38.1%) and spleen (18.8%). Most of the fish affected by a single class of parasite presented an infection confined to a single organ (22.1% for metacercariae and 14.6% for Myxosporea).Mugil cephalus showed a higher prevalence of TM in the heart (54.5%) and in the kidney for MP (36.3%) but without any significant statistical association. In C. ramada, TM were more frequent in the heart (12.7%), whereas MP mostly affected the kidney . The kidney was the most affected organ in C. auratus for both parasite classes . In Chelon labrosus, MP were mostly found in the spleen . Details on the prevalence of organs affected by granulomas induced by both groups of parasites are shown in Most of the fish affected by a single class of parasite presented lesions confined to a single organ (35.9% for metacercariae and 40.5% for Myxosporea). Details on the percentages of each fish species affected by each of the parasite classes in one, two, three, or four evaluated organs are shown in Ziehl-Neelsen staining was negative for mycobacteria in all examined organs. The microscopic examination of the organs revealed a multifocal, mild-to-severe granulomatous inflammation around the parasites and three different stages of lesions. Out of 75 lesions, 25 were categorized as stage I, 27 as stage II, and 23 as stage III. Most of the selected fish showed the presence of only one stage of granuloma (68.1%), whereas 23.4% showed the presence of two stages, and in 8.5%, all stages were detectable.Stage I was termed as pre-granuloma stage and characterized by an intact parasite surrounded by a thin cyst wall, in association with scarce inflammatory cells. At this stage, positive labeling for anti-cytokeratin antibody was occasionally expressed by the parasite capsule, whereas immunoreactivity to anti-vimentin antibody was negative A–C.Stage II was termed as intermediate-stage granuloma and characterized by parasites with loss of somatic details or partially destroyed parasites, but that were still recognizable in the center of the lesion, and often intermingled with a scarce to moderate amount of granular eosinophilic material and nuclear debris (necrosis). Outer encircling layers of flattened cells with a moderate to abundant eosinophilic cytoplasm, an ovoid vesicular nucleus with prominent, basophilic, and central nucleolus were detected around the degenerated parasite and necrosis. Due to these morphological features and to their strong and diffuse positive cytoplasmic signal to anti-cytokeratin CKAE1-AE3 antibody, these flattened cells were identified as epithelioid cells D–F. MoreStage III was designated as late-stage granuloma and characterized by a central eosinophilic core of necrosis, with parasite remnants only occasionally visible, and increased layers of epithelioid cells. The external wall was constituted by increased layers of fibroblasts strongly positive to anti-vimentin antibody G–I.Scattered macrophages were detected near parasite cyst walls in stage I lesions and/or intermingled with necrotic material or scattered in the surrounding parenchyma in later stages. Aggregates of macrophages, frequently showing pigment-laden cytoplasm, were observed in association with the external layers of granulomas in 25/75 granulomas H,I.At histological examination, parasites were described based on characteristic features of the parasite classes, clearly discernible at stage I. Trematode metacercariae were detected in all examined organs, ranging from 150 to 400 microns in diameter, without body cavity and surrounded by a light eosinophilic capsule. Oral and ventral suckers, identifiable by the presence of a radially arranged musculature, were often recognizable, as well as a cuticle with a thickness of 2–4 microns with occasionally detectable cuticle spines that appeared as distinct eosinophilic linear structures A.Histozoic plasmodia of myxozoans were found encysted in the parenchyma of the heart, liver, spleen, and kidney. Plasmodia, containing a variable number (until hundreds) of mature spores, were round to oval, ranging from 100 to 450 μm, and were delimited by a thin eosinophilic wall B.In granulomas, collagen fibers were recognizable as blue filaments with Masson’s trichrome staining in the external layer of intermediate- and late-stage granulomas C.Mast cells (MCs) appeared as ovoid cells with distinct borders and abundant cytoplasm containing numerous eosinophilic granules, brightly evidenced by Giemsa staining, and a central nucleus A.Mast cells also showed a strong cytoplasmic positive signal to anti-vimentin antibody B. In botRodlet cells were observed in scarce numbers in all stages, mainly disposed near the parasite capsule or in the surrounding parenchyma. They were also observed near the main vascular structure both in parasitized and healthy samples D.The results of the structural components of granulomas and associated immune cells, as well as the description of histological parasite characteristics, were combined in a general scheme of possible developmental stages of granuloma formation .2 (corrected for ties) = 62.686, p = 0.00010; Dunn’s post hoc test, pre-granuloma vs. intermediate p = 0.000002; pre-granuloma vs. late p = 0.000000; intermediate vs. late p = 0.000450). In the intermediate stage, ECs covered a mean area of 13.42% (± 7.19% SD) of the total area of the granuloma, whereas in the late stage, they reached approximately 1/3 of the total area of the granuloma (mean area 30.85% ± 14.68% SD).Epithelioid cells showed a statistically significant positive correlation with granuloma progressive stages, showing an increase in parallel to the host immune response progression  = 38.963, p = 0.00010; Dunn’s post hoc test, pre-granuloma vs. intermediate p = 0.000842; pre-granuloma vs. late p= 0.000000, intermediate vs. late p = 0.002460). Fibroblasts covered averagely 2.94% (± 3.85% SD) of the total area of the granuloma in stage II, reaching a mean area of 7.15% (± 5.71% SD) in the late stage.The fibroblast layer increased with granuloma progression  = 36.791, p = 0.00010) . Moreover, collagen showed a statistically significant association with the fibroblast layer in the intermediate-stage granuloma (ρ =0.6257 p < 0.05). The collagen layer covered a mean area of 0.82% (± 1.43% SD) in the intermediate stage and a mean area of 3.58% (± 2.92% SD) in the late-stage granuloma.Statistical analysis evidenced that collagen had a significant increase in intermediate- and late-stage granulomas  =29.531, p = 0.00010 . Moreover, a significant positive correlation between the collagen area and the number of mast cells was observed . Evaluation of the association between the number of mast cells and the granuloma proximity to vascular structures revealed no significant statistical association.Mast cells were observed in all stages of the lesion development, showing an increase from the pre-granuloma stage, with a mean number of 0.68 (± 1.86 SD), to the intermediate- (2.89 ± 3.33 SD) and late-stage granulomas (9.86 ± 18.98 SD). Statistical analysis of MCs revealed a significant increase in number in the pre-granuloma and intermediate-stage granuloma  in the pre-granuloma stage, 0.28 (± 0.71 SD) in the intermediate stage, to 0.65 (± 1.19 SD) in the late-stage granuloma. Moreover, conversely to MCs, no statistically significant difference in the number of the RCs was observed in the different stages of host immune response.Macrophages aggregates were found associated in 16% of the pre-granuloma stage, 44.4% in the intermediate stage, and 39.1% in the late-stage granuloma. However, no statistically significant variation was observed between their presence and the different stages of granulomas.p > 0.05).Quantification of granuloma’s proximity to vascular structures gave the following results: 33.3% of lesions were considered in the “close” category, 33.3% fell in the “near” category, and 33.3% in the “far” category. The association between the distance of granulomas from vessels and granuloma stages was not statistically significant . Heterophyids, which have a fish-eating bird or mammals as the definitive host, are widely encountered parasites of mullets in Sardinian brackish waters [Inflammatory lesions associated with TM and MP were detected in (71.9%) of the examined fish, confirming the observation of other authors that reported Mugilidae as a widely parasitized species . The parh waters ,59,60 ash waters ,42,48. Ph waters ,63,64,65Myxobolus spp. infection in many Mugilidae species from the Mediterranean area, which described encysted plasmodia in different tissues [Myxozoan parasites, especially the Myxobolidae family, have been extensively described in mugilids worldwide ,67,68,69 tissues ,73,74,75 tissues .Parasite infection in fish can be the result of a single contact with intermediate hosts or can be due to multiple exposures to parasites, leading to the formation of younger parasite cysts near to older and almost resolved infection . The graThe response of the fish immune system after parasite invasion is variable and reliant on a multitude of factors , with a different outcome in terms of damage extent to host tissues ,77,78.Myxobolus spp., respectively [In the pre-granuloma stage (stage I), parasites were detected encysted in the parenchyma of several organs, generally surrounded by a compact eosinophilic layer, composed of compressed parenchymal cells because of the cyst pressure. The inflammatory response in this phase was absent or mild, and when present, mainly characterized by a limited number of mast cells close to the parasite wall or scattered in the surrounding parenchyma, and by occasional macrophages. This feature has been reported by other authors in early encysted TM ,64 and hectively .The hallmark of the intermediate-stage granuloma (stage II) was the presence of epithelioid cells that encircled the parasites. These cells were discernible with IHC staining by the strong positive labeling to anti-cytokeratin antibody (CKAE1/AE3), as was reported by studies in other fish species ,28. The Lastly, late-stage granuloma (stage III) represented the late phase of chronic inflammation with the highest quantity of immune cells, which led to the resolution of parasite infection. In this stage, due to the presence of central necrosis and the lack of parasite remnants, the results of Ziehl-Neelsen staining excluded the presence of mycobacterial infection.p < 0.001) could be explained by the fact that in this phase, both fibroblasts and collagen were present in similar proportions. However, in stage III, even if both categories were numerically increased, collagen production augmented fourfold compared to the twofold rise in fibroblasts. The biological explanation for this difference could be that in late stages, fibroblasts have accomplished their role and their number start to decrease, whereas collagen, which represents fibroblast’s final product, is at the highest levels. There is evidence that fibroblasts, although not proper immune cells, make an important contribution in the host response against parasites, by crosstalk with other immune cells, as demonstrated in mammals and fish species [Epithelioid cells and fibroblasts, which are recently considered strictly associated with immune response, were found in the largest amount in the late-stage granuloma. Fibroblasts were more prominent and evidenced by a diffuse membrane immunoreactivity to anti-vimentin antibody, a marker for the intermediate filament of mammalian mesenchymal cells that is preserved in some fish species . Fibrobl species ,92. An i species ,33. Mast species ,93. Thisp < 0.05) reinforced the hypothesis that action of MCs could be more specifically addressed to the regulation of fibroblasts in collagen production, rather than to fibroblast recruitment in the site of infection, in intermediate- and late-stage lesions.In this work, microscopic examination of stages II and III granuloma revealed the presence of mast cells grouped around the external layer, adherent to fibroblasts, thus leading to the hypothesis that a transgranulation mechanism of MCs towards fibroblasts could be plausible. The statistically significant association of MCs with the collagen layer  infected by nematode larvae [The results of the present work suggest that the role of mast cells as promoters of collagen production may not be the only mechanism in which MCs are involved against histozoic parasites. Indeed, the low number of MCs observed in the pre-granuloma stage and their distribution around the parasite capsule suggest that their action could be involved, synergistically with macrophages, in promoting host response, perhaps by the recruitment of other immune cells at the site of infection. This mechanism, recognized in mammalian mast cells during parasite infection, has been supposed in teleosts, where MCs are recognized as first effectors during parasitism . Mast cee larvae .Another group of immune cells, known as rodlet cells, that represent a unique element of the teleostean immune system ,96, was Macrophage aggregates were inconsistently found at the periphery of the granulomas, sometimes containing brownish cytoplasmic pigments. Aggregates of pigment-laden macrophages (MMAs) are normally present in the liver and spleen of many teleosts and can vary in number and dimension under the influence of several factors, such as age, stress, diseases, and pollution . MMAs haThe present study underlined that host immune response against the two groups of parasites revealed a substantial superimposability, leading to a common model of development of the immune response against trematode metacercariae and myxozoan parasites.Parasitic disease is a relevant issue for fish health and represents a challenge between the attempt of parasites to survive and the host’s action aimed to prevent the spread of infection.This study, by combining histopathology with immunohistochemical methods, represents an effort to expand the knowledge of inflammatory reactions associated with parasites in fish organs and their staging. This makes it conceivable that the identified histological patterns could be reliable tools to staging the granulomatous response associated with histozoic parasites and represent an effort to classify granulomas of different origins.Further analyses should be conducted to understand mechanisms of granuloma formation induced by different species of parasites in various experimental models, as well as the development of early detection methods, especially in species of great commercial interest for human consumption.


Chronic obstructive pulmonary disease (COPD) is a common, preventable disease of airflow limitation that accounts for the third leading deaths of any disease process in the worldwide. Health benefits of liuzijue qigong (LQG) on patients with stable COPD has been assessed. This study was designed to perform a systemic review and meta-analysis of the effect of Liuzijue breathing exercise on patients with stable COPD.Published articles from 1970 to December 2020 were conducted using electronic searches. Two independents reviewers conducted data extraction. The Cochrane risk of bias assessment tool was used to evaluate the quality of the included studies.P < .00001), FEV1% , the ratio of forced expiratory volume to forced vital capacity in the first second (FEV1/FVC [%]) , 6 minutes walking distance (6MWD) , health-related quality of life  and modified medical research council dyspnea scale (mMRC) . The observed effect was more pronounced for short term and medium-term duration interventions of study. It also showed improvements in the secondary outcome measures by LQG.A total of 16 eligible trials with 1039 patients with stable COPD were identified. Compared with control group, the pool meta-analysis of LQG showed a significant improvement in forced expiratory volume in one second (FEV1) (MD = −0.16, 95% CI , In this systematic review and meta-analysis, LQG can improve lung ventilation function, exercise endurance and health-related quality of life of patients with stable COPD.This study is a systematic review and it does not involve harming to the rights of participants. Ethical approval will not be require for this study. The research results may be published in a peer-reviewed journals. Additionally, the medical burden of COPD is significant ranking fifth in the world's economic burden of disease. According to statistics, the U.S. government spent nearly $50 billion on treatment of COPD in 2010. In China, about 1.5 million people die from COPD every year. COPD presents a significant challenge to the health care provider worldwide.–9Chronic obstructive pulmonary disease (COPD) is a respiratory system disease that is characterized by persistent respiratory tract symptoms and fixed airflow limitation. Non-drug treatments mainly comprised oxygen therapy and pulmonary rehabilitation training. Pulmonary rehabilitation training is widely used in COPD, with the purpose of reducing symptoms, improving quality of life, reducing medical burden, and increasing social participation. Its efficacy is widely recognized.,9,11The Global Initiative for Chronic Obstructive Lung Disease (GOLD) recommends that patients with acute exacerbation of COPD are mainly treated with drugs and oxygen therapy, while extra non-drug treatment should be used for COPD patients in stable phase, in addition to drugs.–14 Despite the potential benefits of LQG for COPD management, various design and methodologic weaknesses have consistently been identified across studies. In addition, there lacks a systematic review and meta-analysis of clinical therapeutic effect about LQG on patients with COPD. So this study is a systematic review and meta-analysis of published literature on the application of LQG in patients with stable COPD, in order to provide high-quality evidence synthesis and decision basis for the rehabilitation of COPD patients.Liuzijue qigong (LQG) is a traditional Chinese method of fitness based on breath pronunciation. As a part of the traditional fitness qigong series launched by Chinese Health Qigong Association, LQG performs the actions of inhaling and exhaling though different mouth patterns to control and regulate the rise and fall of the breath in the body, and completing the practice of “xu, he, hu, si, chui, xi” with breathing and pronunciation. These exercises plays a positive role in regulating respiratory system, exercise endurance and quality of life on patients with stable COPD.22.1https://www.crd.york.ac.uk/prospero/PROSPERO registration number: CRD42020209191).This systematic review and meta-analyzes has been registered on PROSPERO  terms were chronic obstructive pulmonary disease (COPD), liuzijue qigong. The keywords were traditional Chinese exercise; qigong; health qigong; liuzijue; Liuzijue respiratory gymnastic; randomized controlled trial; chronic obstructive pulmonary disease (COPD). A details of Search Strategies are showed on Appendix. The search strategy of this study uses a combination of Mesh terms and keywords, and is determined after repeated pre-checks, supplemented by manual search, and retrospectively included references when necessary.2.42.4.11.Only RCTs regarding the efficacy of LQG for COPD were included.2.Patients were diagnosed with COPD in a stable state according to the Global Initiative for Chronic obstructive pulmonary disease (GOLD), and no acute exacerbation occurred in all patients within 6 months before entering the trial.3.The baseline interventions included routine basic treatment of western medicine, oxygen therapy, usual care, conventional respiratory therapy, health education and were equally implemented in both groups.4.The experimental group participated in LQG, while the control group received health education, or conventional breathing exercise or or no intervention.2.4.21.The papers with unscientific and unrigorous experimental design are excluded2.The full text which could not obtained through various channels was excluded;3.Qualitative studies, animal experiments, case reports and conference abstract reviews were excluded;4.Documents with incomplete data or data problems were excluded;5.Documents with inconsistent main outcome indicators were excluded;6.Patients with COPD accompanied by other complications were excluded, these complications include bronchial asthma, bronchiectasis, bronchial tumors, tuberculosis, acute coronary syndrome, severe heart and kidney failure, poorly controlled diabetes and blood glucose, as well as patients with severe blood system diseases and mental disorders that cannot be treated with treatment;7.Patients with COPD are in an unstable phase were excluded.2.5Data were extracted independently by 2 reviewers (PG and FT) according to inclusion and exclusion criteria, and then cross-checked. If there was a dispute, it was settled through discussion. The extracted contents included:1.The basic materials of the literature, such as the author, the year of publication, etc.;2.Specific details of experimental design, such as randomization, allocation and hiding, blind method, basic data, intervention measures, outcome evaluation indexes, intervention time and follow-up time of the study subjects.If the research data is found incomplete, the author was contacted by phone or email to obtain the data. If the relevant data is not obtained in the end, the article will be excluded. When RCT with multiple studies was involved, the experimental group and control group related to this study were extracted.2.6The primary outcome included [forced expiratory volume in one second (FEV1)], , the ratio of forced expiratory volume to forced vital capacity in the first second (FEV1/FVC [%]), exercise endurance . And the secondary outcome comprised Health-related quality of life (St. George's Respiratory Questionnaire (SGRQ) and COPD Assessment test (CAT), Dyspnea index .2.7 with a total score of 7 points. Scores <4 were considered as low quality studies, while scores ≥4 were considered as high quality studies. The risk of bias was assessed using the evaluation criteria recommended by the Cochrane Handbook 5.1.0, “low risk bias,” “high risk bias” and “unclear” (lack of relevant information or uncertainty of bias) were assessed for each of the included literatures. The quality evaluation of literatures were conducted independently by 2 reviewers (PG and YM). Any controversy occurring during the evaluation process were discussed with a third reviewer (WL) and resolved by consensus.Jadad score was used to evaluate the methodological quality of each RCTs included,2.82 test and I2 test, if P < .05, I2 ≤ 50% indicate that there is homogeneity among the studies, and fixed effect model was used for analysis. If P ≤ .05, I2 > 50%, indicating statistically heterogeneity, random effect model was used for analysis. Finally, we used the funnel plots and Egger's regression asymmetry test to detect publication bias. To prove the reliability of our meta-analysis results, a sensitivity analysis were conducted by removing each study one by one to evaluate the consistency and quality of results. If variables in the studies included in this meta-analysis were continuous, we used the mean difference (MD) and 95% confidence interval (CI) to analyze the studies, otherwise, standardized mean difference (SMD) was used when variables were inconsistent. We considered P values less than .05 to be statistically significant.The RevMan5.3 and Stata14.0 software were used for meta-analysis. As a priori analysis, we also analyzed the property of data across 3 intervention durations, defined as short-term (≤3 months), medium-term (6 months), and long-term (12 months). Heterogeneity test: judged by Chi33.1The flow diagram of the selection process is summarized in Figure 3.2–33 involving 1039 individuals were selected for this meta-analysis, 1 studies were published in English, and 15 studies–22,24–33 were published in Chinese, six of which were dissertations.–29,31–33 All eligible studies were published from 2008 to 2019. There were 13 studies with a sample size ≥40 participants, and the average age of participants is >60 years old. The quality of each study was assessed using the Jadad scale, 5 studies were high-quality,,29,31,33 10 studies were low-quality.–28,30,32 The basic characteristics of the included studies were shown in Table A total 16 studies3.3–28,30–32 and one was high risk. There was high risk of bias in the domain of blinding of participants and personnel, only 3 studies,29,33 used single-blind method, but no specific method of blinding was mentioned in these studies. Only five of them were shown to blind their outcome assessment.–24,32–33 All trials reported methods with a low risk of incomplete outcome data and 38 studies were at low risk of bias. With regard to selective outcome reporting bias, 10 studies were determined as low risk and the remaining were determined as unclear risk. All studies were graded as unclear risk of other bias. These results were summarized in Figure The methodological quality of all included studies were evaluated according to the bias risk assessment tools provided by the Cochrane Collaboration. All of the included trials described randomized allocation, and they were low risk in the fields of randomized allocation. Twelve studies were classified as having an unclear risk in the fields of allocation concealment3.43.4.1,23,26–29 used FEV1 to evaluate the therapeutic effect of LQG on stable COPD patients. The fixed effects model was utilized to integrate the results (I2 = 7%). The results showed that, overall, LQG significantly improved the FEV1 compared with the control group . The effect size of the studies–24,26–33 showed that Liuzijue exercise could significantly improve the FEV1% . The effect size of the studies–29,31–33 showed that Liuzijue breathing exercise could significantly improve the FEV1/FVC (%)  are important component of pulmonary function. Seven trials3.4.2I2 = 43%). The overall effect of the studies–25,33 found that LQG was associated with significantly improvement the 6MWD compared with the control group , and a random effect model was used for merge the results. The overall effect of the studies,29–31,33 showed significantly improvement the health-related quality of life compared with the control group . The results showed that LQG significantly lowered the mMRC compared with the control group , the study of FEV1/FVC (%), study of health-related quality, the sensitivity analyses showed obvious changes in the statistical significance of outcomes Fig. Some results of this study had high heterogeneity for example, FEV1 (%), FEV1/FVC (%) and health-related quality, by removing single studies for example the study3.8P = .764 for FEV1; P = .436 for FEV1%; P = .076 for 6MWD; P = .348 for health-related quality, respectively), only the outcome of FEV1/FVC% (P = .048) was showed publication bias.As shown from the Egger asymmetry tests, there was little indication of publication bias on the these outcomes  for COPD patients.In our study, we created subgroups based on the different intervention duration time of LQG to evaluate the overall effect and each subgroup's effect, which would informed COPD patients of the effect of different intervention duration times. Generally, compared with conventional therapy, our study found that LQG was helpful for COPD rehabilitation as assessed by FEVI, FEV1 (%), FEV1/FVC (%), 6MWD, mMRC, and health-related quality.–39 In our study, we found the distance of the 6MWD in the LQG group was increased compared with the distance in the control group. In subgroup analysis, whether it was a short-term intervention or a medium-term intervention, LQG had a positive effect for improving 6MWD of COPD patients. However, long-term intervention has no obvious effect, the main reason is that there is only 1 literature included. Declining of exercise endurance and lung function are the main characteristics of COPD, and there are numerous reasons for the decreasing of patients’ exercise ability. It is generally believed that the patient's movement is restricted to airflow obstruction, lung hyperinflation and gas exchange barriers during the activity process,–41 furthermore, the movement restriction makes the patient unable to engage in related sports which causes the patient's exercise ability to further decline, then this will form a vicious circle. LQG contains breathing exercises and limb movement, which is not only beneficial to the flexibility and functional coordination of the upper limb muscles, but it also strengthens the function of the lower limbs. Thus LQG could prolong the 6MWD of COPD patients.6MWD has been used as a simple and valid evaluation parameter for exercise tolerance of COPD patients. In our study, LQG improved patients’ pulmonary function  compared with control group by the pool effect of our meta-analysis. In subgroup analysis, LQG had a good effect on these index of lung ventilation function  in COPD patients regarding short-term or mid-term duration intervention. This finding may be because LQG can enhance the strength of respiratory muscle, Also “Xu, Si and He” word tactic of LQG can extend expiratory time, improve the airway pressure, avoid premature closure of the airway, increase pulmonary ventilation function in patients with COPD. However, our finding is contrary to Tong HX's research viewpoint that the Liuzijue of traditional health qigong cannot improve the lung function of COPD patients. For this reason, we carefully read Tong's study of meta-analysis and found that there is only 1 article about Liuzijue's intervention in COPD patients included in his study of meta-analysis, which is not sufficient to demonstrate the effectiveness of LQG intervention in COPD patients. In the actually practice of LQG, the breathing method is beneficial to improve the abdominal muscle tension, increase movement range of the diaphragm rise and fall, enhance the strength of respiratory muscle, and thus obtain the greatest improvement of lung function. So patients with COPD could choose LQG for improving respiratory function.FEV1, FEV1%, FEV1/FVC (%) are important indicators to reflect the degree of airway ventilation and obstruction of COPD patients, which can assess the severity of the patient's clinical symptoms and disease severity. As a form of traditional fitness exercise, limb training of LQG can effectively relieve dyspnea symptoms during activities while adjusting the respiratory function and relaxing the whole body function, so it has better effects on health-related quality of life of COPD patients.–48Currently, improving COPD patients’ dyspnea and enhancing their exercise endurance were the main target through LQG. Declining of exercise endurance and respiratory function directly affects the quality of life of COPD patients. Therefore, the assessment of the health-related quality of life of patients should be an important part of the effect of treating patients. CAT respiratory questionnaire and the SGRQ respiratory questionnaire were widely used for assessing the quality of life of patients with COPD. In our study, the overall effect size showed that LQG significantly improved the health-related quality of life compared with the control group. Additional, good evidence was found in our study that LQG decreased mMRC score of dyspnea. The underlying mechanism is that LQG has a positive effect on the T cell immune function of patients with stable COPD and prevent patients from getting sick more easily,In subgroup analysis of our study, LQG also had better evidence of the effect on FEV1, FEV1 (%), FEV1/FVC (%), 6MWD and health-related quality of life in both short term and medium-term duration of study. However, the long-term intervention duration of LQG had no effect, and it may be that there is too little literature to prove the evidence. From our study, we suggested that the intervention duration of LQG lasts at least 3 to 6 months.4.1Athough we have comprehensive analysis and assessed all eligible studies, it still has some limitation. First, 15 of 16 RCTs in this meta-analysis were published in Chinese and little of relevant foreign RCTs, there may be publication bias that the result of this study were regional. Secondly, in our study, some results had high heterogeneity with regards to intervention intensity, duration, and frequency that may have contributed to unwanted heterogeneity and may have further influenced the outcomes. Even though our classification of intervention durations , the relatively small number of studies included in each category did not allow us to effectively explanation the heterogeneity underlying the different studies in our random effect models. Thirdly, most of the studies showed only the randomized trials, but no specific methods of random sequence generation, RCTs of allocation concealment, and blinding of outcome assessment. There were only 3 studies which reported single blinding. The methodological quality of many of the included RCTs was generally low and might have a high risk of bias.4.2,49–50 so we should try to improve the exercise intensity of the traditional fitness method in the research. Thirdly, the mechanism of COPD is still under further exploration at the present, so it is necessary to add some other index to comprehensively evaluate the efficacy of COPD patients, such as acute exacerbation of COPD, BODE index, peripheral muscle strength and cellular immune factors.First, research design should be carried out with stable COPD patients as the inclusion objects, high-quality, large sample RCT. Secondly, LQG is a kind of aerobic exercise with medium and low intensity,5In summary, this meta-analysis of RCTs suggested that LQG had positive effects in field of lung function, exercise endurance, health-related quality of life and dyspnea of patients with stable COPD.Data curation: Pincao Gao, Fang Tang.Formal analysis: Kai He.Methodology: Pincao Gao, Weiguo Liu, Yu Mo.Project administration: Kai He.Software: Pincao Gao, Kai He, Yu Mo.Supervision: Fang Tang, Weiguo Liu.Writing – original draft: Pincao Gao.Writing – review & editing: Pincao Gao, Fang Tang, Kai He.