Endocrine diseases have a considerable impact on public health from an epidemiological point of view and because they may cause long-term disability, alteration of the quality-of-life of the affected patients, and are the fifth leading cause of death. In this extensive review of the literature, we have evaluated the prevalence of the different disorders of endocrine interest in the world and Italy, highlighting their epidemiological, clinical, and economic impact. Data from the European Observatory of Health Policies and Systems show that in Italy cardiovascular diseases (40% in women and 35% in men) followed by oncological diseases (24% in women and 33% in men) represent the most frequent causes of subsequent mortality events. Endocrine diseases (5% in women and 4% in men) rank fifth on this list, after respiratory (6% in women and 8% in men) and neurological (8% in women and 5% in men) diseases . This imBased on these assumptions, we conducted an overview of the prevalence and incidence of the main disorders of endocrine interest, highlighting their real impact on daily clinical practice. The data of prevalence and incidence were extracted, when possible, from national and international registers, such as the Istituto Nazionale di Statistica (ISTAT), the Istituto Superiore di Sanità (ISS), and ARNO Observatory, World Health Organization (WHO). When the former was not available, guidelines of international societies or reviews summarizing the epidemiological evidence collected in different geographical areas of the world and Italy were used. Many of the studies included were conducted in the last twenty years (2000-2020), but those conducted in the last four years (2017-2020) were preferred in the selection of the epidemiological data. This research strategy allowed us to extract the most recent epidemiological data available in the literature, although the lack of solid prevalence studies on some of the diseases addressed in some continents such as Africa, Asia, and Oceania only allows us to estimate the worldwide prevalence, but not to have a precise estimation.The most common pituitary disease is pituitary adenomas. Early diagnosis and eventual treatment of these masses are clinically essential as they can secrete hormones and cause a mass effect . The general prevalence is about 1 case/865-2688 people even though autopsy studies report the presence of small pituitary adenomas in about 10% of people . ApproxiThe other pituitary disease of clinical interest is hypopituitarism. This disorder is characterized by a partial or complete deficiency of pituitary hormone secretion resulting in variously combined clinical pictures of adrenal insufficiency (AI), hypothyroidism, hypogonadism, growth hormone deficiency, and less frequently diabetes insipidus. Patients with hypopituitarism, especially those with multiple hormonal deficits, represent an important source of health care costs and indirect costs (due to disability and workdays lost). In addition, they require the intervention of specialized centers to guarantee the best quality of life (QoL). The prevalence of the disorder is about 45 cases/100,000 with an annual incidence of 4 cases/100,000 inhabitants .Among the diseases due to a reduced pituitary hormone secretion, GH deficiency (GHD) represents the most important endocrine cause of short stature with an estimated prevalence of 1:3500-1:4000 children. In ˜50% of cases, the deficit is idiopathic, in the remainder it is associated with hypopituitarism, central nervous system tumors, cranial irradiation, or other physiological causes . This diHowever, the impact of GHD in the adult population is poorly understood as many children with GH deficiency are often not re-examined when they reach adulthood. Furthermore, the deficiency is often not sought in adult patients with risk factors such as vascular or traumatic brain injury. The annual incidence is estimated at 12 cases/1,000,000 inhabitants in France and 14-19 cases/1,000,000 inhabitants in Denmark. The scarce epidemiological information available today makes it necessary to increase the effort for the diagnosis and management of these patients .AI due to ACTH deficiency is another dysfunction present in approximately one-third of the patients with hypopituitarism. It seems to have a higher prevalence than the primary forms estimated in about 150-280/1,000,000 inhabitants even if this figure is only based on few prevalence studies. Adrenal insufficiency caused by chronic glucocorticoid administration, which results in adrenal cortex atrophy due to suppression of the hypothalamic-pituitary-adrenal axis, is the one with the highest prevalence. However, to date, no data are available on its prevalence .Pituitary disorders generally represent a significant clinical burden for the patient and an economic burden for the health care system since they are chronic diseases. In fact, even after treatment, the disease sequelae require medical treatment and compromise the patient’s QoL. For example, acromegalic patients may develop osteoarthritis, obstructive sleep apnea syndrome, hypertension that may persist even after surgical treatment. Even in Cushing’s disease, fractures, hypertension, and cardiovascular events constitute elements of long-term disability. Moreover, the side effects resulting from the administration of drugs used for the management of these diseases should not be overlooked. Finally, surgery and radiation therapy used for the treatment of these diseases can lead to hypopituitarism with all the consequences associated with this clinical condition .Thyroid dysfunctions are common and easily treatable. However, if undiagnosed, they can significantly affect health due to the vital role that thyroid hormones play in regulating heart and gastrointestinal function, brain development and function, physical development, and cellular metabolism . The mosThe second most prevalent thyroid disease is chronic autoimmune thyroiditis, which is the leading cause of hypothyroidism in iodine-sufficient countries. The incidence of this disease is extremely variable depending on the geographical area, affecting from 30-150/100,000 people/year with a frequency 4-10 times higher in women than in men. The prevalence of hypothyroidism in patients with autoimmune thyroiditis is about 3.5-5/1000 in women and 0.6-1/1000 in men and it increases with advancing age . In ItalConcerning subacute thyroiditis, lymphocytic and granulomatous forms are distinguished . The lymFinally, a mention should be made the acute suppurative thyroiditis, an extremely rare condition but with a mortality rate of 7.8%. It accounts for 0.1-0.7% of all thyroid diseases. A recent systematic review that collected all the data published from January 2000 to January 2020 identified 148 studies on this topic, with a total of 200 cases described. The vast majority of the cases are due to bacterial infections (Streptococcus spp. and Staphylococcus spp). In countries with a high incidence of tuberculosis, acute tubercular thyroiditis is extremely frequent. Finally, in immunocompromised patients, forms caused by fungi can also occur with a mortality rate that can reach up to 33%. Among the predisposing factors, the most important is immunosuppression, followed by the presence of the pyriform sinus fistula, diabetes mellitus, and disseminated infection. Complications and sequelae include airway obstruction, dysphagia, esophageal perforation or fistula, Horner’s syndrome, an extension of the abscess resulting in mediastinitis, pericarditis, thrombophlebitis, sepsis, thyroid dysfunction associated with thyroiditis (hyper- or hypothyroidism), and death .Another very prevalent thyroid dysfunction, affecting about 1-2% of the general population in iodine-sufficient countries, is ypothyroidism. In particular, the prevalence ranges from 0.2-5.3% in Europe and 0.3-3.7% in USA according to the definition used and the population studied . The maiFinally, we have to consider hyperthyroidism that affects about 0.2-1.3% of the general population in iodine-sufficient countries, with an annual incidence of about 51 cases/100,000 inhabitants. The main cause of hyperthyroidism is represented by the Graves-Basedow disease, followed by toxic multinodular goiter and Plummer adenoma. Less frequent are instead the forms resulting from thyroiditis, TSH-secreting pituitary adenoma, and drug-induced forms . In ItalThe socio-economic impact of thyroid diseases must not be overlooked. For example, goiter represents the 32nd most common sequelae of disease in humans. Furthermore, thyroid cancer is responsible for approximately 836,000 disability-associated life years (DALYs), while iodine deficiency represents the 85th major contributor to DALYs globally, with an even greater impact in some regions such as South Asia and Central Sub-Saharan Africa  is significantly higher, with 3 cases/1000 in the general population, increasing to 21 cases/1000 in the population aged 55-75 years . PHPT isThe most prevalent adrenal disease is adrenal incidentaloma. By definition, adrenal incidentaloma is an adrenal mass that is incidentally diagnosed during imaging performed for other reasons. Autopsy studies estimate the prevalence of this disease to be around 2% of the general population ranging from 1% to 8.7%. The frequency increases with advancing age. Instead, the prevalence is 3% with an increase of up to 10% in the older population in the context of imaging diagnosis . In 75% Congenital adrenal hyperplasia (CAH) is a group of disorders caused by the mutation of one of the genes that encode steroidogenesis enzymes. CAH recognizes the deficiency of the 21α-hydroxylase enzyme in 90% of cases. Depending on the type of mutation and the enzyme involved, the clinical presentation of the disease is extremely variable from severe and life-threatening forms with salt loss crises (8.8 cases/100 patients) to virilizing forms (2.5 cases/100 patients) , 42. TheAnother important condition is Addison’s syndrome. This disorder affects approximately 82-144/1,000,000 inhabitants with an annual incidence of 4.5 cases/1,000,000 . In the Given the chronic nature of AI, the economic and social costs are very high. In fact, it is associated with a decreased QoL, absenteeism resulting in reduced productivity, premature mortality, and long-term morbidity including cardiovascular disease, infections, anxiety, and depression. In addition, they require constant monitoring of the therapy to avoid over-treatment associated with comorbidities such as obesity, osteoporosis, and impaired glucose tolerance. Finally, the diagnostic delay of AI must also be taken into account as the diagnosis is made at the onset of an adrenal crisis . A UK stThe opposite condition characterized by endogenous cortisol overproduction is Cushing’s syndrome. The prevalence of this disease is about 10-15 cases per 1,000,000 inhabitants  and the Male hypogonadism is a disorder characterized by reduced testicular function resulting in a deficient secretion of androgens, inhibin B, anti-müllerian hormone (AMH), and spermatogenic failure. The clinical features vary according to the age of hypogonadism onset. Before puberty, an impaired testicular function can be diagnosed based on low AMH and inhibin B levels. At the time of puberty, hypogonadism manifests as a pubertal delay. Congenital hypogonadism may become clinically manifest at birth with genital abnormalities 1 case/4500 births). The main cause of genetically determined primary hypogonadism is represented by Klinefelter syndrome with a prevalence of 1/500-1000 births. Among the acquired forms there are those secondary to chemotherapy and radiotherapy and functional forms associated with chronic diseases that have an annual incidence of 1/10,000-100,000. The most prevalent acquired form, closely related to age, is represented by late-onset hypogonadism (LOH). This condition affects about 2.1% of the male population aged 40-79 years and is related both to the physiological decline of testosterone production that occurs throughout life, the weight gains, and the development of chronic diseases that often characterize the elderly . Another0 births.The most prevalent male sexual dysfunction is premature ejaculation (PE). This affects about 30% of the male population worldwide . In ItalThe onset of ovarian dysfunction under the age of 40 years is called premature ovarian failure (POF) and it has a prevalence of 1%. POF is mainly due to autoimmune causes or genetic abnormalities. There are also acquired forms resulting from chemotherapy, radiotherapy, ovariectomy for ovarian cancer . This laThe ovarian dysfunction of the fertile age with the highest prevalence is represented by polycystic ovary syndrome (PCOS). This disorder has a considerable clinical impact because it not only causes menstrual irregularities, hyperandrogenism, and polycystic ovaries but can also be associated with alterations of glucose (insulin-resistance and impaired glucose tolerance) and lipid metabolism, which in turn are important cardiovascular risk factors . Women wSexual dysfunctions have a great epidemiological impact. It is estimated that about 40% of women worldwide have sexual dysfunction, but only 12-25% of them cause personal distress . The mosThe economic burden of female sexual dysfunction is poorly studied. However, significant is the impact in terms of anxiety, depression, interpersonal difficulties, broken relationships, and difficulties to conceive. All these aspects in turn contribute to increasing the economic impact of sexual dysfunction. Therefore, increased attention should be reserved for the diagnosis and treatment of these dysfunctions .Another condition of great endocrine interest and partly related to the previously listed disorders is represented by infertility. It is estimated that 15% of couples are infertile (about 48.5 million couples worldwide). Infertility recognizes a male factor in about 30% of cases, a female factor in 50%, and both partners are involved in the remaining 20% of cases . TherefoTo understand the economic impact of infertility on the healthcare system and patients, we have to consider the costs of a single ART cycle. The costs vary from $2,500 up to $10,000 based on the direct and indirect costs and also on the costliness of the underlying healthcare system. Direct costs include medical consultations, ovulation stimulation drugs, laboratory and embryology services, ultrasound scanning, oocyte retrieval, and embryo transfer, hospital charges, nursing and counseling services, and administrative and overhead charges. Indirect costs, on the other hand, are mainly related to the increased risk of multiple pregnancies associated with these procedures, resulting in an increment of the costs for the management of pregnancy and associated complications .Gender identity disorders or gender dysphoria affects between 0.005 and 0.014% of biological males and 0.002-0.003% of biological females according to the DSM5 . AlthougDisorders of puberty include premature puberty and pubertal delay. Precocious puberty is a condition characterized by the appearance of secondary sexual characteristics before the age of 8 years in girls and before 9 years of age in boys. It affects about 1/5000 children with a female-to-male ratio of 10:1 . DelayedThe most prevalent metabolic alteration is represented by dyslipidemia and in particular by hypercholesterolemia. This condition is associated with a twice greater risk of developing cardiovascular events compared to unaffected patients . For thiThe other metabolic condition with health, social, and economic implications is DM. This disorder affects more than 425 million people worldwide , of which only 5-10% can be attributed to type I DM . ApproxiThe main condition, which is partly associated with the metabolic alterations described above, is represented by obesity. The prevalence and incidence of this phenomenon are increasing due to evolutionary, biological, psychological, sociological, economic, and institutional factors . AccordiNeuroendocrine tumors (NETs) are relatively rare tumors whose incidence, probably due to an improvement in diagnostic ability, seems to be increasing. In particular, the annual incidence varies from 3.4 cases per 100,000 inhabitants in Germany to 10.3 cases per 100,000 inhabitants according to the Kentucky registry . In ItalIn most cases, NETs are sporadic, although in 20% of cases genetic abnormalities are associated with their occurrence. These include MEN 1 and 2, Von Hippel-Lindau (VHL) syndrome, neurofibromatosis, and tuberous sclerosis . NETs loPulmonary NETs include typical and atypical carcinoids, large-cell neuroendocrine carcinomas (LCNEC), and small-cell lung carcinomas (SCLC). The latter is the most aggressive and also the most frequent among lung NETs with a prognosis of few months. It is also the one most frequently associated with paraneoplastic syndromes, such as syndrome of inappropriate antidiuretic hormone, Cushing’s syndrome, hypercalcemia from parathyroid hormone-related peptide secretion, and neurological syndromes  .Osteoporosis is associated with high morbidity and mortality. Each year, osteoporosis causes more than 8.9 million fractures, equivalent to one fracture every 3 seconds. In detail, 1 out of 3 women older than 50 years and 1 out of 5 men older than 50 years develop fractures from osteoporosis . In EuroMEN syndromes are characterized by the occurrence of multiple neoplasms of endocrine glands that can be benign and malignant. There are basically 3 main variants  and some new entities, such as MEN 4 considered a variant of MEN 1 and familial medullary thyroid cancer (FMTC) considered a variant of MEN 2A .MEN1 gene that encodes for menin, a protein involved in the regulation of cell proliferation and differentiation, apoptosis, endocrine-metabolic functions, and maintenance of genomic stability through DNA repair. The endocrine glands more frequently involved in the neoplastic transformation are parathyroids, pancreatic islets, and anterior pituitary gland. In particular, the main disease present in almost 100% of the patients is primary hyperparathyroidism. Gastroenteropancreatic endocrine (GEP) tumors occur in 70-80%. In more than half of them, they are gastrinomas that, along with foregut carcinoids, are the leading cause of morbidity and mortality in these patients. Finally, pituitary tumors are found in 10-60% of patients and mainly they are prolactin-secreting pituitary macroadenomas . In addition to MTC, patients with MEN 2A can develop pheochromocytoma  in 50% of the cases and parathyroid hyperplasia in 25% of the cases. More rarely, amyloid cutaneous lichen (10%), generally in the interscapular region, and Hirschsprung’s disease (2%) (agangliosis of the intestine) may be present. On the other hand, in patients with MEN 2B, in addition to MTC, pheochromocytoma can be observed in about 50% of cases, and in almost 100% of cases, there is marfanoid habitus and mucosal neurinomas. In FMTC, MTC is present alone , 96.The analysis of the RET gene is crucial not only for the diagnosis of MEN 2 but also for the prognostic role given the important genotype-phenotype association present in this disease. In fact, MEN 2A is the most frequent form and is associated with an intermediate aggressiveness of the disease. MEN 2B is the rarest but also the most aggressive. Finally, FMTC is considered the mildest variant since it does not present the other manifestations of MEN 2A –97.The QoL of patients with these syndromes is obviously impaired. Particularly in patients with MEN 1, the persistence of hyperparathyroidism after surgery, the need for numerous medical appointments, and sometimes the need to travel to reach centers specialized in the treatment of these rare diseases, represent the factors that mostly affect the QoL of these patients . In addiAPS is characterized by the development of multiple autoimmune endocrine diseases associated or not with autoimmune disease of non-endocrine organs. The association between these diseases is not random but configures 4 main clinical pictures. In detail, APS-1 is mainly characterized by the presence of at least two among chronic candidiasis, chronic hypoparathyroidism, and Addison’s disease. APS-2 associates Addison’s disease with autoimmune thyroid diseases, and/or type 1 DM. APS-3 is characterized by autoimmune thyroid diseases associated with other autoimmune diseases (excluding Addison’s disease and/or hypoparathyroidism). Finally, APS-4 presents combinations not included in the previous groups .APS-1 is a monogenic disease due to a mutation in a gene called autoimmune regulator with a prevalence ranging from 1:9000 inhabitants among the Iranian Jewish community, 1:25,000 in Finland, 1:80,000-90,000 in Norway . In ItalAPS-2 is more common than APS-1 with an estimated incidence of 1.4-4.5 cases per 100,000 inhabitants. It predominantly affects women (F/M ratio 2.7-3.7) and is very rare in children . Since aAPS-4 is rare and there are no studies that have evaluated its true incidence and prevalence .Another condition is the X-linked immune dysregulation, polyendocrinopathy, and enteropathy (IPEX), an extremely rare inherited syndrome characterized by early-onset type 1 DM, intractable diarrhea, and malabsorption due to autoimmune enteropathy and various forms of dermatitis . It is due to mutation of the FOXP3 gene expressed in regulatory T cells involved in the control of the immune response. It occurs in childhood with a prevalence of about 1,000,000 inhabitants .APS are diseases with considerable clinical impact since they require multidisciplinary interventions, multiple hormone replacement therapies, and the management of complications such as adrenal (APS-1 and 2) and hypocalcemic crises (APS-1), cancer of the esophagus and mouth (APS-1), complications of DM (APS-2), and infections (IPEX). Certainly, there is a need for further research in this area, especially to identify genetic mechanisms and environmental triggers to tailor the therapy to the patient and identify immunomodulatory treatments that block the autoimmune process before the irreversible organ damage occurs .Hypertension is a disorder that affects approximately 30% of the adult population. In most cases, hypertension is primary, also referred to as essential, whereas it is secondary in about 10% of cases. More than 15 endocrine diseases can cause secondary hypertension. These include primary aldosteronism, pheochromocytoma, Cushing’s syndrome, thyroid disease, and hyperparathyroidism, acromegaly, and mineralocorticoid excess. Primary hyperaldosteronism is the most frequent form believed to be responsible for 5% of all cases of hypertension and 20% of cases of hypertension resistant to drug therapy . For theKnowledge suggests on the correlation between endocrine disorders and COVID-19 is still quite scarce. Some evidence suggests a strong correlation between SARS-CoV-2 infection and DM. Indeed, SARS-CoV-2 infection aggravates inflammation and alters immune system responses, leading to difficulties in glycemic control and worsening insulin resistance. The viral infection increases also the risk of thromboembolism and cardiorespiratory failure more in patients with DM than in patients without it. This helps, in part, to explain the poor prognosis of patients with DM .The sex-related difference in the severity of COVID-19 has an endocrine explanation. Indeed, host entry of SARS-CoV-2 is mediated by transmembrane serine protease 2 (TMPRSS2), whose transcription is, in turn, promoted by the activation of the androgen receptor. For this reason, men have a more facilitated viral entry into host cells. Accordingly, conditions associated with increased sensitivity of the androgen receptor, such as androgenetic alopecia and prostate cancer, have been correlated with worse COVID-19 outcomes and hospitalization . FurtherThe correlation between sexual function, particularly ED and COVID-19 has also been recently investigated. In fact, Sansone and colleagues in a study conducted on 100 patients, showed that 25 patients who were infected with SARS-CoV2 had a higher prevalence of ED than the 75 healthy patients who were not infected. Furthermore, regression analysis confirmed that COVID-19 has a significant effect on the development of ED independently of other variables affecting erectile function, such as psychological status, age, and BMI . This asEvidence on COVID-19 and pituitary and adrenal function are still scarce. Undoubtedly, patients with AI require more careful monitoring and increased doses of the replacement therapy in case of SARS-CoV-2 infection. However, to date, there is no evidence of direct pituitary or hypothalamic effects by COVID-19 . The posFinally, a recent systematic review of 9 reviews concluded that patients with COVID-19 may develop thyroid dysfunction that includes thyrotoxicosis, hypothyroidism, and non-thyroidal illness syndrome. However, it is not clear whether this association is directly attributable to the virus or is merely a consequence of the disease and/or its treatment. In fact, the use of heparin can increase the levels of non-esterified fatty acids that displace thyroxine and triiodothyronine from the binding proteins, leading to increased levels of FT4 and FT3. Corticosteroid therapy can also decrease TSH levels, reduce thyroxine-binding protein levels resulting in increased FT4 levels, and inhibit the conversion of FT4 into FT3. Furthermore, patients with thyroid disease do not have an increased risk of SARS-CoV-2 infection and do not require COVID-19-adapted follow-up. Some cases of subacute thyroiditis have been reported with a late consequence after SARS-CoV-2 infection, but further studies are needed to better clarify this aspect .. In detail, gestational DM affects about 7% of pregnancies and its inadequate management is associated with important maternal-fetal complications such as gestational hypertension, placental abruption, intrauterine growth retardation with intrauterine death, and congenital malformation  and hypofunction could compromise fertility. Indeed, very rare are the cases described in the literature of acromegalic women that got pregnant since the excess of growth hormone causes anovularity. The first case of normal pregnancy in an acromegalic patient was described in 1954. Hypopituitarism is also extremely rare and the few cases described in the literature refer to women who have undergone ART cycles .The prevalence of primary hyperparathyroidism in pregnancy is around 0.15%. Hypoparathyroidism is also rare occurring mainly as a consequence of thyroidectomy. As evidence of the rarity of the event, the first case of hypoparathyroidism in pregnancy was described in 1942 .As far as adrenal diseases, to date, about 200 cases of Cushing’s syndrome have been described in pregnant women (both ACTH-dependent and ACTH-independent). The rarity of this disease in the gestational period derives from the fact that the hypercortisolism and hyperandrogenism present in Cushing’s syndrome alter fertility. Addison’s syndrome is also quite rare with a prevalence of approximately 1 case per 30,000 pregnancies. Even rarer is pheochromocytoma with a prevalence of 0.007% of pregnant women .. In particular, based on the graphical elaboration of the data, it does not a surprise that metabolic diseases have the greatest impact on clinical practice and economic costs. Therefore, the role of the endocrinologist in the adequate management of these patients is prominent.Endocrine dysfunctions are chronic diseases with a relevant negative impact on the patients affected and considerable social and economic burden for the health system. Taken into account the data of prevalence above-reported to the Italian population or, for some diseases, to specific reference populations , we evaluated the real epidemiological impact of the various diseases of endocrine interest . As above-mentioned considering the close associations of these conditions with systemic diseases and that sometimes they may escape diagnosis and treatment, the importance of andrological and gynecologic endocrinology results clear in recognizing these patients to improve their QoL and to prevent future morbidity.Gonadal and sexual disorders rank second in this list Conceptualization: AC and SV. Writing—original draft preparation, AC. Writing—review and editing, AEC, AA, SV. Visualization: EG. Data curation: LM, RC, EG. Supervision, RAC, AA. Project administration, AEC and SV. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Brassica oleracea L. var. capitata. GLS are important in food flavor, plant antimicrobial activity, insect resistance, disease resistance, and human anti-cancer effects. Sulfur is an important raw material of GLS, directly affecting their synthesis. However, the mechanism of sulfur regulation of GLS biosynthesis in cabbage is unclear. In the present study, cabbage was treated with sulfur-free Hoagland nutrient solution , and normal Hoagland nutrient solution . Through joint transcriptomic and proteomic analyses, the effect of exogenous S on GLS synthesis was explored. S application induced GLS accumulation; especially, indole glycosides. Transcriptome analysis showed that +S treatment correlated positively with differentially expressed genes and proteins involved in amino acid biosynthesis, carbon metabolism, and plant hormone signal transduction. Compared with −S treatment, the mRNA expression of GLS synthesis genes  and those encoding transcription factors  were upregulated significantly in the +S group. Combined transcriptome and proteome analysis suggested that the main pathway influenced by S during GLS synthesis in cabbage is amino acid biosynthesis. Moreover, S treatment activated GLS synthesis and accumulation.Glucosinolates (GLS) are important anionic secondary metabolites that are rich in thiocyanin in cabbage,  Brassica oleracea L. var. capitata (2n = 18), originated from the Mediterranean to the North Sea, and is a widely planted vegetable worldwide, with high economic and consumption values. According to data released by the Food and Agriculture Organization (FAO), the global cabbage planting area has increased from 282,943 to 286,762 hg/ha from 2016 to 2019. As living standards continue to increase, people’s nutritional quality and health care requirements for vegetable crops are increasing. Cabbage is recognized one of the anti-cancer vegetables, and its anti-cancer activity is mainly derived from thiol glucosides, including isothiocyanate [Sulfur is a macronutrient required for plant growth ,2. It isGLS are sulfur-rich plant metabolites in the order Brassicales that function in the plant defense against stress and pathogens, and have anti-cancer activities . GLS areAs shown in As shown in The differential gene expression analysis was performed using the DESeq2 software, and the results are show in To functionally annotate the differentially expressed genes (DEGs), GO functional annotation was performed using all reference genes as the background. The GO classifications of the 1848 DEGs were annotated, of which 541 were annotated as GO items. The annotated genes were divided into three major functional categories: Biological process (BP), cellular component (CC), and molecular function (MF) . DEGs weKEGG pathway enrichment analysis was performed on the DEGs . We mappTo further explore the role of sulfur in cabbage, we analyzed DEGs that were enriched in biosynthesis of amino acids and plant hormone signal transduction. Exogenous S treatment resulted in the differential expression of 60 genes involved in biosynthesis of amino acids. We focused on the six amino acid synthesis genes that had good sequencing results or are related to GLS synthesis and can regulate GLS synthesis genes A. CysteiThe GLS biosynthetic pathway has a strong influence on GLS synthesis. Through GO enrichment, and non-redundant (NR) annotation and Swiss Prot annotation, 14 DEGs representing genes involved in the biosynthesis of GLS were identified. Among them, six genes were upregulated and eight genes were downregulated C. ConsisTo verify the reliability of these DEGs identified by the analysis of the transcriptome using RNA Seq, the expression levels 20 genes were assessed using quantitative real-time revsere transcription PCR (qRT-PCR), including 10 GLS biosynthesis genes and 10 randomly selected genes (five upregulated and five downregulated genes). The qRT-PCR results were consistent with RNA-seq data, which indicated that the gene expression profiles from RNA-Seq were reliable.To further study the molecular mechanism of S on GLS synthesis in cabbage seedlings, quantitative proteomic techniques were used to analyze the differences in protein abundance between the +S and –S groups. In this study, a 1.2-fold change in protein abundance was used as the criterion to identify differentially abundant proteins. Compared with the −S group, 95 proteins were differentially abundant under +S conditions, with 29 upregulated and 66 downregulated.Sulfur is an important element in plants. The disulfide bond in proteins plays an irreplaceable role in stabilizing the spatial structure of proteins and regulating their activity. To better reveal the effect of sulfur on the growth of cabbage seedlings, we performed gene ontology (GO) classification analysis on the 95 differentially abundant proteins, which revealed that they are mainly associated with metabolic processes, unicellular processes, cellular processes, binding, and catalytic activity A. AccordThe relationship between mRNA and protein expression levels is complex; therefore, in the present study we analyzed the correlations between transcriptional regulation and protein regulation from the relationship between differentially abundant proteins and mRNA expression trends  . A totalGO functional classification was carried out for the differentially abundant proteins whose level was consistent with their encoding mRNA expression trend. Among the cellular components category, they were mainly represented in the organelle subcategory. In the molecular function category, they were mainly associated with catalytic activity and binding. In the biological processes category, they were mainly associated with response to stimulus, single-organism process, metabolic process, and cellular process. The GO function analysis results for differentially abundant proteins whose level showed the opposite trend to the mRNA expression are shown in To further explore the biological pathways by which the DEGs and differentially abundant proteins play their roles, KEGG pathway enrichment analysis was conducted. The upregulated differentially abundant proteins were mainly enriched in phenylpropanoid biosynthesis and protein processing in the endoplasmic reticulum, while the genes were mainly enriched in glutathione metabolism, protein processing in the endoplasmic reticulum, and limonene and pinene degradation . The dowThrough the correlation analysis between the mRNA level and the protein level, 39 proteins were identified in the combined analysis in cabbage seedlings, among which 38 had the same expression trend as the mRNA and 1 had the opposite expression trend. Among the 38 differentially abundant proteins with the same expression trend, 7 were upregulated and the remaining 31 were downregulated. The one differentially abundant protein with the opposite expression trend was upregulated at the mRNA level and downregulated at the protein level. The main pathways involved were: Tryptophan metabolism (ko00380) and valine, leucine, and isoleucine degradation (ko00280). Through the joint analysis of the 39 differentially abundant proteins, we found that they were enriched in 25 different pathways, eight of which were pathways of amino acid biosynthesis ; and tryptophan metabolism, beta-alanine degradation, histidine degradation, lysine degradation, and arginine and proline metabolism. These results showed that S treatment mainly affects the synthesis of GLS by modulating the biosynthesis and metabolism of amino acids.As shown in In recent years, the incidence of cancer has been increasing. Studies have found that isothiocyanate and other glucosinolates (GLS) degradation products show good defense against cancer. GLS have become the first choice for natural anti-cancer bioactive substances. Hence, investigation GLS has become a research hotspot. Medicinal and edible vegetables with high GLS contents have become a research direction in breeding and cultivation. There are great differences in the types and contents of GLS in different cruciferous crops and in crops grown using different cultivation methods. Therefore, to breed and cultivate cruciferous crops with a high GLS content, many scholars have studied the exogenous substances that control the accumulation of GLS in cruciferous crops. During the growth of Cruciferae, sulfur fertilizer provides a direct source of sulfur for the synthesis of GLS. Inorganic sulfur is absorbed by the root system and transported to the crop body. After the amino acid side chain is extended, the core structure of GLS is improved. The formation and secondary modification of the side chain ultimately generates the variety of GLS. In this study, the content and types of GLS in the leaves of cabbage seedlings treated with sulfur were more than those in the −S group, manifesting that exogenous sulfur has a remarkable effect on the synthesis of GLS. As shown in The transcriptome and proteome data showed that +S and −S treatments mainly affected amino acid metabolism, hormone metabolism, and biosynthetic pathways of GLS in cabbage seedlings. Amino acids are precursors of GLS synthesis. Zhai’s results showed that the addition of amino acids could increase the content of GLS in cabbage, and Cys could significantly increase the content of indole GLS . In the Arabidopsis in regulating the synthesis of indole GLS [Arabidopsis [For the biosynthetic pathways of GLS, Ida. summarized glucosinolates synthesis and control genes in the process, including BCAT, MAM, IPMI, CYP, GSTU, SUR, UGT, FMO, AOP, and other members of their gene families . Bednaredole GLS ,34. Intebidopsis ,34,35. Hbidopsis . TherefoArabidopsis and the results showed that overexpression caused a significant increase in the aliphatic GLS content in plants, and silencing had the opposite effect [Arabidopsis resistance study, gene CYP81F2 which regulates GLS synthesis in the WRKY18/WRKY40 double mutant was significantly up-regulated [Transcription factors respond to various biotic and abiotic stresses in plants by activating downstream gene expression. Studies have shown that MYB, AP, bHLH, AUX/IAA, and WRKY ,39,40,41e effect . At the e effect . Wittstoe effect . Studiese effect ,49. Henne effect . In addie effect . Accordiegulated . In thisBrassica oleracea L.) plants were grown in a greenhouse in Gansu Agricultural University from August to September 2020. The cabbage seeds are sowed in the seedling bowl, and they are allowed to grow in the seedling bowl for ten days without any treatment, after which the seedlings were grown in 1/8 nutrient solution. The seedlings were planted at two plants per 400 mL pot. Water Hoagland nutrient solution every five days. The treatments consisted of two S supply levels . Except for sulfur, the rest of the nutrient solution had the same composition in the two groups. The experiment was set up in three replicates, and 30 pots of cabbage were planted in each replicate. The plants were harvested 30 days after treatment initiation. The leaves were immediately frozen in liquid nitrogen and freeze-dried.Cabbage . The injection volume was 20 µL, the chromatographic column was a C18 reversed phase column , the column temperature was 30 °C, the mobile phase was water and acetonitrile, the gradient was 0–45 min, the acetonitrile gradient was 0–20%, the detection wavelength was 229 nm, and the flow rate was 1 mL/min. The GLS content was calculated based on the internal standard value.Glucosinolates (GLS) were extracted according to the methods of Yang et al. , with so® UltraTM RNA Library Prep Kit for Illumina Step one plus . After library construction, an Agilent 2100 instrument and a real-time PCR System were used to check the library . Clusters were generated on the cBot using HiSeq PE Cluster Kit v4-cBot-HS . The double-ended sequencing program (PE) was run on a HiSeq4000 sequencing platform. Clean Data was obtained by filtering the data with double-ended sequencing reads of 150 bp. Bowtie V1.0.1 (Johns Hopkins University) was used to build the reference genome library, and then the clean data were compared with the reference genome using TopHat V2.0.12  to obtain the location information of the gene on the reference genome, and the annotation information for the associated genes. The clean reads were then subjected to further analysis and annotation.First, 0.1g sample for each replicate is ground in a mortar containing liquid nitrogen and placed in a centrifuge tube. Then, 1 mL of TRizol reagent  was added and vortexed immediately at 25 °C; next, chloroform was added and centrifuged, the supernatant was retained, and isopropyl alcohol was added, incubated for 10 min, and centrifuged again. One milliliter of 75% ethanol was added and mixed, and the tube was centrifuged again. Finally, the supernatant was discarded, and the pellet was dried and resuspended in a suitable amount of RNase-free water. The RNA concentration was detected using a Nanodrop spectrophotometer . A 3 µg sample of total RNA was taken from each sample as the starting material to construct the transcriptome sequencing library, which was performed according to the instructions of the NEBNext p-value obtained was corrected to control for false positives according to Benjamin and Hochberg methods. After correction, the FDR threshold was set as 0.05. The Log2 (Fold Change) threshold was set as 1.5. After screening and identifying the DEGs, GO, KEGG, NR, Pram, and Swiss-PROT analyses were used to identify the functions of the DEGs.DEG Seq V1.18.0 (Bioconductor Open Source Software for Bioinformatics) was used to analyze the DEGs on the basis of their expression levels, and the p < 0.05. Through a searchable database of this experiment transcriptome database, to GO ultimately identified protein function. The database search software is Proteome Discover 2.4. [Select the third leaf from the growth point of the cabbage from the inside to the outside. The plant tissue samples were ground under liquid nitrogen and transferred to A 1.5 mL centrifuge tube. Then, 4 mL PE Solution A, 4ml PE Solution B and 4 mL PE Solution C were successively added. The supernatant was discarded overnight and centrifuged for 15 min. Acetone was added to the precipitate and centrifuged for 5 min to collect the precipitate. Then, acetone was added again to collect the precipitate and 4 mL PE Solution D was added to obtain the supernatant as a protein solution. Isopropyl alcohol (70 μL) was added to each tube, vortexed for 1 min, centrifuged and restored the labeling reagent to room temperature. The labeling reagent was added to the pre weighed peptide segments (about 100 μg) of each sample, and different samples were labeled with isotopes of different sizes. Protein samples were labeled as 126, 127N, 128C, 129N, 130C, 131. After labeling, 10 μg samples were mixed and then analyzed using high precision LC/MS spectrometry (Thermo fisher—Q—Exactive—Orbitrap). After scanning the mass spectrometry signal, the general layout was completed, and then converted it to a MGF file using Mascot software (Mascot 2.3.01) for quantitative retrieval and qualitative analysis. The relative quantification of peptide was calculated according to the relative abundance of ions, and the relative quantification of protein was calculated according to the relative quantification of peptide. Differences in protein screening criteria: the same protein in a relative to the other group of ratio greater than 1.2 times (up) or less than 1.2 times (down), and ver 2.4. ,54,55.®IIIRT . Trans Start ® Top Green qPCR Super Mix (AQ131) from Transgen Biotechnology Co, Ltd.  was used for qPCR detection. The reaction system consisted of 10 µL of qPCR Mix, 1 µL of upstream primer (10 µmol l−1), 1 µL of downstream primer (10 µmol l−1) and 2 µL of cDNA template and sufficient ddH2O added to the mixture for a total volume of 20 µL. The qPCR reaction and data collection were performed on a Roche Light Cycler 96 instrument . The qPCR reaction conditions were 95 °C for 30 s; followed by 40 cycles of 95 °C for 5 s, 60 °C for 30 s, and 72 °C for 10 s. After the reaction was completed, the specificity of the reaction product was analyzed using melting curve analysis, and the PCR products were sequenced to determine the specificity of the reaction. The relative gene expression was calculated using the 2−△△Ct method [According to the nucleic acid sequences of the DEGs, primers for quantitative real-time PCR were designed by PrimerPremier6 software , and synthesized by the Lanzhou branch of Beijing Kinco Biotechnology Co., Ltd. The cabbage actin gene (ACTIN) was used as the internal reference gene. The mRNA of cabbage leaves was extracted for first-strand cDNA synthesis using Superscriptt method .The present study confirmed that S can promote the accumulation of glucosinolates in cabbage seedlings. In addition, on the basis of transcriptomic analysis, that found that most of the key genes related to amino acid metabolism, hormone metabolism, and biosynthesis of GLS responded to S treatment in cabbage. Through proteome analysis and proteome and transcriptome conjoint analysis, we found that the synthesis and metabolism of amino acids affect the biosynthesis of GLS. Several possible transcription factors related to GLS synthesis, such as RLK, BHLH, AP2, MYB, AUX/IAA, and WRKY, were identified. The results of the present study increase our understanding of the mechanism GLS synthesis under S treatment at the molecular level.


PCA biplots of soil samples showed that the carbonate rock area was associated with the most species of PTEs in the study area including Pb, Cd, Hg, As, and Zn. The clastic rock area was associated with Cu and Ni, and the lime and cement plants were associated with CaO, pH, Corg, TC, and aggravated PTE pollution around factories. In high-level PTE areas, rice was planted. Two out of 52 rice grain samples contained Cd and 4 out of 52 rice grain samples had Cr concentrations above the Chinese food safety standard pollutant limit . Therefore, the PTEs from parent rocks are already threatening rice safety. The government should therefore plan rice cultivation areas accordingly.Potentially toxic elements (PTEs) in Chinese agricultural soils, including those in some heritage protection zones, are serious and threaten food safety. Many scientists think that these PTEs may come from parent rock. Hence, at a karst rice-growing agricultural heritage area, Babao town, Guangnan County, Yunnan Province, China, the concentrations of eight PTEs  were determined in 148 surface soil, 25 rock, and 52 rice grain samples. A principal component analysis (PCA) and hierarchical cluster analysis were used to divide the surface soil into groups, and inverse distance weighting (IDW) was used to analyze the spatial distribution of PTEs. Soil pollution was assessed with the geoaccumulation index (Igeo). The results show that Cd, Hg, Zn, and Cr were polluting the soil (average Igeo > 0). The highest concentration of PTEs was distributed in the southwest of Babao town in the carbonate rock area, which had the highest pH and soil total organic carbon (Corg), Mn, and TFe Cr, Ni, Cu, Zn, Cd, Pb, Hg, and As are the potentially toxic elements (PTEs) with the highest levels of toxicity in the environment . They arSouthwest China is the largest karst region in China, and it has a wide range of carbonate rocks. In southwest China, the background concentrations of some PTEs, such as Cr 133.3 mg/kg), Cd (1.2 mg/kg), As (28.5 mg/kg), Zn 135.9 mg/kg), and Ni (66.7 mg/kg) in Guizhou province and Cu (48.2 mg/kg) in Yunnan province  mg/kg, a, mine ex3.3 mg/kgBabao town is one of the vital rice planting conservation areas in Yunnan Province, Southwest China and is one of world cultural heritage sites of United Nations Educational, Scientific, and Cultural Organization (UNESCO). The town is located in a typical karst zone and is famous for its high-quality rice. Historically, the rice was used as a tribute. Now, the rice has become a geographical indication product . Due to http://www.weather.com.cn/cityintro/101290607.shtml accessed on 11 March 2021), and the average annual rainfall is 1104.65 mm (based on Funing station observations from 2001–2015): ideal climate conditions for rice cultivation. The farmland has a fertile texture with limestone soils that are fit for rice growth. Hence, Babao town has a history of planting rice of more than 700 years.The study area was located in Babao town, Guangnan County, Wenshan Zhuang-Miao Autonomous Prefecture of Yunnan Province, China  . The annBabao town is a typical peak-cluster depression area with elevation ranging from 1100 to 1300 m, and rice planting is mainly concentrated in three areas a–c. The 2 sampling unit. Samples of rice were then dried and 500 g rice grain samples were placed into polyethylene plastic bottles for sample delivery.The following sampling and analysis methods were used: “Specification of Multi-Purpose Regional Geochemical Survey  (DZ/T 0258-2014)”, “Analysis Methods for Regional Geochemical Sample (DZ/T 0279-2016)” issued by Ministry of Land and Resources of the People’s Republic of China, and “Technical requirements for analysis of samples for ecological geochemical evaluation (DD2005-03)” issued by the China Geological Survey. These articles also adopted the sampling and testing methods promulgated by the China Geological Survey and Ministry of Land and Resources of the People’s Republic of China ,20. The 3–HCl–HClO4–HF acid mixture in a Teflon digestion vial and then heated. They were then extracted with hydrochloric acid and diluted to a certain volume. Then, the Cd, Pb, Zn, Cu, and Mn contents were determined . The As, Cr, and Hg contents were measured using atomic fluorescence spectrometry . The Ni, CaO, MgO, and TFe2O3 contents were measured with an X-ray fluorescence spectrometer . The soil pH value was determined with a pH meter, and the soil–water ratio was 2.5:1. The soil total organic carbon (Corg) was determinated by titration with burette and total carbon (TC) contents were determined with a High frequency infrared carbon sulfur analyzer, Model CS-206 .An elemental analysis of samples was carried out by the Guangdong provincial geological experimental testing center and the Hubei provincial geological experimental testing center. Before testing, the rice grains were hulled, crushed, sifted through 40–60 mesh screens. The method used to determine the total PTE content in the soil, rice grain, and rock samples was as follows: Samples  were digested in HNOTo ensure the accuracy of the sample analysis, 5% of the samples were randomly selected for parallel testing, and the repeat test pass rate was 100%. Reagent blanks, standard reference soil/rock samples (GBW07443), and standard rice samples (GBW10010) were obtained from the Center of Standard Materials of China to monitor the determination quality. The recovery of samples spiked with standards ranged from 89.8% to 107.9%. The LOD (limit of detection) ranged from 0.0005 mg/kg (Hg of soil sample) to 3 mg/kg (Cr of soil sample). The Cu and Pb contents were below the detection limit  in only a few rice grain samples. The values for these samples were set to half the detection limit.The principal component analysis (PCA) is a multivariate statistical method that uses the idea of dimensionality reduction and divides multiple indicators into several components on the premise of losing little information. The Kolmogorov–Smirnov test was used to assess the normality of the data set distribution. The results show that most elements did not have a normal distribution. PCA was performed before the data was log-ratio transformed (clr), and the numerical output with the eigenvalues was ≥1.The hierarchical cluster analysis was conducted with Origin 2019, and a z-score was used for the standardized data analysis. The proposed approach is based on sampling sites observed using the Ward Method with the Euclidean distance and the determination of clustroids by the sum of distances. Similar sampling points belong to the same cluster and are different from data within other clusters . The selThe inverse distance weighting (IDW) method was used in this study to analyze the distribution of PTEs. Both IDW and kriging interpolation methods can provide high accuracy for predicting the average concentrations of elements in the soil. Each of them has its shortcomings. IDW enlarges the estimated pollution area . The kriiC is the soil content of the element, i, and iB is the geochemical background value in the soil. The background values used in the study were based on the soil background values in Yunnan Province [Igeo ≤ 0, practically unpolluted; 0 < Igeo ≤ 1, slightly polluted; 1 < Igeo ≤ 2, moderately polluted; 2 < Igeo ≤ 3, heavily polluted; Igeo > 3, extremely polluted [The geoaccumulation index (Igeo) takes the influences of human activities and natural geological processes of background values into account and can quantitatively assess the extent of PTE contamination of sediments or other materials . TherefoProvince . The clapolluted .Sampling sites were mapped using ArcGIS 10.6 . The data were processed in Excel and SPSS v22 . Charts, and graphs of data were produced using Origin 2019 .The descriptive statistics of soil characteristics in Babao Town are shown in The average values of all PTEs were higher than the background values for Yunnan, which indicated this area is enriched with PTEs . The aveThe surface soil was divided into groups by the hierarchical cluster analysis. The numerical output with eigenvalues ≥1 from the principal component of soil are listed in 2O3, Cu, Ni, Mn, and As. PC3 has positive contributions from MgO, TFe2O3, Cu, Ni, and Cr, and negative contributions from Zn, Mn, and As. The surface soil samples of cluster 2 (surface soil samples affected by clastic rock) were associated with MgO, TFe2O3, Cu, and Ni and occupied the +PC3 quadrant  and those with mudstone and sandstone as the main rock types  displayed different chemical characteristics. All mean PTE concentration values in clastic rocks were higher than those in carbonate rocks; however, the mean values of Cd in carbonate rocks and clastic rocks were almost equal, and the median value of Cd in carbonate rocks was higher than that in clastic rocks , MgO , and CaO  , Cu , Mn , Ni , Zn , TFen 0.51%) . Cluster highest . Cu, Ni,trations .Carbonate rock areas were mainly found in the southwest part of Babao town  and had 2O3 content in the soil (The soil types present in carbonate rock area are brown soil and scattered brown silt/sand soil. This may be why Cluster 3 (soil samples affected by carbon rock) had the second-highest Corg and TC values . Soils ithe soil . Hence, The highest average CaO, TC, Corg, and pH zones were found in cluster 4. Apparently, the use of carbonate rocks by plants has resulted in elevated levels of calcium in the soil. The pH of the soil is mainly controlled by calcium . A high In addition to enrichment of the concentrations of PTEs (such as Cd) in the soil by lime and cement factories, atmospheric deposition and fertilizer application are important ways to increase the soil PTEs . Howeversoil/PTEs rock) was calculated under the assumption that the soil in the investigated area was directly derived from the chemical succession of the bedrock [soil/PTEs carbonate rock ratio was calculated using sampling points of cluster 3, and PTEs soil/PTEs clastic rock ratio was calculated using sampling points of cluster 2. A ratio of >1 indicates enriched soil. The ratios of PTEs soil/PTEs rock in both two types of rocks were above 1, indicating that soils generated by carbonate rock and clastic rock were both rich in PTEs.The ratio of the mean rock and soil concentration (PTEs  bedrock . PTEs sosoil/PTEs rock of carbonate rock was 2.3(Ni)-32.8(Zn) times as much as that of clastic rock (soil/PTEs rock of carbonate rock ranged from 6.64 (Ni) to 83.53 (Zn), while in clastic rock, it ranged from 1.06 (Pb) to 3.30 (Cu). The reason for the higher PTEs soil/PTEs rock ratio in carbonate rock area is because of the large rock/soil volume change that occurred during the process of weathering carbonate rock into the soil [2O3 concentrations . In this study, areas of excess Cd were mainly located in P2y and D3g  in France [soil to be of a high level  > Cd . The highest Cr and Cd concentrations were 1.92 and 1.52 times the limit values of 1 and 0.2 mg/kg, respectively. However, the Cd concentration in rice grains was not above the limit of 0.4 mg/kg from the Food and Agriculture Organization of the United Nations/World Health Organization (FAO/WHO) standards [Due to the high pollution risk of Cd and Hg in this area, the study area may not be suitable for growing crops. However, rice planting areas were located in areas with high-levels of Cd, Hg, Zn, and Cr . We fountandards .Areas exceeding the concentration limit of Cd in rice grains were located around the rock samples R24 and R20 and soil samples B128 (Cd 9.67mg/kg) and B136 (Cd 12.31 mg/kg) . The sam2O3. These good conditions led to the Cd concentrations of surface soil in the study area were as high as 26.70 mg/kg.This research aimed to examine the PTE concentration and spatial variation and the soil–rice system quality in Babao town. We found that the average values of all PTEs were higher than the background values of Yunnan, and the surface soil was mainly threatened by Cd and Hg. The area southwest of Babao town (the carbonate rock area) had the highest PTE content, and this was related to the parent rock and human activities in this area. In the study area, 7 out of 17 carbonate rocks had Cd concentrations above 1 mg/kg, and carbonate area had high pH value and was riched with Corg, Mn, and TFeThe lime plant and cement plant also caused increments in the PTE content in soil. Hence, these factories need to strengthen their supervision and management, especially in rural areas, where the factories are sometimes unsupervised.2O3, other factors such as repeated rainfall in humid southern climates and irrigation water also affected PTE concentrations in soil and rice. However, these factors were not examined in this study. These factors should be further investigated in the study area.In this area, only 2 out of 52 rice grains had Cd and 4 out of 52 rice grains had Cr concentrations slightly above the Chinese standards, which was related to the pH and Corg values. The areas with excess Cd of rice grains were located in low pH areas and the areas with excess Cr of rice grains were located in high pH and Corg areas. In addition to pH, Corg, Mn, and TFe


Due to the high rate of spontaneous abortion (SAB) in porcine pregnancy, there is a major interest and concern on commercial pig farming worldwide. Whereas the perturbed immune response at the maternal–fetal interface is an important mechanism associated with the spontaneous embryo loss in the early stages of implantation in porcine, data on the specific regulatory mechanism of the SAB at the end stage of the implantation remains scant. Therefore, we used high-throughput sequencing and bioinformatics tools to analyze the healthy and arresting endometrium on day 28 of pregnancy. We identified 639 differentially expressed lncRNAs (DELs) and 2357 differentially expressed genes (DEGs) at the end stage of implantation, and qRT-PCR was used to verify the sequencing data. Gene set variation analysis (GSVA), gene set enrichment analysis (GSEA), and immunohistochemistry analysis demonstrated weaker immune response activities in the arresting endometrium compared to the healthy one. Using the lasso regression analysis, we screened the DELs and constructed an immunological competitive endogenous RNA (ceRNA) network related to SAB, including 4 lncRNAs, 11 miRNAs, and 13 genes. In addition, Blast analysis showed the applicability of the constructed ceRNA network in different species, and subsequently determined HOXA-AS2 in pigs. Our study, for the first time, demonstrated that the SAB events at the end stages of implantation is associated with the regulation of immunobiological processes, and a specific molecular regulatory network was obtained. These novel findings may provide new insight into the possibility of increasing the litter size of sows, making pig breeding better and thus improving the efficiency of animal husbandry production. Spontaneous abortion (SAB) of embryos in porcine pregnancy remains a global and primary concern in commercial pig farming. Previous studies have shown that around 20–30% of porcine embryos are lost between days 12 and 30 of pregnancy during the peri-implantation stage . PreviouLong non-coding RNAs (lncRNAs) are a large class of RNAs with a nucleotide length of >200, low conservation and expression level, and tissue specificity . MultiplA total of 701,720,602 raw reads were generated from the 4 paired endometrium porcine samples using Illumina Hiseq 2500 platform. Out of the total raw reads, 695,694,564 were clean , accountTo further understand the differences between the lncRNAs and mRNAs in the HE or AE, we compared and analyzed data on gene structure and expression patterns. Notably, both the length of the lncRNAs and the number of exon and open reading frames (ORF) of the lncRNAs E,F were Using an FDR < 0.05 and |FoldChange| > 2, we identified 639 DELs in both the HE and AE. There were 528 upregulated and 111 downregulated DELs . HierarcAnalysis of the differential expression identified 2357 DEGs, including 1415 upregulated and 942 downregulated genes . HierarcTo further determine whether the DEGs between the AE and HE were related to the immunological functions, 139 immunobiological process gene sets were downloaded from MSigDB, and then gene set variation analysis (GSVA) was performed based on 139 GO terms . The twoIn addition, we downloaded a total of 1793 immune genes from Immport , an immuTo identify the lncRNA implicated in SAB, we performed a lasso regression analysis of the DELs and identified the following 8 lncRNAs: ENSSSCT00000004780, TCONS_00042638, TCONS_00051274, TCONS_00071236, TCONS_00108310, TCONS_00161675, TCONS_00177102, and TCONS_00215223 A,B. We tIn addition, in order to evaluate whether the constructed immunological ceRNA network was related to SAB, we downloaded 930 endometrial spontaneous abortion genes (ESABGs) from the GeneCard database . The datDue to the low conservation of the lncRNAs, using lncRNAs identified in different species are generally not extrapolated for other species. To assess whether our constructed ceRNA mechanism for SAB of the embryos could be applied to different species, we analyzed the sequence conservation properties of the lncRNA TCONS_00161675, with the most connectivity in the ceRNA network using NONCODE BLAST. Surprisingly, we found a 716 bp region matching with NONHSAT211839.1 (human lncRNA), and 650 bp with NONMMUT056947.2 (mouse lncRNA) . This dePrevious studies have shown that miRNAs function mainly through the seed sequences at nucleotide positions 2 through 7. Besides the seed sequences indicating that the miRNAs target genes could be the same B 20], o, o20], oPorcine animals are a unique SAB model, where up to 45% of embryos are spontaneously lost as embryos develop, which allows detailed study of the specific molecular regulation mechanisms . The higThe implantation in pigs begins on days 15–16 of pregnancy and ends on days 27–28 of pregnancy. Previous studies have shown that around 20–30% of porcine embryos are lost between days 12 and 30 of pregnancy , anotherPrevious studies associated the loss of embryo with recruitment of lymphocytes and angiogenesis in the porcine maternal–fetal interface during the early stage of implantation. Whereas there was increased transcription of angiogenic genes in healthy implantation sites, the arresting fetal sites showed rapid elevation in the expression of proinflammatory cytokines, Fas and Fas ligands . AlthougIn addition, growing evidence indicated that many lncRNAs play a vital role in the regulation of cellular growth and disease development ,28. As aInterestingly, our analysis found that the highly conserved lncRNA TCONS_00161675 overlaps with the transcription factor HOXA3 in the genomic location. Compared with the known Homo HOXA-AS2, we associated the conserved gene with HOXA-AS2 in pigs. Previous studies have shown that HOXA3 plays an important role in embryonic development, including control of distinct genetic programs for differentiation and morphogenesis in different cell types ,35. In aThis study was approved by the Ethics Committees of the Laboratory Animal Center of South China Agricultural University (permit number: SYXK-2019-0136).Tibetan gilts with an average litter size of 7–8, which have been artificially fed and bred for multiple generations on farms in Guangdong, China, were slaughtered at a local slaughterhouse on day 28 of pregnancy and the uteri were quickly removed and transported to the laboratory in an icebox. The uteri samples were then opened longitudinally along the anti-mesometrial side and the healthy and arresting embryos were analyzed. The embryos were obtained from 4 different pigs and the analysis was based on embryo length, weight, and vascularity of the placental membranes . Subsequ® spectrophotometer . Furthermore, RNA integrity and quantity were measured using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system , and the RIN (RNA integrity number) values were above 7.60.Total RNA of endometrium tissues was extracted using TRIzol reagent  following the manufacturer’s instructions. The RNA purity and concentration were assessed using NanoPhotometerThe library was prepared by an rRNA depletion method , and conAfter the library was constructed, the concentration of the library was adjusted to 1 ng/ul. Agilent 2100 Bioanalyzer was deployed to detect the insert size of the acquired library. Last, qPCR was used again to check the exact concentration of the cDNA library. The library preparation was completed, and the libraries were sequenced on Illumina PE150.http://asia.ensembl.org/Sus_scrofa/Info/Index, accessed on 3 October 2020) with the software HISAT2 (v.2.0.5) and the reads alignment results were transferred to StringTie (v.1.3.3) for transcript assembly [Raw reads were trimmed by removing adapter sequences, reads with more than 10% ploy-N, reads with ploy-A/T/G/C, and reads with more than 50% nucleotides with Qphred ≤ 20. Besides, Q20, Q30, and GC contents of the clean reads were calculated. All downstream analyses were based on the high-quality clean reads. Clean reads for each sample were mapped to the reference genome  software, and then, based on the characteristics of lncRNAs, lncRNAs were identified from the assembled transcripts. First of all, the transcripts with FPKM < 0.1, length < 200 bp, and exon numbers < 2 were removed. Cuffcompare (v.2.2.1) was used to compare transcripts with the reference to remove the annotated transcripts. Whereafter, we analyzed the coding potential of the transcripts by three software (CNCI (v.2) , Pfam  of lncRNAs and mRNAs was calculated by StringTie (v1.3.3). Then, a paired t-test was used to analyze the differential expression of lncRNAs and genes, and the R method . FDR < 0http://kobas.cbi.pku.edu.cn/kobas3, accessed on 15 October 2020). Moreover, the protein–protein interaction (PPI) network was analyzed using STRING database , and Cytoscape  was used for further visualization.The genes within 100 kb upstream and downstream of lncRNAs were used as the nearest target genes of lncRNAs . GO enrihttp://www.oligo.net, accessed on 11 December 2020), and all reactions were run in triplicate . Briefly, we synthesized cDNA using a PrimeScript™ RT reagent Kit with gDNA Eraser . We then performed quantitative RT-PCR PowerUp™ SYBR™ Green Master Mix  on the Step One Plus Real-Time PCR System . We adopted the following qRT-PCR protocol: 95 °C for 10 min, 50 cycles of 95 °C for 15 s, 60 °C for 15 s, and 72 °C for 20 s. Primers were designed using NCBI Primer-BLAST and Oligo 7 . Subsequently, gene set variation analysis (GSVA) and gene set enrichment analysis (GSEA) were conducted to explore the enrichment of DEGs in the functional gene sets of immunobiological processes. The immune genes (IGs) were acquired from the Immport database  and the endometrium spontaneous abortion genes (ESABGs) were downloaded from the GeneCard database .Functional gene sets of immunobiological process were obtained from the molecular signature database . Based on the ceRNA hypothesis, we predicted the interaction between lncRNAs and miRNAs, and miRNAs and mRNAs by three computational target prediction algorithms (miRanda (v.3.3) [p-value was corrected by the FDR method. Then, we selected the lncRNA-mRNA relationship pair with a correlation coefficient > 0.8 and FDR < 0.05 to construct the ceRNA network.To identify the potential lncRNAs associated with SAB, we performed a lasso regression analysis on DELs. The sequence information of miRNAs was downloaded from the miRBase database  , Targets (v.3.3) , and RNA (v.3.3) ). The cohttp://www.noncode.org, accessed on 15 February 2021) for blast comparison analysis to understand the conservation of lncRNA among different animal species, and visualized through the UCSC genome browser . In addition, the conservation of miRNA was comprehended by analyzing the consistency of the miRNA seed region sequence.The sequence of lncRNA was submitted to the NONCODE database . A p-value of less than 0.05 was considered to be statistically significant.The data are presented as the mean ± the standard error of the mean (SEM). The group data was compared using the Student’s In conclusion, we profiled the whole transcriptome expression of HE and AE in porcine on day 28 of pregnancy and obtained some lncRNAs and mRNAs involved in SAB. Our data demonstrated a correlation between SAB at the completion stage of implantation and the immunobiological process, but showed different molecular mechanisms from the beginning stage of the implantation. Finally, an immunological ceRNA network related to SAB was constructed and HOXA-AS2 in pigs was identified. Further studies are, however, required to verify the constructed ceRNA network and its applicability among different species, as well as the specific molecular mechanisms of action of the identified HOXA-AS2 in pigs. These novel findings will provide new targets for increasing the litter size of sows, making pig breeding better and thus improving the efficiency of animal husbandry production.


Hydrochoerus hydrochaeris, is the largest extant rodent of the world. To better understand the correlation between size and body mass, and biochemical parameters of capybaras from areas with different degrees of anthropization , we sampled free-ranging capybaras from areas of natural landscapes (NLs) and human-modified landscapes (HMLs) in Brazil. Analyses of biometrical and biochemical parameters of capybaras showed that animals from HMLs were heavier (higher body mass) than those from NL, a condition possibly related to fat deposit rather than body length, as indicated by Body Condition Index (BCI) analyses. Biochemical parameters indicated higher serum levels of albumin, creatine kinase, cholesterol, fructosamine and total protein among capybaras from HMLs than from NLs; however, when all adult capybaras were analyzed together only cholesterol and triglycerides were positively correlated with body mass. We propose that the biochemical profile differences between HMLs and NLs are related to the obesity condition of capybaras among HMLs. Considering that heavier animals might live longer and reproduce more often, our results could have important implications in the population dynamics of capybaras among HMLs, where this rodent species is frequently represented by overgrowth populations that generate several levels of conflicts with human beings.The capybara,  Hydrochoerus hydrochaeris, is a mammal belonging to the order Rodentia, family Caviidae. It is native to South America, where it occurs in all countries except for Chile [The capybara, or Chile . In Brazor Chile ,3. CapybCapybaras live in social groups always with more females than males because when male capybaras reach sexual maturity they are expelled from the group by the dominant adult male . The groIn Brazil, the destruction of native forests for the implantation of monocultures and cultivated pastures has been a historical process over the centuries, especially during the last 50 years . In soutThe advance of sugarcane crops caused a decrease in the habitat areas of native fauna. Along with these conditions, the state of São Paulo has been experiencing both horizontal and vertical expansion of capybaras populations during the last decades. In the first case, capybaras have started to occupy many areas where they did not exist before; in the second case, population densities have increased significantly in places with already established populations ,9.In natural landscapes, as in the Brazilian Pantanal, during the water season capybara select food and ingest smaller quantities, while in the dry season the selection is fewer, but the intake is higher . In the Several lines of study have demonstrated that loss or reduction of biodiversity tends to increase the transmission of pathogens and the emergence or reemergence of diseases. This pattern has occurred in several ecosystems, with different pathogens, hosts and vectors. Different studies have confirmed the theory of the correlation between environmental changes and the reduction of biodiversity and disease transmission: West Nile fever ,14,15, hGiven the primary importance of capybara in the epidemiology of Brazilian spotted fever and other types of human conflicts, this rodent species is becoming an important object of study, which leads us to believe that a better understanding of its biometric and biochemical profile, and the correlation between both, can be important to better understand the ecological aspects of this rodent among different landscapes. To better understand the correlation between size and body mass, and biochemical parameters of capybaras from areas with different degrees of anthropization , in the present study we sampled free-ranging capybaras from areas of natural landscapes (NLs) and human-modified landscapes (HMLs). Our main hypothesis is that the high food supply among HMLs would impact on the body conditions and healthy status of capybaras when compared to the NLs in which capybaras live under minimal anthropic interference.Captures of free-ranging capybaras were carried out during 2015–2019 in seven municipalities of the state of São Paulo (HMLs), and in two municipalities (NLs), one in the state of Mato Grosso and another one in the state of Mato Grosso do Sul . Among H2 corrals baited with sugar cane and green corn. Once closed in the corral, animals were physically restrained by a net catcher and anaesthetized with an intramuscular injection of a combination of ketamine (10 mg/kg) and xylazine (0.2 mg/kg). In Mato Grosso do Sul, corrals were not effective; thus, capybaras were captured by anesthetic darting via a CO₂-injection rifle  using the same chemicals listed above [Capybaras were captured by using 16 to 20 med above . Under aed above .Only adult animals weighing > 35.0 kg . For eacThe total length of the animals was measured from the end of the muzzle to the end of the last vertebra, and the height was measured from the highest point of the withers . The neck perimeter was measured at the angle of the mandible. The chest perimeter was measured at the height of the armpit and the circumference of the abdomen at the umbilical line . For allWeighing was done with the animal wrapped in a nylon net (weight 2.4 kg). The scale used was a digital dynamometer with a precision of 0.1 kg ; it was attached to the net through a metal hook and then the net/animal set was suspended using a 1.5 m-aluminum bar.-BM/TL: Body mass (BM) divided by total length (TL) ,29.-2: Body mass (BM) divided by the square of the total length (TL2) [BM/TLth (TL2) ,31.-3: Body mass (BM) divided by the cube of the total length (TL3) [BM/TLth (TL3) .-2: Residual of linear regression of body mass (resBM) divided by the square of total length (TL2) [resBM/TLth (TL2) .-3: Residual of the linear regression of the body mass (resBM) divided by the cube of the total length (TL3) [resBM/TLth (TL3) ,34.It has been pointed out that the calculation of the body condition of animals is a common goal for several studies in order to compare absolute size (or other measurement) with body mass; however, authors have used several terminologies to indicate the same thing ,26. In tAn assumption of fat amount was estimated by calculating the residual index, which is the regressed body mass over body size. The residual index was calculated after data were appropriately transformed to meet regression assumptions. The residual distance from the point of the individual to the regression curve works as an estimate of the body condition, i.e., the distance from the point to the curve can be a good estimate of the extra mass accumulation, in this case, the fat .Blood samples (5 to 10 mL of venous blood) were collected through the cranial cava, saphenous or cephalic vein of each adult capybara. These samples were immediately stored in a vacuum tube containing separator gel for subsequent serum centrifugation. In the laboratory, sera were transferred to two 1.5 mL polypropylene tubes in order to keep all the samples in duplicates, and then frozen at −20 °C for further analyses.® biochemical analyzer, Labmax 240 model. To verify the presence of possible obesity-related metabolic disorders, the biochemical profile of adult capybaras was performed. The following serum elements were analyzed: Triglycerides, cholesterol, fructosamine, albumin, total protein, aspartate aminotransferase, alkaline phosphatase, creatine kinase, urea, calcium and phosphorous. All protocols were performed according to the manufacturer’s instructions of each kit.Biochemical variables were determined on a Labtest®–11531, Barcelona, Spain) and alkaline phosphatase  were performed. Obese patients tend to develop liver steatosis, which would lead to increases in liver enzymes, aspartate aminotransferase (AST) and alkaline phosphatase (ALP). Even if there is no clinical condition of steatosis, the simple deposition of excess fat can lead to increases in these enzymes [For liver profile, measurements of aspartate aminotransferase  were performed. Obese patients tend not to have large changes in serum urea concentrations, but since the intent is an overview of the animal’s general picture, it is important to determine the animal’s renal function. Urea is directly related to muscle mass and obese patients have less muscle mass, which may lead to decreased or fluctuating serum urea levels [For renal profile, measurements of urea  and triglycerides  were performed. These parameters were chosen because they tend to be increased in obese animals [®–21790, Barcelona, Spain).To analyze fat metabolism, measurements of cholesterol . Fructosamine is glucose conjugated with blood albumin [Because of the possible insulin resistance in obese animals, we also measured the amount of serum fructosamine  and albumin . Albumin is related to fructosamine. In addition, the World Health Organization considers obesity to be a pro-inflammatory or inflammatory state [Protein metabolism analyses were performed by measuring total protein  [Overweight patients have metabolic dysfunction, which may lead to alterations in the hypothalamus-pituitary-adrenal axis, leading to increases in cortisol. Cortisol, by definition, is a catabolic hormone, causing increased muscle catabolism rates, which, again, may increase serum CK concentrations. Increased blood CK may cause proximal tubular damage to the kidneys, causing changes in serum phosphorus levels (Labtest Brazil) .®—11570, Barcelona, Spain).Finally, obese animals have lower bone and muscle densities when compared to non-obese individuals, so there might be changes in the total blood calcium concentrations of obese animals, registering lower levels . Becauset-test for parametric distributions or non-parametric Wilcoxon test. Normality was verified by the Shapiro–Wilk test and for all analyses 5% was used as the significance level. For statistical analysis, by standardization, only the most recent recaptures were included in the calculations. Preliminarily, data were analyzed considering all adult capybaras  or only adult female capybaras . As variables presenting statistically significant differences were the same by both analyses, only the data with all adult capybaras is presented.Biometrical and biochemical data were evaluated using descriptive statistics, using violin-type density graphs and statistical summaries describing the groups through means and standard deviations for group comparison (HMLs versus NLs). Differences between capybaras from HMLs and NLs were assessed using the Student’s Correlation between serum biochemical data  and capybara body mass or BCI values were tested by linear regression analyses, considering each biochemical variable independently. For this purpose, adult capybaras were analyzed all together, regardless of whether they were from HMLs or NLs.All analyses were performed using R software .A total of 218 adult capybaras were sampled, 174 in HMLs and 44 in NLs . Among tOverall, the body mass range of adult capybaras ranged from 35 kg to 105.6 kg. Among HMLs, the mean body mass was 54.5 kg (range: 35.4 kg–83.4 kg) for males, and 63.1 kg (35 kg–105.6 kg) for females. Notably, three female capybaras weighed 105.6, 104.8, and 100.6 kg, and were the overall heaviest animals, all captured in the HML of Pirassununga municipality. Among NLs, the mean body mass was 48.6 kg (range: 38.6 kg–63.6 kg) for males and 54.8 kg (35.7 kg–80.4 kg) for females. Overall, mean weights for adult capybaras were 61.2 kg for HMLs and 54.2 kg for NLs.p = 0.005). In contrast, there were no significant differences (p > 0.05) in body height and total length to explain the body mass differences , there were no significant differences between body mass and body measures of male and female capybaras. The body mass of capybaras from HMLs was, on average, 7 kg higher than the body mass of animals from NLs  between the two capybara groups. Significant differences (p < 0.05) between animals from HMLs and NLs were observed for albumin, CK, cholesterol, fructosamine and total protein. Some parameters were borderline: Calcium (p = 0.0523) and AST (p = 0.0597).The biochemical variables were measured in a total of 161 free-ranging adult capybaras, 129 from HMLs and 32 from NLs. The results of the statistical analyses of the biochemical parameters compared between the two groups (HMLs and NLs) are shown in p > 0.05) between body mass and the parameters fructosamine, total protein, AST, CK, urea, calcium or phosphorous. However, there were positive correlations (p < 0.05) between capybara body mass and factors related to fat metabolism, namely, cholesterol and triglycerides. In addition, there was a negative correlation (p < 0.05) between body mass and the amount of circulating albumin or ALP  with cholesterol (BM/TL2) and triglycerides (BM/TL and BM/TL2). In addition, there were negative correlations (p < 0.05) between the amount of circulating phosphorous and all five BCI types, and between AST and all BCI types but BM/TL3  than those from NL, although the body total length and height did not differ statistically. Biochemical parameters indicated significant differences between animals from HMLs and NLs for albumin, CK, cholesterol, fructosamine and total protein; however, when all adult capybaras were analyzed together only cholesterol and triglycerides were positively correlated with body mass, and albumin negatively correlated with body mass. Our samples of adult capybaras were characterized by a predominance of females over males, in either HMLs or NLs. This condition is in agreement with the typical social structure of capybara groups . Anyhow,Previous records of body mass of adult capybaras from areas with low anthropic alterations, such as the Venezuelan Llanos, reported a 35 kg–65 kg range , whereasThe five body indexes showed differences between the sampled groups, revealing that capybaras in the HMLs had higher BCI than capybaras in the NLs and therefore higher fat reserves. We can infer that the extra weight of animals from HMLs comes from excess fat, since most of the fat in a capybara is subcutaneous . The meaPuma concolor), a natural predator of capybaras in HMLs [Based on individual energy reserves, we can infer that capybaras from HMLs have better survival conditions than animals from NLs. Firstly, this is due to the vast supply of food offered by crops  and cultivated grasses throughout the state of São Paulo . Secondl in HMLs , heavier in HMLs . In addi in HMLs ,50. The  in HMLs . A trait in HMLs .There are studies with domestic cattle considering body condition and reproductive efficiency, the higher the BCI, the better the reproductive efficiency . This asRegarding the blood biochemistry patterns, the present ALP values  were lower than the values reported for free ranging capybaras in Paz de Ariporo (235.75 ± 144.83 U/L), Colombia . The reaEndocrinological studies indicate that overweight and obese populations have lower serum concentrations of vitamin D, due to its sequestration by adipose tissue . VitaminThe urea was compared with results available in the literature by converting the results found by our group to blood urea nitrogen (BUN), which consists of multiplying the value found for serum urea by 0.467 , since that found in the literature for the species is the BUN. Therefore, converted from urea to BUN we have the following results: HMLs = 12.18 ± 7.79 mg/dL; NLs = 13.3 ± 4.85, which in both cases, are above the results recorded by Matus and Pulido .The values found for serum albumin in this study  are close to those found in free-living capybaras (2.54 ± 0.25 g/dL) by Álvarez-Méndez and Barragán ; howeverPanthera onca), puma (Puma concolor), caiman alligators (Caiman spp.) and anacondas (Eunectes spp.) [Notwithstanding the difference in diet, capybaras from NLs are subjected to the presence of natural predators—jaguar (es spp.) ,60,61,62es spp.) . The prees spp.) . This ines spp.) . In addies spp.) , food dees spp.)  and conves spp.) , all of Previous studies dealing with stress-associated changes in plasma immunoglobulin levels demonstrated that chronic stress increases blood levels of immunoglobulins, leading to an increase in total protein ,69. In aIn our study, NL capybaras had a serum cholesterol value (37.6 ± 15.4 mg/dL) close to those recorded by Matus and Pulido  for captTo our knowledge, this is the first work to measure fructosamine in capybaras. As previously mentioned, fructosamine gives a seven- to ten-day overview of blood glucose behavior. Since obese patients have insulin resistance, our hypothesis that capybaras from HMLs, exposed to high-calorie diets, have higher fructosamine values than capybaras from NLs , is confFasciola hepatica, in capybaras of at least two of the HMLs of the present study (Piracicaba and Tatuí), as recently reported [As previously defined, aspartate aminotransferase (AST) increases due to cell death for several reasons, including hepatic steatosis, but it can also increase due to muscle damage . A recenreported . This coAmblyomma sculptum [This study demonstrated that there were significant differences for some biometrical and biochemical parameters of capybaras between HMLs and NLs. In HMLs, where more energy-rich food sources are available for capybaras, their higher body mass is related to fat deposit rather than body length, as indicated by BCI analyses. The presumed obesity condition among HMLs could also be the reason for higher values of some biochemical parameters that are usually related to obesity, such as higher blood levels of cholesterol, triglycerides and fructosamine, when compared to capybaras from NLs. Considering that heavier animals could live longer and reproduce more often, the present results could have important implications in the population dynamics of capybaras among HMLs, where this rodent species is frequently represented by overgrowth populations that generate several levels of conflicts with human beings ,74,75,76sculptum ,76,77,78


Long noncoding RNAs (lncRNAs) perform indispensable functions in cancer pathologies and are involved in the onset and progression of multiple cancers. Multiple platforms were performed to comprehensively analyze the head and neck squamous cell carcinoma (HNSCC) for determining molecular subtypes. Molecular subtypes were clustered and analyzed by the “ConsensusClusterPlus” R package. The Limma software was utilized to screen for differentially expressed genes (DEGs). Functional enrichment analyses, including Gene Set Enrichment Analysis (GSEA), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Ontology (GO), were performed on the three database resources. Seventeen lncRNAs were determined as HNSCC-specific immune lncRNAs that were dysregulated. Our research identified and redefined two distinct molecular subtypes, C1 (230 samples) and C2 (269 samples). Moreover, the C1 subtype had a higher survival rate than the C2 subtype in HNSCC samples, as well as a prolonged median survival duration with activated immune response. 1531 DEGs, including 529 upmodulated genes and 1002 downmodulated genes, were identified in the above two subtypes. Functional enrichment analysis revealed that upmodulated genes in C2 were associated with tumorigenesis and development, while downregulated genes in C2 were associated with immune response. By comparing with the existing immunophenotyping group, it found that C1 had more overlaps with the existing Atypical and Basal, and C2 and Classical and Mesenchymal had a high degree of coincidence. On the basis of lncRNA, there were significant differences in the aspect of prognostic and immunological characteristics in the two identified molecular subtypes of HNSCC. Head and neck tumors have ranked sixth among the most common kind of malignant tumors worldwide, accounting for 16–40% of all malignancies . In receLong noncoding RNAs (lncRNAs) are well-recognized noncoding RNAs that exceed 200 nucleotides in length . So far,In our research, a coexpression network on the basis of the immune-related lncRNAs and mRNAs was firstly formed, and we obtained 17 lncRNAs associated with the immune and prognosis. Next, we identified two immune-related molecular subtypes of lncRNA in HNSCC samples. Moreover, analysis of their functional enrichment and characteristics of the immune microenvironment was performed, and the relationship between molecular subtypes and known molecular subtypes was investigated.https://portal.gdc.cancer.gov/) database containing their latest lncRNA expression level and clinical follow-up information [https://www.gencodegenes.org/human/) to divide the expression spectrum into two parts of lncRNA and mRNA and then converted the Ensembl IDs of these genes into Symbol form. In the TCGA-HNSCC dataset, we firstly deleted those genes with 0 expression values in HNSCC samples to maintain those gene sets with biological significance. Then, we obtained 13,628 lncRNAs' expression profiles and 19,500 protein-coding genes altogether. The following steps were used to process the TCGA RNA-seq data. Those samples without expression spectrum, clinical follow-up information, survival data, and survival status were all completely eliminated. We only stayed the solid tumor samples. The overall work flowchart of the study is shown in On April 20, 2020, we obtained HNSCC patients' data from the TCGA GDC API .In order to figure out whether lncRNA connected with immune function, we detected the enrichment connection between lncRNA and 17 kinds of pathways that were involved in immune function by the GSEA method . We acquBased on the connection of lncRNA and mRNA, according to the correlation scores calculated by the above equation, all related mRNAs were arranged in ascending order from small to large for every lncRNA. At the same time, based on the connection of lncRNA and immune function, the GSEA method was performed to exactly compute the enrichment significance between every lncRNA and every immune function-related pathway, as well as the lncRES scores between the above two items . On the For analyzing the degree of enrichment between dysfunctional lncRNAs related to immune function in HNSCC samples and different immune cells, we gathered 24 kinds of immune cells marker gene collection by the ImmuCellAI, including Gamma_delta, CD8_naive, Neutrophil, Cytotoxic, Macrophage, Tr1, B cell, iTreg, MAIT, Th2, Effector_memory, Tfh, Central_memory, Th1, NKT, nTreg, DC, Exhausted, Monocyte, CD4_naive, NK, Th17, CD8_T, and CD4_T. Then, we extracted those aberrant immune lncRNAs, which obviously correlated to marker genes of 24 different immune cells, and further analyzed the obvious enrichment connection between extracted aberrant immune cells and immune lncRNAs by the method of the hypergeometric enrichment analysis. The calculation formula for the hypergeometric is as follows:i denotes the enrichment value of lncRNA marker genes; j denotes immune cell; n denotes the number of mRNAs obviously connected with lncRNA in HNSCC samples; M denotes the number of marker genes present in immune cells; K denotes the quantity of obviously connection genes between immune cell and lncRNA; and N denotes 19,498, total mRNAs. Moreover, it will be regarded as having no significance when the K value is less than 3. On the basis of the calculation formula, the expression connection of each potential lncRNA and 24 immune cells and enrichment significance were calculated, and all candidates' aberrant immune lncRNA regulatory factors and immune cells' dramatically enrichment connection were also identified on the basis of the threshold p < 0.05. Eventually, we filtered out aberrant immune lncRNAs specific to HNSCC, which were apparently enriched in at least 12 immune cells.The meanings of every alphabet were described detailed as follows: In accordance with the aberrant immune lncRNAs' expression values in cancer samples, HNSCC samples were classified by the ConsensusClusterPlus . We did In accordance with the survival time , as well as the survival status in different sample categories, the samples were subjected to survival analysis. The survival rate, as well as the median survival time, was estimated by the method of Kaplan-Meier . At the http://www.webgestalt.org/option.php) [p < 0.05, and FDR < 0.25 were used as the threshold for screening.The Limma software was employed to detect the DEGs in different molecular subtypes . The scrion.php)  for diffFor figuring out the differences of biological characteristics in various cancer samples, various characterizations of TCGA-HNSCC samples were acquired from existing articles , which ip < 0.05, 2616 important lncRNA-immune cell pairs were determined, and 17 lncRNAs considerably enriched in a minimum of 10 immune cells were further screened out as lncRNA collections for the immune dysregulation in HNSCC  and only 29.42% in tumor samples (1637/5564), which showed that the lncRNA-immune function pathway relationship between normal samples and tumor samples was very different . Similarin HNSCC .p=0.079) on OS   on OS  and sign Figures , with HNp < 0.05)   , and there were apparently higher expression levels of these lncRNAs in the C1 subtype than those in the C2 subtype (DEGs between C1 and C2 molecular subtypes were identified by the Limma (3.40.6) package. There were altogether 1531 DEGs; specifically, 1002 genes were downmodulated, and 529 genes were upmodulated Figures . The dif subtype . The R s subtype , 68 path subtype , and 36  subtype . A total subtype . The R s subtype , 59 path subtype , 40 path subtype , and a t subtype , includip < 0.05, FDR <0.25)  . The resPathway genes related to tumor development and immunity were selected from the gene set for c2.cp.kegg.v7.0.symbols. Then, we compared the expression level of those selected genes in the 2 molecular subtypes; the results indicated that the level of tumor-related pathway genes expression for EMC, Cell Cycle, WNT, and P53 was substantially elevated in the C2 subtype in contrast with the C1 subtype expression, while immune-related NK Cell, B Cell, T Cell, and Chemokine pathway genes were considerably elevated in the C1 subtype in contrast with the C2 subtype . ImmunotCompared with the existing molecular subtypes Atypical, Basal, Classical, and Mesenchymal, what was interesting was that we found that the proportion of Atypical and Basal in the C1 subtype samples has increased significantly, accounting for 61.38%, which was related to a good prognosis; the C2 subtype contains Atypical and Basal, accounting for only 49.56%, which were also the reason for the poor prognosis of C2 Figures . These fTCGA is an outstanding database, and it attempted to use genome analysis technology to map out the genome variation maps of all human cancers and conduct systematic analysis. At present, TCGA has performed various crucial molecular characterizations of unique histological cancer types, including HNSCC , 27. A uIncreasing amounts of evidence show that lncRNA exerts an enormous function on tumor progression and tumorigenesis, including HNSCC, and immune-related lncRNA has been reported in tumor typing. Two different microenvironment-based subtypes with the characterization of exhausted or active immune response markers in HNSCC were identified . ImmLnc Although there are important discoveries revealed by these results, there are still limitations. First, lncRNA needs to be researched with more methods and from more different aspects, which may offer more new therapeutic targets to treat patients who suffer from HNSCC. Secondly, it is not quite enough to just validate the relationship of immune-related lncRNA and HNSCC with independent patient data. Thus, it is absolutely critical to verify the effect of immune-related lncRNA in HNSCC with more patient datasets for accelerating clinical application.Overall, we have identified two molecular subtypes that were closely related to clinical outcomes in HNSCC patients based on the immune lncRNA.