Tropheryma whipplei has been suggested as one of the causative agents of diarrhea among children worldwide. Limited data is available on the prevalence of T. whipplei among children with diarrhea in most countries such as Iran. This study was conducted to evaluate the prevalence of T. whipplei in children with acute diarrhea in Iran.Recently, T. whipplei using the SYBR Green Real-time PCR method. Positive T. whipplei samples were finally confirmed by PCR Product sequencing.In this study, the stool samples were collected from 130 children under 10 years old with acute diarrhea from children's hospitals in Tehran city. Genomic DNA was extracted from stool samples and was tested for the presence of DNA of T. whipplei, which were confirmed by sequencing. 66.67% of positive cases were males. The duration of diarrhea in infected children with T. whipplei (83.3%) was significantly longer  compared to children with negative results (45.8%). Other demographic factors and clinical signs had not a statistically significant relationship with T. whipplei infection.The mean age of participants was 32.5 months, and 54.6% of children were female. Using the SYBR Green Real-time PCR, 9.23% (12/130) of samples were positive for T. whipplei was detected in stool samples of children with acute diarrhea. The results indicated that T. whipplei could be associated with childhood diarrhea in Iran. The health care system and physicians should be aware of the presence of T. whipplei infection in Iran, especially in childhood diarrhea.In this study, The online version contains supplementary material available at 10.1186/s12879-022-07198-5. Tropheryma whipplei is a road shape Gram-positive bacterium that causes Whipple’s disease. The human intestine is the only known natural site of this microorganism. After the colonization of T. whipplei in the intestinal mucosa, it is taken up by macrophages cells and then replicates inside them [ide them .T. whipplei has been detected in various biological specimens of infected people, including saliva, urine, blood, heart valve, myocardium, synovial fluid, skeletal muscle, feces, skin, lymph nodes, lung, bronchoalveolar fluid, stomach, spleen, liver, larynx, small intestine, colon, maxillary sinus, cerebrospinal fluid, and aqueous humor. T. whipplei can persist in vectors for several years, and asymptomatic carriers can become a large reservoir for infecting others [T. whipplei infection in humans, including classic Whipple disease, chronic localized infection, acute infection, and asymptomatic form [T. whipplei is often without gastrointestinal involvement. However, it can be associated with encephalopathy, endocarditis, respiratory problems, lymphadenopathy, osteoarticular, and uveitis [T. whipplei caused acute infections, including gastroenteritis, bacteremia, or pneumonia [T. whipplei infection, diagnosis is often difficult because there are no specific endoscopic findings, and this bacterium is not detectable by routine stool culture. The histopathology and PCR methods are the most well-known T. whipplei diagnostic approaches [T. whipplei in various specimens , and it is a fast, specific, sensitive, and cost-effective method [T. whipplei in patients. However, misdiagnosis or improper treatment, which especially occurs in patients without the classic symptoms, can lead to lethal outcomes or irreversible neurological damage. Sometimes, patients with early symptoms may also be hard to treat due to the presence of bacteria in an inaccessible niche for the antibiotic, antibiotic resistance, or reinfection in late relapses after antibiotic treatment. Where combination therapy with antibiotics is helpful, immunosuppressive treatment can be a risk factor for displaying or aggravating Whipple's disease [T. whipplei is one of the infectious causes of diarrhea in children. The results of these studies showed that the prevalence of T. whipplei was more in the stool of children with diarrhea than healthy children as a control group. For instance, using the qPCR method, stool prevalence of T.whipplei was 27.5% among children with diarrhea from Ghana. That case group carried T.whipplei in their stool twice as frequently as controls without diarrhea [T. whipplei was identified in 15% of stool samples collected from children with gastroenteritis using the qPCR method. In addition, T. whipplei antibody was reported in the case group. None of the control cases were positive for the presence of bacterium or antibody. Interestingly, T. whipplei was not detected in the stool samples of children after recovering from diarrhea [T. whipplei should be considered as one of the potential causes of diarrhea in children. Since clinical symptoms are not sufficient for T. whipplei diarrhea diagnostic, molecular tests are essential to confirm this disease. In Iran, similar to most countries of the world, there is no comprehensive survey on the prevalence of T. whipplei, particularly in children with diarrhea. Therefore, the present study was performed to evaluate the presence of T. whipplei in stool samples of children with acute diarrhea using molecular methods.g others . Whippleg others , 4. Thertic form . The clatic form . All the uveitis . Recentlneumonia . Althougproaches , 7. Howee method . In addie method . Early d disease , 9, 10. diarrhea . In anotdiarrhea . TherefoOne hundred and thirty children under 10 years old with acute diarrhea who were referred to three hospitals in Tehran province  from May 2018 to November 2018 were included in this cross-sectional study. The questionnaire form containing demographic, clinical, and laboratory information was completed at admission time.Diarrhea stool samples were obtained from included children with acute diarrhea. Sampling was performed under an identical process and according to the hospital guidelines and regulations. A healthcare professional explained to the parents how to collect the stool of their children. Briefly, fresh diarrhea stools (10 gr) were collected inside a completely clean, dry, and sterile container with a tight screw lid. Urine was not mixed up with stool.Stool samples were transferred to the laboratory under the cold chain conditions immediately. Future analysis was performed as soon as possible; otherwise, specimens were sore in −20 until the investigation.Genomic DNA was extracted from stool samples using the QIAamp DNA Stool Minikit , according to the manufacturer's recommendation. 220 mg of each stool sample was used for DNA extraction.To determine quality control of extracted DNA from stool samples as well as control DNA, the DNA absorbance at 260 nm and 280 nm was absorbed using a microplate spectrophotometer . Absorbance ratios of 260/280 nm around 1.8 were considered as good quality. Higher or lower values were considered as RNA or protein contamination. In addition, extracted DNAs were be run on gel agarose to determine quality.The extracted DNA was stored at −20 ℃ until the future test.T. whipplei.All extracted DNA samples were tested using SYBR Green quantitative real-time PCR (qPCR) for the diagnosis of the WiSP protein family gene of T. whipplei DNA was provided from IRCCS Sacro Cuore Don Calabria Hospital in Italy and applied as the positive control. Sterile distilled water was used as the negative control. The mixture was run in the Corbett 6000 Rotor-Gene system  under the following temperature program: Initial denaturation step at 95 °C for 15 min, and then 40 cycles of 95 °C for 15 s, 30 s at 60 °C, and 72 °C for 30 s. After amplification, the melting protocol was followed by raising the temperature slowly from 65 to 97 °C to differentiate the specific and non-specific amplified amplicons. The samples with suitable amplification and melting (80 ± 0.5 °C) curves were considered positive for T. whipplei.Tws1F (5′-AGAGAGATGGGGTGCAGGAC-3′) and Tws1R (5′-AGCCTTTGCCAGACAGACAC-3) were used as forward and reverse primers , respectively. The qPCR mixture included 10 μl RealQ Plus 2 × Master Mix Green Low ROX™ , 1 μl from each primer (900 nmol), and 4 μl of extracted DNA (11). The mixture was reached to 20 μl with sterile distilled water. T. whipplei positive samples, PCR products were sequenced using the Sanger sequencing method. Finally, The nucleotide sequences were blasted in GenBank (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and compared with the deposited wips genes of T. whipplei in the GenBank.To confirm Statistical analysis was performed using SPSS software version 18  for the data of this study. Chi-squared and Fisher exact tests were used to compare the variables. A p-value less than 0.05 was considered statistically significant.One hundred and thirty children with acute diarrhea were included in this study. The mean age of children was 32.5 ± 31.6 months; 45.4% were male, and 54.6% were female. The mean frequency of diarrhea was four times a day. The mean duration of diarrhea was 14.6 days, and the median was 3 days. Also, 42.3% of children had a fever, and 5.38% had an underlying disease (Table T. whipplei was detected in 9.2% (12/130). Then, the positive results were sequenced to confirm. The amplification and melt curves obtained from SYBR Green real-time PCR for Tropheryma whipplei detection based on the wips gene are shown in the Additional file Extracted DNAs had good quality in 260/280 measurement (ratios around 1.8) and on gel agarose. Using the SYBR Green qPCR method, T. whipplei, 66.7% were male, 58.3% were less than 32 months, and 83.3% had diarrhea for more than 3 days. Also, 66.7% of positive children had diarrhea more than four times a day. People with positive results (83.3%) had significantly more diarrheal days than people with negative results (45.8%) . No correlation was found between positive results and other demographic factors and clinical signs  during 2018 were positive for T. whipplei using the qPCR method. As far as we know, except for a recently reported study, there have not been any studies on T. whipplei in Iran so far. In that study, 10% of immunocompromised children in a province (Qom) in Iran had T. whipplei DNA (using qPCR) in their stool samples [T. whipplei is a hard-growing bacterium, bacterial culture requires a long incubation time that is not available in many laboratories. Therefore, molecular methods are a preferred method to identify T. whipplei infection in patients [T. whipplei in stool specimens [Although  disease . T. whip samples . Since Tpatients , 15. Forpecimens .T. whipplei is much lower than similar studies conducted in other countries, including Ghana 27.5% [T. whipplei in children is significantly higher in tropical regions, particularly in countries with low levels of health [T. whipplei infection with history of travel, contact with animals, family history of diarrhea, and a set of clinical and laboratory findings. However, diarrhea for ≥ 3 days was observed among 83.3% of children with T. whipplei infection. In contrast, only 45.8% of children with negative T. whipplei experienced prolonged diarrhea. According to our results, it seems that T. whipplei might cause prolonged diarrhea compared to previously known bacterial agents causing diarrhea.This study shows that the prevalence of na 27.5% , France na 27.5% , Gabon 4na 27.5% , Laos 48na 27.5% , and Senna 27.5% . VariousT. whipplei is an unknown causative agent of acute diarrhea in children among physicians and laboratories in Iran, our results, even with this reported prevalence, can be a warning to inform physicians and laboratories of the existence of this disease in Iran. Considering that T. whipplei may colonize all ages asymptomatically, future epidemiological studies are required to assay the association of T. whipplei with acute diarrhea in children in different parts of the world, including Iran. In addition, diagnosis, control, and treatment of T. whipplei should be considered by health systems in Iran and even other countries.Since T. whipplei in patients with diarrhea. In addition, this study did not include a control group (children without diarrhea) which made result interpretation more difficult. Another limitation was that we did not measure possible antibodies produced against T. whipplei. Furthermore, no other clinical microbiology diagnosis was performed to identify other enteric pathogens in positive samples of T. whipplei. These data could help to interpret of obtaining results in the present studies.One of the limitations of the study is the small number of samples. Future studies among a larger population with different demographic and clinical characteristics can determine comprehensive knowledge on the frequency of T. whipplei among children with acute diarrhea in Iran was 9.23% using the qPCR method. Significantly, children with T. whipplei infectious diarrhea had prolonged diarrhea than children without T. whipplei infection (83.3% vs. 45.8%). Therefore, T. whipplei could be a potential cause of acute diarrhea in children in Iran, and physicians and laboratories should be aware of the presence of this disease in Iran. However, comprehensive studies are required to evaluate the epidemiology and pathogenicity of T. whipplei in pediatric diarrhea in Iran. This study showed the importance of qPCR in the diagnosis of T. whipplei infection as a possible cause of diarrhea in children. Therefore, molecular diagnosis of T. whipplei is recommended, especially when diarrhea lasts more than 3–4 days.In this study, the stool prevalence of Additional file 1: Figure S1. Representative the amplification plot (A) and melting curve (B) of SYBR Green real-time PCR for Tropheryma whipplei detection based on the wips gene in specimens. Positive and negative controls were run in all runs. A) The cycle number is shown on the x-axis and change in fluorescent intensity is shown on the y-axis. B) Positive control and samples have a melt pick in 80 ± 0.5 °C in melting curves.


Hodgkin's lymphoma (HL) with skin involvement is reasonably rare. It typically occurs late in the course and is associated with a poor prognosis; however, it may also be indolent in some cases.We report a case of a 15‐year‐old previously healthy male with Hodgkin's lymphoma who presented with multiple lymphadenopathies of axilla and serpiginous ulcerative nodular lesions involving pectoral skin. A lymph node biopsy was performed following an initial diagnostic workup for a suspected active infectious disease, which revealed a neoplastic invasion from a mixed cellularity classical HL with skin involvement. A total of six cycles of ABVD  chemotherapy regimen was administered to the patient.In comparison to other studies, this case demonstrates that a good response is possible with standard ABVD chemotherapy. This was associated with an on‐and‐off fever, night sweats, and anorexia with more than a 6‐kg loss of weight over the previous 3 months. The patient also had bilateral cervical and axillary lymphadenopathy, showing no signs of improvement after a broad‐spectrum antibiotics treatment prescribed by the general practitioner. He also took anti‐tubercular treatment for approximately 2 months but failed to find any relief. It is important to note that there was no significant family history of a similar disease.2.22 to 6 × 5 cm2 on the anterior chest wall, protruding right mammary region, collection intercostals space  with neutrophilia (77%), platelets (612 000/mm3), C‐reactive protein , and erythrocyte sedimentation rate . The viral screen was negative for human immunodeficiency virus (HIV) and hepatitis B surface antigen (HBsAg). No acid‐fast bacillus (AFB) was seen on AFB testing (Table Laboratory data reported an increased white blood count (23 860/mmatelets 62 000/mm32.42) and left axillary node (3.2 × 3.0 cm2). Multiple ill‐defined soft tissue deposits were seen in the anterior chest wall muscles, and necroses were noted within these deposits. Multiple well‐defined cutaneous soft tissue deposits were also seen in the chest wall, and the largest measure was 4.0 × 2.5 cm2. Ulceration was noted within a few of the lesions. Nodular pleural thickening was also observed in the bilateral upper hemithorax. No evidence of pleural effusion was found.A whole‐body CT scan revealed prominent bilateral level II and V nodes in head and neck regions. Multiple enlarged left supraclavicular, bilateral axillary, subpectoral, deep pectoral, bilateral internal mammary, and prevascular nodes in the chest were observed where the largest left internal mammary nodal mass extending into the left chest wall (6.0 × 5.5 cmPositron emission tomography (PET) scan revealed increased 18‐fluorodeoxyglucose (FDG) uptake involving enlarged left supraclavicular node (SUV max 10.4), axillary/subpectoral , prevascular (SUV max 8.6), paraaortic (SUV max 7.1) anterior cardiophrenic (SUV max 9.1), and supradiaphragmatic (SUV max 8.1) nodes, large pleural based deposit in the left internal mammary region (SUV max 11.5), pleural based deposits in both hemithorax (SUV max 9.3), bilateral pectoralis muscle , and cutaneous and subcutaneous deposits in the anterior chest wall (SUV max 9.3) Figure . Imaging2.5Histopathological examination of the right axillary lymph node and nodular lesions on the chest wall by excision biopsy got the impression of atypical lymphoid infiltrates, consistent with lymphoma Table . Immunoh2.6doxorubicin, bleomycin, vinblastine, dacarbazine), that is, doxorubicin 25 mg/m2, bleomycin 10 mg/m2, vinblastine 6 mg/m2, and dacarbazine 375 mg/m2 as days 1 and 15 schedules and to be repeated after every 28 days for six cyclesThere is no specific treatment for HL with skin lesions. Standard treatment is chemotherapy with ABVD/ BEACOPP/Stanford V with or without involved field radiation depending on the stage and bulk of the disease.2.7Overall, HL has a good prognosis with greater than 80% 5‐year survival.3HL is responsible for 0.58% of all diagnosed cancers, where a slight male predominance exists (1.1:1). HL incidence has a bimodal peak depending on age, early peak, from 25 to 30 years of age, and a second peak, from 75 to 80 years of age.The extent of intrathoracic spread can be measured by CT scanning of the chest. An abdominal and pelvic CT scan should be performed to assess enlarged nodes in the retroperitoneal area, hepatosplenomegaly, or focal nodules in the spleen or liver.While cutaneous Hodgkin lymphoma is fairly rare, it should be considered among the atypical causes of non‐specific papules, plaques, and ulcers, necessitating the consultation of a hematopathologist. In contrast to the majority of recorded cases of HL affecting the skin, our case shows that a successful response can be achieved with appropriate therapy.The authors declare no conflicts of interest.Conceptualization, Su.G., Sa.G., N.S.P., S.C.; Data Curation, Su.G., Sa.G.; Validation, Sa.G., R.J.A., F.C.; Writing ‐ Original Draft, Su.G.; Writing ‐ Review & Editing, Sa.G., R.J.A., F.C., N.S.P., P.P., S.C.; Visualization, S.C.; Supervision, P.P., S.C.All authors had full access to the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient has given his consent for his images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published, and due efforts will be made to conceal their identity.Data S1. Supporting information.Click here for additional data file.


Scutellonema brachyurus was recovered from soil samples collected around the rhizosphere of wild grass in the North West and Limpopo provinces. This species characterized by a hemispherical lip region with four to six annuli, basal lip’s annuli with longitudinal incisures, body length 696–904 µm , stylet 21–27 µm length, tail rounded with 10–19 µm length and spermatheca nonfunctional and male absent. The nblast analysis based on the D2-D3 segment of 28 S rDNA placed South African populations of S. brachyurus with 98% similarity to Greece (KU059494) and 99% similarity to South African (JX472052) S. brachyurus. Besides, nblast of COI of mtDNA showed 98% similarity of the test species with South African populations of S. brachyurus . The phylogenetic analysis put the South African populations of S. brachyurus together with other S. brachyurus with a 100 posterior probability support. Besides, the measurements, line illustration, and scanning electron microscopy photographs are provided for S. brachyurus from South Africa.During a survey on plant-parasitic nematodes from South Africa,  Scutellonema was established by Scutellonema with its type species S. bradys  to study new populations of S. brachyurus using morphology, and (2) to study the phylogenetic position of South African S. brachyurus using 28 S rDNA and mtDNA.The present paper reports Rhizosphere soil samples were collected from the natural grass from North West and Limpopo provinces of South Africa. Nematode extraction was achieved using the Measurements were taken of specimens mounted on permanent slides, and Specimens preserved in glycerine were selected for observation under SEM, according to S. brachyurus populations, principal component analyses (PCA) with different morphological traits were conducted. PCA analyses were carried out in XLSTAT , and ddH2O for a final volume of 30 μ l. The amplification was processed using an Eppendorf master cycler gradient , with the following program: initial denaturation for 3 min at 94°C, 37 cycles of denaturation for 45 s at 94°C; 56°C and 52°C annealing temperatures for 28 S rDNA and COI, respectively; extension for 45 s to 1 min at 72°C, and finally an extension step of 6 min at 72°C followed by a temperature on hold at 4°C. After DNA amplification, 4 μ l of product from each tube was loaded on a 1% agarose gel in TBE buffer  for evaluation of the DNA bands. The bands were stained with RedGel and visualized and photographed on a UV transilluminator. The amplicons of each gene were stored at –20°C. Finally, the PCR products were purified for sequencing by Inqaba Biotech (South Africa). Also, Hoplolaimus galeatus , MW504475  and MW504476 . The partial COI of mtDNA was deposited in GenBank under accession number MW509624 .DNA extraction was done using the Chelex method . Five sp,000 rpm . The sup,000 rpm , and JB3,000 rpm , were usgaleatus  Thorne, galeatus  and aliggaleatus . The lengaleatus . The GTRgaleatus . The selsoftware . The parMaterial examined: eight females in good condition.Measurements: see ca eight blocks , without sperm, ovaries with a single row of oocytes. Intestine not overlapping the rectum. Rectum 13–15 µm long. Scutellum moderate, 3.9 ± 0.6 (3.2–4.4) µm, two to three annuli anterior or three to six annuli posterior to the anus. Tail short, rounded, 12.8 ± 1.4 (10.5–14.0) µm long, with 7–14 annuli at ventral side. Hyaline at tail tip 3.5 ± 0.7 (2.8–4.6) µm wide.Body spiral after heat relaxation. Lip region slightly offset with body contour, with bearing three  to five Not found.S. brachyurus were collected in North West Province , and Limpopo Province , around the rhizosphere of natural grass.Specimens of S. brachyurus with the original description , o (r = 0.913), and DGO (r = 0.816) were responsible for the significant variability of the F1 (r = 0.931) had the most contribution to the variability and showed a positive and high correlation  of S. brachyurus. Compare with a population of S. truncatum (acc. nr: KX959272) showed a 90% similarity. Regarding COI of mtDNA, Nblast showed 98% similarity with South African populations of S. brachyurus . Compared with S. truncatum from Benin (MG973138), our sequence showed 79% similarity, whereas another population of S. truncatum (KX959307) from Botswana showed 82% similarity with tested S. brachyurus from South Africa. Genetic pairwise distance  having 0.01–0.03, whereas the genetic distances among the type B (South African populations) ranges from 0.06–0.07. In addition, S. clavicaudatum showed the same genetic distance estimation with type B of S. brachyurus.The genes 28 S rDNA and COI of mtDNA for distance  for 28 SScutellonema species  S. transvaalenseS. paralabiatumS. truncatumS. clavicaudatum and S. brachyurus with 1.00 posterior probability.The Bayesian inference tree of 28 S rDNA of  species  grouped Scutellonema species  S. clathricaudatum with 1.00 posterior probability; (III) S. cavenessi with 1.00 posterior probability; (IV) S. paralabiatum with 1.00 posterior probability; (V) S. truncatum with 1.00 posterior probability; (VI) S. transvaalense with 1.00 posterior probability; and (VII) S. brachyurus with 0.63 posterior probability.The Bayesian inference tree of COI of mtDNA of  species  grouped S. brachyurus populations, together with the other S. brachyurus. Additionally, based on 28 S rDNA phylogeny, S. brachyurus and S. clavicaudatum place together in a clade with support of a 1.00 posterior probability value. However, they clearly distinguish in lip region (lips annuli present in S. brachyurus vs lips annuli absent in S. clavicaudatum) and tail . The lip region is differentiated into small blocks in S. brachyurus, whereas six large rectangular blocks are present in S. clavicaudatum  and S. clavicaudatus share the same range (0.07–0.08). Based on ITS and 28 S rDNA phylogeny, these two species are placed within a clade  and B (0.06–0.07) are different. Overall, the molecular study using 28 S rDNA and COI of mtDNA indicated that different populations of S. brachyurus place under the same clade. However, populations that belong to this species make a cryptic species need to study the populations deeply. The 28 S rDNA and COI of mtDNA markers demonstrated an admirable option for diagnosing species belong to Scutellonema. However, type A and B phylogenetic position remains unresolved, as indicated by S. brachyurus were deposited in the Nematology laboratory collection of the University of Limpopo, South Africa. In conclusion, the ecological behavior of S. brachyurus in terms of the economic impact on the crops needs to be investigated.The PCA results  also sho


Voc) deficit, which affected their energy conversion efficiency. Strategies to improve on the properties of this material such as alloying with other elements have been explored and have yielded promising results. Here, we report the synthesis of CZTS and the partial substitution of S with Te via anion hot injection synthesis method to form a solid solution of a novel kesterite nanomaterial, namely, copper zinc tin sulfide telluride (CZTSTe). Particle-size analyzed via small angle X-ray scattering spectroscopy (SAXS) confirmed that CZTS and CZTSTe materials are nanostructured. Crystal planes values of 112, 200, 220 and 312 corresponding to the kesterite phase with tetragonal modification were revealed by the X-ray diffraction (XRD) spectroscopic analysis of CZTS and CZTSTe. The Raman spectroscopy confirmed the shifts at 281 cm−1 and 347 cm−1 for CZTS, and 124 cm−1, 149 cm−1 and 318 cm−1 for CZTSTe. High degradation rate and the production of hot electrons are very detrimental to the lifespan of photovoltaic cell (PVC) devices, and thus it is important to have PVC absorber layer materials that are thermally stable. Thermogravimetric analysis (TGA) analysis indicated a 10% improvement in the thermal stability of CZTSTe compared to CZTS at 650 °C. With improved electrical conductivity, low charge transfer resistance (Rct) and absorption in the visible region with a low bandgap energy (Eg) of 1.54 eV, the novel CZTSTe nanomaterials displayed favorable properties for photovoltaics application.Metal chalcogenides such as copper zinc tin sulfide (CZTS) have been intensively studied as potential photovoltaic cell materials, but their viability have been marred by crystal defects and low open circuit potential ( Eg) value of 1.5 eV, which is optimal for semiconductor materials for photovoltaic (PV) application [Eg of the material which is brought about by the change in the anion species [Eg of the solid solution has the advantage of matching the energy corresponding to the maximum efficiency in the Shockley–Queisser plot for a single absorber cell [Eg of the material from 1.5 to 2.1 eV makes it suitable for constituent-element substitutions [Eg values of the kesterite materials of chemical composition CZTX . The Eg trend is as follows: Eg > Eg > Eg. This type of Eg trend has been observed in germanium-substituted kesterite materials (Cu2ZnGeX4) for which Eg(Cu2ZnGeS4) > Eg(Cu2ZnGeSe4) > Eg(Cu2ZnGeTe4) [Eg value. For example, Eg > Eg [The use of kesterite, which is a metal chalcogenide , as in the development of the absorber layer of a solar cell has been extensively explored ,2. The mlication . Among tlication ,8,9. Witlication . The anilication ,12,13. Plication . This hi species . The Eg ber cell . The tunitutions ,17. Thisitutions ,19. In rZnGeTe4) . HoweverTellurium has been used specifically in the solar cell industry due to its metalloid and semiconductor properties ,22; for All the reagents were of analytical grade and no further purification was performed. The following reagents used were purchased from Sigma-Aldrich, Johannesburg, South Africa: copper (II) chloride , zinc chloride , tin (II) chloride , isopropanol , diethylene glycol , sodium borohydride , Te powder , purity 99.5%), and S powder .et al. [This work reports a modified hot-injection technique of Dong et al.  for the −1 with a sample size of 1 mg. The structure of the synthesized materials was studied with a Spectrum II Perkin-Elmer Fourier Transform Infrared (FTIR) Spectrometer  for 400 - 4500 cm−1. An Auriga field emission scanning electron microscope (FESEM) that was fitted with an energy-dispersive X-ray spectrometer (EDS)  and operated at an acceleration voltage of 200 kV, was used for morphological and elemental and morphological analyses of samples. A Field Electron and Ion Company (FEI) Tecnai G2 F20X-Twin MAT 200 kV Field Emission Transmission Electron Microscope  with energy-dispersive X-ray spectrometry (EDS) and selected area electron diffraction (SAED) capabilities, was used to perform high-resolution transmission electron microscopy (HR-TEM) experiments. All the electrochemical data were obtained with PalmSens 4 , which was connected to a 0.071 cm2 glassy carbon disk working electrode, platinum wire (Pt) counter electrode and silver–silver chloride  reference electrode in a 10 mL electrochemical cell. Electrochemical analysis of each of the nanoparticles (CZTS or CZTSTe) was performed in 3 mL 0.1 M tetrabutylammonium phosphate-lithium perchlorate (TBAP-LiClO4) electrolyte.Absorption studies in the wavelength region of 300–900 nm at room temperature were observed via Varian Cary 300 Ultraviolet-Visible- Near-infrared (UV-Vis–NIR) Spectrophotometer . X-ray diffraction (XRD) analysis was performed using a Philips PW 1830 X-ray diffractometer  with Cu Kα radiation at wavelength of 1.5406 Å. MATCH and DIAMOND softwares  were used in analyzing the XRD data, as well as the simulation of the crystal structures of the synthesized nanoparticles. Simultaneous thermogravimetric analysis (TGA) and differential thermal analysis (DTA) analyses were performed with a TGA 4000 Thermogravimetric Analyzer from Perkin-Elmer . The TGA/DTA measurements were performed at a temperature range of 30 - 600 °C under nitrogen gas atmosphere and a heating rate of 10 °C minThe discussion of the experimental results of CZTS and CZTSTe nanomaterials are categorized into two. The first section  involves1.92Zn1.0Sn0.8S4.26 for CZTS and Cu1.91Zn1.0Sn0.644 for CZTSTe. These chemical compositions were in close conformity to the general chemical composition of Cu2ZnSnS4 for CZTS and Cu2ZnSn4 for CZTSTe [In the synthesis of nanoparticles, it is important to control the growth and dispersity . Emphasir CZTSTe .Significant morphological data can be obtained by utilizing the small-angle X-ray scattering (SAXS) technique ,33. SAXSν(O–H) 3753.93 cm−1) [ν(C–H) 2897.60 cm−1 [ν(C–C) 1620.50 cm−1 [ν(C–O) 1107.04 cm−1 [ν(S–S) 569.78 cm−1 [ν(O–H) 3434.03 cm−1, ν(C–H) 2897.60 cm−1, ν(C–C) 1620.50 cm−1, ν(C–O) 1107.04 cm−1, ν(Te=O) 715.79 cm−1 [ν(S–S) 569.78 cm−1. The presence of S in both nanomaterials can be seen from the vibrational band at 569.78 cm−1. The Te=O vibrational band at 715.79 cm−1 in the spectrum of CZTSTe, confirms the successful integration of Te into the structure of the newly synthesized nanomaterial. The vibration modes of the organic functional groups the spectra are due to the remnants of the solvents (diethylene glycol and isopropanol) used for synthesis [Fourier transform infrared spectroscopy (FTIR) was used for the determination of bonding characteristics of the kesterite nanomaterials . The vib93 cm−1) , ν(C–H) .60 cm−1 , ν(C–C) .50 cm−1 , ν(C–O) .04 cm−1 ,32 and ν.78 cm−1 ,36. CZTSynthesis . The sol2θ = 28.37°, 32.94°, 47.23° and 56.31°, which correspond to the crystal interplanar spacing or crystal lattice spacing (d-spacing) values of 3.1346, 2.7091, 1.9230 and 1.6406 Å, respectively. The patterns for CZTSTe had major peaks at 2θ = 28.26°, 32.84°, 47.19° and 56.08°, with d-spacing values of 3.1330, 2.7072, 1.9225 and 1.6379 Å, respectively. These peaks were identified using Joint Committee on Powder Diffraction Standards (JCPDS) card no. of 00-026-0575. These peaks corresponded to the 112, 200, 220 and 312 planes of the kesterite phase, respectively, which has a tetragonal modification [a, b and c  and were α, β and γ  were evaluated for the nanomaterials. The values obtained for CZTS were a = b = 5.43682 Å and c = 10.8388 Å, while CZTSTe had values of a = b = 5.44221 Å and c = 10.8578 Å. The α, β and γ values were 90° for CZTS and CZTSTe. These values of the lattice parameters are in good agreement with values for the tetragonal kesterite phase for which a = b = 5.430 Å and c = 10.8680 Å [The most versatile technique employed for determining the crystal structure of materials is X-ray diffractometry (XRD). This spectroscopic method allows for the identification of chemical compounds from their crystal structure. fication ,39. The XRD data can provides useful the information for estimating the crystal size of materials. For this purpose, the Debye–Scherrer equation (Equation (1)), was used to calculate the crystal size of the materials:D is the mean size of the crystal in Å; k is a dimensionless shape factor with value of ~ 0.9; λ is the X-ray wavelength; β is the line broadening, i.e. the full width at half maximum (FWHM) of the peak in radians; and θ is the Bragg angle in radians. The calculated crystallite size for the most prominent peak (112 planes) of both nanomaterials was 106.6 Å (~11 nm) for CZTS and 310.2 Å (31 nm) for CZTSTe. Parameters used for calculating the crystal size are summarized in In Equation (1) 2θ value) as binary, secondary and ternary phases, such as for CuS, ZnS and Cu2SnS3 [−1, 281 cm−1, 347 cm−1 and 400 cm−1, which are attributed to SnS, CZTS, CZTS, and ZnS, respectively [−1, 149 cm−1, 318 cm−1 and 395 cm−1, which originated from CZTTe, CZTTe, CZTS and ZnS, respectively [Earlier studies have shown that XRD is not enough to categorically establish the presence of the desired kesterite phase, as its characteristic peaks appear at the same peak position  is a useful technique for structural and crystal defects analyses. Monocrystalline materials give rise to SAED micrographs that have distinct spots and well-defined lattice fringes corresponding to specific planes . The SAE−1 in argon atmosphere. The initial weight loss of about 9% for CZTS and 4% for CZTSTe at about 100 °C, is attributed to the evaporation of water. Further 4% weight loss for CZTS and 5% for CZTSTe at about 200 °C is due to the evaporation of isopropanol and diethylene glycol which were synthesis solvents. Further heating to 400 °C resulted in a ~14% weight decrease for CZTS and ~13% for CZTSTe due to the loss of superficial chalcogens. The loss of Sn by the sublimation of SnS at temperatures above 400 °C, caused the weight to decrease by 9% and 8% for CZTS and CZTSTe, respectively. These finding are similar to the pattern reported in previous studies [MP = 449.51 °C, BP = 988 °C and κ = 3 W/mK) is more thermally stable than S  [MP, BP and κ are melting point, boiling point and thermal conductivity, respectively.  studies . The TGA05 W/mK) ,43,44; wUltraviolet visible spectroscopy (UV–Vis) technique was used to study the absorption properties of the nanoparticles. The spectra obtained are plotted in αhν)2 vs. hν, where hν is the photon energy and α is the absorption coefficient of the material, is used to determine their bandgap (Eg) values. The Tauc plots for CZTA and CZTSTe are presented in Eg values are 1.76 eV and 1.54 eV, respectively. The incorporation of Te to kesterite nanomaterial narrowed the band gap towards the optimum Eg value (1.5 eV) required for solar cell application. It has been reported that a high S content in kesterite increases the Eg value, due to its insulating properties [Eg value obtained by the inclusion of Te in the kesterite, implies that it allows very good absorption of the visible light as required for photovoltaic application [Eg value by the addition of Te and other metal chalcogenides into the kesterite structure [Eg value obtained for CZTSTe nanomaterial, was due to quantum confinement effects which increases the absorption wavelength [For direct band gap materials such as kesterites, the Tauc plot  and cathodic (1c) peaks, and an addition anodic peak (2a) for CZTSTe. The anodic peak potential (paE) values for CZTSTe are 0.97 V and 1.32 V for 1a and 2a, respectively; while the cathodic peak potential (pcE) value is 0.79 V for 1c. The paE values of 0.97 V corresponds to the oxidation of copper [2a) corresponds to the oxidation of Te2- to Te(IV) [pcE values of 0.79 V is attributed to the reduction of copper. For CZTS paE and pcE values are 0.96 V (for 1a) and 0.82 V (for 1c), respectively, and refer to the oxidation and reduction of Cu. The value of the anodic peak current/cathodic peak current, paI(1a)/pcI(1c), is ~1 for both materials, which indicates a reversible fast electron transfer process for the Cu+/Cu2+ electrochemistry. However, the formal potential, 0E′ = (paE + pcE)/2, values for CZTS and CZTSTe are 0.89 V and 0.88 V, respectively, and the currents density of CZTSTe is by a factor of 1.3 larger than that of CZTS, showing an improvement in the rate of the charge transfer reaction, which is possibly, due to the inclusion of Te in the kesterite [f copper ,56 and to Te(IV) ,58. The esterite ,60. 2+ to Cu+ (as discussed for 2−.The square-wave voltammetry (SWV) was used to study the electrochemical stability of the materials . The resRs is solution resistance, constant phase element is the CPE and Rct is charge transfer resistance. The values obtained for the parameters as written in Rct values obtained for CZTSTe and CZTS are 3.9 kΩ and 9.8 kΩ, respectively, implying greater conductivity properties of CZTSTe. The Bode −phase angle (−θ) and log , log Z, plots of the EIS data are contained in the Z value calculated for CZTS (16.6 kΩ) is significantly higher than that of CZTSTe (5.5 kΩ). The −θpeak values of CZTS (67.5°) and CZTSTe (60°) confirm that both materials are semiconductors [νpeak) values are CZTS (45.71 Hz) and CZTSTe (21.38 Hz). The lower νpeak value for CZTSTe indicates its greater propensity for interfacial faster charge transfer reaction compared to CZTS.The electrochemical impedance spectroscopy (EIS) data of CZTS and CZTSTe are presented in nductors ,51. HoweRct, νpeak and Z values obtained from both the Nyquist and the Bode analyses, point to an improved electro-conductivity of CZTSTe compared to CZTS. A similar trend was observed in the CV analysis of the materials, where the cathodic and anodic peak current densities of CZTS were by a factor of 1.3 lower than those of CZTSTe, due to the greater conductivity of the Te-containing material. Literature antecedents also, confirmed that the conductivity of metal chalcogenide materials improved as elements down the group 16 of the periodic table are incorporated in the material, with Te being the most effective [The ffective . A novel CZTSTe nanostructed kesterite material was successfully synthesized through the anion hot injection method, which is a non-toxic solid solution process. The size, shape and structure of the material were confirmed with SAXS, SEM, TEM, Raman and XRD spectroscopy experiments. The incorporation of Te in the CZTSTe nanomaterial improved the band gap energy, electro-conductivity, rate of charge transfer reaction and thermal stability; and brought about a redshift in its absorption characteristics. The improvement in the photophysical properties implies that the novel kesterite material holds much promise as an excellent material for solar cell application. The improved electrochemical impedance spectroscopy characteristics of CZTSTe, as deduced from the values of its Bode-phase angle and Bode-total impedance parameters, make it a very viable material for photovoltaic and photocatalytic applications.


Eupatorium fortunei Turcz. is a widely used Chinese herbal medicine in China. In this study, a gas chromatography-triple quadrupole mass spectrometry (GC-MS/MS) method was developed and validated to simultaneously determine the contents of p-cymene, thymol, neryl acetate, and β-caryophyllene in roots, stems, and leaves of Eupatorium fortunei Turcz. harvested at different growth periods. All four constituents could be detected in leaves, three could be detected in stems except β-caryophyllene, and only thymol could be detected in roots. The order of the total contents of four constituents in different parts was leaves > stems > roots. It indicated that the leaves could be the proper medicinal parts of Eupatorium fortunei Turcz. The content of four constituents in leaves varied a lot among different growth periods and showed an M-shaped change trend with the growth of Eupatorium fortunei Turcz. The four constituents accumulated to the highest values in early July followed by mid-September. Accordingly, the best harvest time of Eupatorium fortunei Turcz. is early July and mid-September. Eupatorium fortunei Turcz., one species of the genus Eupatorium (Compositae), is a commonly used aromatic traditional herbal medicine in China. It has been applied for relieving nausea, vomiting, and poor appetite due to dampness obstruction since thousands of years ago [Eupatorium fortunei Turcz. is stipulated in Chinese Pharmacopeia, and the total volatile oil is consequently used as an important index to evaluate its quality [Eupatorium fortunei Turcz. from different regions and harvest time varied a lot [Eupatorium fortunei Turcz.ears ago . Previouears ago , 3. The  quality , while ved a lot –7. Accorp-cymene and neryl acetate were the earliest separated and identified compounds from the volatile oil of Eupatorium fortunei Turcz. [p-cymene possesses an effect on dispelling phlegm [β-Caryophyllene was reported to be the constituent with the highest content in the volatile oil and has been studied as an indicator constituent to establish the content determination method of Eupatorium fortunei Turcz. [p-cymene, thymol, neryl acetate, and β-caryophyllene  and identified by Prof. Xiaofeng Niu. Ten plants were harvested at each stage. Each plant was separated into leaves, stems, and roots. Then, the leaves, stems, and roots were separately collected together and dried.p-Cymene (purity: 99.5%) and neryl acetate (purity: 95%) were purchased from Shanghai Aladdin BioChem Technology Co., Ltd. . Thymol (purity: 99%) and β-caryophyllene (purity: 90%) were purchased from J&K Scientific Ltd. .Ethanol, ethyl acetate, petroleum ether (60°C∼90°C), and hexane were of analytical grade. μm film thickness) and triple quadrupole mass spectrometer was used (Japan). The helium of high purity (99.9999%) was the carrier gas at a flow rate of 1 mL·min−1. Samples were injected in a split mode with a ratio of 10 : 1. The temperature procedure of the column was set at 70°C for 3 min, increased to 80°C at a rate of 10°C·min−1, to 100°C at a rate of 20°C·min−1, to 130°C at a rate of 2°C·min−1, and to 280°C at a rate of 20°C·min−1, and maintained for 2 min. An electron ionization system was used with ionization energy of 70 eV. Injection, interface, and ionization temperature were set at 250°C, 250°C, and 230°C, respectively. We used standard compounds of each volatile under the MRM mode to assay the contents of four target components [A Shimadzu GC-MS 8040 equipped with SH-Rxi-5Sil MS capillary column .All measurements were performed using triplicate samples. Data were expressed as mean ± SD. All data were statistically processed using GraphPad Prism 7  and analyzed by Tukey's multiple comparisons test to calculate the difference significance at a 5% level , respectively. LODs and LOQs were calculated at a signal-to-noise of 3 and 10. Accuracy was measured by recoveries. The percentage recoveries were 105.31%, 106.41%, 99.61%, and 102.24% for p-cymene, thymol, neryl acetate, and β-caryophyllene, respectively. The repeatability of the method was determined by analyzing the standard solution 6 times. The relative standard deviations (RSD) for p-cymene, thymol, neryl acetate, and β-caryophyllene were 1.00%, 2.72%, 1.55%, and 1.59%, respectively. Six homogeneous samples were prepared and analyzed to evaluate the precisions. The RSD values of four analytes varied from 0.98% to 3.38%. For stability, the same sample was assayed separately at 0, 2, 4, 6, 8, and 12 h after extraction. The RSD values were less than 1.36%.Linearity, range, accuracy, precision, limits of detection (LOD), and limits of quantification (LOQ) were evaluated under the optimal conditions . p-Cymenp-cymene, thymol, neryl acetate, and β-caryophyllene in Eupatorium fortunei Turcz.The results demonstrated that the developed method was accurate and repeatable which could be a reliable tool for identification and quantification of The influence factors, including extraction solvent, method, and duration were investigated through single-factor experiments. Extraction solvents including ethanol, ethyl acetate, petroleum ether (60°C∼90°C), and hexane were compared, and hexane resulted in a preferable peak and baseline. Two classical extraction methods, ultrasonic extraction, and reflux, were investigated. The ultrasonic extraction showed better extraction efficiency. Then, we set the extraction duration at 15 min, 30 min, 45 min, and 60 min. The contents of compounds were increased with the prolongation of extraction time . Therefop-cymene, thymol, neryl acetate, and β-caryophyllene in different parts of Eupatorium fortunei Turcz. are listed in Samples from different growth periods and parts were assayed. The contents of p-cymene, thymol, neryl acetate, and β-caryophyllene in leaves were ranged from 1.51 ± 0.056 to 25.25 ± 2.600 μg·g−1, 20.81 ± 0.688 to 132.21 ± 3.169 μg·g−1, 13.77 ± 2.600 to 59.90 ± 1.006 μg·g−1, and 13.36 ± 2.611 to 144.21 ± 1.365 μg·g−1, respectively. β-caryophyllene could not be detected in stems. The concentration of p-cymene, thymol, and neryl acetate in stems were within the range of 1.79 ± 0.034∼5.90 ± 0.659 μg·g−1, 5.48 ± 0.043∼19.10 ± 1.729 μg·g−1, and 12.51 ± 0.115∼40.94 ± 0.182 μg·g−1, respectively. Only thymol could be detected in roots, and the contents ranged from 5.42 ± 0.547 to 32.60 ± 0.409 μg·g−1. In a previous study, it has been found that the compounds from the volatile oils extracted from Eupatorium fortunei Turcz. with the three methods were different [Eupatorium fortunei Turcz. [The contents of i Turcz. .Eupatorium fortunei Turcz. Higher content of essential oil in leaf than other organs of a plant has been previously reported for other species [Eupatorium fortunei Turcz.All the constituents could be detected in leaves, and only thymol exists in roots. The order of the contents of four constituents in different parts was leaves > stems > roots. Volatile oil exists in glandular hairs, oil rooms, oil tubes, secretory cells, or resin ducts. Volatile oil exists in different parts of different plants. Some of them are found in the whole plant, while others are found in some organs of flowers, fruits, leaves, roots, or rhizomes. The location of volatile oil varies with plant species. Hence, one can see that the studied four compounds mainly existed in leaves of  species , 23. Basp-cymene, thymol, neryl acetate, and β-caryophyllene in different parts of Eupatorium fortunei Turcz. with growth time were also analyzed. The contents of all constituents in leaf showed a trend of increase-decrease-increase-decrease with the plant growing. The content of p-cymene accumulated to its highest value on July 15. After an 80% reduction, the content rose again to 86% of the highest value approximately on September 15. Thymol reached its first peak value on August 15 and then reached the highest level on September 15. The highest value of thymol was about 6.5 times and 3.3 times higher than that on September 1 and October 1. The content of neryl acetate was the highest from July 1 to July 15, and it was also high on September 15, which is about 74% of the highest value. The yield of β-caryophyllene was the highest on July 1 and then decreased sharply. The content of β-caryophyllene increased again from 17.86 μg·g−1 on September 1 to 44.38 μg·g−1 on September 15. The contents of four constituents in the stem at each harvest stage showed different patterns. The yield of p-cymene was the highest on August 15. After a 63% reduction, p-cymene content rose again to a higher value on October 15. While the content of thymol was highest on April 15, it then decreased with the plant growth. Similarly, the content of neryl acetate reached the highest value on April 15 and had the second highest content on July 15, which was about 90% of the highest value. Only thymol could be detected in roots from June 1 to August 15, and the content was the highest on June 1.The concentrations change of Eupatorium fortunei Turcz. reached the highest value in early July, followed by mid-September. These two time points could be considered as the best time to harvest the plant.In summary, the contents of four constituents in different parts of Eupatorium fortunei Turcz. and the content of essential oil extracted from it. In other studies, it has been found that the contents of many biological active compounds in plants depend on growth time; the yields of compounds usually reach the highest at summer or flowering stages (July or August) [To the best of our knowledge, there are no studies regarding the relationship between the growth stages of  August) , 19.p-cymene, thymol, neryl acetate, and β-caryophyllene in Eupatorium fortunei Turcz. by GC-MS/MS. The contents of the four constituents in samples from different parts were assayed and distribution was analyzed. All constituents could be detected in leaves. The contents order of constituents in different parts was leaves > stems > roots. From that, we could deduce that leaves should be the best medicinal part of Eupatorium fortunei Turcz. The growth stages of the plant strongly influence the content of four analytes in Eupatorium fortunei Turcz. The four constituents accumulated to the highest value in early July followed by mid-September. Accordingly, the best harvest time of Eupatorium fortunei Turcz. is early July and mid-September.A quantitative determination method was developed and validated to assay the contents of


Nature Communications 10.1038/s41467-021-21710-6, published online 9 March 2021.Correction to: 2”. This has been corrected to “persons/0.01 km2“. The colour scale of Fig. 1b and Supplementary Figs. S3a, b, and c were previously only labelled at the boundaries between groups, and only alternate labels were provided . This has been updated so that all groups have a separate label and the range is provided ”, “”). The background colour of Fig. 1b was incorrectly shaded white, which suggested zero population density. The Figure has been updated with the correct shading of light yellow to indicate a population density of <1 person per 0.01 km2. The colour scale of Fig. 1c was previously only labelled at the boundaries between groups and only alternate labels were provided . This has been updated so that all groups have separate labels and ranges are provided . These changes have been made in both the PDF and HTML versions of the Article.The original version of this Article contained errors in Fig. 1 and Supplementary Fig. S3. The population density of Fig. 1b and Supplementary Figs. S3a, b, and c were given the incorrect units of “persons/kmSupplementary information.


Clausena lansium (Lour.) Skeels seeds have been shown to have diverse beneficial medical value due to their unique active components. This study analysed the composition of essential oils (EOs) of C. lansium seeds and investigated their potential antifungal effects against Candida strains. A total of forty-six components were identified in all samples by gas chromatography-mass spectrometry (GC–MS). The main components were sabinene, β-phellandrene and 4-terpineol. Thirteen EOs of C. lansium seeds were classified into three clusters based on their components. Cluster analysis showed that the difference between the tropics and subtropics was the greatest. These EOs and the three main chemicals showed different antifungal activities against five Candida species . The antifungal activity against C. glabrata and C. krusei was higher than that against other Candida strains. EOs of C. lansium seeds displayed noteworthy antifungal activity against both sensitive and fluconazole-resistant strains, with inhibition zone diameters in the range of 9.4–23.4 mm. Comprehensive analysis illustrated the importance of sabinene, β-phellandrene and 4-terpineol to antifungal activity, and there may be some synergistic effects with other components. These results represent the first report about the correlation between the chemical composition of EOs of C. lansium seeds and antifungal activity. Taken together, the results obtained provide scientific evidence for the traditional use of C. lansium seeds waste. Recently, candidiasis has become one of the most common fungal infections, and its incidence has increased rapidly.5. Although Candida albicans is the main yeast species causing infections, non-albicans Candida species, including Candida glabrate, Candida krusei, Candida parapsilosis and Candida tropical, have caused an increasing number of infections over the past decade7. With a flush pipeline of investigational antifungals, the clinician must identify appropriate roles for currently available therapies. However, many antifungal agents have been found to have different degrees of side effects including allergic reactions, hepatotoxicity, myelosuppression, nervous system disorder, etc.10. Early on the 80’s, fluconazole (FLZ) became one of the most efficient agents to treat Candida infections due to its broad-spectrum activity, high bioavailability, strong tissue penetration, significant curative effect and fewer adverse reactions. It was extensively used on oral candidiasis in HIV patients and with the extensive application of FLZ, there has been a notable increase in drug resistance among infectious yeast species7. Thus, many efforts have been made to isolate pure natural products for medicinal use.Invasive fungal infections have been associated with significant morbidity and mortality in clinical infections11. EOs are secondary metabolites found in many plants. They may exist in fruits, seeds, flowers and leaves. They have many beneficial biological activities, such as antioxidant and antimicrobial, anti-inflammatory, antitumour, and analgesic activities. They have already become a focus because of their advantages such as their abundance, low toxicity, broad-spectrum activities and diverse mechanisms of action14.Natural plant components, especially essential oils (EOs), have attracted much attention from the scientific communityClausena lansium (Lour.) Skeels is a tropical species of the Rutaceae family and is widely distributed in China, in provinces such as Hainan, Guangxi, and Guangdong15. Almost all parts of the plant, including the fruit, root, bark, leaf, peel, seed and pulp, have been reported to have medicinal values18. Hainan Province, a subtropical and tropical region of China, is one of the main geographic regions for growing C. lansium. In Hainan, C. lansium fruits have been used for treating digestive disorders, gastrointestinal diseases and bronchitis diseases, along with the pericarps and seeds19. A likely mechanism is that EOs from C. lansium fruits can help release gastrointestinal gas, eliminate stagnation, dissipate heat and relieve pain. The pulp of C. lansium can be eaten fresh or made into preserved fruit or jam, but the weight of seeds in the fruit is high and is the residual product of pulp processing, resulting in not only a waste of resources but also pollution of the environment. It is a current focus make use of waste seeds to achieve their maximum application value.C. lansium seeds contain volatile components, alkaloids, and coumarins21. They have attracted great attention due to their extensive biological activities, including anti-inflammatory, anticancer, insecticidal, antidiabetic, and antioxidant effects23. Considering the unique active components in C. lansium seeds and their medicinal and agricultural value, increasing research on C. lansium seeds has been carried out in recent years. In a previous study, EOs of C. lansium seeds from different areas were obtained and analysed27. However, there has been no comparative study on the components and contents of EOs of C. lansium seeds from different areas. Moreover, the correlation between EOs of C. lansium seeds and antifungal activity has not been reported.C. lansium seeds of different cultivars and from different areas in Hainan and to investigate the in vitro antifungal activity against Candida species, including C. albicans, C. glabrata, C. krusei, C. parapsilosis and C. tropicalis. The correlation between EOs of C. lansium seeds and the antifungal activity was studied. The results of this study may provide support for further study of the resource development and utilization of C. lansium seeds in Hainan.The antifungal activity of EOs is closely related to their components and contents. The aim of the current study was to comparatively analyse the chemical components of EOs of C. lansium EOs extracted by hydrodistillation were white to light yellow in colour. The yields ranged from 0.43 to 0.91% (v/w). The colour and yield of EOs are listed in Table P > 0.05). It is impossible to come to a conclusion from the colour and yield of EOs. To compare the compositional differences, each EO was subjected to gas chromatography-mass spectrometry (GC–MS) for analysis. Figure C. lansium seed. The chemical composition and content results are shown in Table C. lansium seeds from different cultivars and different areas in Hainan. These components were monoterpene hydrocarbons, oxygen-containing monoterpenes, sesquiterpene hydrocarbons and other compounds. The main components were monoterpene hydrocarbons, such as sabinene and β-phellandrene, and oxygen-containing monoterpenes represented by 4-terpineol. The differences and relationships of these EOs cannot be easily seen from these complex data. Thus, a hierachical cluster analysis was conducted using SPSS (version 17.0) to investigate the similarities and differences among these extracts in EO profiles. The variables were the chemical components and contents of EOs of C. lansium seeds. The clustering results are shown in Fig. According to the GC–MS analysis, almost thirty components were identified in each EO, amounting to 97.10% to 98.64% of the total components. Differences existed in the components and contents of EOs of C. lansium seeds in the three clusters.Samples were clustered into two clusters: QZ1, QZ2 and other samples. Geographically, QZ1 and QZ2 were both located at 110.02°E, 18.90°N, which were closest to the tropics. This result indicates that the difference between the tropics and subtropics is the greatest. Based on the cluster analysis, three clusters were classified. Table The dominant EOs in each of the three clusters are listed below:β-phellandrene ranged from 13.39% to 16.19%. HK3, HK4, HK1 and HK2 were classified into a small cluster, which contained more 4-terpineol with relative percentages of 9.37%, 9.92%, 11.28% and 11.83%, respectively. This result is probably because the four places are geographically close (both are in Haikou city). QH1 and TC2, which are both Hainan local wampees, were classified into a small cluster.Cluster I: sabinene. HK3, HK4, HK1, HK2, QH1 and TC2 belonged to this cluster. Sabinene in this cluster had a high percentage of almost 50%. The percentages of β-phellandrene. These samples were LG1, TC1, WC1, QZ3 and LG2. Although sabinene was the most predominant component in this cluster, with a relative percentage of approximately 40%, the β-phellandrene percentage was relatively higher than that in cluster I (more than 20%). LG1 and TC1, chicken heart wampees were classified into a small cluster. WC1, QZ3 and LG2, Hainan local wampees, were classified into a small cluster. Hence, we infer that although the cultivar was not a very clear cause of the different components and contents, some relationships were evident.Cluster II: sabinene and β-phellandrene. QZ1 and QZ2 were classified into this cluster. They clustered differently from other samples, possibly because QZ1 and QZ2 are closest to the tropics. The percentages of β-phellandrene were the highest in all samples, at 33.24% and 25.91%, respectively. They contained similar percentages of sabinene, with 27.70% in QZ1 and 33.24% in QZ2.Cluster III: C. lansium seeds was examined using the filter paper disc diffusion method. Since sabinene, β-phellandrene and 4-terpineol are the main components in these EOs, the antifungal activities of three pure chemicals were also examined. As shown in Table β-phellandrene, 4-terpineol and FLZ were in the ranges of 9.4–23.4 mm, 13.7–25.7 mm, 15.6–22.1 mm, 26.7–35.6 mm, and 0–35.3 mm, respectively. Statistical analysis results showed that there was significant difference between FLZ and EOs of C. lansium seeds (P < 0.05). In all samples and chemicals, 4-terpineol exhibited the greatest antifungal effect against all strains.The antifungal activity of EOs of C. glabrata and C. krusei was higher than that of the other five Candida strains and there were signifcant differences (P < 0.05).Table C. albicans, the antifungal activity efficacies of both EOs and the constituent chemicals against the FLZ-resistant strain  were better than those against the FLZ-sensitive strain . The efficacy order of the three pure chemicals was as follows: 4-terpineol > β-phellandrene > sabinene. Among the EOs, HK1, HK2, HK3 and HK4 in cluster I exhibited the greatest antifungal effects and were rich in 4-terpineol. The antifungal activity of QZ3 in cluster II was also higher, which may be because it contained higher contents of 4-terpineol and β-phellandrene. Cluster III showed the weakest effect against C. albicans.For C. lansium seeds exhibited the greatest antifungal effect against C. krusei, with inhibition zone diameters of 17.7–23.4 mm. The efficacy order was 4-terpineol > sabinene > β-phellandrene > cluster I > cluster II > cluster III > FLZ. In EOs, cluster I exhibited the greatest antifungal effect, which may be due to the high contents of 4-terpineol and sabinene. The percentage of 4-terpineol and sabinene in cluster III were relatively lower. Therefore, even if the percentage of β-phellandrene was the highest, the antifungal effect of cluster III was still the worst against C. krusei.The EOs C. glabrata, the inhibition zone diameters of FLZ, sabinene, β-phellandrene, and 4-terpineol were 23.0 mm, 22.3 mm, 22.1 mm, and 28.1 mm, respectively. Statistical analysis results showed that the antifungal activity efficacies of sabinene and β-phellandrene were similar (P = 0.687). The antifungal activity of 4-terpineol was higher than FLZ and there was significant difference (P = 4.99 × 10−17). EOs in HK1-4 and QZ2 showed greater antifungal effects, while QH1, TC1, TC2, WC1 and LG2 showed weaker effects against C. glabrata. Considering their compositions, we infer that these differences may be related to the contents of monoterpenes containing oxygen: the higher the contents of monoterpenes containing oxygen, the higher the antifungal effect.For β-phellandrene > sabinene against C. parapsilosis, with inhibition zone diameters of 33.6 mm, 15.6 mm, and 15.0 mm, respectively. Among the EOs of C. lansium seeds, the antifungal activities of clusters I and II were similar, and cluster III showed the weakest effect against C. parapsilosis.The antifungal activity efficacy order was 4-terpineol > C. tropicalis, the antifungal activity of 4-terpineol was also greatest in these samples and chemicals. The inhibition zone diameters of EOs were in the range of 12.1–13.6 mm against the sensitive strain  c2f.) and 9.5–11.4 mm against the resistant strains . There was no significant difference in the antifungal effects among the three clusters (P > 0.05).For C. lansium from Changjiang County, Hainan, China were obtained by hydrodistillation and were analysed by GC-MS24. The oil yield was 0.5% (v/w). Twenty-six components were identified, and the main components identified in the seed EO were phellandrene (54.8%), limonene (23.6%) and p-menth-1-en-4-ol (7.5%). Using the same hydrodistillation method, fifty-three components were identified in both fresh and dried fruit samples from Thailand25. The main components were sabinene (33.68–66.73%), α-pinene (9.57–13.35%) and 1-phellandrene (5.77–10.76%). Comparison revealed that the components and percentages were different from different areas. The reasons for the differences may be due to the fruit containing not only seeds but also pulp and pericarp.In a previous study, EOs of v/w) in the supercritical fluid extraction (SFE) of C. lansium fruits from Guangdong, China26. Thirty-six components were identified, and the main components were 4-terpineol (26.94%), γ-terpinene (14.39%), β-phellandrene (8.24%) and sabinene (5.58%). Chokeprasert et al.27 analysed the volatile components of C. lansium fresh seeds in Thailand by headspace (HS) GC–MS. Twenty-five components were identified in the seeds, and the major components were sabinene (83.56%) and α-pinene (4.26%). In our previous study, volatile components of C. lansium seeds from different areas in Hainan, China, were detected using ionic liquid-based HS-GC-MS21. Thirty-three components were detected. The volatile components were rich in sabinene (17.76–43.81%) and were accompanied by β-bisabolene (5.83–22.25%), caryophyllene (6.75–16.62%) and β-sesquiphellandrene (3.20–22.25%). The components and percentages obtained by SFE and HS were quite different from those obtained by hydrodistillation.The oil yield was 0.1%  were evaluated using the disc diffusion method. C. lansium EOs of pericarps displayed higher antifungal activities than EOs of leaves. Higher contents of β-phellandrene, β-sesquiphellandrene and β-bisabolene in EOs of pericarps were likely responsible for the high antifungal activity.Few studies have reported the antifungal activity of 30 studied the chemical compositions and antibacterial activities of the leaves and berries of Algerian Juniperus phoenicea EOs and found that higher contents of β-phellandrene and α-pinene were positively correlated with the antibacterial activity. A study of the antibacterial activity of Piper species EOs from distinct rainforest areas in southeastern Brazil showed that was β-phellandrene was responsible for the higher antibacterial effects31. Melaleuca alternifolia (tea tree) EO was particularly rich in 4-terpineol (40.44%) and γ-terpinene (19.54%) and showed significant antifungal activity against all Candida strains tested32. The antifungal activity could, in part, be attributed to the major component 4-terpineol.In previous reports, Abdelli et al.C. lansium seeds showed significant activity against all the strains. There were differences among the different types of Candida strains. Notably, they were active towards FLZ-resistant strains . Moreover, the antifungal activity of EOs against C. glabrata and C. krusei was higher than that of the other five Candida strains, and C. glabrata and C. krusei were the main Candida spp. that might have fluconazole resistance (with lower inhibition zone diameters). Therefore, it comes to a conclusion that the natural EOs of C. lansium seeds might be more effective against FLZ-resistant strains. Comprehensive analysis illustrated the importance of sabinene, β-phellandrene and 4-terpineol to antifungal activity, and there might be synergism between the pure chemicals. In addition, antifungal activity may not entirely depend on the main chemical components. Other chemical components present at lower concentrations may also have antifungal activity or may have synergistic effects with other components. More detailed in vivo studies are still necessary.In this study, the EOs of C. lansium (Lour.) Skeels seeds by GC–MS. The main components were sabinene, β-phellandrene, and 4-terpineol. Cluster analysis results showed that the differences in the components were most significantly contributed by different harvest area, followed by different cultivar. EOs of C. lansium seeds showed significant antifungal activity against all seven tested Candida strains. Through comprehensive comparison and analysis of chemical composition and antifungal activity, higher contents of sabinene, β-phellandrene and 4-terpineol may be positively correlated with antifungal activity. There were differences in the antifungal activity efficacies among the different types of strains. Notably, EOs of C. lansium seeds were active towards FLZ-resistant strains. This study may provide a basis for the development of EOs C. lansium seeds as new antifungal agents with high efficiency, broad-spectrum activity, low toxicity and low cost. It may also provide new solutions for clinical infections caused by drug resistance problems.In summary, this study identified a total of forty-six components in the EOs of C. lansium were collected from Hainan, China, in May and June 2020. The details of the source, location, and cultivar of C. lansium samples employed in this study are listed in Table n-alkanes (500 mg·L−1 in hexane) were purchased from America o2si , sabinene (75%) from Sigma-Aldrich (Shanghai) Trading Co., Ltd. , β-phellandrene (96%) from Toronto Research Chemicalstrc  and 4-terpineol (98%) from Shanghai Aladdin Biochemical Technology Co., Ltd. . Other chemicals and their suppliers are listed as follows: Sabouraud’s dextrose agar (SDA) and Mueller–Hinton agar (MHA) powder , fluconazole (FLZ) , and C8-C40 n-hexanes .The fruits of Candida strains included the standard reference strains  c6e and C. tropicalis CMCC(F) c2f.) and clinical strains . C. albicans ATCC 10231 and C. parapsilosis ATCC 22019 were obtained from Guangdong Huankai Microbial Sci. & Tech. Co., Ltd. , C. krusei ATCC 6258 from Wenzhou Kangtai Biotechnology Sci. & Tech. Co., Ltd. , C. glabrata CMCC(F) c6e and C. tropicalis CMCC(F) c2f. from National Center for Medical Culture Collection . The clinical strains were obtained from the oral mucosa of patients with oral candidiasis at Hainan Wenchang General Hospital and Sanya General Hospital, located in Wenchang and Sanya, respectively, China. The sampling methods were carried out in accordance with relevant guidelines and regulations. All experimental protocols were approved by Hainan Medical University and informed consent was obtained from all subjects. The clinical isolates were previously identified based on ITS gene sequence analysis. The obtained yeast isolates were stored in a − 80 °C freezer until use. The culture media used were SDA and MHA.The 29.Measurements were carried out on a GC–MS-QP2010-Plus system  in electron impact ionization (EI) mode. The column was DB-5MS fused silica  from Agilent, USA. Other instrumentation included an FW-80 high-speed grinder , an AL104 electronic balance , a Tanon-4100 automatic digital gel image analysis system , and an SPX-250B-Z biochemical incubator C. lansium were extracted from the fruits, washed and then dried in an oven at 40–50 °C. Then, the seeds were roughly crushed with a grinder and soaked in water with the ratio of material to solvent 1:8 (w/v) for 5.0 h prior to hydrodistillation. The EOs were extracted by hydrodistillation for 3.0 h in a Clevenger-type apparatus. The volume of extracted EOs was read from the scale of apparatus. The yield was calculated by the ratio of extracted volume to weight. The obtained EOs were stored at 4 °C in an airtight container and dried using anhydrous sodium sulfate before being analysed.The seeds of −1 in split mode (1:20). The column temperature was maintained at 60 °C for 3 min and then programmed to 140 °C at a heating rate of 5 °C·min−1, where it was held for 2 min, after which it was increased to 210 °C at a rate of 10 °C·min−1 for the remaining 7 min. The temperatures of both the injector and the connector were maintained at 250 °C. The operating parameters of MS were EI mode at 70 eV with a mass scanning range of 50–500 amu and a source temperature of 280 °C. C8-C40 n-alkanes were used as reference points in the calculation of relative retention indexes (RIs). The percentages of compositions were obtained from the electronic integration of peak areas. The identity of each compound was determined by comparing its RI to n-alkanes and the NIST library database (NIST08 and NIST08s). The percentage of composition was computed from peak areas without applying correction factors29.Samples were analysed by the GC–MS EI method with a DB-5MS column. Helium was used as a carrier gas at a flow rate of 1.0 mL·minC. lansium seeds was first investigated using the paper disc diffusion method33. All strains were streaked onto the SDA plates. A single colony was streaked again to ensure the viability and purity of the strains. Then about 5 single colonies were picked up into 5 mL saline solution. Microbial suspensions of 0.5 McFarland standard were prepared equivalent to approximately 106 CFU·mL−1 yeast and evenly dispensed onto the MHA plates to form the microbe lawn. Pieces of papers were placed on the medium with sterile tweezers and keep the space between the paper more than 24 mm. The diameters of MHA plates and the paper were 120 mm and 6 mm, respectively. The antifungal drug FLZ (2.5 mg·mL−1) was used as a positive control. Ten microlitres of EOs of C. lansium seeds, the constituent chemicals  and positive control were added onto the paper. The plates were incubated at 30 °C for 24 to 48 h. The antifungal activity of the samples was evaluated by measuring zones of inhibition of microbial growth surrounding the paper discs. The zones of inhibition corresponding to diameters were measured with an antibiotic zone scale (Cecon-Brasil) in mm. All the experiments were carried out in triplicate.The antifungal activity of the EOs of t test. Statistical significance was defined as *P < 0.05. GC–MS data and chromatograms were processed with Shimadzu GC–MS Solution software.Data analysis was performed using SPSS software (version 17.0). Data represent the mean ± standard deviation (SD) from at least 3 distinct experiments. Statistical differences were determined using a 2-sample equal variance Student’s


This study aims to investigate the effects of water soluble particulate matter (WSPM) on the viability and protein expression profile of human lung adenocarcinoma cell A549 in the Bayou Obo rare earth mining area, and explore the influence of WSPM on the A549 cell cycle.RPL6, RPL13, or RPL18A gene expression was inhibited, A549 cells were arrested in the G1 phase, affecting the expression of Cyclin D1, p21, RB1, Cyclin A2, Cyclin B1, CDC25A, CDK2, CHEK2 and E2F1. Furthermore, the La3+, Ce3+, Nd3+ and F- in WSPM also inhibited the viability of A549 cells. After 24 h of exposure to 2 mM of NaF, A549 cells were also arrested in the G2/M phase, while the other three compounds did not have this effect. These four compounds affected the cell cycle regulatory factors in A549 cells, mainly focusing on effecting the expression of CDK2, CDK4, RB1, ATM, TP53 and MDM2 genes. These results are consistent with the those from WSPM exposure.It was found that WSPM can inhibit the viability of A549 cells and induce cell arrest in the G2/M phase. Compared with controls, exposure to WSPM10 and WSPM2.5 induced 134 and 116 proteins to be differentially expressed in A549 cells, respectively. In addition, 33 and 31 differentially expressed proteins were further confirmed, and was consistent with the proteomic analysis. The most prominent enrichment in ribosome-associated proteins were presented. When 3+, Ce3+, and F- are probably the main toxic substances in WSPM, and may be regulate the A549 cell cycle by affecting the expression of genes, such as MDM2, RB1, ATM, TP53, E2F1, CDK2 and CDK4. These results indicate the importance for further research into the relationship between APM and lung cancer.These results revealed that WSPM from rare earth mines decreased the viability of A549 cells, and induced cell cycle G2/M phase arrest, and even apoptosis, which may be independent of the NF-κB/MYD88 pathway, and be perceived by the TLR4 receptor. The dysfunction of the cell cycle is correlated to the down-expression of ribosomal proteins (RPs). However, it is not the direct reason for the A549 cell arrest in the G2/M phase. LaThe online version contains supplementary material available at 10.1186/s12989-021-00416-z. The experiments for control and exposure groups were each performed in triplicate.6 cells were placed in 6-well plates at 37 °C with 5 % CO2 for 24 h. Cells were detached by trypsinization, and collected and fixed in 5 ml of 75 % cold ethanol at 4 °C for two hours. Then, these cells were incubated with 0.5 ml of propidium iodide  at 37 °C for 30 min. DNA content was detected using a flow cytometer .Cells were treated with WSPM10 after 10After treatment, the A549 cells were washed for three times with cold PBS, and scraped and centrifuged at 1,500 g for three minutes. Then, the resulting pellets were homogenized in 0.55 ml of lysis buffer , vortexed for one minute, sonicated for five minutes, and unlysed debris by centrifugation at 20,000 g for 30 min. Afterwards, the protein was reduced , alkyated  and precipitated with the addition of 4 volumes of cold acetone . Next, the protein was collected by centrifugation , and the precipitates were redissolved in 0.30 ml of buffer , sonicated [Labeling solutions were provided by the iTRAQ 8-plex reagent kits . According to manufacturer protocol, the fresh vial of iTRAQ reagent 8-plex was required to reach room temperature. Then, 70 µl of isopropanol were added to each reagent vial, and vortex-mixed for one minute. Afterwards, the peptides were labeled with the iTRAQ label reagent, and 8-plex labeling was performed for two hours at room temperature. All labeled peptides, in which the quantity was determined from each tag, were combined in one tube. Finally, the pooled peptides were dried under vacuum –52.2PO4, pH 3.0 with phosphoric acid) and centrifuged . Then, the supernatant was loaded and washed isocratically for 40 min at 0.5 ml/min to remove the excess reagent. The samples were eluted with a gradient of 0-2 M KCl  over 36 min at 1.0 ml/min, with fractions collected at one minute intervals.The combined peptide mixture was separated using a strong cation exchange column . The labeled peptides were solubilized with 1 ml of loading buffer . The Strata-X cartridge were washed with 1 ml of methanol and 1 ml of water prior to use, and were equilibrated with 5 % acetonitrile. Then, the sample was solubilized in 1 ml of ultrapure water, loaded onto the Strata-X cartridge, and desalted with 1 ml of 5 % acetonitrile. The peptides were eluted using 1 ml of acetonitrile from the column, and the solvent was removed by vacuum drier under 4 °C.− 1.The desalted peptide mixture was redissolved with 0.1 % formic acid (FA). Peptide separation was performed on a nano-chromatography system . The sample was injected and captured onto a C18 column  and eluted onto a C18 analytical column . The peptides were eluted using an automated gradient from 95 % (v/v) buffer A (0.1 % FA in water) to 80 % (v/v) buffer B (0.1 % FA in acetonitrile) over 48 min at a flow rate of 400 nl·min5, and an isolation width of 2 Da. In order to evaluate the performance of the mass spectrometry on the iTRAQ labeled samples, higher collision energy dissociation (HCD) was employed. In order to optimize the MS/MS acquisition efficiency of HCD, normalized collision energy (NCE) was systemically examined at 28, stepped 20 %. The MS survey spectrum was measured.The eluted peptides from the C18 column were directly entered to the Q-Exactive MS  through a capillary tip for electrospray, which was set in positive ion mode. The electrospray voltage was 1.8 kV, and the capillary temperature was 320 °C. For each run, 1 µl of sample was loaded, and each sample was analyzed in duplicate. Full MS scans were acquired in the Orbitrap mass analyzer within 350–2000 m/z, with a full scan resolution of 70,000 (m/z 200) and an MS/MS scan resolution of 17,500 (m/z 200). The MS/MS scan had a minimum signal threshold of 1 × 10All .raw files were converted to MGF format using Proteome Discoverer 1.3/1.4 . MASCOT 2.3.01  was used for the database search against the Uniprot human database . The corresponding reversed sequences were also appended to the database for estimating the false discovery rate (FDR) of the peptide identification. The database search parameters included up to one missed cleavages allowed for full tryptic digestion, a mass tolerance of 15 ppm for the precursor, and a mass tolerance of 20 mmu for fragment ions. For all experiments, the carbamidomethylation of cysteine residues was set as a fixed modification, while methionine oxidation, and N-terminal acetylation of protein and iTRAQ modification of peptide N termini were set as variable modifications.The result from each run indicated less than 1 % FDR for peptide identification. For identifications where multiple peptides met these criteria, the results indicated a less than 0.1 % FDR. On the protein level, there was a minimum number of peptide 1 for each protein, in which only rank 1 peptides were counted, and peptides only counted in top scored proteins were applied for all data filtration. In addition, protein grouping was enabled, and a strict maximum parsimony principle was applied. Therefore, if multiple proteins were identified from same peptides, these proteins were grouped into one protein group, and each protein group has at least one unique peptide. The protein groups and peptides identified by UHPLC ESI-MS/MS analysis from A549 cell lysate digest with the MASCOT database search are listed in the supporting material.P < 0.05) to assess the accuracy of the protein ratio, and a minimum of three spectra for quantifying a protein. The iTRAQ ratios were normalized to the control group.The protein quantification settings were as follows: protein ratio type of ‘median’, normalization method of the ‘median’, outlier removal of ‘automatic’, minimum peptide threshold of ‘1’, a 95 % confidence interval (http://www.omicsbean.cn) was utilized to analyze the GO distribution of the obtained differential proteins. The distributions of each protein in the biological process (BP), cellular components (CCs) and molecular functions (MF) were based on the Gene Ontology (GO) categories. A Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis (http://www.genome.ad.jpkegg/pathway.html) was performed to enrich the high-level functions in the defined biological systems.The identified proteins were classified according to the annotations obtained from the UniProt knowledgebase. OmicsBean  at 15.00, ion source gas 2 (GS2) at 0.00, curtain gas (CUR) at 35.00, ionspray voltage floating (ISVF) at 2,400.00 V, interface heater temperature (IHT) at 150.00 °C, Q1 and Q3 at unit resolution, scan speed at 10 Da/s, collision gas (CAD) at high, entrance potential (EP) at 10, and collision cell exit potential (CXP) at 10.For each differentially expressed protein in the iTRAQ data, one unique tryptic peptide of length of 6–20 amino acids were chosen for the MRM. Peptides were excluded when they had a missed cleavage, had potential ragged ends, or contained amino acids that were susceptible to variable modifications, including cysteine and methionine. Precursor ions were monitored for confident identification of the peptides. Candidates for control proteins were identified from iTRAQ, according to the following criteria: if they had a fold change of 1 ± 0.2, the coefficient of variation (CV) was < 10 % in the iTRAQ data. Next, the samples were trypsin digested. For the supernatant samples, strong cation exchange was performed using SCX tips (Thermo Fisher Scientific) after digestion. The MRM assays were carried out using a QTRAP 6500 mass spectrometer  coupled to an ekspert NanoLC 425 system . The mobile phases consisted of solvent A, 2 % acetonitrile with 0.1 % FA and solvent B, and 98 % acetonitrile with 0.1 % FA. Each sample was loaded onto a NanoLC trap column . The peptides were separated using a NanoLC column  at 0.3 µl·min2F1-antibody . β-actin  or GAPDH  was used as the loading control. The secondary antibodies were incubated with goat IgG-HRP  at room temperature for one hour. The blots were detected using a ChemiDoc MP chemiluminescence imaging system .Cells were collected in RIPA lysis buffer, and the proteins were quantified using a BCA Protein Concentration Assay Kit . The protein was isolated on a 10 % SDS-PAGE gel , and transferred onto PVDF membranes. Then, the membranes were blocked with 5 % (w/v) nonfat dry milk in PBS and 0.05 % Tween. The primary antibodies used as follows: anti-FAK antibody , anti-CDC27 antibody , RPRD1A-antibody , RPL34-antibody , RPL24-antibody , EIF6-antibody , p53-antibody , MDM2-antibody , p21-antibody , CDK2-antibody , CDK4-antibody , RB-antibody , ATM-antibody , EBased on the gene sequence in GenBank and siRNA design principles, the siRNA sequences were designed and chemically synthesized by Shanghai Shenggong Biotech. The constructs are listed in Table S6 cells per well. Then, these cells were exposed to WSPM, LaCl3, CeCl3, NdCl3 and NaF, respectively, for 24 h. Subsequently, these cells were trypsinized and collected. Afterwards, the adherent cells were collected. The total RNA was extracted using the TRIzol reagent , according to manufacturer’s instructions.A549 cells were seeded in 6-well plates at a density of approximately 1 × 10The total RNA of A549 cells was extracted using a TRIzol™ RNA reagent , according to manufacturer’s protocol. The mRNA levels for modulated genes were determined by the reverse transcription of total RNA, followed by qRT-PCR, on a LightCycler 480 real-time PCR  using PrimeScript reverse transcription reagent kits , according to manufacturer’s protocol. The primers were designed for the modulated genes. These are presented in Supplemental Table SThe levels of related cytokines in A549 cells were measured using the Human Caspase 3, 6, 8 and 9 ELISA Kit , according to manufacturer’s instructions. Each sample was assayed in triplicate.P < 0.05 were considered statistically significant.The data (except for the fold change of protein expression) were all expressed as the mean ± standard deviation (SD) of triplicate experiments. Significant differences among multiple groups were determined using one-way analysis of variance (ANOVA), followed by the least significant difference post hoc test. Probabilities of Additional file 1: