Curvuluria lunata is a melanized fungus pathogenic to both plants and animals including humans, causing from mild, febrile to life-threatening illness if not well treated. In humans, it is an etiological agent of keratomycosis, sinusitis, and onychomycosis in immunocompromised and immunocompetent patients. The development of multiple-drug-resistant strains poses a critical treatment issue as well as public health problem. Natural products are attractive prototypes for drug discovery due to their broad-spectrum efficacy and lower side effects. The present study explores possible targets of natural antifungal compounds  against C. lunata via gene expression analysis, molecular docking interaction, and molecular dynamics (MD) studies. Curcumin, berberine, eugenol, and α-pinene exhibited in vitro antifungal activity at 78 μg/ml, 156 μg/ml, 156 μg/ml, and 1250 μg/ml, respectively. In addition, treatment by these compounds led to the complete inhibition of conidial germination and hindered the adherence when observed on onion epidermis. Several pathogenic factors of fungi are crucial for their survival inside the host including those involved in melanin biosynthesis, hyphal growth, sporulation, and mitogen-activated protein kinase (MAPK) signalling. Relative gene expression of velB, brn1, clm1, and pks18 responsible for conidiation, melanin, and cell wall integrity was down-regulated significantly. Results of molecular docking possessed good binding affinity of compounds and have confirmed their potential targets as THR and VelB proteins. The docked structures, having good binding affinity among all, were further refined, and rescored from their docked poses through 100-ns long MD simulations. The MDS study revealed that curcumin formed a stable and energetically stabilized complex with the target protein. Therefore, the study concludes that the antifungal compounds possess significant efficacy to inhibit C. lunata growth targeting virulence proteins/genes involved in spore formation and melanin biosynthesis. Curvularia genus is one of the major groups of opportunistic human pathogenic dematiaceous filamentous fungi  is in the growing list of emerging fungal pathogens in humans  and polyenes (amphotericin B) are antifungals that have widely been used to control invasive human fungal infections for more than 4 decades . The clifections . C. lunan plants , and to n plants . Transfen plants  as some n plants .C. lunata depicting alteration in growth, sporulation, and secondary metabolite pathways  and brn1 gene responsible for mechanical strength of appressorium required for penetration . The collected samples were placed in sterile polythene bags properly tagged with date, time, and location  method (www.ncbi.nlm.nih.gov.in) using basic local alignment search tool (BLAST) analysis, and identification was confirmed when 99–100% sequence identity was observed.Genomic DNA was extracted from ) method . Molecul) method . The PCRThe compounds, namely, α-pinene, curcumin, berberine, and eugenol were procured from Sigma-Aldrich (India). The compounds were solubilised in dimethyl sulfoxide (DMSO) to make a stock solution of 100 mg/ml, except berberine (20 mg/ml in methanol). For working solution, stock solution was further diluted in potato dextrose broth (PDB). The final concentration of dimethyl sulfoxide (DMSO) never exceeded the amount with any detectable effect in assays .4 conidia/mL in PDB , observed, and counted using a haematocytometer under a light microscope. The final conidial suspension was adjusted to 10L in PDB . Minimumprotocol . Two-folprotocol .2 strips. Sections of onion peel were floated on 4 ml distilled water in 60-mm Petri plates for treated and untreated samples. Freshly harvested conidia were washed with sterile water followed by centrifugation at 4500 g for 10 min and resuspended in sterile distilled water to the final concentration of 1×104 conidia/ml. Only conidial suspension (10 µl) was placed on a single strip as a positive control. MIC of α-pinene (1250 μg/ml), curcumin (78 μg/ml), berberine (156 μg/ml), and eugenol (156 μg/ml) was added with 10 µl of the conidial suspension on individual strips of onion peel epidermis. Each experiment was independently conducted in triplicate. The strips were incubated at 28 ± 2°C for 24 h. After 24 h of inoculation, extra suspension was removed from the peel, and 30% methanol was applied to prevent further penetration during observation  was performed by the modified method of 50) of α-pinene, curcumin, berberine, and eugenol in a 12-well cell culture plate. Inhibitory Concentration-50 (IC50) of α-pinene, curcumin, berberine, and eugenol were 625 μg/ml, 39 μg/ml, 78 μg/ml, and 78 μg/ml, respectively, where 50 % growth of C. lunata was inhibited. The extracted melanin was resuspended in 100 mM borate buffer, and absorbance was recorded in the wavelength range (250–800 nm) on a UV-visible spectrophotometer. Also, 100 mM borate buffer was used as a blank. The experiment was conducted in triplicate.1) Melanin quantification: Isolation of melanin from C. lunata conidia were harvested in 1× PBS from treated and untreated samples, and their absorbance was set to 0.3 using a spectrophotometer (at wavelength 630 nm). Hexadecane (500 µL) was added to conidial suspension and vortexed for 2 min at an interval of 30 s; then, for the hydrophobic phase, separation tubes were kept at room temperature for 10 min. At 630 nm, absorbance of the aqueous phase was determined and compared to the initial absorbance, that is, 0.30.2) Conidial cell surface hydrophobicity (CSH) and conidiation: Using two-phase partitioning with hexadecane as the hydrocarbon phase, hydrophobicity assay was conducted (C. lunata conidia using the formula:Percentage reduction in cell surface hydrophobicity (%CSH) was calculated for treated as well as untreated 3 mycelial mat of treated and untreated cultures and resuspended in 1 ml of 1× PBS supplemented with 0.25% Tween-20. A volume of 100 µl of conidial suspension was placed on the surface of the counting chamber of the haemocytometer and covered with a cover slip. The number of conidia was counted from square grids in the counting unit of the haemocytometer. The conidia concentration was calculated.The effect of compounds on conidia formation was analysed by counting the number of spores using a haemocytometer . Conidiabrn1, velB, clm1, pks18, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene sequences were downloaded from the NCBI (https://www.ncbi.nlm.nih.gov/pubmed) database for designing the primers for expression studies. The primers were designed by Primer3 software (http://primer3.ut.ee/) and were analysed for potential hair pin formation and self-complementarity . The details of primers are given in The The expression of the genes of interest was quantified by quantitative real time-PCR (qRT-PCR) , 2022. MGAPDH gene was set as the reference gene. Relative expression was estimated using the 2−ΔΔCt formula, whereThe relative quantification of individual gene expression was performed using the comparative threshold cycle method. The amplification program used for real time was 95°C for 3 min, 40 cycles at 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s. To check the specificity of the PCR product, the melting curve was analysed at 95°C for 15 s, 60°C for 60 s, 72°C for 30 s, and holding stage 10 s. The results were analysed using ABI QuantStudio 3 software, and the genes were considered differentially expressed if they were at least two-fold up- or down-regulated.C. lunata were retrieved from the NCBI (https://www.ncbi.nlm.nih.gov/). To obtain the 3D structure of proteins, homology modelling was performed using SwissModel via the ExPaSy web server (https://swissmodel.expasy.org/). The Self-Optimized Prediction Method with Alignment (SOPMA) server was used to speculate the secondary structure of VelB and THR proteins (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_sopma.html). The best template having maximum percentage identity with the target and modelled structure was then evaluated via PROCHECK. For model protein preparation such as charge assignment, solvation parameters, and fragmental volumes, Swiss-PdbViewer version 4.10 (SPBDV-4.10) was used (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4702940/) in the spatial data file (SDF) format. The PubChem compound identifier (CID) was 969516 (curcumin), 2353 (berberine), 3314 (eugenol), and 6654 (α-pinene). The SDF file format was further converted to the PDB file format using the Open Babel tool  for molecular docking via the AutoDock4.2.3 tool (http://www.swissadme.ch/index.php)  was not available in the Protein Data Bank (PDBhence FASTA sequence of virulence proteins velvet protein B (VelB) with accession number ARH19411 and 1,3,8-trihydroxynaphthalene reductase (THR) with accession number QTG11042—proteins of was used . The 3D 2.3 tool . The absdex.php) .via Discovery Studio Visualizer programs . The amino acid residues that displayed interactions with the ligand are documented in Molecular docking was performed using the AutoDock4.2.3 tool  to predi+Cl−). All the MD simulations were performed under physiological conditions (All-atom molecular dynamics (MD) simulation was performed to understand the conformational stability of VelB and THR proteins bound with the docked molecules in comparison to the unbound state of proteins. Atomic coordinates of VelB and THR protein complexes with docked molecules were used to generate the simulation trajectory using GROMACS v5.1.4 . During nditions . The prenditions  and the nditions  algorithnditions  were empvia qRT-PCR and melanin and CSH percentage for biochemical assays. The experiment was conducted in technical and biological triplicate. Statistical analysis was also performed using GraphPad Prism software 8.0.2.263 version and Microsoft Excel. p < 0.05 was considered statistically significant.Statistical analysis was performed using the one-way analysis of variance (ANOVA) for the comparison of results of gene expression analysis Curvularia based on its colony morphology, viz., fluffy and velvety mycelia, greyish black colour, black pigment on the reverse side, and straight to pyriform conidia having three to four cells, with a large and curved central cell with smooth-walled in a lactophenol cotton blue mount. Morphological characteristics of the isolated fungus were in agreement with Rice plants with symptoms of blast disease specifically leaf lesions were collected from Yamuna Nagar, Haryana, India . The funC. lunata from the GenBank database. The sequence was submitted to the NCBI with GenBank accession number OL757869 .Furthermore, molecular characterisation and identification of the fungal isolate at the species level were conducted and confirmed by the amplification and sequencing of 18 S ITS1 and ITS4 regions. The obtained sequence showed 100% similarity with C. lunata was in the range of 1250–78 μg/ml: curcumin (78 μg/ml), berberine (156 μg/ml), eugenol (156 μg/ml), and α-pinene (1250 μg/ml). Among all, curcumin inhibited the pathogen hyphal and conidial germination at a very low concentration. At IC50 of α-pinene, white fungal morphology was observed in vitro, whereas IC50 of other compounds inhibited the growth of fungi to 50% with minor changes in its morphology. IC50 of the compound referred when the growth of microorganism was suppressed by 50%.The isolated fungal pathogen was susceptible to polyenes and azoles. The calculated MIC of bioactive compounds against epidermis, in the presence of bioactive compounds when observed after 24 h of incubation. No visible conidial germination was observed at MIC of α-pinene, curcumin, berberine, and eugenol. In the control sample, conidia with dense hyphal growth were observed on the onion peel under a light microscope as well as SEM as depicted in Conidia failed to adhere to onion peel C. lunata culture showed significant reduction in the quantity as compared to that of control.1) Melanin content: The overall characteristic absorption of melanin was observed at 205 nm which was 0.604, 0.401 0.274, 0.247, and 0.184 for control, berberine, eugenol, α-pinene, and curcumin, respectively (C. lunata as compared to the control (2) CSH percentage: A statistical decrease was observed in the biochemical CSH value of treated cultures as compared to the untreated positive control. The calculated CSH percentage was 87.05%, 79.57%, 75.54%, and 58.73% for α-pinene-, eugenol-, berberine-, and curcumin-treated 50 of compounds to check the number of conidia formed, and a 50% reduction in conidiation was observed in treated cultures in comparison with the control.3) Culture was grown at ICbrn1, velB, pks18, and clm1 of C. lunata was investigated by reverse transcription followed by qRT-PCR for differential gene expression. α-pinene, curcumin, berberine, and eugenol treatment led to a significant down-regulation of velB, brn1, clm1, and pks18 gene transcripts in comparison with the control (untreated)  . The expPrediction of the secondary structure of THR  resultedC. lunata was performed via SwissModel. The PDB ID 1YBV was used as the template to model the 3D protein structure of the THR protein sequence and PDB ID 4N6R chain B for VelB protein. The stereochemical quality and accuracy of the model were tested using PROCHECK. Results from PROCHECK were reported as the Ramachandran plot. For THR protein, 89.0% residues were in most favoured regions, 11.0% residues were in additional allowed regions, and no residues were in disallowed regions. Similarly, in protein VelB, 75.7% residues were in most favoured regions, 19.8% residues were in additional allowed regions, 2.1% residues were in generously allowed regions, and 2.5% residues were in disallowed regions. Both modelled proteins obtained by homology modelling were of good quality on the basis of the Ramachandran plot , berberine (156 μg/ml), eugenol, (156 μg/ml) and α-pinene (1250 μg/ml). In the studies conducted elsewhere, α-pinene showed significant antifungal activity, with greater inhibitory activity against Candida parapsilosis, and proved to be effective in inhibiting fungal growth (C. lunata (T. rubrum strains (64–512 μg/ml)  . The extntration , and ant.2 mg/ml  and Cand–2 mg/ml .C. lunata begins with the adherence of conidia onto the leaf surface . The spore germination and its pathogenicity were studied on the onion epidermis layer, where the compound-treated  conidia failed to adhere and germinate on the surface. The invasive hyphae of C. lunata were observed when untreated conidia were observed under light and electron microscope. Another study on spore germination of Curvularia maculans reported that berberine affected the germination process of the fungus  furan-2-carboxylate toxin production, respectively. Molecular docking of compounds with THR and VelB proteins showed significant negative binding affinity using AutoDock4.2.3. Polyketide synthase plays an important role in the pathogenicity, which synthesised THR, another crucial step for the biosynthesis of DHN-melanin in both mycelia and conidia , an  conidia . The vel conidia . The mai conidia . Curcumi conidia . CurcumiC. lunata. The natural bioactive compounds possess less toxicity and associated side effects, which makes them a suitable candidate for drug discovery. Among all, curcumin exhibited effective antifungal properties to inhibit the growth of the fungus. Conidia adherence and their germination were restricted by the activity of these compounds. It was shown that the compounds down-regulate brn1, velB, pks18, and clm1 gene expression, leading to decreased cell wall integrity and sporulation. The correlation between the virulence gene down-regulation and in silico molecular docking interaction represented the potential antifungal activity of bioactive compounds and highlighted virulence proteins THR and VelB as a possible drug target.Plant-derived compounds  are effective against


Plasmodium falciparum while overlooking other Plasmodium species such as P. malariae. A better understanding of the role of Plasmodium species other than P. falciparum is needed to strengthen malaria elimination initiatives. The aim of the present study was to elucidate the contribution of P. malariae to malaria transmission in Cameroon.Malaria control efforts are highly skewed towards Plasmodium spp. was also determined by quantitative PCR, and dried blood spots were collected from 156 participants with the aim to detect different Plasmodium species by nested PCR.The study was conducted in the Ngatti Health District, a forest–savannah transition area in the Adamawa Region, Cameroon. A total of 497 individuals aged from 1 to 85 years were diagnosed with malaria in November 2020 using a rapid diagnostic test (RDT) and microscopy. Adult mosquitoes were collected between September 2019 and March 2020 by indoor aspiration and identified morphologically and molecularly. The infection status of Plasmodium prevalence was 50.3%, 51.8% and 64.7%, as detected by microscopy, the RDT and PCR, respectively. Based on the PCR results, P. falciparum was the most prevalent species (43%); followed by co-infections P. falciparum/P. malariae (17%), P. falciparum/P. ovale (1.3%), P. falciparum/P. ovale/P. malariae (1.3%); and then by P. malariae mono-infection (2.5%). The same trend was observed using microscopy, with 35% of participants infected with P. falciparum, 11% co-infected with P. falciparum/P. malariae and 4% infected with P. malariae. The prevalence and parasite density of malaria infection varied significantly with age group (P < 0.05), with the highest prevalence rate observed in children aged 6–10 years (P = 0.0001) while the density of Plasmodium infection increased significantly in children aged < 5 years compared to the other age groups (P = 10−3). Among the 757 Anopheles mosquitoes collected, 737 (97.35%) were An. funestus sensu stricto, 15 (1.9%) were An. gambiae and 5 (0.6%) were An. hancocki. The Plasmodium species recorded at the head/thorax level were P. falciparum and P. malariae, with a sporozoite infection rate of 8.4%; the highest sporozoite infection rate was recorded at Mibellon village (13.6%).The overall P. malariae, in addition to P. falciparum, to the high malaria transmission rate in this region. These findings highlight the need to deploy initiatives to also tackle this Plasmodium species to eliminate malaria in the region.The results of this study reveal the significant contribution of  The past decades have been witness to a substantial reduction in the number of malaria cases and deaths due to malaria worldwide by 37% and 60%, respectively, between 2000 and 2015 [Anopheles species can be considered to vectors of malaria disease to humans in Cameroon [Anopheles gambiae sensu stricto (An. gambiae s.s.), An. coluzzii, An. arabiensis, An. funestus, An. nili and An. moucheti [Plasmodium falciparum and, to a certain degree, Plasmodium vivax, making them the most studied Plasmodium species responsible for severe cases of malaria disease. However, Plasmodium malariae and Plasmodium ovale, which have been reported to be widely distributed in most malaria-endemic regions of the world, are less studied and difficult to identify by light microscopy because the low level of parasitaemia is often below detection thresholds of microscope readers. This failure is the result of sub-microscopic malaria, which accounts for 20–50% of all human-to-mosquito transmissions in sub-Saharan Africa [P. malariae infection is usually benign, but could cause morbidity such as severe anaemia, acute renal failure and splenomegaly [P. malariae and P. falciparum, it has been reported that P. malariae infection can reduce the burden of parasitaemia of P. falciparum infection by up to 30% and contribute to the reduction of the pathology due to P. falciparum as the people become even more asymptomatic [P. malariae prevalence when the prevalence of P. falciparum decreased. These results demonstrate that there is a need for more information regarding the epidemiology of non-falciparum species in the context of the distribution of malaria vaccines targeting only the P. falciparum antigen. Four Plasmodium species have been recorded to circulate in Cameroon: P. falciparum, P. ovale, P. malariae and P. vivax [Plasmodium falciparum remains the predominant Plasmodium species followed by P. ovale and P. malariae, with average prevalence rates of infection of 95%, 3% and 1%, respectively [P. falciparum malaria and especially on symptomatic malaria cases in patients aged < 15 years [Plasmodium in a patient with an axillary temperature of < 37.5 °C). In the study reported here, we first conducted an entomological survey to identify the vector competent to transmit P. malariae. Then we used molecular, microscopic and RDT techniques to evaluate the dynamics of P. falciparum and P. malariae infection transmission in all age groups of the study population.Despite past and ongoing efforts to eliminate malaria, the disease remains endemic in some parts of the world, with > 90% of cases recorded in the sub-Saharan region of Africa. This is largely due to the increasing resistance of mosquitoes to insecticides, which has diluted the effectiveness of malaria control measures . Hence, Cameroon –7, with moucheti , 8. To dnomegaly , 11. It nomegaly . In the ptomatic –15. A stptomatic  in TanzaP. vivax –19. PlasP. malariae/P. ovale or P. vivax infection rate of 4% in the mosquito population of Mibellon village, one of the villages in our study area.The entomological survey was performed by collecting mosquitoes in six locations between September 2019  and March 2020  /m2 [Z-score (a Z-score of 1.96 was used with a 95% confidence level), P = the percentage of population probability (assumed to be 50% = 0.5) and m = the margin of error (taken to be 5% = 0.05). All people from year 1 and thereafter were considered for blood collection.The sample size of the human population was calculated following the formula: s = z − P)/m2 , where sDuring the survey, malaria diagnosis was carried out using thick blood smears, RDTs  Combo, Access Bio Inc. 65 Clyde Road. Somerset, NJ, 08873). and PCR analyses. Body weight, temperature, gender and age of subjects sampled in the study were taken into account. The blood was collected on slides in all the localities and on Whatman FTA cards  only in Mibellon and Moinkoing villages.Plasmodium parasites. Parasite density was defined as the number of Plasmodium parasites per microlitre of blood, counted against 200 leukocytes and expressed according to the WHO guidelines as the number of asexual parasites per microlitre of blood, assuming a leukocyte count of 8000 cells/μl. Samples of blood drops were deposited on Whatman FTA cards and dried for 24 h at room temperature, following which they were stored in clean zippered bags until DNA extraction.Thick blood smears collected from the finger of each subject sampled in the study were air-dried and stained with 10% Giemsa solution for 20 min. The slides were screened by direct microscopy (100×) to visualise the different species of DNA was extracted from blood on Whatman paper using the Chelex protocol. Briefly, 400 µl of 5% chelex100 was added to an Eppendorf tube containing a blood sample on filter paper. The tubes were placed in a shaking dry heater and incubated at 56 °C for 1 h, following which it was incubated for 30 min at 95 °C in a shaking heater. The tube was then removed and centrifuged at a maximum of 13,500 rpm for 10 min. The supernatant was then transferred into a labelled tube and stored at − 20 °C .Plasmodium species infections were performed with all samples using the nested protocol with some minor modifications to differentiate the various species of Plasmodium [Plasmodium was amplified. In the first reaction, PCR amplifications were performed in a 15-μl volume containing 1.5 μl PCR buffer A, 0.75 μl 25 mM MgCl2, 0.12 μl 25 mM dNTPs, 0.51 μl forward and reverse primers (10 mM), 0.12 μl Kapa Taq, 10.49 μl ddH2O and 1 μl DNA. A 5-μl sample of the first reaction’s product was used as a template in a second nested reaction to specifically differentiate P. falciparum, P. vivax, P. ovale and P. malariae products. The temperature profile for the second PCR was: 5 min at 95 °C, followed by 25 cycles of 1 min at 94 °C, 2 min at 58 °C, 2 min at 72 °C; with a final cycle at 72 °C for 10 min. The cycles were repeated 30 times for the second reaction at the same cycling parameters. After electrophoresis in 1.5% agarose gel and staining with Midori green, the amplicon products were visualized under UV light. Plasmodium identification was made based on amplicon sizes.Real-time PCRs (RT-PCRs) for the identification of asmodium . A speci Anopheles mosquitoes collected were morphologically identified using morphological keys [Female mosquitoes were captured inside houses using an electric aspirator during the early morning hours of between 05.30 a.m. and 08.00 a.m. Femalecal keys . These mcal keys . Anopheles mosquitoes were dissected individually into between heads, thoraxes and abdomens. Genomic DNA (gDNA) was extracted from each body part (head + thorax and abdomen) using the LIVAK method [Plasmodium sporozoite infection rate was assessed using the TaqMan assay [P. ovale, P. vivax and P. malariae species [All femaleK method , and thean assay . As a la species .χ2). Parasitaemia was log transformed before analysis. Means were compared using an independent sample t-test and analysis of variance where appropriate; Kruskal–Wallis tests were used to make associations between P. falciparum, P. malariae and mixed infection and other variables of interest. P-values < 0.05 were considered to indicate significance, and a 95% confidence interval (CI) of geometric means that did not overlap was considered to indicate a significant difference.Data analysis was conducted using the Microsoft Excel sheet , GraphPad Prism V8.02R software . Data were summarised into means and standard deviations (SD), and percentages were used in the evaluation of the descriptive statistics. Proportions were compared using the Chi-square test  , Anopheles gambiae sensu lato (An. gambiae s.l.)  and Anopheles hancocki .A total of 757 Plasmodium sporozoites was 8.4%, recorded in An. funestus s.s. and An. gambiae s.l. , P. OVM  (2.5%) and mixed infections  (0.4%) were recorded in Mibellong village. Mosquitoes infected with P. OVM were found only in the Mibellong and Tibati localities, and two mosquitoes were found to be infected with a mixed infection  in Mibellong and Meng  was observed only in Mibellong (1.8%) and Mbougam (1.1%).The P.OVM were P. malariae for those with successful amplification .For the microscopy analyses, samples were collected from 497 people  enrolled in this study. Due to some amplification issues, only 156 samples were analysed by PCR, at two locations (Mibellon and Moinkoing), from 67 (43%) males and 89 (57%) females, respectively. The prevalence of malaria infection assessed by microscopy, RDTs and PCR on the same set of samples (156) was 47.4%, 43.7% and 64.7%, respectively. The general prevalence of infection taking into account the entire study population was 51% (252/494) for microscopy and 51.8% (252/484) for microscopy  of the individuals sampled were asymptomatic. Microscopy, RDTs and PCR showed that 44.8% (95% CI: 37.0–59.1), 43.7% (95% CI: 33.9–55.6) and 62.5% (95% CI: 37.0–59.1) of asymptomatic people, respectively, were infected with P. falciparum, four (2.5%) for P. malariae, 27 (17.1%) for P. falciparum/P. malariae, two (1.3%) for P. falciparum/P. ovale and two (1.3%) for P. falciparum/P. ovale/P. malariae  for P. malariae, 57 (11%) for P. falciparum/P. malariae and two (0.4%) for P. falciparum/P. ovale , Moinkoing (54.6%) and Ngatti (51%) villages was high, while the lowest prevalence of 12% was recorded in Mbougam village  was recorded in Mibellon (25.7%)  tested positive for .3% testeP. falciparum prevalence of 47.7% (95% CI: 34–64) was observed among older children aged 6–10 years, followed  by teenagers aged 11–15 years , infants aged 1–5 years , young adults aged 16–20 years  and adults, with the latter age group showing the lowest prevalence of 22% (95% CI 19–26%)  followed the same trend, with the highest prevalence of 27% observed in children aged 5–10 years, followed  by subjects in the age groups 1–5, 10–14, 16–20 and > 21 years, with 22%, 14.2%, 4.5% and 1% infection rates, respectively  was recorded in subjects aged 16–20 years, followed a prevalence of 7.1% in subjects aged 11–15 years. The prevalence of P. malariae obtained in subjects aged 6–10, 1–5 and 21+ years was 3.4%, 3.3% and 2%, respectively  was highest in children aged 6–10 years , while the younger adolescents (aged 10–14 year) were more infected with P. malariae with a prevalence of 9%.According to age groups, the highest  95% CI: 4–64 was  95% CI: 4–64 was  95% CI: 4–64 was P. falciparum and P. malariae parasitaemia was 4354.7 ± 7991.6 and 606.1 ± 572.9 parasites/μl, respectively. The highest parasitaemia was recorded in children aged 1–5 years, at 5873.9 ± 8910.2 and 854.4 ± 608.4 parasites/μl for P. falciparum and P. malariae,, respectively. The lowest parasitaemia was observed in adults (age group: 21–85 years), at 1901.8 ± 2389 and 215 ± 119.8 parasites/μl for P. falciparum and P. malariae, respectively .The overall geometric mean P. falciparum and P. malariae infections in mosquito and human populations. The study produced strong evidence that malaria remains highly prevalent in the study locations in Cameroon.In this study we assessed the transmission dynamics of An. funestus s.s., An. gambiae s.l. and An. hancocki, of which An. funestus s.s.(97.3%) was the most prevalent species. Our results are similar to those obtained by Tchouakui et al. in Mibellon [An. funestus prevalence. Our findings vary slightly from those of Tchouakui et al. [An. funestus s.s. in the same village and from those of Menze et al. [Three anopheline species were identified in the study area: Mibellon , where ti et al.  who recoe et al.  who obseAn. funestus is the dominant species in this locality and that the prevalence could vary according on the season since the different collections did not take place during the same season. To the contrary, in Tibati, we recorded 94.4% An. funestus prevalence compared to 55.2 %and 16% prevalence in collections from 2015 and 2017, respectively, by Feufack-Donfack et al. [An. gambiae development become scarce.The focus on the collection of indoor blood-fed mosquitoes may have prevented us from collecting the outdoor resting species of subfamily Anophelinae. Otherwise, our results showed that k et al. . This diAn. funestus and An. gambiae were infected with Plasmodium spp. A slight increase in the overall infection rate was observed in An. funestus in the present study compared to samples collected in 2017 in Mibellon by Menze et al. [X2 = 3.43, P = 0.06) and also to samples collected in Tibati in 2017 by Feufack-Donfack et al. [X2 = 3.14, P = 0.07). Although not significant, this increase in sporozoite infection rate could be due to the reduction in efficacy of LLINs (approx. 3 years) that were massively distributed in this part of the country in 2015. The prevalence of infection was also low in another location.Both e et al.   and, in particular, the infection rate by P. malariae  of mosquitoes collected in 2019 strongly support the role of physical barriers played by the mosquito midgut and immune genes in the process of oocyst migration to the salivary gland [P. falciparum and P. malariae could also be due to the lifespan of mosquitoes, which is generally no longer than 3 weeks in nature. In fact, P. malariae has a longer sporogonic cycle (21 days) [P. falciparum (14 days), which may explain the reduced likelihood of finding P. malariae sporozoites in the mosquitoes. The study site of Mibellon had a high infection rate with P. falciparum and P. malariae; this can possibly be explained by the perennial presence of the malaria vector, mainly An. funestus, during the year in Mibellon. The high infection rate of oocysts and sporozoites was recorded among the mosquito population of Mibellon village despite the human factors, vector immunity, midgut microbiota [P. malariae in the human population.The significantly higher overall infection rate found in the abdomen of the mosquitoes compared to the head + thorax   than P. crobiota , all of Plasmodium spp. infection rate recorded in the present study could be due to several factors, such as the sampling season and the molecular method used for species identification. The PCR test used in this study targeted the 18S rRNA gene [ cox III [ cox III method is able to detect an additional 10–50% more cases with high sensitivities for all four Plasmodium species due to the high number of parasite copies (20–150) found in cox III compared to 18S (4–8 copies) [The high prevalence of infection and high parasitaemia indicate that the Ngatti health district is a high-risk zone for malaria transmission. This is supported by earlier reports of the prevalence of malaria infection in the Adamawa Region varying from 8.1% to 10.6% in symptomatic children aged between 2 and 9 years , 20, 31.RNA gene  whereas  cox III  in the P copies) .P. falciparum and Plasmodium mixed) infection was recorded in children in the age group 6–10 years. Regarding this result, it should be noted that malaria control campaigns target children aged < 5 years of age and pregnant women, with parents advised to ensure that children aged < 5 years, who are considered at high risk of contracting malaria, sleep under a LLIN. This possibly explains the reduction in the prevalence of infection among children in this young age group. However, the good use of ITNs for children aged < 5 years may impede or delay the development of malaria protective immunity in children aged 6–10 years [P. falciparum decreased significantly with age from 10 to 85 years, which could be accounted for by the acquisition of protective immunity by adolescents and adults following multiple exposures to malaria infection [The results also revealed an association between the prevalence of malaria and age at infection. The highest prevalence of malaria  and, not surprisingly, the highest parasitaemia was recorded in children aged < 5 years. This result is explained by the low levels of immunity in young children and increasing naturally acquired immunity in older children through repeated exposure to the parasite [An estimation of malaria parasite density is necessary for patient management. Our study revealed that asymptomatic people coming from different villages in the Ngatti health district tolerated the high burden of asexual parasitaemia of up to 56,600 parasites/μl blood for parasite , 42.P. malariae obtained by microscopy was low, although higher than the general prevalence observed in Cameroon (3%) [P. falciparum in the lower age groups can be assumed to allow non-falciparum species, such as P. malariae, to become more important as P. falciparum is eliminated. In general, high transmission areas constitute a significant infectious reservoir of malaria parasites due to the potentially high percentage of asymptomatic malaria parasite carriers in this location [The prevalence of oon (3%) . The higlocation , 14, whiP. falciparum as well as P. malariae, in the asymptomatic population. The findings revealed that malaria endemicity in the Adamawa Region of Cameroon is driven by multiple Plasmodium species, although P. falciparum remains the most prevalent. In addition, P. malariae infection and P. falciparum/P. malariae co-infections are both transmitted to humans by An. funestus in the studied localities and play an important role in malaria transmission. We found that older children were the age group most infected with malaria. Thus, beyond the control of malaria targeting only the under-fives, pregnant women and P. falciparum, a comprehensive policy targeting all age groups of the population should be put in place to eliminate malaria and its reservoir species more effectively.The aim of this study was to examine the prevalence of malaria infection, targeting both


Wuliangye‐flavor liquor (WLFL), a specific Chinese traditional liquor, one of the major type of global distilled spirits, offers a unique flavor system acquired across thousands of years of development. Free amino acids (FAAs), as major health factors, are considered to be primarily derive from the hydrolysis of protein from the Zaopeis (ZPs) by microbial populations during fermentation. Here, we investigated the changes of microbial communities in ZPs and FAAs in raw liquor (RL) directly related ZPs from different ages of WLFL fermentation pits by phospholipid fatty acid fingerprint (PLFA) and high performance liquid chromatography method. Results indicated that the total PLFAs of 20‐ and 50‐year ZPs were significantly higher (p < .05) than 1‐ and 5‐year ZPs. Gram‐positive bacteria (G+), anaerobic bacteria, and fungi were dominant in the ZPs. Furthermore, the total of FAAs content was highly increased (p < .05) in RLs of aged fermentation pit  compared with young , while, the different ages of RLs could be distinguished according to 9 biomarkers of FAAs , including tyrosine, aspartic acid, leucine, glutamic acid, citrulline, alanine, proline, glycine, and valine. Particularly, the biomarkers of FAAs were positively correlated with gram‐negative bacteria (G−) and fungi, but negatively correlated with G+. This is the first report to uncover the microbial communities in zaopeis, free amino acids in RL, and their correlations for Wuliangye‐flavor raw liquor production. + bacteria increased as the age of the pit increased for Wuliangye‐flavor raw liquor production. The biodiversity of microorganisms in the zaopeis of aged fermentation pit and lower layer higher than the young fermentation pit and upper layer. The differential free amino acids were positively correlated with G−and fungi, while negatively correlated with G+.The total biomass, fungal biomass, and the biomass of G Therefore, it was necessary to reveal the correlation between FAAs of WLFL‐RLs and microbial communities in ZPs directly related to WLFL‐RLs.In fact, the special flavor of WLFL can be affected by the raw materials, Lactobacillus acetotolerans was identified as the main genera in WLFL fermentation , aerobe bacteria and fungi predominated in the ZPs of strong‐flavor liquors. However, few reports have focused on the spatial distribution of microbial communities and their biodiversity in WLFL's ZPs from different fermentation pits.In the latest two decades, culture‐independent approaches were developed and employed to investigate the microbial communities, especially the bacterial community in WLFL's ZPs, and zaopeis, free amino acids in RL, and their correlations for WLFL‐RL production. The results can be used as reference to understand the correlation between FAAs and microbial communities of WLFL‐RL in the later stage and broaden the application range of health factors.Consequently, this study aimed to investigate the FAAs in WLFL‐RLs of different fermentation pit ages by high performance liquid chromatography (HPLC) and the relationship between FAAs and microbial communities in various ZPs directly corresponding to WLFL‐RLs. Multivariate statistical techniques were used to investigate biomarkers of FAAs components of WLFL‐RLs in different fermentation pit ages and to understand the correlation between biomarkers of FAAs and microbial communities in ZPs. To the best of our knowledge, this is the first report to uncover the microbial communities in 22.1Citric acid, sodium citrate, chloroform, acetone, toluene, acetic acid, potassium hydroxide, trichloroacetic acid, orthohexane, sodium acetate, acetic acid, methanol, acetonitrile and tetrahydrofuran were purchased from Chengdu Cologne Chemical Reagent Factory. The standards (≥98.0%) of glutamate (Glu), aspartate (Asp), citrulline (Cit), threonine (Thr), glycine (Gly), arginine (Arg), serine (Ser), methionine (Met), leucine (Leu), proline (Pro), isoleucine (I‐leu), alanine (Ala), tyrosine (Tyr), cysteine (Cys), valine , histidine (His), phenylalanine (Phe), lysine (Lys), and phthalformaldehyde, o‐phthalaldehyde, 9‐fluorenylmethylchloroformate were purchased from Sigma‐Aldrich. Deionized water was used in the EPED‐T purification system (Yi Pu Yi Da Co.) and was degassed under vacuum and filtered through a 0.45 μm nylon membrane before use for HPLC analysis.2.2Wuliangye‐flavor liquor producing company in Yibin, Sichuan, China in July 2019. 36 RLs in the four fermentation pit from commercial level workshop were obtained; the pit ages were 1, 5, 20, and 50 years; the sample was the heart cut of distillation (head cut were dispensed), and corresponding to them were 36 ZPs, which were collected from the upper layer, middle layer, and lower layer in the same fermentation pit, respectively, and three ZPs sample were collected from each layer  to determine the FAAs content after precolumn derivatization with o‐phthaldialdehyde‐3‐ mercaptopropionic acid (OPA) and 9‐fluorenylmethyl‐chloroformate (FMOC), as described previously , and centrifuged at 10,000 2.5p < .05 were deemed as potential biomarkers. A valid co‐occurrence was selected as a strong correlation if the Spearman's correlation coefficient (ρ) was >.6  and HPLC analysis were initially processed by one‐way analysis of variance (ANOVA) at the 5% significant level using IBM SPSS Statistics 23.0 (SPSS Inc.) to compare the differences statistically. Heat maps were generated and cluster analyses of the PLFAs and FAAs compounds were obtained with the TBtools software , with the level of 59.21 nmol/g, followed by 20‐year‐ZPs: the dominant PLFAs were a16:0, i16:0, a15:0, i15:0, i13:0, 14:0, and 13:0. In the middle‐layer (M), the total PLFAs were increased corresponding to the pit ages, the level from 33.51 to 63.06 nmol/g: the dominant PLFAs were i15:0, a14:0, a12:0, 17:0, 15:0, 18:1ω6, 16:0, and 18:0. In the lower‐layer (L), 50‐year‐ZPs had the highest total PLFAs (p < .05), with the level of 75.30 nmol/g, followed by 20‐year‐ZPs. The dominant PLFAs were cy19:0, cy17:0, a8:0, i9:0, 10Me16:0, 10Me18:0, 20:1ω6, 9Me14:0, α‐OH‐10:0, 18:1ω9, and 18:2ω6,9. Simultaneously, the upper layer had the highest total PLFAs (p < .05), followed by the middle layer. This indicates that the relative abundance of microorganisms gradually increased from the upper layer to the lower layer. This may be due to the fact that the brewing microorganisms in the fermentation pit produced a large amount of nutrients such as bacterial autolysates, humus, and organic acids during the production process, which gradually penetrated into the yellow water, enabling the more brewing microorganisms to grow and multiply better in bottom of the pit mud and the lower layers of ZPs  and gram‐negative bacteria (G−), anaerobic bacteria (Anaer‐bac), aerobic bacteria (Aer‐bac), as well as fungi. Of them, G+, Anaer‐bac, and fungi were dominant in the ZPs, and these findings were in accordance with the results of previous studies conducted using culture‐dependent and culture‐independent technologies , fungi increased from 3.10% to 21.77% (Y1–Y50), Aer‐bac decreased from 16.36% to 9.68% (Y1–Y50), and Anaer‐bac decreased from 48.56% to 39.85% (Y1–Y50), respectively , fungi increased from 5.24% to 25.97% (Y1–Y50), Aer‐bac decreased from 10.52% to 4.65% (Y1–Y50), and Anaer‐bac decreased from 62.26% to 49.24% (Y1–Y50), respectively. The microorganisms in the lower layer have also exhibited a similar trend  using Euclidean distance  in aged fermentation pit of RLs (Y20 and Y50), compared to young fermentation pit of RLs (Y1 and Y5). Moreover, in lower layers, the total FAA content increased significantly (p < .05) in accordance with the pit ages (from 26.46 to 37.15 mg/g). As shown in Figure p = .02), Met (p = .002), Tyr (p = .01) were increased significantly (p < .05) corresponding to pit ages, and their contents were increased from 1.22 to 2.22 mg/g (Y1–Y50), from 0.01 to 0.05 mg/g (Y1–Y50), and from 0.92 to 1.70 mg/g (Y1–Y50), respectively. In the middle layer, the contents of Thr (p = .03), Gly (p = .02), Met (p = .001), Tyr (p = .01) were increased significantly (p < .05) corresponding to pit ages, and their contents were increased from 1.88 to 2.93 mg/g (Y1–Y50), from 2.50 to 3.75 mg/g (Y1–Y50), from 0.02 to 0.06 mg/g (Y1–Y50), and from 1.09 to 2.12 mg/g (Y1–Y50), respectively. In the lower layers, all the FAAs were increased significantly (p < .05) in Y50 compared to that of Y1. Moreover, the lower layer had the highest total FAA content, follow by the middle layer. Some of the microorganisms grown in ZPs have been reported to produce acidic carboxyl proteases. This activity of this enzyme is important in the degradation of proteins and their conversion into amino acids, which in turn enhances microbial growth and the formation of trace levels of aromatic compounds  using multivariate data, and compared to PLS‐DA, OPLS‐DA can filter out noise unrelated to classification information and thereby improve analytical ability, ensuring the validity of the models, and maximizing the differences between the groups within the models  were detected in our experiment Figure . BesidesIn the lower layer of RLs, the parameters for the classifications were observed between Y1 and Y5 ; Y5 and Y20 ; as well as between Y20 and Y50  between the FAAs and microbial communities, and |ρ| with >0.6 are shown using a network diagram  with all FAAs (except of Ser), while G+'s biomarker a12:0 was negatively correlated to some FAAs (green edges), including Thr, I‐leu, Ala, Gly, Glu, and Arg. Thus, G−, anaerobe, and fungi can be beneficial to form the overall sensorial quality  of RLs in this study, which was consistent with previous reports (Wang et al., −'s biomarker 20:1ω6 were negatively correlated to Gly, Glu, and Arg (Figure In order to reveal the relationship between FAAs and microbial communities, we calculated the Spearman correlation coefficients (m Figure . As shows Figure . Fungal 4+ bacteria increased as the age of the pit increased. The diversity of the microbial community structure and their shift with pit age was explained by PCA and CA analysis. Young ZPs and aged ZPs were well separated from each other, as well as from different layers. Samples from year 1, year 5, year 20, and year 50 of RLs were analyzed using PCA, OPLS‐DA models. Based on OPLS‐DA analysis, twelve RLs can be distinguished according to the contents of the potential biomarkers of FAAs, such as Asp, Leu, Glu, Cit, Ala, Pro, Tyr, Gly, and Thr. More importantly, the differential FAAs were positively correlated with G− and fungi, but negatively correlated with G+. Overall, this study uncovered the microbial communities in zaopeis, free amino acids in RL, and their correlations for WLFL‐RL production. More research is also needed to determine the main source of FAAs produced in the WLFL brewing process, and traceability research on the microorganisms and enzyme from microorganisms that produce FAAs are of considerable significance for laying a theoretical foundation to stabilize the quality of WLFL and increase WLFL output.In this study, microbial communities' characteristics and differences of ZPs with different pit ages were elucidated by PLFA. PLFA revealed that bacteria and fungi were included in the ZPs. The total biomass, fungal biomass, and the biomass of GThe authors declare that there are no conflicts of interest.Fig S1Click here for additional data file.Fig S2Click here for additional data file.Fig S3Click here for additional data file.Table S1Click here for additional data file.Table S2Click here for additional data file.Table S3Click here for additional data file.


Mycobacterium tuberculosis is a deadly pathogen, currently the leading cause of death worldwide from a single infectious agent through tuberculosis infections. If the End TB 2030 strategy is to be achieved, additional drugs need to be identified and made available to supplement the current treatment regimen. In addition, drug resistance is a growing issue, leading to significantly lower treatment success rates, necessitating further drug development. Vanoxerine (GBR12909), a dopamine re-uptake inhibitor, was recently identified as having anti-mycobacterial activity during a drug repurposing screening effort. However, its effects on mycobacteria were not well characterized. Herein, we report vanoxerine as a disruptor of the membrane electric potential, inhibiting mycobacterial efflux and growth. Vanoxerine had an undetectable level of resistance, highlighting the lack of a protein target. This study suggests a mechanism of action for vanoxerine, which will allow for its continued development or use as a tool compound. Bacterial cultures were grown at 37°C and 180 rpm (or static for BCG) until mid-log (OD600 = 0.5–1.0), and kanamycin (50 μg/ml) was added when required.Bacterial strains  was added. An intermediate compound plate was made using vanoxerine, performing a 1/2 or 2/3-fold serial dilution into dimethyl sulfoxide (DMSO). Each drug concentration (1 μl) was transferred into a 96-well polystyrene plate, followed by addition of diluted cell culture (99 μl). The plates were wrapped in foil and incubated . Resazurin  was added to each well and the plate re-incubated . The resazurin fluorescence  was measured in a BMG Labtech POLARstar Omega plate reader, normalized and cell survival calculated. For A. baumannii, C. glutamicum, E faecium, K. pneumoniae, P. aeruginosa, and S. aureus, the OD600 was measured following 24-h incubation, to calculate the percentage growth. For checkerboard MICs, each well contained two drug compounds or DMSO, for a maximum 2% final concentration of DMSO. The fractional inhibitory concentration (FIC) was calculated  = FIC. If the FIC < 0.5, this represents synergy, 0.5 < FIC < 4, is indifference, and FIC > 4, is antagonism , while the pET28a vector was amplified to linearize at the point of insertion (forward primer - ATGATGATGATGATGGCTGCTGCCC and reverse primer - CACAGCAGCGGCCTGGTG). The NEBuilder® HiFi DNA Assembly Cloning Kit was used, with a 1:2 ratio of vector to insert, and the reaction product transformed into E. coli XL10 competent cells, selecting on LB agar with kanamycin (50 μg/ml). The gene insertion into pET28a was confirmed by Sanger sequencing and the plasmid transformed into the E. coli BL21 (DE3) expression strain. An overnight culture (10 ml) of E. coli BL21 (DE3) was used to inoculate Terrific broth (1 liter) with kanamycin (50 μg/ml). The culture was grown  until mid-log growth was reached. Then IPTG (1 mM) was added and grown overnight . The cells were harvested by centrifugation . The pelleted cells were resuspended in 500 mM NaCl, 30 mM imidazole, and 50 mM Tris–HCl buffer pH 7.8 (25 ml). The cells were lysed via sonication (3 min) and then centrifuged . The clarified lysate was filtered (0.22 μM) and then loaded into a gravity-flow column containing HisPur Ni-NTA Resin (5 ml). The resin was washed with 500 mM NaCl, 30 mM imidazole, and 50 mM Tris–HCl buffer pH 7.8, before elution by increasing the imidazole concentration stepwise to 400 mM. The fractions containing MtAroB were pooled and dialyzed overnight into 200 mM NaCl, 50 mM Tris–HCl buffer pH7.8. The protein was concentrated and flash frozen for storage at –80°C.The 2.4.Mt-AroB protein  was added to 200 mM NaCl, 50 mM Tris–HCl buffer pH7.8 (400 μl) in a 1 cm quartz cuvette. This was equilibrated for 10 min (25°C), before tryptophan fluorescence  measurements were undertaken. Compounds of interest were added  and fluorescence was measured. The change in fluorescence (∆Fluorescence) was calculated relative to the initial baseline fluorescence. The fluorescence changes associated with the buffer or DMSO (10%), were subtracted from the report values.2.5.8 cells of M. bovis BCG. The plates were then incubated at 37°C until the plates dried out, approximately 3 months.Agar plates  containing 2×, 2.5×, 5× and 10× MIC of vanoxerine were each inoculated with 1 × 102.6.600 = 0.8) with tween-80 (0.05%) and ethidium bromide (0.625 μg/ml). For accumulation, glucose [0.4% (w/v)] was added, and the diluted cell culture was immediately added to the 96-well plate. For efflux, verapamil (50 ug/ml) was added, and the diluted culture was then incubated for 1 h at 37°C. The ethidium bromide-loaded cells were centrifuged , and resuspended in cold PBS  with tween-80 (0.05%). The cells were either added directly to the 96-well plate or supplemented with glucose [final concentration 0.4% (w/v)] before addition. In both assays, the fluorescence was then measured, in a BMG Labtech POLARstar Omega plate reader, every 60 s for 1 h , at 37°C. Method adapted from A 96-well plate was prepared containing 1 μl of either DMSO, verapamil, or vanoxerine, for final concentrations of 1%, 25 ug/ml, or 3.3–52 μg/ml, respectively. Mycobacterial cultures were washed and resuspended in PBS  with tween-80 (0.05%) and DiOC2(3) (30 μM). The diluted culture was incubated , before being dispensed (99 μl) into a 96-well plate. The fluorescence was measured every 90 s for 9 min in a BMG Labtech POLARstar Omega plate reader , at 37°C. The plate was then removed from the plate reader, 1 μl of either DMSO, CCCP, verapamil, vanoxerine, or bedaquiline , was added to each well. The plate was then returned to the plate reader and readings continued for a further 50 min. Method adapted from Mycobacteria cultures were washed and resuspended in PBS  containing either no drug, 13, 26, or 65 ug/ml vanoxerine, alongside C14 acetic acid (0.5 μCi per ml). These cultures were incubated at 37°C for 24-h, before cells were harvested by centrifugation . The cell samples were stored at-20°C until required. The pelleted cells were thawed and CH3Cl/MeOH/H2O  was added. The resuspension was incubated , followed by addition of CH3Cl (1.75 ml) and H2O (0.75 ml). The lower organic phase was taken and washed twice with CH3Cl/MeOH/H2O . Then the lower organic phase was transferred to a new tube and dried. For TLC analysis, the lipids were resuspended in CH3Cl/MeOH  and spotted (5–20 μl) onto silica TLC plates corresponding to 10,000 counts. TLC was performed in CH3Cl/MeOH/H2O . The lipids were visualized by exposure of the TLC plate to an X-ray film for 1-week.2.9.Mycobacterium bovis BCG Pasteur was cultured to an OD600 0.4 before exposure to DMSO, 15 or 30 ug/ml vanoxerine for 8 h in four biological replicates each. Cells (108) were pelleted, flash frozen in liquid nitrogen, and stored at −80°C and then sent to Genewiz (Azenta Life Sciences) for RNA extraction and sequencing. The raw transcriptomic outputs were mapped onto the M. bovis BCG Pasteur (GCA_000009445.1) and M. tuberculosis H37Rv (GCA_000283295.1) genomes. Annotation and clustering of the significantly dysregulated gene transcripts was performed using the DAVID server  and Gram-positive (E. faecium and S. aureus) species, alongside testing Corynebacterium glutamicum, to determine the MIC  inducible expression, both in M. smegmatis. These plasmids were transformed in M. smegmatis, and vanoxerine percentage survival curves repeated, comparing empty vector controls to the AroB overexpression plasmids . This suggested the rate of resistant generation is below 108. In addition, an M. bovis BCG strain with a recG mutation was used, due to its increased mutation rate  led to more ethidium bromide accumulation than the DMSO control. The effect was also concentration dependent, with 2× MIC99 (52 μg/ml) of vanoxerine causing 2.6× more fluorescence than DMSO and 1.2× more fluorescence than 0.5x MIC99 (13 μg/ml) across the 60-min, due to ethidium bromide accumulation. As there could be several explanations for these results, including membrane disruption/lysis, efflux inhibition, or cell energetic effects, further work was undertaken.During the 60-min assay window, vanoxerine caused a significant difference in the accumulation of ethidium bromide inside both ovis BCG . Vanoxer99 against M. smegmatis and M. bovis BCG was greater than 100 μg/ml, having limited impact on metabolic activity at the highest tested concentration. Following ethidium bromide accumulation in mycobacteria, verapamil was removed. The efflux could then be monitored as loss of fluorescence. In the absence of vanoxerine or verapamil, a clear decrease in fluorescence could be observed, approximately 65%  was used to monitor the electric potential (∆ψ) of the membrane in response to vanoxerine treatment, as ∆ψ disruption would prevent the majority of efflux (2(3) partitions across phospholipid membranes proportionally to the ∆ψ present (2(3) membrane partitioning. Hence, M. bovis BCG was pre-incubated with DiOC2(3) for 2 h prior to drug addition, to allow the dye to equilibrate across the membrane proportional to the ∆ψ. The red/green fluorescence ratio fluctuated around 1.75 in the absence of compound, reducing to 1.60 across the 60-min assay. Vanoxerine was able to disrupt the dye partitioning in a concentration-dependent manner, with 52 μg/ml vanoxerine reducing the red/green fluorescence to 1.09 after 50 min, suggesting disruption of the ∆ψ (2(3) dye partitioning at a slower rate compared to CCCP. The assay was specific to the ∆ψ disruption, rather than general proton motive force (PMF) disruption. Bedaquiline confirmed the specificity as it is known to only disrupt the ∆pH of the PMF, and hence had a response similar to DMSO in this assay, a red/green fluorescent ratio of 1.63 (2(3) partitioning, with a red/green fluorescent ratio of 1.63 after 50 min of incubation, suggesting a general bactericidal response is less likely to explain the dye’s disruption.As vanoxerine was able to inhibit efflux of ethidium bromide, the voltage-sensitive dye DiOCf efflux . DiOC2, the MIC99 of rifampicin, clofazimine, and FNDR-20081 were reduced by 2× . The evidence provided indicates vanoxerine does not interact with AroB and it is unlikely to be the target.The absence of a shift in the MIC following overexpression of the AroB protein  results , howeverreatment , do not M. smegmatis  is an indicator of membrane potential disruption , and rv3161c (bcg_3185c), as responses to membrane stress, increasing membrane transporter/efflux and a putative dioxygenase and its regulator, respectively (fasI and fasII) and peptidoglycan biosynthesis (mur) genes , except for mpt70. Overall, the high similarity of the transcriptional responses of mycobacteria to vanoxerine and 2B8 provides further evidence that vanoxerine is impacting the PMF and cellular energetics  genes . The maiergetics , 2019.M. smegmatis showed loss of TMM and TDM following vanoxerine treatment, the evidence suggests that vanoxerine does not directly target mycolic acid biosynthesis. This is supported by the fact that vanoxerine inhibits C. glutamicum, for which mycolic acids are not essential, with a comparable MIC99 to M. tuberculosis. Vanoxerine also had equal activity against C. glutamicum wild-type and the ∆pks mutant. It is more likely that the PMF dissipation caused by vanoxerine has an indirect effect on the PMF-dependent transporter MmpL3, which transports TMM across the inner membrane  can be found at: AK designed the experiments, analyzed the data, and wrote the initial draft manuscript. AK and A-RM-J performed the experiments. AK, SB, and GB critically reviewed the manuscript. All authors contributed to the article and approved the submitted version.This work was funded in part by the Wellcome Trust Doctoral Training Program Antimicrobials and Antimicrobial Resistance  and Microbiology Society . For the purpose of open access, the author has applied a CC BY public copyright license to any Author Accepted Manuscript version arising from this submission.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Boletus edulis collected from geographically diverse regions in Poland were studied in 14 composite samples that were derived from 261 whole fruiting bodies. Individual REE median concentrations ranged from 0.4–95 µg kg−1 dry weight (dw). The summed REE concentrations varied widely, with a median value of 310 µg kg−1 dw and a range of 87 to 758 µg kg−1. The Sc and Y median concentrations (dw) were 35 and 42 µg kg−1, respectively. Ce, La, and Nd, with median values of 95, 51, and 32 µg kg−1, respectively, showed the highest occurrence. B. edulis collected from a forested area formerly used as a military shooting range—possibly a historically contaminated site—had an elevated summed REE content of 1796 µg kg−1. REE concentrations were generally low in Polish King Bolete. Dietary intake from a mushroom meal was negligible, posing no health risk to consumers. Mining/exploitation and commercial applications of the rare-earth elements  in the past 3 decades have raised concerns about their emissions to the environment, possible accumulation in food webs, and occupational/environmental health effects. The occurrence and distribution of REEs Y and Sc in the fruitbodies of  The term rare-earth elements (REEs) includes La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, and Lu—all of the lanthanides series . ScandiuPhytolacca americana) and two species of ferns (Dicranopteris dichotoma and Athyrium yokoscence) were observed to be good accumulators of REE Sc and Y compared to other species investigated, while the species Ph. americana closely reflected their pattern in the soils .ubescens . In MacrB. edulis ranged from 0.08 to 1.5, and for Leccinum scabrum (Rough-stemmed Bolete) from 0.003 to 0.027) [Macrofungi are rather poor Ca bio-concentrators and this is even worse for REEs. For example, the Ca bioconcentration factor  for a large collection of o 0.027) ,51.Amanita pantherina, Lactarius hatsudake, Russula mariae, Suillus granulatus, and Tricholoma flavovirens) showed BCF values in the range from 0.003 to 0.027 (La) and from 0.0003 to 0.025 (Ce) [The little available data for REEs (such as La and Ce) in mushrooms (025 (Ce) .B. edulis. Mushrooms collected from the Morąg site in the Warmia region showed an order of magnitude greater quantities of Y, La, Ce, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, and Lu than specimens from other sites (B. edulis (and some other species from the Morąg site) relative to other locations also showed higher concentrations of other metallic elements  [Amanita muscaria was not studied for Ba, Ga, Pb, or Sr (and the REE) and Paxillus involutus had only data for Hg [Cerium was the most abundant REE determined in er sites . If we e and Zr) , though a for Hg ,54. Amanita rubescens and Amanita flavorubescens collected from different locations [Aruguete et al. earlier discussed the possible impact of geochemical background composition on the abundance of REEs in mushrooms. In their study, differences were observed in the uptake and sequestration of La, Ce, and Nd in fruiting bodies by edible ocations . B. edulis with elevated level of REEs—when compared to other sites in this study—was from the Augustów Primeval Forest site (id 6) with 758 µg kg−1 dw (M. procera and Cantharellus cibarius (Yellow Chanterelle), from the Augustów Primeval Forest site, which had REE levels well above the median values for these mushroom species collected from other sites across Poland [Apart from the Morąg site, the next pool of  kg−1 dw . Interess Poland . −1 dw) for these subgroups were 121.4, 12.8, and 5.6 in the caps, 218, 19.1, and 9.2 in the stipes, and 187.7, 17.6, and 8.7 in the whole fruiting bodies. The quotient of LREE/HREE was similar for the morphological parts of B. edulis, i.e., 21.7 for the caps, 23.7 for the stipes, and 21.5 for the whole fruiting bodies. In work by Bau et al., the quotient of LREE/HREE for a single B. edulis specimen (collected in Germany) studied there was 19.6 [Amanita flavorubescens and A. rubescens), with Nd differing from one to three orders of magnitude, and La and Ce up to two orders of magnitude [The light REEs  occurred in mushrooms in higher concentrations when compared to the middle REEs (Sm to Dy) and heavy REEs (from Ho to Lu) . Their mwas 19.6 . Aruguetagnitude . Hence, the number of individual mushroom specimens collected and tested onsite or contained in a composite sample/pool (sample size) remains essential in characterizing the ability of fungi to bioconcentrate REEs and other minerals and the extent to which they are enriched in the fruiting bodies.B. edulis based on the median concentration values as well as results for the Augustów (id6) and Morąg (id8) sites and for a specimen examined/reported by Bau et al. [B. edulis showed minor differences in patterns between some of the sites. REE Sc and Y, as non-essential elements, are considered to be co-absorbed by the mycelium alongside Ca and accumulation patterns in mushrooms more or less follow the natural pattern in soil substrata. Thus, some of the detectable differences in the normalized pattern could potentially be reflecting the local soil for a given REE(s).u et al. ,43. DetaB. edulis, and including the literature data), Y showed a small positive deviation in all the plots which in general exhibited an almost flat, unchanging profile. A similar decoupling was observed in studies of mushrooms M. procera and Suillus luteus [B. edulis from the Augustowska Primeval Forest  for the whole fruiting bodies  weight  in mushrooms (whole fruiting bodies), a meal of 100 g (whole weight) at one time would expose a consumer to 0.0031 mg of REEs, and consumption of 300 g by high level consumers would result in an intake of 0.0093 mg of REEs. The consumption of such mushroom meals daily for a week would result in the intake of 0.0217 and 0.0651 mg, respectively. Considering the highest ∑REE value of 758 µg kg−1 dw (0.0758 mg kg−1 ww) observed for the Augustów Primeval Forest site (id 6), the consumption of 100 g and a 300 g mushroom meals would respectively result in daily intakes of 0.0076 mg and 0.0227 mg of REEs. Weekly, the consumption of 100 g and 300 g meals of King Bolete collected at Augustów Primeval Forest site would result in intakes of 0.0531 mg and 0.159 mg, respectively.The sum of REEs (∑REE) in the King Bolete varied from 86.7–758 µg kgg bodies  excludin−1 day−1 for consumption of 100 g mushroom meal and from 0.043–0.379 µg kg−1 day−1 for consumption of a 300 g mushroom meal. Even for the Warmia–Morąg site, with a highly elevated ∑REE value of 179.6 µg kg−1 ww, the EDI would be 0.299 µg kg−1 day−1 for 100 g and 0.898 µg kg−1 day−1 for consumption of 300 g mushroom (whole weight) meal. These intakes are much lower than the EDI reported in the literature that would be damaging to human health (100–110 µg kg−1 day−1) [−1 ww for whole fruiting bodies, a consumer would need to eat about a 100 g mushroom meal (EDI of 0.0516 µg kg−1 day−1) daily for about 1953 days (279 weeks) to exceed the EDI threshold (100 µg kg−1 day−1), while for a child this amounts to about 970 days (138 weeks) (EDI of 0.103 µg kg−1 day−1).The effect of culinary processing of mushrooms, including the King Bolete, on the fate of the REEs is unknown. It is anticipated that, as with other elements for which there are data, that depending on a culinary treatment, the concentration in mushroom meals may be smaller, similar, or slightly greater than in the raw unprocessed  product. Thus, the assessment is made on the basis of data for the raw (unprocessed) fruiting bodies. The estimated dietary intake (EDI) of REEs from the consumption of King Bolete for an adult of weight 60 kg ranges from 0.014 to 0.126 µg kg1 day−1) ,46. Cons−1 day−1 for consumption of a 100 g mushroom meal and 0.087–0.758 µg kg−1 day−1 for consumption of a 300 g mushroom meal. Moreover, for children consuming boletes from the Warmia–Morąg site, the EDI would be 0.599 µg kg−1 day−1 for a 100 g mushroom meal and 1.796 µg kg−1 day−1 for consumption of a 300 g mushroom meal. Therefore, the health risk assessment of intake of the studied King Bolete shows that the mushroom presents no health risks from REEs, though it would be unusual to see an adult eat as much as a 300 g quantity, even during the peak of the mushroom picking season. Culinary processing of mushrooms  lowers the amount of the metallic elements in mushroom meals (wet weight basis) [137Cs, and 40K [For a child of weight 30 kg that is able to consume a similar amount as an adult, the EDI ranges from 0.029 to 0.253 µg kgt basis) ,57,58, w and 40K ,61. EvenB. edulis), foraged across Poland. Results of this study show rather low contents of REEs in the whole mushroom and in the separate morphological parts (caps and stipes). Available data also show that the possibility of contamination of the fruiting bodies of B. edulis by rare-earth elements from their increased usage worldwide in the last decades seems unlikely. Thus, from the point of view of REE contamination, intake of this mushroom, even for high level consumers, would not present any known toxicological risks.The literature on REE in foodstuffs, and mushrooms in particular, is scarce. This study documented the rare-earth element contents of the popular and valued edible mushroom, the King Bolete (


This study examines cognitive abilities, number of people in a social network, and presence of depressive symptoms among the “youngest old”, “middle old”, and “oldest old”, by ethnicity. A sample of 600 people; 200 Blacks, 200 Whites, and 200 non-white Arabs were recruited for the survey. Those with missing data were omitted from analysis, which left 435. Data were collected via a computer assisted telephone survey. Data were analyzed using two-way ANOVAs. We found significant effects on cognition by ethnicity f=2.71, p<0.05 and age group f=11.24, p<0.001. There was a significant effect of the number of people in social networks on cognition across all groups f=10.4, p<0.01. The presence of depressive symptoms was not a mediator. There was no significant interaction between ethnicity and age group, or ethnicity and social network structure.


Parecoxib sodium is a widely used parenteral cyclooxygenase 2 selective inhibitor to relieve acute postoperative pain following gynecologic laparotomy surgery. To ensure the quality of the drug, a detailed quality specification is indispensable. Nevertheless, it is unavoidable to introduce inorganic impurities during the drug preparation process and how to assess and control them matters. This study proposed an analytical procedure for the determination of elemental impurities  in parecoxib sodium, where an easier and safer digestion protocol, graphite digestion, combined with an inductively coupled plasma-mass spectrometer (ICP-MS) was adopted when compared with microwave digestion. Moreover, the study also discussed whether should they be listed in specification to comply with ICH Q3D guidelines after test of process validation batches. Limit of quantitation (LOQ) of the above elemental impurities reached to 0.05, 0.125, 0.375, 0.075, 0.125, 0.25, 0.5, 6.25, 2.25 and 7.5 ppm, respectively, and recovery in accuracy item ranged from 90.2% to 129.9%, reflecting a sensitive and accurate method. Parecoxib sodium, which served as an injectable COX-2-specific inhibitor, was extensively employed for the analgesic efficacy and safety of single intravenous doses to relieve acute postoperative pain in patients after gynecologic laparotomy surgery –3. GivenAnalysis of elemental impurities in drugs, foods, plants, and crude oil using microwave oven digestion combined with an inductively coupled plasma-mass spectrometer (ICP-MS) has been previously reported though –11, therBased on the manufacturing process of parecoxib sodium and ICH Q3D guidelines, 10 selected elemental impurities, including Cd, Pb, As, Hg, Co, V, Ni, Li, Sb, and Cu, should be considered and controlled within acceptable limits after a risk assessment , 13. The3) was purchased from Reagecon (Ireland). ICP standard cadmium 1000 mg/L, ICP standard Lead 1000 mg/L, ICP standard arsenic 1000 mg/L, ICP standard cobalt 1000 mg/L, ICP standard vanadium 1000 mg/L, ICP standard nickel 1000 mg/L, ICP standard copper 1000 mg/L, and ICP standard lithium 1000 mg/L were purchased from Reagecon (Ireland). ICP standard mercury 1000 mg/L, ICP standard antimony 1000 mg/L, and ICP standard gold 1000 mg/L were obtained from National Non-ferrous Metals and Electronic Materials Analysis Center (China). Argon (99.999%) and helium (99.999%) gases were provided by Jinan Deyang Special Gas Co., LTD. . Parecoxib sodium was obtained from New Time Pharmaceutical Co. Ltd, Shang Dong, China.Deionized water produced by a Milli-Q Integral 10 system  was used to prepare all solutions. Optima grade concentrated nitric acid  and analytical reagent 30% (m/m) hydrogen peroxide  were employed in the sample preparation. ICP Multi-Element standard  HNOμg/L was prepared by serial dilutions of ICP Multi-Element standard in 10% (v/v) HNO3. The standard stock solution was prepared by dilutions of 10 ICP standard elements into a mixture in 10% (v/v) HNO3, containing 0.04 mg/L of Cd, 0.1 mg/L of Pb, 0.3 mg/L of As, 0.06 mg/L of Hg, 0.1 mg/L of Co, 0.2 mg/L of V, 0.4 mg/L of Ni, 5 mg/L of Li, 1.8 mg/L of Sb, and 6 mg/L of Cu. The standard solution was prepared by diluting 0.25 mL standard stock solution plus 20 μL ICP standard Gold 1000 mg/L into 25 mL in 10% (v/v) HNO3. All plastic volumetric apparatus used were immersed in 10% (v/v) HNO3 for 24 h and rinsed with deionized water.Internal standard solution at a concentration of 25 μL of ICP standard gold 1000 mg/L, and 3 mL of 30% (m/m) hydrogen peroxide were then added into the tank. After mixed, they were heated on a graphite furnace  at 100°C for 30 min and transferred into a 25 mL plastic volumetric flask after cooling. Finally, they were diluted with deionized water to volume and mixed.Parecoxib sodium  was weighted accurately and placed in a digestion tank; 3 mL of nitric acid, 20 μL of ICP standard gold 1000 mg/L, 3 mL of 30% (m/m) hydrogen peroxide, and 0.25 mL of standard stock solution were then added into the tank. After mixed, they were heated on a graphite furnace at 100°C for 30 min and transferred into a 25 mL plastic volumetric flask after cooling. Finally, they were diluted with deionized water to volume and mixed.Parecoxib sodium  was weighted accurately and placed in a digestion tank; 3 mL of nitric acid, 20 +, 202Hg+, 59Co+, 51V+, 60Ni+, 7Li+, 121Sb+, and 65Cu+, were determined in kinetic energy discrimination (KED) mode. 209Bi+, 115In+, 45Sc+ and 89Y+ were determined in KED mode as internal standards. Dwell time was 0.1 seconds and resolution were selected as normal.ICP-MS experiments were carried out using an iCAP RQ mass spectrometer  equipped with an ASX-560 autosampler (Teledyne Cetac Technologies). Argon was used for plasma generation and worked as nebulization and auxiliary gas. Helium was used as collision gas. Plasma power was set as 1550 W, uptake time 80 seconds at a rate of 40 rpm/min, wash time 60 seconds, a number of sweeps 20 times, main runs 3 times, plasma gas 14 L/min, Nebulizer gas 5.4 mL/min, and Auxiliary gas 0.8 L/min. The most abundant isotopes, 111Cd+, 208 Pb+, 75 AsThe method was validated as a quantitative procedure to determine elemental impurities in parecoxib sodium and system suitability, specificity, linearity, the limit of quantitation, solutions stability, accuracy as well as precision of the method were monitored. System suitability was determined using the injection of standard solution, every 9 injections, to evaluate the relative standard deviation (RSD%). Specificity was determined by injecting blank, standard solution, sample, and spiked sample solutions to observe the interference of diluent and sample. Linearity was performed by preparing standard solutions of different concentration levels (levels 1–5), including 25%, 50%, 100%, 150%, and 250% standard solutions, and determined by constructing the calibration plots by taking standard solutions of above concentration levels. The stability of both spiked sample and the standard solution was tested for 4 h using freshly prepared solutions at room temperature. For the investigation of stability, each solution was injected into the ICP-MS system and analyzed for intact elements. Changing rate of these samples was compared to that of freshly prepared samples to determine solutions stability. Accuracy was assessed by the recoveries of spiking solutions with drugs corresponding to three concentration levels , recorded for each concentration in triplicate. Precision was demonstrated by interday and intraday studies on spiked sample solutions in sextuplicate, via calculating the standard deviations and %RSD of recoveries.μL of ICP standard gold 1000 mg/L was added before heating, ultimately.The final digestion condition was selected after several experiments with different digestion processes. Initially, 5 mL of nitric acid plus 2 mL of 30% (m/m) hydrogen peroxide were added to 0.1 g parecoxib sodium, the liquid mixture stayed clear after being heated in a graphite furnace at 100°C for 30 min. Nevertheless, it cost a lot of nitric acid and a higher concentration of nitric acid did harm to the environment, minor nitric acid was preferable. When the volume of nitric acid was reduced to 2 mL and other parameters remained, the liquid mixture went from clear to muddy left overnight. Then, 2 mL of nitric acid plus 2 mL of 30% (m/m) hydrogen peroxide were added to 0.05 g parecoxib sodium, the liquid mixture was clear after being heated in a graphite furnace at 100°C for 30 min and went from clear to muddy left overnight, which indicated that 2 mL of nitric acid was not sufficient to digest 0.05 g parecoxib sodium. Finally, 3 mL of nitric acid plus 3 mL of 30% (m/m) hydrogen peroxide were added into 0.05 g parecoxib sodium, the liquid mixture stayed clear after heated on graphite furnace at 100°C for 30 min. To prevent recovery of Hg from being lower than 70% caused by volatilization in digestion, 20 μg/g, respectively. When the concentration of the parecoxib sodium sample was confirmed (2 mg/mL), the standard solution concentration could be calculated. Furthermore, sample, spiked sample, and standard solution were injected into ICP-MS to evaluate the sensitivity and accuracy of the method preliminarily. Once responses of the elemental impurities to be measured were too low to test, a higher concentration of standard solution would be necessary. Moreover, if recoveries of the elemental impurities calculated were out of the specified range (70%–150%), further optimization of graphite digestion should be carried out.By ICH Q3D guidelines, parenteral permitted daily exposures for elemental impurities  should be 2, 5, 15, 3, 5, 10, 20, 250, 90, and 300 micrograms per day . MoreoveThe developed method was validated as per ICH guidelines for analytical performance parameters including system suitability, specificity, linearity, the limit of quantitation, accuracy, precision, and solutions stability. Moreover, the results showed a selective, highly sensitive, accurate, and reproducible method.System suitability parameters were evaluated from standard solutions every 9 injections in sample list. The % RSD of 10 elements  intensities from five replicated injections were 1.4%, 4.4%, 3.2%, 3.6%, 3.3%, 4.2%, 3.8%, 3.9%, 4.0% and 2.7%, respectively, and satisfy the system suitability parameters (%RSD of intensities NMT 20%), which showed a stable system and that validation could be continued.The specificity of the method was determined from blank, sample, and spiked sample, and standard solutions revealed that there were no other impurities intensity or interference found in each individual analyte selected; hence, it implied the developed method was a specific method.f (x) = 142.8612 x + 2.8318 for Li, f (x) = 13957.8354 x + 39.3971 for V, f (x) = 41037.1883 x + 51.79 for Co, f (x) = 11106.8984 x + 1350.5993 for Ni, f (x) = 27747.0868 x + 1395.775 for Cu, f (x) = 1288.2543 x + 8.1465 for As, f (x) = 5711.2262 x + 9.5109 for Cd, f (x) = 9127.4006 x + 41.9995 for Sb, f (x) = 14472.4198 x + 209.8530 for Hg, and f (x) = 102465.0097 x + 2135.6275 for Pb (f (x) = ax + b), obtained from calibration curves. The obtained calibration plots are depicted in The method linearity was determined from regression equations μg/L, respectively. The procedure described that the framework for the quantification of the 10 elements had a reasonably high sensitivity.It was observed that LOQ values for elements Cd, Pb, As, Hg, Co, V, Ni, Li, Sb, and Cu in this method were 0.1, 0.25, 0.75, 0.15, 0.25, 0.5, 1, 12.5, 4.5 and 15 The accuracy studies were performed for three concentration levels: 50, 100, and 150%. The mean percentage recovery of Cd was 92.8%, Pb was 104.0%, As was 115.6%, Hg was 105.7%, Co was 104.3%, V was 111.1%, Ni was 107.3%, Li was 110.0%, Sb was 94.1%, Cu was 103.1%, and all found to be within the permissible limits ranged from 70% to 150%, which showed that the proposed method was accurate. The data of recovery studies are presented in The precision of the analytical method was demonstrated by interday and intraday studies (repeatability and reproducibility) by calculating the recoveries of 10 elements in each injection as well as their % RSD. % RSD of recoveries was found to be 2.0 (Cd), 1.8 (Pb), 1.6 (As), 2.0 (Hg), 1.7 (Co), 1.7 (V), 2.2 (Ni), 1.2 (Li), 1.2 (Sb), and 2.1 (Cu). The value of % RSD in all samples were less than 20, and the results are presented in The stability of both spiked sample and standard solution were tested over a period of 4 h using freshly prepared solutions at room temperature. For the investigation of stability, each solution was injected into the ICP-MS system and analyzed for intact compounds. Intensities changing rate of these elements compared to that of freshly prepared samples ranged from 0.2% to 9.3%, not more than 20%, which manifested solutions were stable.The validated procedure was adopted to detect elemental impurities in 3 process validation batches and 3 commercial batches. Impurities of all batches were less than 30% of permitted daily exposure that was defined as the control threshold, indicating additional controls were not required in the specification by ICH Q3D and ICH Q6 guidelines for the data had been assessed appropriately and controls on elemental impurities demonstrated adequately , 14. If The linearity, specificity, precision, and recovery found according to ICH guidelines and the development of a graphite-digestion combined with an ICP-MS system for quantitative elemental impurities measurement in parecoxib sodium have been validated. In addition, this article has expanded on how to evaluate the elemental impurities and determine the limit on the base of ICH Q3D and whether the control of elemental impurities should be defined in drug specification.Moreover, the procedure was easier with regard to a briefer operation and shorter duration and safer without a higher pressure compared with microwave digestion, allowing this analytical approach for elemental impurities quantification in drug substances to be used efficiently and conveniently.


To investigate the relationship between the preoperative paraspinal Goutalier grade of fatty infiltration and postoperative cervical sagittal alignment in patients undergoing anterior cervical discectomy and fusion (ACDF).A total of 101 patients who underwent single-level ACDF with the Zero-profile implant system between March 2011 and April 2020 were included in this study. Cervical sagittal alignment parameters, including the C2-C7 Cobb angle, functional spinal unit (FSU) angle, cervical sagittal vertical axis (SVA), and T1 slope (T1S), were assessed. Preoperative magnetic resonance images were used to classify patients according to Goutalier grade. Clinical outcomes including Neck Disability Index (NDI) scores, Japanese Orthepaedic Association (JOA) scores and Visual Analogue Scale (VAS) scores were collected and analyzed.P = 0.007). At the last follow-up, the C2-C7 Cobb angle, FSU angle, and T1S improved after the surgery among the groups. Although there were varying degrees of loss of curvature among the different groups during the follow-up period, the postoperative cervical sagittal alignment parameters demonstrated no statistical differences among the three groups (P > 0.05). In addition, patients in all groups experienced significant relief of their symptoms, and the clinical scores were comparable among the groups (P > 0.05).According to the Goutalier grade, 33 patients were classified as Goutalier 0–1 (Group A), 44 were classified as Goutalier 1.5–2 (Group B), and 24 were classified as Goutalier 2.5–4.0 (Group C). The mean age among the three groups showed significant differences (The complex nature of anterior cervical surgery requires surgical attention both in decompression and sagittal alignment. Our study demonstrates satisfactory postoperative cervical sagittal alignment of patients despite different grades of fatty infiltration of the multifidus muscle following single-level ACDF. Based on our results, the improvement and maintenance of cervical sagittal alignment after ACDF remains a complex problem that spine surgeons should consider before surgery. The lordotic curvature of the cervical spine plays an important role in compensating for the spine’s balance, transmitting axial load and maintaining mechanical function –4. For pPostoperative cervical sagittal alignment is affected by many factors, such as the type of implant system, distraction degree, intraoperative procedure, and paraspinal muscle status –14. TheoUnlike posterior surgical procedures, anterior cervical discectomy and fusion (ACDF) can avoid injuring the posterior structures of the cervical spine , 21, 22.This retrospective study included patients who underwent single-level ACDF with the Zero-profile implant system at our center between March 2011 and April 2020. Patients enrolled in this study provided written informed consent. The same senior surgeon performed the surgeries. The Ethics Committee of West China Hospital of Sichuan University approved this study. The inclusion criteria were as follows: patients over 18 years old, single-level CDDD causing symptomatic radiculopathy or myelopathy, failure of conservative treatment for at least 6 weeks, spinal cord or nerve root compression confirmed by magnetic resonance imaging (MRI) and followed for at least 12 months. Patients who had prior cervical spine surgery, ossification of the posterior longitudinal ligament, tumor, active infection, or ankylosing spondylitis were excluded.The radiological evaluations were performed by lateral X-ray and magnetic resonance imaging (MRI) in all patients. The preoperative T2 MRI scans at the C5–6 level of all patients were obtained to evaluate the paraspinal muscle status . Fatty iStatic and dynamic lateral X-ray images were used to measure the cervical sagittal alignment and the parameters included C2-C7 Cobb angle, functional spinal unit (FSU) angle, range of motion (ROM) of C2-C7, the sagittal vertical axis (SVA) and T1 slope(T1S) . The C2-Postoperative complications were also recorded. The occurrence of subsidence was defined as the loss of FSU height of more than 2 mm . The radClinical evaluation was performed preoperatively and the last follow-up. We routinely collected Neck Disability Index (NDI) scores, Japanese Orthepaedic Association (JOA) scores and Visual Analogue Scale (VAS) scores. The NDI and JOA scores were used to thorough evaluate the neck function and neurological status recovery. The VAS scores were used to evaluate neck and arm pain severity relief.t test or the Wilcoxon signed-rank test was used to compare the preoperative and postoperative parameters. P values < 0.05 were considered significant.Statistical analyses were performed using IBM SPSS Statistics Version 25.0. Continuous variables were summarized as the mean ± standard deviation, and categorical variables were summarized as the rates and ratio index values. The distribution of the parameters was checked by conducting a Shapiro-Wilk test. Based on the distribution of variables, one-way analysis of variance (ANOVA) and the Kruskal–Wallis tests were performed to analyze significant differences among the groups. The Chi-squared test was used for categorical variables. The paired P = 0.007). Patients with less fatty infiltration in the cervical paraspinal muscles were younger than patients with more fatty infiltration in the cervical paraspinal muscles. No significant differences were observed regarding sex, surgical level, body mass index (BMI), intraoperative time, blood loss or smoking status (P > 0.05).In total, there were 101 patients in this study. All patients were followed for at least 12 months and had detailed radiological and clinical data. The mean age was 50.89 (range 21–78) years old, and the mean follow-up duration was 18.52 (range 12–75) months. According to the fatty infiltration of the multifidus, the patients were classified into three groups . In Group A, the Goutalier grade was 0–1; in Group B, the Goutalier grade was 1.5–2; and in Group C, it was 2.5–4. There were 33 patients in Group A, 44 patients in Group B and 24 patients in Group C. The patient characteristics of the three groups are summarized in Table 2 for multifidus, 312.81 ± 101.98 mm2 for semispinalis cervicis, 350.21 ± 155.17 mm2 for semispinalis capitis, and 394.24 ± 147.50 mm2 for splenius capitis. In Group B, the mean CSA was 223.77 ± 72.05 mm2 for multifidus, 308.50 ± 107.80 mm2 for semispinalis cervicis, 354.69 ± 131.13 mm2 for semispinalis capitis, and 396.73 ± 150.95 mm2 for splenius capitis. And in Group C, the mean CSA was 241.86 ± 82.08 mm2 for multifidus, 335.05 ± 102.48 mm2 for semispinalis cervicis, 359.36 ± 102.50 mm2 for semispinalis capitis, and 418.89 ± 137.87 mm2 for splenius capitis. There were no significant differences regarding the mean CSA of the extensor muscle among the three groups (P > 0.05).The mean CSA of multifidus, semispinalis cervicis, semispinalis capitis and splenius capitis are presented in Table P > 0.05) , while no significant difference was found in Group B, and the trend was similar regarding T1S. The FSU angle had significantly improved in the three groups by the last follow-up (P < 0.05). The SVA was almost the same preoperatively and postoperatively. At the last follow-up, no significant differences were observed among the groups regarding the four parameters. The anterior and posterior FSU height improved significantly immediately postoperatively among the three groups, and significant differences were observed. The FSU height decreased slightly during the follow-up, while no significant differences were observed postoperatively. The subsidence rates were 15.15%, 15.91% and 16.67%, and the ASD rates were 21.21%, 22.73% and 16.67% in Groups A, B and C, respectively. Both complication rates were comparable among the groups.The preoperative and postoperative cervical sagittal alignment parameters are summarized in Table 05) Fig. . CompareP = 0.408); the mean VAS score was 1.55 ± 0.62 in Group A, 1.77 ± 0.61 in Group B, and 1.83 ± 0.70 in Group C (P = 0.131); and the mean NDI score was 9.82 ± 1.51 in Group A, 9.91 ± 1.82 in Group B, and 9.67 ± 1.74 in Group C (P = 0.941). Compared with the preoperative scores, the scores improved in all groups, and significant differences were observed. However, no significant differences were found among the three groups  was introduced , measureThis study was limited by several factors. First, this is a retrospective, single-center study. The possible selection bias is an inherent limitation. Second, postoperative MRI is not a routine radiological examination during the postoperative follow-up. Thus, postoperative MRI images are not collected and analyzed. Third, this study only included the Zero-profile implant system, and the sample size and follow-up period were relatively small. Thus, our study could be improved with a prospective study of a larger sample size, a longer follow-up period and more implant systems in the future.The complex nature of anterior cervical surgery requires surgical attention both in decompression and sagittal alignment. Our study demonstrates satisfactory postoperative cervical sagittal alignment of patients despite different grades of fatty infiltration of the multifidus muscle following single-level ACDF. Based on our results, the improvement and maintenance of cervical sagittal alignment after ACDF remains a complex problem that spine surgeons should consider before surgery.