Three prognosis-related autophagy lncRNAs were identified via univariate Cox regression, least absolute shrinkage and selection operator analysis, and multivariate Cox regression analysis. We also constructed a prognostic model based on these autophagy-related lncRNAs and evaluated its ability to accurately and independently predict the prognosis of HNSCC patients. The area under the curve (AUC) was 0.864 (3-year) and 0.836 (5-year), and our model can independently predict the prognosis of HNSCC. The prognostic value of the three autophagy lncRNAs was confirmed via analysis of samples from five databases. To further identify the functions of the three lncRNAs, a co-expression network was constructed and pathway analysis was performed. In that analysis the lncRNAs were correlated with 189 related genes and 20 autophagy-related genes, and these lncRNAs mainly involved homologous recombination, the Fanconi anemia pathway, the autophagy-related pathway, and immune-related pathways. In addition, we validated the expression levels of three lncRNAs and autophagy markers  based on TIMER, Oncomine, and HPA database analysis. Our results indicated that TTTY15 was increased in HPV positive and HPV negative HNSCC patients, and three autophagy markers were up-regulated in all HNSCCC patients. Lastly, association between three lncRNAs and autophagy markers was performed, and our results showed that TTTY15 and MIF-AS1 were associated with autophagy markers. Collectively, these results suggested that three autophagy-related lncRNAs have prognostic value in HNSCC patients.Head and neck squamous cell carcinoma (HNSCC) has a poor prognosis. Considerable evidence indicates that autophagy and non-coding RNA play essential roles in the biological processes involved in cancers, but associations between autophagy-related long non-coding RNAs (lncRNAs) and HNSCC remain unclear. In the present study, HNSCC RNA sequences and autophagy-related gene data were extracted from The Cancer Genome Atlas database and the Human Autophagy Database. A total of 1,153 autophagy-related lncRNAs were selected  Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer in the world and accounts for more than 90% of head and neck cancers . AdvanceAutophagy is a self-degradative process that maintains cellular homeostasis. It plays complex roles at different stages of tumorigenesis, and it may promote oncogenesis in some contexts but participate in tumor suppression in others . Clarifyvia the mediation of autophagy-related gene (ATG) expression (Long non-coding RNAs (lncRNAs) are defined as non-coding RNAs that are ≥ 200 nucleotides in length, and they are reportedly associated with HNSCC prognosis , 7. Prevpression , 9. Incrpression .via analyses incorporating five databases. To putatively predict the function of these three lncRNAs, a co-expression network was constructed and pathway analysis was performed. Moreover, the mRNA and protein expression of three lncRNAs and autophagy markers ATG12, BECN1, and MAP1LC3B were measured through analysis in Tumor Immune Estimation Resource (TIMER), Oncomine, and Human Protein Atlas (HPA) database. Lastly, we analyzed the correlation between three lncRNAs and autophagy makers.The aim of the present study was to identify autophagy-related lncRNAs, and evaluate their prognostic value with respect to HNSCC. HNSCC RNA sequencing and ATG data from The Cancer Genome Atlas (TCGA) database and the Human Autophagy Database were extracted and analyzed to identify autophagy-related lncRNAs , 12. Unihttps://portal.gdc.cancer.gov/) (http://www.perl.org) (http://asia.ensembl.org/index.html)  . These Herl.org) . Proteinex.html) . Prior thttp://www.autophagy.lu/)  were downloaded from the Human Autophagy Database (agy.lu/) . Pearsonp < 0.05. LASSO regression was performed based on the results of univariate Cox regression analysis using the survival software and the “glmnet” software package (https://cran.rproject.org/web/packages/glmnet/index.html). After the identification of prognosis-related lncRNAs via LASSO regression analysis, multivariate Cox regression analysis with stepwise selection to identify optimal prognostic autophagy related lncRNAs according to Akaike information criterion (AIC) value (n = 77) and low-risk (n = 77) groups based on the median risk score. To investigate relationships between prognosis and expression levels of the autophagy-related lncRNAs identified, Kaplan-Meier survival curves were then plotted for three key lncRNAs. Based on average expression levels of candidate lncRNAs, HNSCC patients were divided into high and low expression groups.Univariate Cox regression was used to assess associations between autophagy-related lncRNA expression and overall survival using the “survival” software package in R . A significant difference was defined as C) value . We thenC) value . HNSCC pKaplan-Meier survival curves were plotted to compare the difference in overall survival between the low-risk and high-risk groups using the survival package . Using thttp://gepia.cancerpku.cn/detail.php) is a web server tool based on TCGA and GTEx data that can be used to conduct patient survival analysis, differential expression analysis, and correlational analysis, among other things (http://hncdb.canceromics.org/) is a comprehensive source that includes head and neck cancer-related genes and an expression analysis platform. Lnc2Cancer (http://www.bio-bigdata.net/lnc2cancer/) is a database that provides comprehensive experimentally supported associations between lncRNA and human cancer (http://bioinfo.henu.edu.cn/HNSC/HNSCList.jsp) is an online prognostic biomarker analysis tool for head and neck squamous cell carcinoma  is a user-friendly web resource for analyzing cancer OMICS data that allows the user to analyze associations between gene expression and patient survival   were assessed using R software. Pearson’s correlational coefficient > 0.4 and n 3.7.2) . To assen 3.7.2) . Lastly,n 3.7.2) . P < 0.0http://metascape.org/gp/index.html)  . P < 0.0https://cistrome.shinyapps.io/timer/) and Oncomine database (http://www.oncomine.org) (https://www.proteinatlas.org/)  , 26. Theas.org/) . Associaas.org/) , 25.http://www.perl.org). All statistical analyses were conducted using R software . P < 0.05 was considered statistically significant.Data were processed using the PERL language  and low-risk (n = 77) groups based on the median risk score. HNSCC patients were ranked based on risk scores, and survival rate was significantly associated with risk score .Publicly available datasets were analyzed in this study. RNA-seq and corresponding clinical data were available from the TCGA database .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.


Cunninghamia lanceolata (Chinese fir), a member of the conifer family Cupressaceae, is one of the most popular cultivated trees for wood production in China. Continuous research is being performed to improve C. lanceolata breeding values. Given the high rate of seed abortion (one of the reasons being the failure of ovule and pollen development) in C. lanceolata, the proper formation of female/male cones could theoretically increase the number of offspring in future generations. MIKC MADS-box genes are well-known for their roles in the flower/cone development and comprise the typical/atypical floral development model for both angiosperms and gymnosperms.C. lanceolata and divided these genes into separate branches. 27 out of the 47 MIKC MADS-box genes showed differential expression between female and male cones, and most of them were not expressed in leaves. Out of these 27 genes, most B-class genes (AP3/PI) were up-regulated in the male cone, while TM8 genes were up-regulated in the female cone. Then, with no obvious overall preference for AG (class C + D) genes in female/male cones, it seems likely that these genes are involved in the development of both cones. Finally, a small number of genes such as GGM7, SVP, AGL15, that were specifically expressed in female/male cones, making them candidate genes for sex-specific cone development.We performed a transcriptomic analysis to find genes differentially expressed between female and male cones at a single, carefully determined developmental stage, focusing on the MIKC MADS-box genes. We finally obtained 47 unique MIKC MADS-box genes from C. lanceolata, illustrating a potential link of these genes with C. lanceolata cone development. On the basis of this, we postulated a possible cone development model for C. lanceolata. The gene expression library showing differential expression between female and male cones shown here, can be used to discover unknown regulatory networks related to sex-specific cone development in the future.Our study identified a number of MIKC MADS-box genes showing differential expression between female and male cones in  Cunninghamia lanceolata (Lamb.) Hook (Chinese fir) is one of the most commercially important timber trees in the south of China. It has been cultivated for thousands of years because of its outstanding wood properties and high growth rate [C. lanceolata is a monoecious conifer species, with female cones distributed in the upper and middle crown and male cones distributed in the middle and lower crown [C. lanceolata female/male cones differ greatly from classic angiosperm flowers  [SHP, STK) [SEP1, 2, 3, 4) [Flower development is a complex biological process and is affected both by genetic and environmental factors . In angines, AG) . After iHP, STK)  and E   genes – 2, 3, 4) . For exa2, 3, 4) , 16. How2, 3, 4) , 6. Howe2, 3, 4) .AP2 gene, genes belonging to the ABCDE model are members of the MADS-box gene family, which have crucial functions in floral organ development [https://www.pnas.org/content/97/10/5328) “Copyright (2000) National Academy of Sciences, U.S.A.” )[Excepting the elopment . All MADelopment . Both MAU.S.A.” ). A distiU.S.A.” ). The higopyright 00 Nationopyright 00 NationU.S.A.” ). In someU.S.A.” ), 23. TheC. lanceolata has mainly focused on the regulation of cambial activity [C. lanceolata. This study provides a valuable resource for gymnosperm cone development-related genes and may aid in breeding trees with increased seed numbers in upcoming Chinese fir improvement programs.Previous research in activity , EST-SSRactivity –27, geneactivity , celluloactivity  and protactivity . Until nC. lanceolata clone (6421 )[Immature female and male cones were collected in late February from two different living trees  that belong to a single e 6421 ). The tre1 . The tFemale/male cones fixed in 2.5% glutaraldehyde were flushed with 0.1 M phosphate buffer, dehydrated using a series of graded ethanol solutions, dried using a critical point dryer , mounted with double-sided adhesive tape on stubs, and coated with aurum in a sputter coater . Samples were observed on a Quanta 200 scanning electron microscope  .An ethanol precipitation protocol and CTAB-PBIOZOL reagent was used for the purification of total RNA according to the manufacturer’s instructions. Total RNA was quality controlled and quantified by a NanoDrop and Agilent 2100 bioanalyzer . Oligo (dT)-attached magnetic beads were used to purify mRNA. mRNA was then fragmented, after which first- and second-strand cDNA was generated using the First Strand reaction system. Afterwards, the purified cDNA was ligated to specific adapter sequences. Then, cDNA fragments were amplified by PCR, then purified using Ampure XP Beads. An Agilent 2100 Bioanaylzer and ABI StepOnePlus Real-Time PCR System were used for quantification and quality control of the sample library. The library was then sequenced using an Illumina HiSeq 4000 platform  (reads length 151 bp). Sequenced reads were deposited in the NCBI Sequence Read Archive (SRA) with the accession number SRR10161401, SRR10161402, SRR10161403, SRR10161404.The raw data was first filtered to obtain high-quality clean data. Adapter sequences, low-quality reads  and reads with more than 5% of their bases unknown were removed from the raw reads. Clean reads were then quality controlled by FastQC v0.11.7 . Clean rCoding regions of assembled unigenes were annotated by mapping them to several public databases, respectively, using TransDecoder, after which a blastp algorithm  was run Arabidopsis thaliana, Populus trichocarpa, Oryza sativa, and Swiss Prot [A. thaliana and P. trichocarpa. Due to our interest in transcription factors (TFs) specifically, a gene type parameter was added to the annotation process. In all cases, the BLAST algorithm [− 5.Functional annotation of these sequences was performed by running blast against protein sequences from iss Prot . The finiss Prot  annotatilgorithm  was applP-value thresholds were adjusted for false discovery rate (FDR) via a multiple testing approach [Gene expression levels were estimated by mapping clean reads to the Trinity transcript assembly using RSEM  for eachapproach . The conapproach . To deteC. lanceolata MADS-box sequences, two reported Hidden Markov Model profiles SRF (PF00319) and K-box (PF01486) were obtained from Pfam [To identify rom Pfam . Using Hrom Pfam  with therom Pfam .C. lanceolata, lowly expressed MADS-box genes were removed from the DEG list by the edgeR analysis package [To faithfully identify differentially expressed MADS-box genes in female/male cones of  package , leaving package  were dowA. thaliana, O. sativa and Vitis vinifera were obtained from the Plant Transcription Factor Database (http://planttfdb.cbi.pku.edu.cn/index.php), while sequences of Cryptomeria japonica, Picea abies, Pinus taeda (http://congenie.org) were gained separately from three articles by Futamura et al. [V. vinifera, P. abies, P. taeda, C. japonica, and C. lanceolata were selected and showed by Texshade [MADS-box sequences of a et al. , Carlsbea et al.  and Chena et al. . All thea et al. , after wa et al.  was usedTexshade .CleIF3. Expression data were calculated through the Livak calculation method, and show as log (2-ΔΔCt) [Several MADS-box genes were selected to validate our DEGs detection. Total RNA was obtained from immature female/male cones using a Bioteke plant total RNA extraction kit (RP3301), only replacing the lysis buffer by CTAB. Total RNA integrity was determined by gel electrophoresis (1% gel) and RNA concentration was measured using a Nanodrop-2000 spectrophotometer . cDNA was synthesized through a reverse transcriptase approach using the Vazyme HiScript 1st Strand cDNA Synthesis Kit(R211–02), then quantified using a Qubit 2.0 (Invitrogen). Quantitative real-time PCR (qRT-PCR) reactions were performed in triplicate using the Vazyme AceQ qPCR SYBR Green Master Mix (without ROX) (Q121–02) on a LightCycler 480 II (Roche). Gene expression analysis was performed based on three technical and biological replicates and normalized with the reference gene (2-ΔΔCt) .C. lanceolata breeding. To improve breeding values of C. lanceolata, it is necessary to study the molecular mechanisms of cone development. This is only exacerbated by the fact that C. lanceolata is a gymnosperm, and the structure of its female/male cone differs greatly from that of angiosperm flowers  to observe the female and male cones, especially to observe the ovule and pollen. At the stage we sampled, each female cone contains a high number of bract-scales, with an ovuliferous scale at the base of the bract-scale, and 2–3 ovules located at the ovuliferous scale , 18,114,397 , 18,731,606  and 22,054,735  raw reads for each library : AP2, MYB-related, and MADS-box , SEP (class E), AG (class C), STK/SHP (class D), which are involved in flower organ identity [FLC, BS, FUL and AP1 (class A) cannot be found. This may be explained by the low expression of these homologous genes during the selected period. Another possible reason is that the C. lanceolata genome does not contain these genes. Due to not having a certain typical flower structure, floral organ identity related genes, like AP1, which contributes to the sepal and petal formation in angiosperms, could have been lost during evolutionary time. Specific cases of such potential gene loss require further research to illustrate.Most MADS branches can be found in identity . HoweverTM8 genes, are not found in Arabidopsis and Rice, but can be found in C. lanceolata, C. japonica [V. vinifera [TM8 genes were established in the common ancestor of angiosperms and gymnosperms and that they have been lost independently during the relatively recent evolution history of some plant lineages [On the contrary, some MADS branches, like the japonica , V. vinivinifera . These rlineages .AGL15 and AGL12 genes were found in C. lanceolata, and Pinus taeda [A. thaliana [V. vinifera [https://www.ncbi.nlm.nih.gov/) using blastp and found that it was partial identity to AG-like gene. However, the classification cannot be gained in our phylogenetic tree. Thus, we named it with its number: MADS41, which make it a novel candidate gene.We also identified a small number of MIKC MADS-box genes that can be classified into GGM7 branches, and not found in angiosperms . In contus taeda , as wellthaliana  and V. vvinifera , indicatC. lanceolata MIKC MADS-box genes, 27 genes differentially expressed between male and female cones, of which 18 (out of 27) are not expressed in leaves and 9 (out of 27) are not significantly expressed in leaves.  (4) were up-regulated in the male cone, similar to what was found in previous studies performed in other plant species [TM8 genes were clearly expressed at a higher level in the female cone and more likely to be involved in female cone development.We next used our differential gene expression data to identify which MADS-box genes are differentially expressed between female and male cones, using expression data from leaves as a comparison of non-reproductive tissue. We reasoned that genes involved in the development of reproductive organs should be more specifically expressed in those organs. Out of the 47 es. Fig. . Most B- species , 58, whiAG (class C + D) genes are upregulated in male cones and others in female cones, it seems likely that these genes are involved in the development of either cones. We identified three SEP (class E) and four AGL6 genes in C. lanceolata. However, SEP genes showed a very low expression level, which is difficult to determine their differential expression across cones. Nevertheless, we do find two AGL6 genes expressed in both female and male cones. In fact, during the stage we collected, AGL6 genes showed higher expression level in the female cones.Since we found that some GGM7 genes can be subdivided into 2 categories according to the phylogenetic tree: DAL10-like and DAL21-like. They have different expression patterns in female and male cones of P. abies [C. lanceolata. While in A. thaliana AGL15 expressed in leaf, inflorescences, anthers and pollen [SVP expressed in young leaves, floral primordia and early coflorescences [AGL15 and SVP are highly expressed in male and female cones of C. lanceolata, respectively. Besides, MADS41 is a special gene with no obviously classification. But its high expression level in female cones, making it a candidate gene that may be involved in female cone development.P. abies , as welld pollen ; SVP expescences , AGL15 aC. lanceolata MIKC MADS-box genes from the differentially expressed gene list , as well as genes not found in some angiosperms (such as Arabidopsis and rice) (TM8).In order to validate the differences observed between female and male cone libraries, we selected a limited number of t Table S and perft Table S. This seClMADS34 gene in male cones was about 100 times that of female cones, and the expression of ClMADS10 in female cones was almost 10 times that of male cones, which was consistent with the results of the transcriptome data, suggesting the reliability of our transcriptomic profiling data  were mostly up-regulated in the male cone, which is most likely to influence male organ development. Similar results have been reported in angiosperm Quercus suber [C. japonica [CjMADS1 gene (B-type MADS-box gene), expressing in male cone through its development in C. japonica. As is known from Norway spruce, B-type MADS-box genes, which are active in male organ primordia [We found class B, C, D and E genes in us suber  and gymnjaponica , for exarimordia , are homrimordia . These fC. lanceolata, and are expressed in both reproductive organs. AG genes expressed in both cones of P. abies [Gnetum gnemon[Quercus suber (angiosperm), where C-class genes are expressed at a similar level in both male and female flowers [C and D-class genes cannot be separated clearly in P. abies  and Gnetum gnemon  in C. lanceolata expressed in both female and male cones, and even higher in female cones. It reflects that there are both functional conservatism and functional differentiation in genes of different species.Additionally, we identified the expression of . gnemon . GGM7 genosperms . In P. aen cones , and DALAGL15 and SVP gene act as repressor of floral transition in A. thaliana [C. lanceolata, AGL15 and SVP are highly expressed in male and female cones, respectively. It could be an interesting research issue and may imply a similar inhibitor in Chinese fir, restricting the development range of cones.thaliana , 60, whiTM8 genes which were all up-regulated in the female cone and basically not expressed in leaves. Researchers have found that in E. grandis, EgTM8 is expressed in the early and late floral bud [TM8 may be important for ovary and fruit formation [TM8 genes could be found in many gymnosperms, but little research has revealed its function in organ development in gymnosperms. Considering that ovules and pollen of C. lanceolata were still under development at the time of collection, we speculate that these genes are very likely to influence ovule development and can be further studied.Furthermore, we identified several genes which may play an important role in female cone development. We detected oral bud . And in ormation . Gramzowormation  showed tC. lanceolata, more experiments are needed to confirm the function of B(C) genes and rule out the involvement of other genes . In a general way, we study the gene function by overexpressing and knockout this gene in the species. Unfortunately, a mature transgenic system for C. lanceolata has not yet been developed, and performing transgenesis experiments in this species would have the added downside that the flowering of woody plants takes a long time. Thus, other method should be considered, for example, expressed C. lanceolata B-type genes in model organisms such as Arabidopsis, so as to study the degree of functional conservation of those genes. But it must be emphasized that the gene function studies will eventually return to the species itself. Yet considering the difficulty of generating transgenic gymnosperms and their long generation times, these studies would need a lot of time and efforts.Based on our results, we tend to agree with the B(C) model of gymnosperm cone development proposed by Theißen et al. , which ADue to our limitation of material selection, the results were limited to the differential genes between female and male cones at a certain developmental period. Although some noteworthy genes were indeed found through our study, some information for cone development may be lost, and participation of those MADS-box genes in the entire developmental process cannot be obtained. Further research could monitor the entire developmental process, from cone initiation to female cone fertilization, to potentially find all MADS-box genes involved, and perform a more complete interpretation.C. lanceolata and analyzed the gene expression differences between female and male cones. We identified 47 MIKC MADS-box genes in C. lanceolata, and identified some MADS-box genes related to cone development in C. lanceolata, possibly conforming to the previous B(C) model for gymnosperms. We also identified additional genes that may play an important role in female/male cone development. In addition, we provided a library of gene data that shows differential expression between the female and male cones, which can be used as a basis for discovering unknown regulatory networks in the future.In summary, we performed an RNA-Seq analysis of female and male cones in Additional file 1. All reference sequences for phylogenetic tree.Additional file 2 Table S1. Sequencing statistics of C. lanceolata cone RNA-seq libraries. Individual libraries were generated from four specific RNA pools, two from female cones (F3 and F4) and two from male cones (M3 and M4). Table S2. Summary on assembled transcripts and unigenes of all samples. Table S3. Summary on annotation transcription factors (TFs).Additional file 3 Table S4. All C. lanceolata MIKC type MADS-box genes.Additional file 4 Figure S1. Alignment of selected MADS-box genes conserved domains. M (A), I (B), K (B & C), C (D) domain of MADS-box proteins in V. vinifera, C. japonica, P. abies, P. taeda and C. lanceolata (not fully length).Additional file 5 Table S5. Differential expressed MIKC MADS-box genes of C. lanceolata.Additional file 6 Table S6. List of the primers used for qRT-PCR.


Mycobacterium bovis (M. bovis) osteitis is highly suspected. She was treated with antimycobacterium drugs and showed good results. Osteomyelitis can manifest by mimicking bone tumors. Without a biopsy, the pathogen may go undetected. So, interventions such as biopsy are warranted and avoid mass resection without indication. High C-reactive protein (CRP), alkaline phosphatase (ALP), periosteal reaction of radiating spicules, and penumbra sign in magnetic resonance imaging (MRI) are helpful for discriminating osteomyelitis from bone tumor.Chest wall mass in infancy is rare. Malignant lesions are more common than infection or benign tumors. This is a case of a 12-month-old girl who presented with a 2 cm mass at the right costal margin and poor weight gain. Chest radiograph demonstrated a moth-eaten osteolytic lesion at the 8th rib. The resection was performed, and a mass with pus content was found. The positive acid fast stain (AFB) organism was noted. Pathology confirmed caseous granulomatous inflammation compatible with mycobacterial infection. However, QuantiFERON-TB Gold was negative, so  Chest wall mass in infancy is rare . As an iInfection in the form of chest wall osteomyelitis can be caused by bacteria, fungi, and mycobacteria, whether from direct extension of empyema and pneumonia or hematogenous spread . World iH. influenza, and intradermal tuberculosis vaccine at birth.A 12-month-old girl came from Nakornsawan province, who presented with painless progressive palpable round shape mass at the right costal margin. She had no fever. Her appetite was good, but still she had poor weight gain with height and weight both lower than fifth percentile. No history of contact with tuberculosis was found, but her mother had a positive history of TB lymphadenitis and completed treatment more than ten years ago. The natal history was unremarkable. She completed the Thai national immunization program and was given complementary vaccines including pneumococcus, th rib, shown in N: 20.4%, L: 73.1%), PLT: 339,000/mcL, alkaline phosphatase: 201 (150–420) U/L, and hs-CRP: 20.98 (0–5) mg/L. Chest magnetic resonance imaging (MRI) is shown in Physical examination revealed a 2 cm round fixed firm mass at the right anterior lower chest wall. Bacillus Calmette–Guérin (BCG) scar was found at right upper leg. Otherwise, it was unremarkable. Chest radiograph demonstrated a moth-eaten osteolytic lesion at the right anterior 8Mycobacterium tuberculosis complex (MBTC). Pathology, shown in Mycobacterium bovis (M. bovis) osteitis is highly suspected. She was treated with HRZE standard regimen  for 2 months and switched to HR (isoniazid and rifampicin) for a total of 12 months. Outpatient 3-year follow-up demonstrates excellent weight gain, good appetite, and normal wound healing without mass progression.Resection of the mass was arranged. Intraoperative examination revealed a firm round 5 cm mass with signs of osteomyelitis on the rib and pus. The pus was further processed and revealed positive acid fast bacilli stain (AFB) and was reactive of polymerase chain reaction (PCR) for M. tuberculosis. Therefore, other species should be suspected including M. bovis.The diagnostic guidelines recommend that the diagnosis of TB should follow the cautious history of TB contact, symptoms and signs, abnormal chest radiograph, sputum/gastric aspiration smear AFB/culture, and tuberculin skin test . NegativM. bovis infection is more likely to present with extrapulmonary infection [Bacillus Calmette–Guérin (BCG), Tokyo 172-strains, was employed for preventing tuberculosis and has shown excellent results in preventing miliary and meningeal tuberculosis . Althougnfection , 10. Ostnfection . Only 2%nfection .M. bovis to M. tuberculosis. Infected sites are often the rib (6 of 8 cases), and the others are sternum, chest wall, and pulmonary and hematologic dissemination. M. bovis infection was only found in the infant period. Therefore, if age is more than 12 months, M. tuberculosis should be considered first [Review from 8 cases of chest wall mycobacterial infection  shows thed first , 12–18.M. bovis is resistant to pyrazinamide. So, the combination of 2HRZE + 10HR is strongly recommended for osteomyelitis and a longer course is considered in severe disease [Naturally,  disease .Staphylococcus aureus and Klebsiella pneumoniae [Osteomyelitis can manifest by mimicking bone tumors. The most common pathogen in nontuberculous endemic areas are bacteria such as eumoniae . WithoutIt is not possible to distinguish between osteomyelitis and a bone tumor clinically. The majority of osteomyelitis cases did not show concurrent fever and mild or no local reactions . But mulM. bovis should be proven due to difference in drug regimens.This case report shows an atypical finding of a rib mass in infancy. As atypical infection, benign and malignant tumors must be considered. Although infection is in the differential, tissue biopsy or resection may be necessary. CRP, ALP, and penumbra sign on MRI may support the diagnosis of osteomyelitis and favor a biopsy rather than mass excision. For younger patients with mycobacterium osteomyelitis or abscess,


Aspergillus nidulans. Here, the role of HbxB in A. nidulans was further characterized. RNA-sequencing revealed that HbxB affects the transcriptomic levels of genes associated with trehalose biosynthesis and response to thermal, oxidative, and radiation stresses in asexual spores called conidia. A phenotypic analysis found that hbxB deletion mutant conidia were more sensitive to ultraviolet stress. The loss of hbxB increased the mRNA expression of genes associated with β-glucan degradation and decreased the amount of β-glucan in conidia. In addition, hbxB deletion affected the expression of the sterigmatocystin gene cluster and the amount of sterigmatocystin. Overall, these results indicated that HbxB is a key transcription factor regulating trehalose biosynthesis, stress tolerance, β-glucan degradation, and sterigmatocystin production in A.nidulans conidia.Homeobox transcription factors are conserved in eukaryotes and act as multi-functional transcription factors in filamentous fungi. Previously, it was demonstrated that HbxB governs fungal development and spore viability in  Asexual spores are the main reproductive cells in most filamentous fungi ,2. AsexuAspergillus nidulans, as various genetic and molecular techniques have been developed [A. nidulans reproduces primarily through asexual development and produces an asexual-specific structure called conidiophore bearing long chains of asexual spores termed as conidia [The process of asexual spore production in filamentous fungi has been mainly studied in the model fungus eveloped ,7. A. ni conidia . The pro conidia . For con conidia ,12,13,14 conidia ,13,15. W conidia ,18,19,20MATa and MATα form homocomplexes or heterocomplexes and regulate gene expression and yeast differentiation in the model yeast Saccharomyces cerevisiae [Candida albicans, GRF10 is involved in filamentous growth, biofilm formation, and virulence [Aspergillus flavus, Hbx1 plays an important role in fungal differentiation and secondary metabolism [hbx1 affects the mRNA expression of developmental genes and secondary metabolite gene clusters [Aspergillus fumigatus, HbxA, a homolog of Hbx1, is a key regulator for asexual development, secondary metabolism, and pathogenesis [Homeobox domain-containing proteins are found in animals, plants, yeast, and filamentous fungi ,22. Thesrevisiae . In the irulence ,30. In Atabolism . Transcrclusters . In the ogenesis .A. nidulans genome contains eight hbx genes [hbxA and hbxB are essential for appropriate hyphal growth, conidiophore production, and cleistothecia formation in A. nidulans. In addition, hbxB deletion leads to decreased amounts of trehalose, conidia viability, and thermal tolerance in conidia, implying that HbxB plays a key role in conidia maturation. To further test the role of HbxB in conidia, we conducted transcriptomic and phenotypic analyses in this study.A recent study indicated that the bx genes . Among tpyrG89; AfupyrG+) [hbxB deletion mutant , and hbxB-complemented ::hbxB::FLAG3x::pyroA; ΔhbxB::AfupyrG+) [In this study, control  , hbxB defupyrG+)  strains fupyrG+) .hbxB conidia were collected from the plates after 2 days of culture and filtrated through Miracloth . The total RNA from conidia was extracted using Trizol reagent . After RNA extraction, DNase I  was used for the removal of DNA contamination from RNA samples and then further purification using the RNeasy Mini Kit .The whole processes of RNA-seq analysis were performed as described previously . The ΔhbComplementary DNA (cDNA) library preparation and RNA-seq were performed by Theragen Bio Co., Ltd. . Briefly, mRNA from total RNA was isolated from magnetic beads with oligo(dT). The cDNA library for RNA-seq was prepared using the TruSeq Stranded mRNA Sample Prep Kit . The library was evaluated using the Agilent High Sensitivity DNA Kit  and sequenced using an Illumina HiSeq2500 sequencer (Illumina). All RNA-seq data files are available from the National Center for Biotechnology Information BioProject database (PRJNA681980).A. nidulans A4 transcriptome [RNA-seq data were analyzed as reported previously . Brieflycriptome  using thcriptome . Gene excriptome . Gene-lecriptome  with thecriptome  on the bp < 0.001 was considered statistically significant to identify the significant category from the analyzed DEGs.GO term enrichment analysis was shown using the Gene Ontology Slim Mapper at AspGD . A GO-bahbxB, and C’ hbxB conidia were extracted using the method mentioned above. The GoScript Reverse Transcription System (Promega) was used for cDNA synthesis. The iTaq Universal SYBR Green Supermix and the CFX96 Touch Real-Time PCR Detection System  were used for qRT-PCR. The 2−ΔΔCT method was used for calculating the expression levels of the target genes, and β-actin was used as an endogenous control. The gene-specific primers used in this study are listed in For qRT-PCR analysis, total RNAs from control, ΔThe UV stress tolerance assay was carried out as described previously . BrieflyThe amounts of β-1,3-glucan in conidia were measured by the Glucatell assay  as described previously ,42. Brie9 conidia were mixed with CHCl3 and 0.5 mm zirconia/silica beads  and disrupted using a Mini-Beadbeater . After centrifugation, the organic phase was transferred to new vials and evaporated. Each sample was resuspended in CHCl3, spotted onto a TLC silica plate , and resolved in toluene/ethyl acetate/acetic acid . The TLC plates were treated with 1% aluminum hydroxide hydrate . The images of the TLC plates were captured after UV exposure (366 nm). The spot intensities of sterigmatocystin were quantified using ImageJ software.The extraction of sterigmatocystin from 2-day-old conidia was conducted as described previously ,43. AbouhbxB strains were evaluated by Student’s unpaired t-test. The mean ± standard deviation are shown. p < 0.05 was considered significant.The statistical differences between control and ΔhbxB mutant conidia were conducted. The transcriptomic analysis results found that a total of 6230 genes were differentially expressed between control and ΔhbxB mutant conidia  has been mainly studied [Aspergillus species, including Aspergillus nidulans, Aspergillus fumigatus, and Aspergillus flavus, hbxA (or hbx1) deletion affects hyphal growth, conidiophore formation, and secondary metabolite production. The roles of hbxA in asexual development has been demonstrated in other fungi, such as Fusarium graminearum, Magnaporthe oryzae, and Ustilaginoidea virens, suggesting that the roles of HbxA (or HbxA orthologs) are conserved in fungal development [A. nidulans [A. nidulans. In this study, we first reported the function of HbxB in spores through the transcriptomic and phenotypic analyses. These results can provide an insight into the basic knowledge about the function of the HbxB orthologs in other fungal species. Homeobox proteins are conserved in most filamentous fungi and play diverse roles in fungal development and metabolisms ,26. Most studied ,33. In telopment ,27,45. Unidulans . HbxB achbxB conidia , thermal , radiation , and UV  stresses  pathway and the velvet proteins control conidial stress tolerance in A. nidulans [One of the important findings in this study is that HbxB is important for response to various stresses in conidia. RNA-seq results found that the mRNA expression of approximately 150 genes associated with stress response was downregulated in Δ conidia . These astresses . This denidulans . We can hbxB conidia. However, this result is the opposite of a previous result. In dark conditions for sexual development, hbxB deletion decreased sterigmatocystin production and the mRNA levels of aflR, encoding an activator of the sterigmatocystin gene cluster [Another finding in this study is that HbxB is involved in gene expression related to secondary metabolite gene clusters . In part cluster . It was A. nidulans. During the developmental process, HbxB regulates the balance between asexual and sexual development. In conidia, HbxB regulates the mRNA levels of genes associated with stress response, β-glucan biosynthesis, trehalose biosynthesis, and secondary metabolism, thereby governing conidial stress response, primary and secondary metabolism, and conidial maturation (A. nidulans.Overall, this study suggests that HbxB has a multi-functional role in fungal development and metabolism in turation . Althoug


Non-tuberculous mycobacterial pulmonary disease (NTM-PD) is becoming a significant health burden. Recent advances in analysis techniques have allowed the accurate identification of previously unknown NTM species. Here, we report a case of NTM-PD caused by a newly identified mycobacteria in an immunocompetent patient.Mycolicibacterium mucogenicum. We started combination therapy with clarithromycin, moxifloxacin, amikacin, and imipenem/cilastatin, referring to drug sensitivity test results and observed its effect on M. mucogenicum infection. Her symptoms and radiological findings improved significantly.A 44-year-old woman was referred to our hospital due to the frequent aggravation of her chronic respiratory symptoms, with NTM-PD-compatible computed tomography findings. Unidentified mycobacterium was repeatedly isolated from respiratory specimens and we diagnosed her as NTM-PD of unidentified mycobacterium. Subsequent whole-genome analysis revealed that the unidentified mycobacterium was a novel mycobacterium genetically close to Mycolicibacterium toneyamachuris, genetically close to M. mucogenicum. This pathogenic mycobacterium showed different characteristics from M. mucogenicum about clinical presentation and drug sensitivity. The clinical application of genomic sequencing will advance the identification and classification of pathogenic NTM species, and enhance our understanding of mycobacterial diseases.We report a case of NTM-PD caused by a newly identified mycobacteria,  The prevalence of non-tuberculous mycobacterial pulmonary disease (NTM-PD) is increasing worldwide and is becoming a significant health burden . Recent Mycolicibacterium mucogenicum. This novel mycobacterium caused chronic and progressive pulmonary disease in an immunocompetent patient. Furthermore, drug susceptibility and clinical presentation were different from those reported for M. mucogenicum infections [Here, we report a case of NTM-PD caused by a newly identified mycobacteria genetically close to fections .M. mucogenicum . The DNA G + C content of the type strain was 67.18 mol%. The mean nucleotide identity (ANI) to M. mucogenicum and the type strains of M. phocaicum and M. aubagnense by calculating the ANI values and constructing the phylogenetic tree based on core genomes consisting of 455 genes , and 5% Sheep Blood Agar and appeared greyish without pigmentation  Mutual similarity using average nucleotide identity. B) Phylogenetic tree generated by core genome consisting of 455 genes. Supplementary Figure 2. The colony formation of TY81 on Tryptic Soy Agar at 30 °C at day 7. Scale bar indicates 2 mm.


Culicoides transmit a variety of pathogens. Our aim wasto survey the Culicoides species occurring in an Amazonianrural settlement, comparing abundance, richness, and diversity in differentenvironments.  The Culicoides were captured using CDC light traps. TheShannon-Wiener (H’) and Rényi indices were used to compare species diversityand evenness between environments, the equitability (J’) index was used tocalculate the uniformity of distribution among species, and similarity wasestimated using the Jaccard similarity index. A permutational multivariateanalysis of variance was applied to assess the influence of environment onspecies composition. A non-metric dimensional scale was used to representthe diversity profiles of each environment in a multidimensional space. Culicoides were captured, representing 84 species . H’ values showed the following gradient:forest > capoeira > peridomicile > forest edge. The equitability J’was greater in capoeira and forests compared to peridomiciles and the forestedge. The population compositions of each environment differedstatistically, but rarefaction estimates indicate that environments of thesame type possessed similar levels of richness. Species of medical andveterinary importance were found primarily in peridomiciles: C.paraensis, vector of Oropouche virus; C.insignis and C. pusillus, vectors ofBluetongue virus; C. filariferus, C. flavivenula, C. foxi,and C. ignacioi, found carrying LeishmaniaDNA. 6.078 Culicoides can adapt to anthropized environments. This study indicates that diversity was higher in natural environments thanin anthropized environments, while abundance and richness were highest inthe most anthropized environment. These findings suggest that strictly wild Tropical forest ecosystems host two thirds of the Earth’s terrestrial biodiversityand provide significant benefits to the biosphere and the global economyEnvironmental changes caused by anthropogenic interference are associated withincreased health risks. Vectors and pathogens previously found only in forests havebeen observed in human settlements. According to Gottdenker et al. (2014)Hematophagous biting midge populations are affected by anthropogenic interference. Inthe Brazilian Amazon, Castellón (1990)This study was conducted in the settlement of Rio Pardo, Presidente FigueiredoMunicipality, Amazonas State, Brazil. Until recently, human activity in Rio Pardowas limited to subsistence farming, raising livestock, hunting, fishing, andgathering wood for local use, but newly expanded branches of forestry and fishfarming have had a significant impact on the natural environment. Diseasetransmission dynamics in the region may have been altered by the degradation ofnatural habitats causing increased human exposure to forests, and to disease vectorsand their hosts. The increased supply of blood meal sources around peridomiciles anda lack of basic sanitation offer favorable conditions for insect vectors toflourish.Culicoides is comprised of 1.368 species worldwide; 299 ofthese species occur in the Neotropics and 122 species occur in the Brazilian AmazonBasin-Culicoides species transmit viruses such asAfrican horse sickness virus (AHSV), Bluetongue virus (BTV) which infects domesticand wild ruminants, and Oropouche virus (OROV) which infect humansMansonella ozzardi, M.perstans and M. streptocercaCulicoides may also be involved in the transmission ofLeishmaniaThe genus Culicoidesspecies that occur in a typical Amazonian rural settlement, and to compare theabundance, richness, and diversity of Culicoides populationspresent in different environments.Identifying possible disease vectors is therefore of significant epidemiologicalimportance. The aim of this study was to survey the The rural settlement of Rio Pardo (1°49’02.3’’S 60°19’03.5’’W), Presidente FigueiredoMunicipality, Amazonas State, Brazil , was fouCulicoides capture was performed in June, July, and August 2010 onfour consecutive nights per month. Four different environments within the householdbuffer zone were sampled: peridomicile, areas with enclosures for livestock;capoeira, successional areas that have regenerated naturally from both functioningand abandoned agroecosystems; forest edge, areas of transition between capoeira andforest; and forest, comprised primarily of ombrophilous forest or “terra firme”(upland Amazonian forest that never floods) and river margins. Twenty-four householdbuffers were sampled. Four Centers for Disease Control (CDC) light traps perhousehold were used to capture Culicoides, totaling 96 traps. Thesewere installed 1.5 m above the ground and remained in the field from 6:00 p.m. to6:00 a.m. Biting midges were transported in 70% ethanol and slide-mounted in phenol-balsam, asdescribed by Wirth and Marston (1968)Many specimens had morphological variations in common. Unidentified specimens weretherefore grouped into morphotypes and treated as valid species in the diversity andrichness analyses. The Shannon-Wiener (H’) and Rényi indexes were used to comparespecies diversity and evenness between the four environments. The Pielouequitability index (J') was used to calculate the uniformity of distribution ofindividuals among species, and similarity was calculated using the Jaccardsimilarity index (Cj), which qualitatively compares species similarity along anenvironmental gradientCulicoides  werecollected, representing 84 species, comprised of 45 valid species and 39morphotypes. A total of 6.078 Culicoides fusipalpis exhibited the highest abundance(2.178-35.84%), followed by C. dasyophrus (919-15.12%), C.pseudodiabolicus (459-7.55%), C. diabolicus(426-7.01%), C. filarifer (319-5.25%), C.ocumarensi (290-4.77%), C. foxi (231-3.80%), andC. insignis (229-3.77%). The remaining species represented lessthan 3% of the collected specimens  and C. diabolicus (416-11.17%); the most abundantspecies in capoeira were C. pseudodiabolicus (27-31.77%) andC. fusipalpis (20-23.53%); the most abundant species at theforest edge were C. dasyophrus (745-47.79%) and C.fusipalpis (430-27.59%); and the most abundant species in forests wereC. pseudiabolicus (246-34.75%) and C.fusipalpis (87-12.29%) (pecimens .The mos-12.29%) . C. paraensis was found primarily inperidomiciles; C. insignis was most abundant in peridomiciles nearcorrals and chicken coops; a single specimen of C. pusillus wascollected in a peridomicile near a corral; C. filariferus was foundin all environments except capoeira; C. flavivenula exhibited lowabundance in forests and peridomiciles; C. foxi occurred in allenvironments; and C. ignacioi occurred in all environments exceptforests. Of the incriminated and putative vectors recorded : C. paraCulicoides individuals are more equitablydistributed among different species in capoeira and forest environments.Richness between environments varied from 22 to 58 species/morphotypes. Abundancevaried significantly between environments, with values ranging from 85 to 3.725individuals . SpeciesSimilarity was greater between the forest and capoeira than between capoeira andperidomiciles. The population compositions of each environment differedstatistically  .RichnesCulicoides species/morphotypes were collected in therural settlement of Rio Pardo, representing 69% of the knownCulicoides species found in the Brazilian Amazon BasinCulicoides and nine new occurrences,--Eighty-four Culicoides paraensis, which is a vector of OROV inhumans; C. insignis and C. pusillus, which arevectors of BTV in domestic and wild ruminants; C. filariferus andC. flavivenula, which have been found carryingLeishmania amazonensis DNA; and C. foxi andC. ignacioi which have been found carrying Le.braziliensis DNA,-Species of medical and veterinary importance were found primarily in peridomiciles,these include: Culicoidesanthropophily. Of the species identified in this study, the following are known toexhibit anthropophilic behavior: Culicoides batesi; C.benarrochei; C. debilipalpis; C.flavivenula; C. fluviatilis; C. foxi;C. fusipalpis; C. glabellus; C.ignacioi; C. insignis; C. leopondoi;C. limai; C. lutzi; C. paraensis; C.paraignacioi; C. paramaruim; C.pseudodiabolicus and C. pusillus,-. Of these, all except C. glabellus were found inperidomiciles. Santiago-Alarcon et al. (2013)Culicoides species in peridomicile environments.Few studies conducted in the Amazon have examined Culicoides fusipalpis has been observed in high abundancein anthropic environments where it feeds on a variety of blood meal sources,including humans, other mammals and birdsCulicoides toperidomiciles. Santiago-Alarcon et al. (2013)Culicoides feeding behavior in households near anurban forest in Germany where the dominant Culicoides species wereknown to be ornithophilous and found that blood ingested by these midges containedthe mitochondrial DNA of mammals such as cows and humans. Generalist species areable to tolerate a broad set of environmental conditions and make use of a widerange of resources, which allows them to become both widespread and locally abundantAbundance was greatest in peridomiciles, followed by the forest edge, forest, andcapoeira. In Rio Pardo, species diversity was highest in forests, while richness and abundancewas highest in peridomiciles. In a study conducted in Rondônia, Carvalho et al.(2016)Table 1). The high equitability observed in capoeira environmentsis the result of low abundance and a relatively homogeneous distribution of species.The low equitability observed in forest edge environments is likely due to an unevendistribution of species abundance caused by the predominance of C.dasyophrus, which comprised 47.76% of all individuals collected at theforest edge. These findings demonstrate that the local diversity index may decreasein environments where one species is highly dominant.Equitability was highest in capoeira environments, where the number of individualsper species ranged from 1 to 27, and equitability was lowest in the forest edgeenvironments, where the number of individuals per species ranged from 1 to 745(Culicoides fauna tends to adaptto anthropized environments.Species similarity between environments was low (> 50%), which is likely due tothe different degrees of anthropic interference present in each environment.Capoeira-forest edge and forest-forest edge exhibited the highest similarity. Thesefindings suggest that strictly wild We observed that richness differed between environments, but rarefaction estimatesindicate that environments of the same type possessed similar levels of richness.This is probably due to the three-month capture effort occurring during the dryseason, a period that the abundance of general hematophagous dipteria are lowThe data obtained in this study indicate that diversity was higher in naturalenvironments (forest) than in anthropized environments (capoeira), while abundanceand richness were both highest in the most anthropized environment (peridomiciles).It is likely that the concentration of a variety of food sources and the presence ofsuitable breeding grounds in peridomiciles favors the establishment of certainspecies.Culicoides.In settled areas, the presence of domestic and wild animals provides vectors with arich variety of food sources and this fosters their adaptation to new environments.This behavior may alter pathogen transmission dynamics and increase the risk ofdisease transmission by


Solanum lycopersicum), which is an important vegetable crop and serves as a model for fleshy fruit development. In this study, 88 genes were identified in the S. lycopersicum genome and grouped into 12 subfamilies, based on previously identified AATs in Arabidopsis, rice (Oryza sativa), and potato (Solanum tuberosum) plants. Chromosomal localization revealed that S. lycopersicum AAT (SlAAT) genes are distributed on the 12 S. lycopersicum chromosomes. Segmental duplication events contribute mainly to the expansion of SlAAT genes and about 32% (29 genes) of SlAAT genes were found to originate from this type of event. Expression profiles of SlAAT genes in various tissues of S. lycopersicum using RNA sequencing data from the Tomato Functional Genomics Database showed that SlAAT genes exhibited tissue-specific expression patterns. Comprehensive data generated in this study will provide a platform for further studies on the SlAAT gene family and will facilitate the functional characterization of SlAAT genes.Amino acid transporters (AATs) are integral membrane proteins and have several functions, including transporting amino acids across cellular membranes. They are critical for plant growth and development. This study comprehensively identified AAT-encoding genes in tomato ( Solanum lycopersicum) has great global commercial importance owing to its nutritional value and serves as a model for fleshy fruit development and as a reference species for plants in the Solanaceae family. Several genome-wide studies have identified 63, 87, 23, 189, and 72 genes encoding amino acid transporters (AATs) in Arabidopsis thaliana, Oryza sativa, Selaginella, Glycine max, and Solanum tuberosum, respectively [S. lycopersicum. Tomato  [AATs are divided into two families; the first is amino acid/auxin permease (AAAP), which is in turn sub-grouped into eight subfamilies, including amino acid permease (AAP), lysine/histidine transporter (LHT), γ-aminobutyric acid (GABA) transporter (GAT), proline transporter (ProT), like-auxin influx carriers (LAX), aromatic and neutral amino acid transporter (ANT), vesicular aminergic-associated transporter (VAAT), and amino acid transporter-like proteins (ATL) . The secrs (PHS) . In addirs (PHS) .Arabidopsis, which are localized in the plasma membrane and involved in the H+-coupled amino acid uptake system [Arabidopsis, the first two AATs to be characterized, have different substrate specificity with high expression in siliques of Arabidopsis, suggesting their involvement in supplying the seeds with organic nitrogen [Arabidopsis with broad substrate specificities and AAP5 was found to participate in amino acid transport in the developing embryo [Arabidopsis thaliana AAP7 (AtAAP7) is still not functionally characterized. AtAAP6 and AtAAP8 are involved in high-affinity amino acid transport, since AAP8 imports organic nitrogen into developing seeds and AAP6 is expressed in xylem, which has low concentrations of amino acids [Arabidopsis, eight members of AAPs have been identified in S. tuberosum [O. sativa [S. tuberosum [Vicia faba L. and have been shown to transport a broad range of amino acids [A few studies have functionally characterized the members of the AAT superfamily in plants. Eight AAPs have been identified in e system . AAP1 annitrogen . AAP3, Ag embryo . Arabidono acids . Similaruberosum . Three pno acids .Arabidopsis. They were found to be localized in the plasma membrane and were found to import organic nitrogen into the roots, mesophyll cells [Ten LHTs (AtLHT1–10) have been identified in ll cells , and thell cells . AtLHT1 ll cells ,20.Arabidopsis and it was shown that despite the similar localization of the three transporters in the plasma membrane and similar affinity to glycine betaine, each transporter has a different role in Arabidopsis [Unlike AAPs and LHTs, the only proteinogenic amino acid transported by ProTs is proline . Howeverbidopsis . For exabidopsis .S. lycopersicum is a general transporter for compatible solutes [Arabidopsis, it was revealed that AtGAT1 has a high affinity to GABA and is localized in the plasma membrane [ProT1 in  solutes  and tran solutes . Regardimembrane . In addimembrane .Aux1 mutant Arabidopsis plants show defects in the root gravitropic response, and AtAUX1 is expressed in the columella, lateral root cap, epidermis, and stele tissues of the primary root [Arabidopsis [Arabidopsis, the AUX1 gene belongs to a small family consisting of four members, including AUX1 and three LAX genes  [O. sativa [Arabidopsis, which transports arginine, indole-3-acetic acid, and 2,4-dichlorophenoxyacetic acid [Arabidopsis with varying affinities and functions [Camellia sinensis and their expression was revealed to be sensitive to abiotic stress [Arabidopsis, a bidirectional amino acid transporter (BAT), which performs both exporting and importing activities, was reported to exhibit transport activity for alanine, arginine, glutamate, and lysine [ary root . AtAUX1 ary root , and AtAbidopsis . In Arab, and 3) , while t. sativa . Regarditic acid . Of CAT unctions . For exaunctions . Recentlc stress . In Arabd lysine .S. lycopersicum AATs are not fully studied, we aimed to elucidate the entire members of AAT gene superfamily in S. lycopersicum. In this study, a genome-wide identification and phylogenetic analysis of S. lycopersicum AAT (SlAAT) genes were performed for AAT superfamily classification and to explore the evolution of this gene superfamily. In addition, the features of the exon-intron structures, patterns of the conserved motifs, and duplication events, including tandem and segmental duplications within the tomato genome that likely contribute to the expansion of the SlAAT superfamily were explored. The resulting data will be useful in studies on the biological functions of each gene in the SlAAT superfamily.Since the functions of Arabidopsis thaliana, O. sativa, and S. tuberosum as queries in BLAST search at SGN with ‘amino acid transporter’ and ‘amino acid permease’ as keywords, we identified 88 members of SlAATs. All the retrieved protein sequences of SlAATs were subjected to InterProScan (http://www.ebi.ac.uk/Tools/InterProScan/ (accessed on 31 January 2021)), and all the candidate proteins were found to contain AAT domain(s). Information about locus identity number of SlAATs assigned by SGN ), the given nomenclature of S. lycopersicum AATs, number of intron(s) in the SlAAT genes, length of the ORF for SlAATs, protein characterization of SlAATs , and genomic location are presented in SlAATs ranged from 267 to 3376 bp. It was observed that each subfamily member shared a nearly similar gene structure and intron numbers. For example, members of ProT subfamily contained six introns and an ORF length of 1473–1828 bps. The intron-exon regions in each genomic sequence of SlAAT are illustrated in Using ATTs of SlAAT genes are distributed in all the 12 chromosomes in S. lycopersicum ) (S. lycopersicum vs. S. lycopersicum) revealed that 29 SlAAT genes originate from the duplication events and there are 16 gene pairs of SlAATs . The number of transmembrane regions , which wpersicum . An unevf SlAATs . Examinilculated  and the S. lycopersicum, O. sativa and Arabidopsis thaliana, revealed that the 88 SlAATs could be clustered into 12 clades  showed high expression in the roots. The high expression of ProT genes was observed in different organs; for example, SlProT1 and SlProT2 were highly expressed in the flowers and in buds respectively, while SlProT3 and SlProT4 were highly expressed in both S. lycopersicon ‘Heinz leaves and roots. The genes of LAX subfamily were highly expressed in the roots except SlLAX3, which showed a high expression in the fruits . The expression of SlBAT2 and SlBAT3 was the same in all the examined organs. Two genes of the TTP subfamily (SlTTP1 and SlTTP4) exhibited a similar expression pattern: high in the leaves and low in other organs. The expression of S. lycopersicum, Arabidopsis thaliana, and O. sativa demonstrated that the S. lycopersicum AATs are orthologs of a number of O. sativa and Arabidopsis thaliana AAT genes (SlLAT5 and SlLAT6 genes are orthologs of OsLAT7 (LOC_Os01g61044.1), and StLAX2 is an ortholog of OsAUX3 (LOC_Os03g14080), whereas StLAX4 is an ortholog of OsAUX1 (LOC_Os01g63770). Syntenic AAT gene pairs between S. lycopersicum and Arabidopsis thaliana, and between S. lycopersicum and O. sativa are presented in Syntenic analysis performed among the AATs in AT genes . For exaArabidopsis thaliana, O. sativa, and S. tuberosum. However, ATTs in S. lycopersicum have not been identified so far. In this study, 88 AATs were identified in S. lycopersicum and divided into two superfamilies based on their similarity with previously identified AATs in Arabidopsis thaliana, O. sativa, and S. tuberosum plants [Arabidopsis, S. tuberosum, and S. lycopersicum but in O. sativa, this number is more than double (19 members). This expansion in the members of AAP subfamily in O. sativa may be due to segmental and tandem duplication events [O. sativa, ten in Arabidopsis, 11 in S. tuberosum, and 13 in S. lycopersicum. In fact, the largest subfamily in S. lycopersicum is LHT and the smallest subfamilies are ACT and GAT with only three members each , which is known to be primarily expressed in the roots [SlLHT2, SlCAT5, and SlProT4 were observed to be high in the roots. Syntenic analysis showed that SlLHT2, SlAAP2, and SlProT2 are orthologs of AtLHT1, AtAAP5, and AtProT2, respectively, which are reported to play a role in amino acid uptake by the roots [The genes encoding SlLAX1, SlLAX2, SlLAX4, and SlLAX5 are specifically expressed in roots. Syntenic analysis revealed that he roots . Further1 AT2G381, which ihe roots . lCAT6 expression was high in both S. lycopersicon ‘Heinz roots and S. lycopersicon ‘Heinz fully opened flowers. AtCAT6, was revealed to be involved in amino acid uptake into the sink cells of flowers and root primordia [SlAAP8, the ortholog of AtAAP6, was high in S. lycopersicon ‘Heinz unopened flower buds and S. lycopersicon ‘Heinz fully opened flowers. However, AtAAP6 expression is mainly present in the xylem parenchyma cells [In addition, Srimordia . The expma cells . SlAAT genes elucidated in the current study provide foundation for further investigation of the AATs to completely understand their importance and roles in plant physiology and their contribution to plant productivity. Moreover, as AATs are usually induced by abiotic stresses such as salinity and drought [The expression patterns of  drought , it is iS. lycopersicum, those in Arabidopsis, O. sativa, and S. tuberosum [http://solgenomics.net/ (accessed on 31 January 2021)) against the S. lycopersicum genome and protein sequence database (version SL4.0) with default settings. The databases were also queried for homologs using ‘amino acid transporter’ and ‘amino acid permease’ as keywords. The redundant sequences were removed, and the remaining protein sequences were submitted to InterProScan (https://www.ebi.ac.uk/interpro/search/sequence/ (accessed on 31 January 2021)) to scan for AAT domains. Information about gene structure , locus accession, amino acid length, and genomic location were all acquired from SGN. Molecular weight (MW) and the theoretical isoelectric point (pI) were predicted using the Compute/Mw tool (http://web.expasy.org/ (accessed on 31 January 2021)). The gene structure of SlAATs were also analyzed using Gene Structure Display Server (GSDS) (http://gsds.gao-lab.org/index.php (accessed on 31 January 2021)) [http://www.cbs.dtu.dk/services/TMHMM/ (accessed on 31 January 2021)) was used to predict the putative transmembrane regions in each SlAAT protein [http://gsds.cbi.pku.edu.cn/ (accessed on 31 January 2021)).The nomenclature of S. lycopersicum AATs was assigned according to chromosome order and considering their phylogenetic relationship. However, SlLax1, SlLax2, SlLax3, SlLax4, and SlLax5 have been previously named [To identify the AATs in uberosum ,5 were uy 2021)) . The TMH protein . In ordely named .SlAAT genes were identified using the available information at SGN, and mapped onto the 12 corresponding S. lycopersicum chromosomes using MapChart software [The approximate locations of software .http://chibba.agtec.uga.edu/duplication (accessed on 31 January 2021)) was used to download the gene pairs of the S. lycopersicum genome and the related information about synonymous substitution rate (Ks) and nonsynonymous substitution rate (Ka) was also obtained. Genes distributed nearby and separated by five or fewer genes was selected as the criterion for considering tandem duplicates. Divergence time  of the gene pairs was estimated using the following equation: T = Ks/2x (x = 6.56 × 10−9), in which x is the mean synonymous substitution rate for tomato [The Plant Genome Duplication Database (PGDD) (r tomato .https://www.bioinformatics.org/sms2/ident_sim.html (accessed on 31 January 2021)).The sequence similarity between the proteins of duplicated genes was calculated using the sequence manipulation suite programs   was usedsoftware .S. lycopersicum, O. sativa, and Arabidopsis thaliana was performed using MEGA 6.0 software, with the default settings of MUltiple Sequence Comparison by Log-Expectation (MUSCLE) alignment [In order to construct the phylogenetic tree, the multiple sequence alignment AAT proteins of lignment . The phylignment . The roblignment . To anallignment .http://meme-suite.org/tools/meme (accessed on 31 January 2021)) with default settings, except that the maximum number of motifs was defined as 20.The motifs of SlAAT proteins were identified using the MEME tool (SlAAT genes were obtained from RNA sequencing (RNA-Seq) data from the Tomato Functional Genomics Database (http://ted.bti.cornell.edu/ (accessed on 31 January 2021)) using the transcriptomic analysis of various tissues in the S. lycopersicon ‘Heinz and the wild relative S. pimpinellifolium. The search was performed using the locus name as a query. The heatmap was generated using ComplexHeatmap version 2.2.0 package in R [Expression profiles of the age in R .S. lycopersicum was identified and analyzed using a broad range of bioinformatic tools. The SGN was first used to retrieve the nucleotide and amino acid sequences of the SlAATs to obtain general information and sequence characterization of SlAATs. Chromosomal localization and gene duplication events in SlAATs were demonstrated. In addition, the phylogenetic relationships and protein motifs of SlAAts were determined. The expression profiles of SlAAT genes in various tissues of S. lycopersicum were elucidated using RNA-Seq data in the Tomato Functional Genomics Database. This study will facilitate further investigation of the SlAAT gene superfamily and the functional characterization of the members of SlAATs. Further analysis of expression profiles under abiotic stress conditions will reveal their potential roles in response to environmental stress.In this study, the AAT gene superfamily in


Yersinia pseudotuberculosis, Y. enterocolitica and Y. pestis are pathogenic bacteria capable of causing disease in humans by growing extracellularly in lymph nodes and during systemic infections. While the capacity of these bacteria to invade, replicate, and survive within host cells has been known for long, it is only in recent years that their intracellular stages have been explored in more detail. Current evidence suggests that pathogenic Yersinia are capable of activating autophagy in both phagocytic and epithelial cells, subverting autophagosome formation to create a niche supporting bacterial intracellular replication. In this review, we discuss recent results opening novel perspectives to the understanding of intimate host-pathogens interactions taking place during enteric yersiniosis and plague. Yersinia includes several pathogenic bacterial species for humans, which include the food-borne enteropathogens Y. enterocolitica and Y. pseudotuberculosis ..Y. pestiYersinia genus, Y. ruckeri, is a fish pathogen responsible for enteric redmouth disease [Y. ruckeri has the capacity to invade host cells [Y. ruckeri is not able to multiply or survive inside epithelial cultured cells. Interestingly, Ryckaert and colleagues (2010) demonstrated by gentamicin protection assays that Y. ruckeri can invade and survive within rainbow trout macrophages [Y. ruckeri is detected in infected macrophages within double membrane vacuoles, which display autophagosomal features. Intracellular bacteria replicate and survive within these compartments for at least 24 h after infection [Another species from the  disease . Experimst cells ,50,51. Hrophages . Using tnfection . NeverthY. enterocolitica, Y. pseudotuberculosis, and Y. pestis produce potent effectors (Yops) that block internalization within host cells [Y. pseudotuberculosis in macrophages have been shown to be required for bacterial virulence in vivo [Y. pestis [Y. pestis skin infection suggest that transport to draining lymph nodes might not require intracellular transport by macrophages, dendritic cells or neutrophils [Y. pestis [Y. enterocolitica and Y. pseudotuberculosis.st cells ,10, and st cells ,14. Howest cells ,4,5. Con in vivo . Since t. pestis , it has . pestis . Recent trophils ,56. Howe. pestis . This coY. enterocolitica, Y. pseudotuberculosis, and Y. pestis in both phagocytic and epithelial cells clearly converges with the idea that pathogenic Yersinia activate and subvert autophagy to replicate and survive within host cells. Initial experiments from Deuretzbacher et al. (2009) [Y. enterocolitica killing by macrophages. As discussed by Valencia-Lopez and colleagues (2019) [Y. enterocolitica may survive within professional phagocytes, as observed for Y. pseudotuberculosis and Y. pestis. As mentioned, Y. ruckeri has been also described to persist in fish macrophages within double membrane-bound compartments [Yersinia species.Independent evidence generated with the three species . (2009)  suggestes (2019) , the useartments , suggestYersinia to subvert autophagy have not been identified. The work of Valencia-Lopez et al. [Y. enterocolitica. The plasmidic T3SS is dispensable, as shown by experiments in which pYV- or pCD1-cured bacteria are able to induce the formation of spacious autophagosomes. Interestingly, some Y. enterocolitica strains encode a chromosomal T3SS named Ysa (Yersinia secretion apparatus) that is required for bacterial intracellular growth in Drosophila S2 cells [Y. ruckeri [Y. pestis also argues against a functional role of this surface protein in promoting a phenotype that seem shared by the three bacterial species discussed in this article. Valencia-Lopez et al. (2019) [Yersinia species, might trigger autophagy by damaging the membrane of the YCVs and exposing bacteria to the cytosol, allowing therefore the recruitment of the phagophore formation machinery. Differences observed between the specific autophagy cascades triggered by pathogenic Yersinia  could be explained by variations in the various sets of chromosomal effectors encoded by each bacterial species which would mediate autophagy induction.The molecular effectors that allow pathogenic z et al.  indicateS2 cells . This sy ruckeri , but its. (2019)  suggest Y. enterocolitica from infected cells. They draw attention to the fact that Rab11b, VAMP3, and VAMP7, which have been associated to the formation of autophagosomes by Y. pestis and Y. pseudotuberculosis, are regulatory elements of the exocytic machinery. It would therefore be interesting to investigate the potential contribution of a general autophagy-related mechanism involved in promoting bacterial escape from host cells.Several additional observations from the work of Valencia-Lopez and colleagues (2019) deserve attention . First, Yersinia species have been instrumental models to explore the molecular interaction between pathogens and host cells. The issues raised by the study of autophagy subversion by Y. enterocolitica, Y. pseudotuberculosis, and Y. pestis  reviewed in here, clearly indicate that these bacteria have still many secrets to unfold.Pathogenic


Mycobacterium tuberculosis in the diagnosis of TB. Many approaches, e.g., whole-cell organisms, subunit, and recombinant vaccines are currently being explored for safer and more efficacious TB vaccines than BCG. These approaches have been successful in developing a large number of vaccine candidates included in the TB vaccine pipeline and are at different stages of clinical trials in humans. This paper discusses current vaccination strategies, provides directions for the possible routes towards the development of new TB vaccines and highlights recent findings. The efforts for improved TB vaccines may lead to new licensed vaccines capable of replacing/supplementing BCG and conferring therapeutic value in patients with active/latent TB.Currently, there is only one licensed vaccine against tuberculosis (TB), the Bacillus Calmette–Guérin (BCG). Despite its protective efficacy against TB in children, BCG has failed to protect adults against pulmonary TB, lacks therapeutic value, and causes complications in immunocompromised individuals. Furthermore, it compromises the use of antigens present in the purified protein derivate of  Mycobacterium tuberculosis (Mtb), is the leading cause of death due to an infectious agent in the world [Mycobacterium bovis Bacillus Calmette–Guérin (BCG) [Tuberculosis (TB), a disease caused by he world . Mtb infhe world . Furtherhe world . Currentin (BCG) , and it in (BCG) . Each yein (BCG) . Vaccinain (BCG) . The BCGin (BCG) . Additioin (BCG)  and lackin (BCG) . Furtherin (BCG) . The BCGin (BCG) .Furthermore, another worldwide epidemic, type 2 diabetes, is associated with a 2–4 times increased risk of developing active TB , and a kMany vaccines are in the pipeline (Tables 1–3) with an aim to produce a new vaccine for TB, and different strategies include replacing BCG, boosting BCG, and therapeutic use . DiffereMycobacterium bovis that causes TB primarily in cows [M. bovis on an artificial medium in vitro for 13 years (1908–1921) and a total of 231 passages. The resulting organism, BCG, did not cause TB lesions in various animal models but was immunogenic and protected the immunized animals against challenges with M. bovis [The only currently licensed vaccine against pulmonary and extrapulmonary TB is the live attenuated BCG vaccine. It was originally derived from the pathogenic  in cows . To atteM. bovis , avoidinM. bovis . Howeveryears 190–1921 andM. bovis . BCG’s dM. bovis . In factγ and TNF-α, compared to healthy infants [The BCG vaccine also poses a risk to immunocompromised individuals ,19. Thos infants . In addi infants .M. tuberculosis and many of them are present in BCG as well, leading to antigenic cross-reactivity and causing a high rate of false positivity [BCG also poses a complication for the diagnosis of active as well as latent TB by using the antigenic preparation of Mtb known as purified protein derivative (PPD) in Mantoux or tuberculin skin test (TST). In this test, PPD is injected intradermally to evaluate cell-mediated immunity in response to Mtb antigens. The intradermal injection of PPD produces a type IV hypersensitivity reaction, and is used for diagnosis of both active and latent TB . Althougsitivity . A reduc+ T cells are activated to control the infection, leading to the formation of a granuloma [+ T cells will control the bacteria from further infection; however, in some cases, the infection can reactivate and develop to an acute, chronic, or extrapulmonary infection [The complexities of Mtb infection in humans create challenges in finding targets of the immune reaction to the pathogen. Mtb bacilli first enter the lungs in aerosol droplets. Due to the innate immune response, macrophages ingest the bacteria and recruit other immune cells . Neutropranuloma . The graranuloma . In the nfection . The disnfection . Reasonsnfection .γ, TNF-α, TGF-β, IL-17, and memory T cells have been shown to be active in the immune response against TB [+ T cell response but did not produce a robust response in the phase 2 trial [+ T cells play a predominant role in protective TB immunity, strategies that boost CD8+ function also enhance vaccine efficacy [+ T cells in immunized monkeys led to reduced protection. Similarly, CD8+ T cell depletion in M. tuberculosis-infected and then antibiotic-treated monkeys led to increased susceptibility to reinfection, indicating their importance in conferring immunity in a vaccination or natural infection setting. Human CD8+ CCR7– CD45RA+ effector memory T cells exhibit significant anti-mycobacterial activity [The cytokines IL-12, IL-8, IFN-ainst TB . While tainst TB . Satti aainst TB  stated t 2 trial . Althougefficacy . In a noactivity .Based on the current understanding of Mtb, there are a number of strategies for the application of new vaccines, i.e., preventive pre-exposure , preventive post-exposure (for latent TB/adolescents and adults), and therapeutic (for active TB) . Pre-expThere are currently at least 23 vaccine candidates in clinical trials in humans, as shown in M. tuberculosis complex (MTBC) known as lineage 4 (L4). MTBVAC is derived from a clinical M. tuberculosis isolate Mt103 after deletion of two virulence-related genes phoP and fadD26. Safety studies in immunocompromised mice with severe combined immunodeficiency (SCID) have shown that MTBVAC is more attenuated than BCG [M. tuberculosis belonging to two other strains of MTBC i.e., L2 (MTBVAC-L2) and L3 (MTBVAC-L3), two of the three modern lineages of M. tuberculosis. The study demonstrated that vaccination with MTBVAC, MTBVAC-L2, and MTBVAC-L3 had similar or superior protection compared to BCG in immunocompetent mice, when the immunized mice were challenged with the three representative strains. This study shows the potential of MTBVAC to protect against the globally diverse strains of MTBC [2auxo [2auxo in comparison with BCG and MTBVAC [The vaccines using the live attenuated approach contain whole Mtb in its weakened or altered form . MTBVAC than BCG . Preclin of MTBC . MTBVAC C [2auxo . In thisd MTBVAC , showingd MTBVAC .M. tuberculosis cells in liposomes , and M. obunese (DAR-901)  .M. vaccae are being used as therapeutic vaccines to reduce drug treatment duration for patients with active TB [A phase 2b vaccine, RUTI, has exhibited significant humoral and cellular immune responses against antigens expressed in actively growing and latent bacilli . It has ctive TB ,38,39.- monocyte phenotype in the spleens of immunized mice [- related mRNA transcripts in splenocytes, producing a monocyte phenotype shift from LyC6+ [- monocytes have been shown to have an anti-inflammatory role [+ monocytes are proinflammatory [A recent study, using an ex vivo mycobacterial growth inhibition assay (MGIA), has shown RUTI vaccine-induced inhibition in mycobacterial counts and a significant shift towards Ly6Czed mice . Additioom LyC6+ . Ly6C- mory role , whereasammatory . TherefoIn comparison to whole pathogen vaccines, subunit vaccines contain selected parts of the pathogen. While this is generally a safer approach, it can produce a less robust immune response . TherefoAluminum-based adjuvants are primary adjuvants used in vaccines to drive a humoral response. Humoral immunity is not considered to have a major role in protection against Mtb, as it is an intracellular pathogen , therefoM. tuberculosis-specific immune response (an indicator of active TB) in individuals immunized with BCG in neonatal age [M. tuberculosis-specific immune response as determined by quantifying the concentration of IFN-γ secreted by using the QuantiFERON-TB Gold In-tube assay (QFT). QFT conversion, shown by the authors as having a higher risk of progression to TB, was determined in both H4: IC31 vaccinated group and BCG revaccinated group. BCG revaccination reduced the rate of QFT conversion (with an efficacy of 45% and 95% CI 6-68) compared to the H4: IC31  [An exciting study with subunit vaccine H4: IC31 was performed in adolescents in a high-risk setting for TB to assess its efficacy in reducing atal age . The resI 16–58) . This stI 16–58) ,14.+ T-cell memory responses [+ T cell response demonstrates the best vaccine take because the induction of these cells correlates with protective immunity in TB [In another study, a phase 2b clinical trial, the subunit vaccine M72/AS01E prevented pulmonary TB in adults already infected with Mtb with an efficacy of 54% . In humaesponses . The abity in TB .Recombinant vaccines are produced using the techniques of genetic engineering. In brief, a piece of DNA encoding an antigen of Mtb is inserted into an appropriate vector to transform either a bacterial, mammalian, or yeast cell. The recombinant vector produces the antigen in large quantities in those cells . BecauseM. bovis BCG, M. vaccae, and M. smegmatis have been used as vectors. M. bovis BCG is commonly used as an expression vector due to its stability, cost-effectiveness, and non-specific immune stimulation [L. lactis as a vector. This recombinant vaccine is at a preclinical stage, and it was able to induce both cellular and antibody responses after mucosal immunization in mice [Recombinant vaccines  can be pmulation ,56,57. A in mice . Many otSeveral live viral-vector based vaccines have also entered into clinical trials . The MVAM. tuberculosis. These antigens may have the same problem of false positivity as BCG, with respect to the diagnosis of TB using PPD. Furthermore, because of the cross-reactivity with environmental mycobacteria, these candidate vaccines may face the problem of masking or blocking effects and may not be useful as booster vaccines in BCG pre-vaccinated individuals [M. tuberculosis-specific antigens as new TB vaccines may overcome these problems of blocking or masking. Therefore, to have TB vaccines better than BCG, development of new subunit and/or recombinant vaccines based on M. tuberculosis-specific antigens is required and is currently in progress [M. tuberculosis-specific regions known as regions of differences (RD) were identified using classical molecular and biochemical techniques, the identification of all M. tuberculosis-specific regions was possible with advances in whole genome sequencing and the comparative genome analysis of M. tuberculosis with other mycobacterial genomes [Mtb [The above stated recombinant DNA vaccines undergoing clinical trials are based on cross-reactive antigens of ividuals . Accordiividuals . The useprogress . Althoug genomes . These R genomes . The com genomes . To iden genomes ,72. Test genomes ,75,76,77mes [Mtb ,79,80.M. tuberculosis-specific antigens ESAT6-CFP10 with an adjuvant has been tested in a phase I clinical trial [γ and IgG antibodies. The immune responses were induced to all three antigens included in GamTBvac [M. tuberculosis-specific ESAT-6 antigen is L. lactis FnBPA+ . When delivered by mucosal route, this vaccine produced a systemic Th1 cell response and significant increase in specific secretory immunoglobulin A production in colon tissue and fecal extracts [γ, IL-6, and TNF-α from spleens of immunized mice [M. tuberculosis-specific antigen with a bacteria L. lactis as a DNA delivery system.In humans, a recombinant subunit TB vaccine candidate, GamTBvac, which contains a dextran-binding domain modified Ag85a and al trial . Safety GamTBvac . This vaextracts . In a bozed mice . These fLemna minor to activate the immune response [BCG is administered intradermally, where it induces a strong systemic response but weak mucosal immune response . This prresponse , and nanresponse . HoweverTB is an ancient disease with worldwide prevalence, but only one licensed vaccine, BCG, is available for use with many concerns. Hence, sincere efforts are being made globally for the development of improved vaccines against TB. Among the strategies to develop new TB vaccines, whole-cell mycobacteria, subunit, and recombinant vaccine strategies have been employed. These efforts have led to the identification of about two dozen vaccine candidates currently in the pipeline at different stages of clinical trials in humans. Furthermore, additional TB vaccine candidates are at the preclinical stage and are being tested in animal models of TB. The progress made for the development of improved vaccines against TB is highly encouraging and it is hoped that new licensed TB vaccines for preventive and therapeutic applications in humans may become a reality in the near future.


Bersama abyssinica (Melianthaceae) stem bark were screened for enzyme inhibitory and antioxidant properties. The water extract possessed the highest concentration of phenols , while the methanol extract was rich in flavonoids , and the ethyl acetate extract possessed the highest amount of saponins . The aim of this study was to investigate the antiproliferative effects against the human colon cancer HCT116 cell line challenged with serotonin (5-HT) as a stimulating-proliferation factor. The level of HCT116 cell-deriving pool of kynurenic acid (KA) was also assessed. The UHPLC results confirmed the presence of 58, 68, and 63 compounds in the ethyl acetate, methanol, and water extracts, respectively. Mangiferin, vitexin and its isomer isovitexin were tentatively identified in all extracts and KA (m/z 190.05042 [M−H]+) was also tentatively identified in the methanol and water extracts. The methanol extract  showed the highest activity in the CUPRAC assay, whereas the water extract (1063.70 mg TE/g extract) showed the highest activity with the FRAP technique. The ethyl acetate extract was the most active acetylcholinesterase  and α-glucosidase  inhibitor. The water extract was able to inhibit 5-HT-stimulated viability of HCT116 cells, and blunt 5-HT-induced reduction of cell-deriving KA. The scientific data generated in this study provide baseline data regarding the biological properties of B. abyssinica stem bark, highlighting its potential use for the development of new pharmaceutic and cosmetic agents.In this study, ethyl acetate, methanol, and water extracts of  Since ancient times, populations around the globe have relied on plants for food and medicinal purposes. Today, medicinal plants remain the most accessible source of therapeutics for the management of multiple ailments. Over the past decades, researchers from academic institutions to pharmaceutical industries have shown significant interest in natural products including plants and this shift has been driven by increased public interest in plants. Compounds from plants can be used as templates for the development of novel pharmaceutical agents, as well as in the form of botanical products or supplements referred to as complementary and alternative medicine for the prevention and management of diseases. One of the major challenges of scientists is to sustain alleged traditional therapeutic properties of medicinal plants and to rationalize proper dosage for safe use. In this context, research plays a pivotal role in the quest for novel candidates from plants. However, there is still a lack of scientific attention on bioactive compounds from many plants .B. abyssinica (Melianthaceae) occurring across regions of Sub Saharan Africa, has been used in traditional medicine for the management of multiple ailments, such as colic, diarrhea, dysentery, intestinal worms, rabies, gonorrhea, syphilis, malaria, diabetes, lumbago, fever, debility, hemorrhoids, epilepsy, cancer, rheumatism, menstrual pain, leprosy, impotence, snake bites, and liver disease [B. abyssinica was reported to be traditionally used for treating patients with malignancies [B. abyssinica showed cytotoxic activity [B. abyssinica alcoholic stem bark extracts, showed cytotoxic activity against human nasopharynx cancer cells [B. abyssinica leaves containing picolinyl hydrazide [ disease ,2,3. B. gnancies . Later sactivity . Hellebrer cells . Anotherydrazide . B. abyssinica stem bark, and therefore this presents a promising avenue for bioprospection. In this study, the enzyme inhibitory properties and antioxidant activity of the ethyl acetate, methanol, and water extracts of B. abyssinica stem bark were determined. Considering the traditional antitumoral use [B. abyssinica for future works. Currently, there is a lack of scientific literature on the possible enzyme inhibitory activity of oral use , antiprooral use ,9,10. ItThe plant samples were collected from the Gontougo region  of the Ivory Coast, in 2018 (summer season), and were identified by the botanist, Dr. Kouadio Bene, from the Laboratoire de Botanique et Phytothérapie, Université Nangui Abrogoua, Abidjan, Côte d’Ivoire. The stem barks were collected from ten plants in the same population. The stem barks were dried at room temperature for ten days. A laboratory mill was used to grind the samples.The extraction procedure was conducted following maceration (for ethyl acetate and methanol) and infusion (for water) methods. Briefly, for maceration, 5 g powdered plant sample was stirred with solvents (100 mL) overnight at room temperature. Then, the solvents were evaporated using a rotary evaporator. For water extracts, 5 g powdered plant sample in boiled water (100 mL) was allowed to stand for 20 min. Then, the aqueous extract was lyophilized and all extracts were kept at +4 °C until use.B. abyssinica extracts was determined using a Dionex Ultimate 3000RS UHPLC instrument. All analytical and chromatographic details are given in The total bioactive compounds were determined colorimetrically, as described previously ,12,13. Tp < 0.05 considered to be statistically significant) for determining differences in the extracts.For antioxidant capacity, we used different test systems, including radical quenching, reducing power, phosphomolybdenum, and ferrous ion chelating. Details of the methods used were described in our earlier paper . ResultsArtemia salina lethality bioassay, brine shrimp larvae were incubated for 24 h with B. abyssinica extracts (0.1 to 20 mg/mL) dissolved in incubation medium . The detailed protocol is described in our previous article [In  article .® CCL-247™, ATCC Company, Manassas, Virginia, USA) was cultured in DMEM (Euroclone) supplemented with 10% (v/v) heat-inactivated fetal bovine serum and 1.2% (v/v) penicillin G/streptomycin in 75 cm2 tissue culture flask , as previously reported [HCT116 cell line -2,5-diphenyltetrazolium bromide (MTT) test. Cells were incubated with extracts (0.1 mg/mL) for 24 h. An aliquot of 10 μL of MTT (5 mg/mL) was added to each well and incubated for 3 h. In the same condition, the kynurenic acid (KA) extracellular level was determined through HPLC-fluorimeter, as recently described [To assess the basal cytotoxicity of escribed .Finally, we tested extracts on HCT116 cell spontaneous migration, in the wound healing experimental paradigm. The details about MTT and the wound healing test are described in our previous paper . p < 0.05.Results of in vitro studies  were expressed as means ± standard deviation (SD) of three experiments performed in triplicate. Statistical analysis was determined through analysis of variance (ANOVA), followed by a post hoc Newman–Keuls comparison multiple test. The level of significance was set at B. abyssinica stem bark extracts for phenols, flavonoids, phenolic acids, flavanols, tannins, and saponins. The main classes of secondary metabolites identified from B. abyssinica stem bark extracts were phenols, flavonoids, and saponins. The water extract possessed the highest concentration of phenolic (230.83 mg GAE/g extract), while the methanol extract was rich in flavonoid (75.82 mg RE/g extract), and the ethyl acetate extract possessed the highest amount of saponin (97.37 mg QE/g). As shown in B. abyssinica stem bark extracts and the results are summarized in B. abyssinica stem bark extracts follow the order water extract > methanol extract > ethyl acetate extract. The reducing potential of the extracts was further determined using the FRAP and CUPRAC methods. The methanol extract (1464.08 mg TE/g extract) showed highest activity in the CUPRAC assay, whereas for FRAP the highest activity was noted for the water extract (1063.70 mg TE/g extract). Radical scavenging assays, namely DPPH and ABTS, revealed that the methanol extract, followed by the water extract showed higher activity as compared with the ethyl acetate extract. The metal chelating evaluation showed that the water extract was the most active. With reference to a previously published paper, B. abyssinica stem bark  [Plants possess an abundance of natural compounds also known as secondary metabolites. Plant secondary metabolites are expressed in hundreds of thousands of combinations in different classes of plants, in their different plant parts, at different maturity stages, and under different environmental conditions . Secondatem bark  assessedctively) .B. abyssinica stem bark  was identified only in the ethyl acetate and methanol extracts. The flavone, vitexin and its isomer, isovitexin, both exhibiting precursor at m/z 433.1135 were identified in all the studied extracts. Some compounds could not be identified, for example, a tannin (C34H26O2) exhibiting fragment ions at m/z 649.0660, 561.0911, 499.0735, 347.0622, and 300.9995; a saponin (C48H76O19) exhibiting fragments ions at m/z 893.4806, 793.4410, 731.4385, 551.3740, and 455.3530. Kynurenic acid (KA)  tentatively identified in the methanol and water extracts, has previously been reported to act as a neuromodulator by interacting with nicotinic and GPR35 receptors and regulated the release of neurotransmitters, such as, acetylcholine [B. abyssinica stem bark could be related to an individual secondary metabolite or the synergistic action of several compounds.In addition to spectrophotometric determination of secondary metabolites, the detailed profiles were elucidated using HPLC-fluorimetric technique. The detailed secondary metabolites profiling provide a better understanding of the secondary metabolites occurrence in herbal extracts and can be used as support for further interaction and mechanisms studies. HPLC-fluorimetric profiling confirmed the presence of 58, 68, and 63 compounds in the ethyl acetate, methanol, and water extracts of B. abyssinica stem bark extracts showed inhibitory action against AChE. In addition, the results presented in B. abyssinica stem bark extracts on tyrosinase. Tyrosinase is a rate-limiting enzyme responsible for the biosynthesis of melanin which protects the body against harmful UV rays. However, excessive production of melanin causes hyperpigmentation-related conditions, such as melasma and freckles. Therefore, controlling tyrosinase activity using inhibitors has been revealed to be an ideal therapeutic strategy for the management of skin hyperpigmentation conditions. Here, it was found that B. abyssinica stem bark extracts inhibited tyrosinase. The methanol extract exhibited higher inhibition against tyrosinase with a value of 136.51 mg KAE/g extract. However, B. abyssinica leaves methanol extract expressed higher inhibitory activity against tyrosinase with a value of 148.94 mg KAE/g extract. Loliolide, monoterpenoid hydroxylactone previously reported to possess anti-melanogenic effects on skin, and other secondary metabolites could be responsible for the observed anti-tyrosinase activity. Enzymes act as catalyst in cellular reactions, and therefore are ideal drug targets . Enzyme B. abyssinica stem bark extracts to inhibit α-amylase and α-glucosidase was determined. These two carbohydrate hydrolyzing enzymes have been targeted for controlling post-prandial glucose peaks as their activity is directly related to the release of glucose from ingested food. Therefore, inhibition of α-amylase and α-glucosidase blunt glucose rise, thereby preventing hyperglycemia. On the one hand, the results from this study showed that B. abyssinica stem bark extracts possessed low inhibition against α-amylase, with acarbose equivalent values per gram of extract ranging from 0.97 to 0.21. On the other hand, the ethyl acetate extract (15.22 mmol ACAE/g extract) showed potent activity against α-glucosidase. It is noteworthy that B. abyssinica stem bark ethyl acetate extract was more active than the ethyl acetate extract of B. abyssinica leaves (1.09 mmol ACAE/g extract) [The ability of B. abyssinica which was selected for further investigation aimed to verify the traditional antitumoral use of the plant. Firstly, the biocompatibility limit was determined through the A. salina brine shrimp lethality test. The nauplii were treated with B. abyssinica water extract, in the range 0.1–20 mg/mL. The lethality test showed a LC50 value >1 mg/mL. On the basis of our previous investigation [B. abyssinica [B. abyssinica water extract blunted the 5-HT-induced downregulation of kynurenic acid (B. abyssinica extract (100 µg/mL)  , suggestigration  ruled ouB. abyssinica stem bark, highlighting its potential use for the development of new pharmaceutic and cosmetic agents. In addition, B. abyssinica stem bark is regarded as a source of active phytochemical compounds which could be isolated and incorporated in drug formulation for the management of Alzheimer’s disease, diabetes type II, and skin hyperpigmentation complications. Finally, this pharmacological study suggests antiproliferative effects of the B. abyssinica water extract that corroborates the traditional use and supports further studies for the confirmation of the antitumoral role in selected colon cancer animal models. Oxidative stress triggers the onset of several pathologies, but key enzymes remain the main targets in the management of Alzheimer’s disease, diabetes type II, and skin hyperpigmentation problems. Developing agents possessing both antioxidant and enzyme inhibitory properties could be ideal for the holistic management of these complications. Scientific data generated in this work provide baseline data regarding the enzyme inhibitory and antioxidant properties of