There is little information regarding the duck enteritis virus (DEV) US10 gene and its molecular characterization.Escherichia coli BL21 cells and used to immunize rabbits for the preparation of polyclonal antibodies. The harvested rabbit antiserum against DEV US10 was detected and analyzed by agar immunodiffusion. Using this antibody, western blotting and indirect immunofluorescence analysis were used to analyze the expression level and subcellular localization of US10 in infected cells at different time points. Quantitative reverse-transcription PCR (qRT-PCR) and pharmacological inhibition tests were used to ascertain the kinetic class of the US10 gene. A mass spectrometry-based strategy was used to identify US10 in purified DEV virions and quantify its abundance.Duck enteritis virus US10 was amplified and cloned into the recombinant vector pET32a(+). The recombinant US10 protein was expressed in The recombinant pET32a(+)/US10 protein was expressed as inclusion bodies, purified by gradient urea washing, and used to prepare specific antibodies. The results of qRT-PCR, western blotting, and pharmacological inhibition tests revealed that US10 is mainly transcribed in the late stage of viral replication. However, the presence of the DNA polymerase inhibitor ganciclovir and the protein synthesis inhibitor cycloheximide blocked transcription. Therefore, US10 is a γ2 (true late) gene. Indirect immunofluorescence analysis showed that US10 proteins were initially diffusely distributed throughout the cytoplasm, but with the passage of time, they gradually relocated to a perinuclear region. The US10 protein was detected in purified DEV virions by mass spectrometry, but was not detected by western blotting, indicating that DEV US10 is a minor virion protein.The DEV US10 gene is a γ2 gene and the US10 protein is localized in the perinuclear region. DEV US10 is a virion component. Alphaherpesvirinae (genus Mardivirus) according to the 10th report of the International Committee on Taxonomy of Viruses (ICTV) [Duck viral enteritis (DVE) is an acute, hemorrhagic, highly contagious disease of waterfowl caused by duck enteritis virus (DEV) , 2. Sincs (ICTV) .The complete sequences of different DEV strains have been published in GenBank, including three field (virulent) strains CHv, 2085, and CSC) and five attenuated strains  , and CSC. The smaDEV US10 homologs were found in MDV, HVT, HSV-1, HSV-2, CeHV-1, EHV-1, EHV-4, VZV, and ILTV. The percentage homology ranges from 18.3 to 31.0% . The US1E. coli BL21 cells, E. coli DH5α cells, the prokaryotic expression vector pET-32a(+), pMD18-T/β-actin, the rabbit anti-DEV serum, and rabbit anti-gC serum were preserved and provided by the Avian Diseases Research Center, College of Veterinary Medicine, Sichuan Agricultural University. The pET32a(+)/US10 vector was constructed by Takara Biotechnology Co. . Monolayer cultures of duck embryo fibroblasts (DEFs) were cultured in Minimal Essential Medium  containing 10% fetal bovine serum  and 100 μg/mL streptomycin. After DEV inoculation, the DEFs were incubated in MEM containing 3% FBS.The CHv strain of DEV (GenBank accession number: JQ647509), All primers Table  were desE. coli BL21 cells, which were then induced using IPTG at a working concentration of 0.6 mM for 6 h at 37 °C. After centrifugation, the cells were resuspended in 20 mM Tris-HCl. The expression of the recombinant protein was determined using SDS-PAGE after the disruption of cells by cold sonication. The reactivity of recombinant proteins was determined by western blot analysis using rabbit anti-DEV serum as the primary antibody and HRP-labeled goat anti-rabbit IgG as the secondary antibody.The recombinant plasmid pET-32a(+)/US10 was transformed into The recombinant US10 protein was mainly expressed as inclusion bodies, purified through urea washing, dialysis, and renaturation, as described by Wu et al. . Six heaThe reactivity of the antibody was determined using western blot analysis . On the −ΔCt method simplified from the 2−ΔΔCt method. To evaluate the efficiency of each assay, standard curves were constructed using tenfold serial dilutions of pMD18-T/US10 and pMD18-T/β-actin.Total RNA was extracted from DEV-infected DEFs at different time points post-infection  using Trizol, followed by DNase treatment during RNA extraction. The quality of the RNA samples was assessed and the samples were reverse-transcribed to cDNA, as described by Li et al. . SubsequPharmacological inhibition tests were performed to confirm the kinetics of DEV US10 . Three bIFA was conducted using a standard procedure . Brieflyg, 20 min, 4 °C) to remove the cell debris. Extracellular DEV virions were harvested by ultracentrifugation  through a 30% (wt/vol) sucrose cushion. Virions were then banded by isopycnic gradient ultracentrifugation in a continuous 30 to 60% (wt/vol) potassium tartrate gradient in TBS . The band containing virions was collected, diluted tenfold in TBS, and pelleted by ultracentrifugation . The pellet was finally resuspended in TBS and stored at −80 °C.DEF cells were mock-treated or infected with DEV CHv strain at an MOI of 5. At 2 hpi, the cells were washed twice with PBS and the medium was replaced by serum-free MEM. At 72 hpi, the medium was collected and clarified by low-speed centrifugation (2000×Purified virion samples were boiled for 10 min in 5 × SDS-loading buffer and separated on 12% gels by SDS-PAGE. Proteins were transferred from the gels to polyvinylidene difluoride (PVDF) membranes, which were then blocked for 2 h in 5% BSA. All primary antibodies were diluted in blocking buffer and added to blots for 2 h. Blots were then washed three times in TBST and probed with HRP-conjugated goat anti-rabbit IgG secondary antibody.Purified virion samples were separated by SDS-PAGE. The whole gel was stained with Coomassie brilliant blue and then sent to Sangon Biotech Company  for liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis. Details of in-gel trypsin digestion, LC-MS/MS, and database searches were as described previously by Loret et al. , Leroy eE. coli BL21 cells and induced by IPTG  was observed by SDS-PAGE after the plasmids pET-32a(+)/US10 and pET-32a(+) had been transformed into PTG Fig. . Lanes 1PTG Fig. . FinallyPTG Fig.  and usedThe harvested antiserum was titered by the agar diffusion assay. The results showed a maximum titer of 1:16 Fig. . The celWestern blot analysis was performed using protein samples collected at 4, 8, 24, 48, 60, 72, 84, and 96 h post-infection (hpi). A specific protein band of approximately 36 kDa was first detected at 8 hpi; expression gradually increased and peaked at 72 hpi, but began to decline at 84 hpi. US10 was still detected at 96 hpi . The US10 protein could only be detected in DEV-infected cells, while gC protein could be detected in both purified virions and DEV-infected cells Fig. .Fig. 9WeP < 0.05). The relative abundance of US10 was low based on the exponentially modified protein abundance index (emPAI). From the results of the above two methods, DEV US10 is a minor component of the virion and the failure of western blotting may be caused by its low abundance.The purified virions were also analyzed by mass spectrometry to identify the protein content. We identified 40 structural proteins in the purified DEV virions (data not shown), including DEV US10 protein Table . One uniHerpesvirus genes are expressed sequentially in three distinctly defined stages: α (immediate-early), β (early), and γ (late) genes , 15. TheWe also observed the subcellular localization of DEV US10 proteins by indirect immunofluorescence. They were abundantly distributed in the cytoplasm, specifically localizing around the nucleus, which was in accordance with earlier observations of Marek’s disease virus (MDV) US10 proteins . MDV US1Recently, mass spectrometry has been widely used to analyze the structural proteome of herpesviruses, such as KSHV, HCMV, EBV, PRV, HSV-1, BoHV-4, and BoHV-1 , 24–28. Overall, our data suggest that US10 is a γ2 gene that probably functions as a virion protein, similar to HSV-1 US10 [Through experiments involving qRT-PCR, western blotting, and pharmacological inhibition tests, the DEV US10 gene was identified as a true late (γ2) gene. In infected cells, DEV US10 protein was initially diffusely distributed throughout the cytoplasm and gradually relocated to a perinuclear region. DEV US10 is detected in mature virions, so it is a virion protein. However, further studies are still needed to explore its functions.


Hibiscus cannabinus L.) is an economically important global natural fiber crop. As a consequence of the increased demand for food crops and the reduction of available arable land, kenaf cultivation has increasingly shifted to saline and alkaline land. To investigate the molecular mechanism of salinity tolerance in kenaf, we performed Illumina high-throughput RNA sequencing on shoot tips of kenaf and identified 71,318 unigenes, which were annotated using four different protein databases. In total, 2,384 differentially expressed genes (DEGs) were identified between the salt-stressed and the control plants, 1,702 of these transcripts were up-regulated and 683 transcripts were down-regulated. Thirty-seven transcripts belonging to 15 transcription-factor families that respond to salt stress were identified. Gene ontology function enrichment analysis revealed that the genes encoding antioxidant enzymes were up-regulated. The amino acid metabolism and carbohydrate metabolism pathways were highly enriched among these DEGs under salt stress conditions. In order to confirm the RNA-seq data, we randomly selected 20 unigenes for analysis using a quntitative real-time polymerase chain reaction. Our study not only provided the large-scale assessment of transcriptome resources of kenaf but also guidelines for understanding the mechanism underlying salt stress responses in kenaf.Kenaf (The online version of this article (doi:10.1007/s10265-016-0898-9) contains supplementary material, which is available to authorized users. Soil salinization has limited the ability to respond to the increase in demand for food crops. Globally, more than 20% of the available agricultural land is affected by salinity, and this area is predicted to double by 2,050  is one of the most important fiber crops, which has multiple uses, including paper pulp, carpet backing, building materials, and livestock forage had different effects on kenaf seed germination and seedling growth. When a 50 mmol/l Na2SO4 solution was applied to kenaf seeds, the germination rate was not significantly different from the control, whereas a 50 mmol/l Na2CO3 solution considerably inhibited seed germination rates  in a plant growth room. After 5 days, the seedlings were divided into two groups: No. 1 were grown under normal conditions; No. 2 were grown in a concentration of 250 mM NaCl. After 2 weeks, we collected 10 shoot tips from each of three biological replicates of each group, the shoot tips were frozen in liquid nitrogen and stored at −80 °C until use. The No. 1 group seedlings were used as the control sample (CK), and the three biological replicates were named as CK1, CK2, and CK3; the No. 2 group seedlings were used as the salinity-stress sample (ST), and the three biological replicates were named as ST1, ST2, and ST3.The shoot tips from each group were powdered in liquid nitrogen using a mortar and pestle and the total RNA was extracted using TRIzol reagent according to the manufacturer’s protocol . The quality of the total RNA was verified using an Agilent 2100 Bioanalyzer before further processing. Each RNA fraction used to prepare a separate cDNA library were CK1, CK2, CK3, ST1, ST2, and ST3 for sequencing and the sequencing was performed at the Beijing Genomics Institute . Poly-(A) mRNA was isolated from total RNA with Magnetic Oligo (dT) Beads. First, the purified mRNA was fragmented into small pieces, and then double-stranded cDNA was synthesized using the SuperScript Double-Stranded cDNA Synthesis kit (Invitrogen) with a random hexamer (N6) primer (Illumina). The double-stranded cDNA was subjected to end-repair and phosphorylation using T4 DNA polymerase, Klenow DNA polymerase, and T4 polynucleotide kinase. These repaired cDNA fragments were 3′-adenylated using Klenow 3′–5′ exo-polymerase, then ligated with Illumina paired-end adapters to the end of these fragments using T4 DNA ligase. The adapter-ligated fragments were separated on an agarose gel and cDNA fragments of approximately 200 bp (±25 bp) were excised from the gel. To enrich the purified cDNA template, The PCR was performed to amplify the cDNA fragments. The amplification protocol comprised a 30 s incubation at 98 °C then a cycle of 98 °C for 10 s, 60 °C for 30 s, 72 °C for 30 s, repeat 14 times, 72 °C for 5 min, and a final 10 °C hold. The cDNA library was constructed with a fragment length of 200 bp (±25 bp). After validation with an Agilent 2100 Bioanalyzer, the cDNA library was sequenced on a PE flow cell using an Illumina Hiseq2000 sequencing platform.Before assembly of the kenaf transcriptome, a stringent filtering process was carried out. The adapter sequences of the raw reads were removed, and low-quality sequences (reads with ambiguous bases ‘N’) and reads with more than 20% Q < 20 bases were removed. Assembly of the clean reads was performed using Trinity software. The Trinity software was used to combine reads with overlapping nucleic acid sequences to form contigs. Subsequently, contigs from the same transcript were assembled and sequences that could not be extended at either end were defined as unigenes. TGICL software was used to eliminate redundant unigenes and to further assemble unigenes to form a single non-redundant set. Unigenes from all libraries were assembled again to acquire non-redundant unigenes (All-Unigene set) that were as long as possible.− 5) against the National Centre for Biotechnology Information (NCBI) non-redundant (NR) database. Additionally, the all-unigenes were aligned to the other five public databases, including NCBI nucleotide sequences (Nt), Swiss-Prot, KEEG, COG, and GO databases. Based on NR annotation we used the Blast2GO program  between the CK and ST groups, the expression levels of all the transcripts analyses were determined using DESeq  from CK and ST samples. To evaluate the validity of comparative transcriptome analysis of the patterns of DEGs, 20 candidate unigenes (14 up- and 6 down-regulated) were selected and validated by quantitative real time PCR (qRT-PCR).2O. Actin was used as the endogenous reference gene. The reactions were incubated at 95 °C for 2 min, followed by 40 cycles of 95 °C for 15 s, 61 °C for 15 s, and 72 °C for 20 s. After the PCR, a melting curve was generated by gradually increasing the temperature to 95 °C to test the amplicon specificity. To determine relative fold differences for each sample, the CT value for each gene was normalized to the CT value for the reference gene and was calculated relative to a calibrator using the DDCT method as described by Livak and Schmittgen (qRT-PCR was performed using SYBR Green qPCR Master Mix (Takara) on a iQ5™ multicolor real-time PCR detection system (Bio-Rad). The volume of the qRT-PCR reaction was 10 µl, containing 2 µl cDNA, 5 µl 2 × SYBR Green qPCR Master Mix, 0.3 µl of the forward and reverse primers, and 2.4 µl ddHhmittgen . All reaTo obtain a global overview of the kenaf gene expression profile and the salt stress genes, total RNAs were extracted from shoot tips of CK and ST plants. To minimize bias from the Illumina sequencing and transcriptome sampling, we constructed 6 cDNA libraries: CK1, CK2, CK3, ST1, ST2, ST3, and sequenced these separately using an Illumina Hiseq2000 genome analyzer.Using the Illumina Hiseq2000 sequencing platform, each sequenced sample yielded 2 × 90 bp independent reads from either end of a cDNA fragment, approximately 159.5 million (CK) and 160.9 million (ST) raw reads were generated. After a stringent quality assessment, 146.4 million (CK), 147.7 million (ST) clean reads were obtained with 97.77% Q20 bases (Table The data were deposited in the NCBI Sequencing Read Archive database (accession number SRP064405). The high quality reads were assembled into one reference transcriptome using the Trinity software.Using the Trinity assembler, 71,318 unigenes were generated with an average length of 1,143 nt, an N50 of 1,784 nt, and a total length of 81,509,2256 nt. The total unigenes included 13,818 (19.38%) of less than 300 nt, 54,085 (75.84%) with lengths from 301 to 3,000 nt, and 3,415 (4.78%) with lengths greater than 3,000 nt , 38,065 (53.37%), 33,807 (47.40%), 22,049 (30.92%), and 51,851 (72.7%) showed significant similarity to known proteins in the Nr, Swiss-Prot, KEGG, COG, and Nt databases, respectively. In total 58,095 unigenes  were successfully annotated in at least one of the above databases  database has three principal categories: biological process, cellular component, and molecular function. On the basis of Nr annotation, 45,855 unigenes were assigned to these three categories, and were distributed across 45 functional categories. For the biological process, 65.29% were cellular process unigenes, and 61.32% were metabolic process unigenes. For the cellular component, 77.62% of the unigenes were assigned to cells and 77.6% to cell parts.The GO classifications of all unigenes are shown in Fig. The COG database was used to classify orthologous gene products. In total, 22,049 unigenes were assigned to the 25 COG categories. Of the 25 COG categories, the most frequently identified classes were general function 35.7%), transcription (20.1%), and signal transduction mechanisms (18.7%); a few unigenes were assigned to the nuclear and extracellular structure categories  in salt-treated kenaf, among these DEGs, 1,702 transcripts were up-regulated and 683 transcripts were down-regulated Fig. .GO annotation and statistical analyses demonstrated that 1,789 tested DEGs (some contigs had more than one GO annotation) were classified in three GO ontologies and 49 terms. Among these GO categories, cell, cell part and metabolic process were significantly enriched among DEGs compared to the whole transcriptome background. In the molecular-function category, 971 tested DEGs which were up-regulated (some contigs had more than one GO annotation) were enriched in 581 terms. The overwhelming majority of the genes were related to “binding”  (Table S3). Through a GO function enrichment analysis, we found that the genes coding antioxidant enzymes were up-regulated, such as those for peroxidase, glutathione transferase, and superoxide dismutase (Table S4).In our analysis, 2,384 tested DEGs mapped to 117 pathways with most mapping to “metabolic pathways” (501 DEGs), “biosynthesis of secondary metabolites” (302 DEGs), and “plant hormone signal transduction” (90 DEGs) (Table S5). Through KEGG enrichment analysis, we found that some unigenes in amino acid metabolism pathway and carbohydrate metabolism pathway were up-regulated (Table S6).We performed a global transcription-factor classification for differentially expressed transcripts and identified 37 transcripts belonging to 15 transcription-factor families (Table S7). Among them, 21 transcription-factors were up regulated, and 14 transcription-factors were down regulated. AP2/ERF domain-containing transcription factor was strongly upregulated under salinity treatment. Of the eight WRKY-family members, more were downregulated under salt treatment.To evaluate the validity of Solexa analysis of the patterns of DEGs, we selected 20 candidate unigenes (14 up- and 6 down-regulated), which responded to salt stress and were detected by qRT-PCR. Solexa sequencing revealed that the expression patterns of 17 out of 20 (85%) unigenes showed general agreement with Solexa sequencing. However, the patterns of arginine decarboxylase, gibberellin-regulated protein, and photosystem I reaction center V unigenes did not agree with the Solexa sequencing. They were detected as identically expressed in the CK and ST plants by qRT-PCR. The confirmation of the expression profiles of selected genes by qRT-PCR is shown in Table With the development of sequencing technology, NGS has enabled a novel gene discovery on a genome-wide scale. As NGS is low cost, with high efficiency and accuracy, it has been widely used to sequence transcriptomes and has been successfully applied to de novo transcriptome sequencing and assembly in many plants, e.g., bamboo, rubber, and ramie  , glutathione transferase, superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR). Under salt-stress conditions in kenaf, catalase and glutathione reductase activity increased with increasing salt concentrations . Our results showed that kenaf is salt tolerant, and that free amino acid and sugar are important compounds for salt resistance in Zhonghongma variety 16.Regulation of gene expression in salinity stress includes a wide array of mechanisms that are used by plants to up- or down-regulate the production of specific gene products (protein or RNA). Transcription factors are considered as the most important regulators that control gene expressions, including bZIP, WRKY, NAC, HD-Zip, and DREB. These transcription factors are capable of controlling the expression of a broad range of target genes by binding to the specific cis-acting element in the promoters of target genes. Johnson et al.  found thIn the present study, we found transcrption factors NAC, BZIP AP2/EREBP, ARF, AP2/ERF, Bhlh, and TCP were up-regulated. But 8 NAC transcription factors and 2 WRKY transcription factors were found to be down-regulated. The results showed that these transcription factors play an important role in the salinity tolerance mechanisms of kenaf.In conclusion, our study suggests that multiple pathways are involved in salinity tolerance in kenaf. The transcriptome sequencing and comparative transcriptome analysis will serve as a valuable resource to support genome analysis, develop expression arrays and will provide useful information for a better understanding of the physiological and molecular processes involved in salt tolerance in kenaf.Below is the link to the electronic supplementary material.Supplementary material 1 (DOCX 14 KB)Supplementary material 2 (DOCX 17 KB)Supplementary material 3 (XLSX 20 KB)Supplementary material 4 (DOCX 45 KB)Supplementary material 5 (DOCX 21 KB)Supplementary material 6 (DOCX 18 KB)Supplementary material 7 (DOCX 18 KB)Supplementary material 8 (DOCX 19 KB)


The retinoic acid-related orphan receptors (RORs) regulate several physiological and pathological processes, including immune functions, development and cancer. To study the potential role of RORs in melanoma progression, we analysed RORα and RORγ expression in nevi and primary melanomas and non-lesional skin and metastases in relation to melanoma clinico-pathomorphological features. The expression of RORα and RORγ was lower in melanomas than in nevi and decreased during melanoma progression, with lowest levels found in primary melanomas at stages III and IV and in melanoma metastases. Their expression correlated with pathomorphological pTNM parameters being low in aggressive tumors and being high in tumors showing histological markers of good prognosis. Higher nuclear levels of RORα and RORγ and of cytoplasmic RORγ correlated with significantly longer overall and disease free survival time. Highly pigmented melanomas showed significantly lower level of nuclear RORs. This study shows that human melanoma development and aggressiveness is associated with decreased expression of RORα and RORγ, suggesting that RORs could be important in melanoma progression and host responses against the tumor. Furthermore, it suggests that RORα and RORγ might constitute a novel druggable target in anti-melanoma management using tumor suppressor gene therapy restoring their normal functions. Melanoma incidence has been increasing during the last few decades with annual increase of incidence ranging from 3% to 7% in white population, especially in older individuals –4. At thRORγ gene generates two isoforms, which exhibit a distinct tissue-specific expression pattern and play important role in immune regulation (reviewed in  were obtained from patients who underwent surgery not related to skin diseases, while primary melanoma samples n=79], melanoma metastases [n=39] and nevi [n=27] were excised from patients treated in the Oncology Centre in Bydgoszcz during the period 2003-2010 , 9. Clin, melanomRORα and RORγ immunostaing was performed on standard formalin-fixed paraffin-embedded 4 μm sections of human samples, as described previously . BrieflyImmunostained sections of normal and pathological skin were analyzed without knowing the detailed histopathological diagnosis, malignancy grade and other clinical data. RORα and RORγ immunostaining intensity of melanomas was scored semiquantitatively, taking into consideration both staining intensity and percentage of immunostained tumor cells or, in normal skin, epidermal cells. The staining intensity was evaluated with reference to immunostaining of control, epidermal cells, scored as strong. The semiquantitative score was calculated as: SQ=mean (IR x SI), where IR is the percentage of immunoreactive cells and SI is the staining intensity from 0 to 3 arbitrary units (A.U.) with 0 as negative (0), weak (1), moderate (2) and strong (3). Then, cases were stratified according to RORs SQ-score as follows: SQ 0.0-10.0=none RORα, SQ 10.1-50.0=low RORα, SQ 50.1-300.0=high RORα, SQ 0.0-50.0=none RORγ, SQ 50.1-100.0=low RORγ, SQ 100.1-300.0=high RORγ.Proliferative activity, melanin content evaluation and TILs were performed as previously described , 78, 79.Clinico-pathological data related to the time between primary treatment  and histopathological diagnosis of melanoma to diagnosis of metastases were obtained from electronic database of the Oncology Center, Bydgoszcz Poland. Metastases were diagnosed by histopathological diagnosis of resected lesions, fine needle aspiration biopsy and/or diagnostic imaging techniques . The date of deaths was obtained from the Department of Registry Office in Bydgoszcz, Poland. All data are presented according to REMARK guidelines .Human SKMEL-188 and AbC1 hamster melanoma cells were cultured in Ham's F10 supplemented with 5% fetal bovine serum (FBS) and 1% antibiotics  as described previously , 14. CelWe have followed protocols described previously in , 14. BriDetailed description of WB method is provided in . Shortlyt-test were used, and for comparison three or more groups – ANOVA. To evaluate the association between RORα and RORγ staining and categorical variables, the Pearson's correlation was used. Survival analysis was performed using Log-rank test. For analysis of survival after adjustment of Breslow thickness and overall stage the Cox proportional-hazards regression analysis was performed using NCSS10 . Data are presented as mean ±SD.Statistical analysis was performed with the Prism 5.00 . Results were considered as statistically significant when P < 0.05. For comparison two groups the Student's


Lactobacillus rhamnosus strains of probiotic potential from healthy human infants.Probiotics provide health benefits to their hosts, including modulation of host immune response, inhibition of colonization by pathogens, modulation of the gut microbiota, and epithelial barrier enhancement. Here, we present the draft genome sequences of two newly isolated  Lactobacillus rhamnosus is a lactic acid bacterium with probiotic properties when associated with the host gut  were identified in this study.We isolated 175 bacterial strains from stool samples from healthy infants (0 to 2 years old) . Each stGenomic DNA from AMC010 and AMC143 was isolated using UltraClean microbial DNA isolation kit (Mo Bio). AMC010 was subjected to sequencing on the Ion Torrent PGM platform , generating sequence data of 485.3 Mb over 3,022,036 reads, with an average read length of 160 bp, providing 43× coverage of the 3.14-Mb genome. AMC143 genomic DNA (gDNA) was sequenced on the Roche FLX Titanium platform, generating 69.4 Mb of sequence over 318,612 reads, with an average read length of 210 bp, providing 23× coverage of the 2.87-Mb genome. Raw reads were quality filtered and assembled using Newbler (Roche) . AssemblL. rhamnosus genomes using the EDGAR genome analysis software  fructose transporter system, PTS lactose transporter system, and a large number of previously uncharacterized hypothetical proteins.AMC010 has a G+C content of 46.6%, 53 annotated tRNA genes, and 3,171 annotated coding sequences (CDSs), with 162 unique genes compared to other sequenced and annotated software . In addiMSTB00000000 (AMC143) and MSTC00000000 (AMC010).This whole-genome shotgun project has been deposited at DDBJ/ENA/GenBank under accession numbers


Mycobacterium avium (MA) belongs to the intracellular parasitic bacteria. To better understand how MA survives within macrophages and the different pathogenic mechanisms of MA and Mycobacterium tuberculosis (MTB), tandem mass tag (TMT) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis have been used to determine the proteins which are differentially expressed in MA-infected and MTB-infected macrophages. 369 proteins were found to be differentially expressed in MA-infected cells but not in MTB-infected cells. By using certain bioinformatics methods, we found the 369 proteins were involved in molecular function, biological process, and cellular component including binding, catalytic activity, metabolic process, cellular process, and cell part. In addition, some identified proteins were involved in multiple signaling pathways. These results suggest that MA probably survive within macrophages by affecting the expression of some crucial proteins. Mycobacterium tuberculosis (MTB) infection, making it a major global health problem. MTB could also be largely found in patients who were affected by HIV [ Mycobacterium avium (MA), one of the Nontuberculous Mycobacteria (NTM), is a ubiquitous organism usually isolated from natural environments such as soil and drinking water and causes infections such as disseminated disease, bacteremia, and severe pulmonary in immunocompromised people, especially in patients who suffered from AIDS [ mycobacterium kansasii, mycobacterium abscessus, mycobacterium xenopi, and mycobacterium malmoense [Tuberculosis (TB) is an infectious disease which is transmitted in the air and caused by bacillusd by HIV , 2. Mycorom AIDS , 4. Besialmoense , 6.γ and (interleukin) IL-12 pathway is more susceptible to MA infection as well [α blocker [Macrophage serves as the first defense response of a host to MTB or MA infections . Several as well , 6. Simi blocker , 6 whoseMTB and MA are facultative pathogens capable of growing inside macrophages. The macrophage probably exerts very strong selective pressure on the mycobacteria residing within it, influencing the expression of gene products essential for the survival of the bacteria within this hostile environment. The induction of survival mechanisms, alongside a range of immunological effector molecules, emphasizes the complexity of the cross-talk that occurs between the macrophage and the mycobacterium. To characterize this cross-talk and to detail the changes which occur following the initial interaction between mycobacterium and macrophage, some researchers have studied the proteins changes of the host cell in the early stages of mycobacterial infection for a long time –19. HoweIn this study, we used a method called TMT technology , which s2 in RPMI 1640 medium supplemented with 10% fetal bovine serum, penicillin (100 μg/mL), and streptomycin (30 μg/mL) until it was grown to a density of 1 × 107 cells per mL. Subsequently, cells were centrifuged, washed, and resuspended in RPMI 1640 supplemented with 10% fetal bovine serum. The cell density was adjusted to 106 cells per mL and 2 mL of the cell suspension was transferred to a 6-well tissue culture plate. MTB was cultured in Lowenstein-Jensen culture medium and MA was cultured in Egg culture medium. Both bacteria were cultured for 15 days and then harvested, washed, and suspended in phosphate buffer saline. The suspension was diluted to a concentration of 1 × 108 CFU/mL to make sure the MOI was 1 : 100. The treated MTB and MA were added to 6-well tissue culture plate which was precultured with U937 cells. The cells were divided into three groups: cells infected with MTB, cells infected with MA, and cells with PBS (control). The cells of each group were cultured at 37°C, 5% CO2 for 24 hours, respectively.The MTB and MA were obtained from Center for Disease Prevention and Control of Guangxi. The U937 cell line was obtained from Obio Technology (Shanghai) Co. Ltd. U937 cell line was cultured at 37°C, 5% COg for 20 minutes. The supernatant was discarded. The cell pellets were lysed in lysis buffer containing protease inhibitor mix and then centrifuged at 4°C, 16000g for 15 mins. The supernatant was transferred to a precooled centrifugal tube. The cell lysate was stored at −80°C until further analysis.The cells of three groups were centrifuged at 4°C, 1000μg of protein was transferred to a new microcentrifuge tube and adjusted to a final volume of 100 μL with 100 mM TEAB. 5 μL of 200 mM TCEP was added and incubated with the sample at 55°C for 1 hour. Another 5 μL of 375 mM iodoacetamide was added and incubated with the sample mixture for 30 mins in the dark at room temperature. At last, 600 μL of cold acetone was used to precipitate proteins at −20°C for about 4 hours. Precipitated protein was centrifuged at 8000g for 10 mins at 4°C and then was dried and stored at −80°C until further use.Bradford method was used to measure the protein concentrations. For each sample, 100 μL of 100 mM TEAB and incubated with 20 μL of the Trypsin Storage Solution for 5 mins. 2.5 μL of trypsin was used to digest 100 μg of protein of each sample overnight at 37°C. The protein sample was dissolved in anhydrous acetonitrile with occasional vortexing. TMT labeling reagent was added and incubated with the sample for 1 hour at room temperature. MA-infected group sample was labeled with reagent 127 and MTB-infected group sample was labeled with reagent 128 while the sample of control group was labeled with reagent 126. 5% hydroxylamine was used to quench the reaction. The labeled samples were stored at −80°C until future analysis.The protein pellet was resuspended with 100 2PO4, 20% v/v acetonitrile, and pH 3.0) and then loaded onto polyethylene A. The peptide mixtures were eluted with a flow rate of 700 μL/min and a gradient of 0–40% mobile phase B (mobile phase A plus 500 mM KCl) for 50 minutes, 40% B–100% B for 10 minutes, 100% B–100% B for 5 minutes, 100% B–100% A for 5 minutes, and 100% A for 10 minutes. The eluates were monitored by absorbance at a wavelength of 254 nm, and the fractions were collected every 2 minutes. The collected fractions of 28 tubes were desalted on C18 reverse phase column.Firstly, we separated polypeptide with C18 reverse phase column. Protein samples labeled with reagents 126, 127, and 128 were air-dried with vacuum followed by enzyme digestion. The peptide mixture was acidified with mobile phase A (10 mM KHμL/min. Mobile phase B (98% ACN + 0.1% HCOOH) was also used and the elution gradient was 95% A for 5 minutes, 95%–65% A for 45 minutes, 35% A–100% B for 2 minutes, and 100% B for 3 minutes. The mass spectrometer data were analyzed using the ESI in the positive ion mode with a selected mass range of 350–2000 mass/charge (m/z) and the survey scans were acquired at a resolution of 60000. The resolution for HCD spectra was set at 15000. Normalized collision energy was 1.5 KV with a collision mode of CID. The instrument was set at data-dependent automatic collecting mode.Each fraction underwent nano-LC-MS/MS analysis using a Q Exactive MS equipped with Easy nLC. The chromatographic column was balanced with mobile phase A (2% ACN + 0.1% HCOOH). The peptide mixtures were separated at a flow rate of 300 P value less than 0.05 were determined as significantly differentially expressed [The primitive data collected by MS was searched and matched with SEQUEST searching material of Proteome Discovery software which uses Xoc BLS256 database of NCBI as its main database. The settings were as follows: trypsin chosen as the enzyme with one missed cleavage allowed; fixed modifications of carbamidomethylation at Cys; variable modifications of oxidation at Met; peptide tolerance set at 0.05 Da and MS/MS tolerance at 0.1 Da; and monoisotopic mass chosen. Proteins with 2-fold or greater and 0.5-fold or less changes between successive comparisons with a xpressed .P value < 0.05 was considered statistically significant.We searched the GO and KEGG database to classify and identify differentially expressed protein. The significant signaling pathway enrichment was examined with the hypergeometric test. A In this study, U937 cells without infection (control) and U937 cells infected with MA and MTB culture, respectively, for 24 hours were collected for protein extraction, digestion, and TMT labeling. Proteomes of both infections were investigated.P value < 0.05). Compared to the identified proteins of MA- and MTB-infected groups, we found that 369 proteins were differentially expressed in MA-infected but not in MTB-infected cells. Among them, 2 proteins were upregulated  while 367 proteins were downregulated . 682 proteins were differentially expressed in MTB-infected but not in MA-infected cells. Among them, 672 proteins were upregulated  while 10 proteins were downregulated .In the sample of MA- and MTB-infected U937 cells, 2269 proteins were identified. Among these proteins, a total of 574 proteins were differentially expressed in MA-infected cells and 887 proteins in MTB-infected cells .To understand the difference between MA infection and MTB infection, the 369 proteins which were only differentially expressed in MA-infected cells are characterized with GO term , catalytic activity (17%), and Poly(A) RNA binding (13%) . In termThe proteins identified in biological process were found to be mostly involved in organonitrogen compound metabolic process 22%), organic substance metabolic process (21%), and catabolic process (6%) . The ide%, organiAmong proteins classified as cellular component, the majority was localized in extracellular membrane-bounded organelle (29%) and cytoplasmic part (17%) . Most ofIt is notable that the only two proteins named P10412 and H0YJ03 were upregulated in MA-infected cells compared to MTB-infected cells. P10412 is histone H1.4 and is necessary for the condensation of nucleosome chains into higher-order structured fibers. It also regulates chromatin remodeling, DNA methylation, and many other functions . H0YJ03 https://david.ncifcrf.gov) and Omicsbean (http://www.omicsbean.cn) to analyze the 369 proteins which were differentially expressed in MA infection but not MTB infection. Hopefully our findings would identify in which pathway these proteins were involved in order to understand the mechanism of MA infection into macrophages. The analysis came up with 152 KEGG orthologues. All these signaling pathways may suggest how MA affects the expression of proteins in MA-infected cells. The KEGG pathways with more differentially expressed proteins involved included Hippo signaling pathway, PI3K-Akt signaling pathway, and phagosome pathway.We used DAVID Functional Annotation Bioinformatics Microarray Analysis (δ/ε), NF2 (Mer), and PPP2R1A (PP2A)) were associated with Hippo signaling pathway , YWHAH (14-3-3), ITGB2 (ITGB2), ACTB (F-actin), PPP2R2A (PP2A), STK3 (Mst1/2), PPP1CA (PP1), CSNK1E , YWHAH (14-3-3), ITGA5 (ITGA), ITGB1 (ITGB), PPP2R2A (PP2A), and PPP2R1A (PP2A) . The PI3α5β1), ACTB (F-actin), ITGB2 (CR3), ITGB1 (β1), TUBA1B (TUBA), ATB6V1B2 (vATPase), SEC22B (Sec22), CANX , RAB5A (Rab5), and FCAR (FcaR)  . FCAR isIn summary, we carried out a proteomic analysis of differentially expressed proteins with TMT technology in MA-infected macrophage. It is the first study to analyze the proteins involved in MA infection. In addition, we profiled the differentially expressed proteins in MTB infections. By comparing the proteomic data of the two different infections, we were able to identify the 369 differentially expressed proteins which were unique in MA-infected cells. These identified proteins were involved in binding, catalytic activity, metabolic process, cellular process, cell apoptosis, phagosome maturation, antimycobacterial response, cellular components, and so forth. In addition, some identified proteins were involved in multiple signaling pathways. The results of this study will help elucidate how MA survives within cells and the different pathogenic mechanisms of MA and MTB infections. It might also provide new ideas for future development of target treatment of MA disease and tuberculosis.


Additionally, treatment with 6% sodium hypochlorite (m/v) resulted in 80.5% decrease in α-helical content and complete digestion of the peptide. The Contrex™ EZ treatment was repeated with three additional α-conotoxins (α-CTxs), α-CTxs LvIA, ImI and PeIA, which verified the decontamination method was reasonably robust. These results support the use of either 1% Contrex™ EZ solution or 6% sodium hypochlorite in biosafety protocols for the decontamination of α-CTxs in research laboratories.Nine publically available biosafety protocols for safely handling conotoxin peptides were tested to evaluate their decontamination efficacy. Circular dichroism (CD) spectroscopy and mass spectrometry (MS) were used to assess the effect of each chemical treatment on the secondary and primary structure of α-CTx MII . Of the nine decontamination methods tested, treatment with 1% ( Conus is comprised of as many as 1000 distinct neurologically active peptides [Conus species, resulting in the staggering potential that 70,000–100,000 unique CTxs exist [50 of CTxs to be as low as 5 μg/kg [The venom of predatory marine snails from the genus xs exist ,3. CTxs xs exist . CTxs ha 5 μg/kg ,5. For t 5 μg/kg . SystemaThe current study is limited to α-CTxs. A survey of literature accessible in the National Center for Biotechnology Information (NCBI) pubmed resource shows that 74% (3385 of the 4564) of published works on CTxs are studies involving α-CTxs. The methods for safely decontaminating and neutralizing α-CTx waste vary greatly in universities where CTx research is conducted; this study evaluated currently employed biosafety protocols used in university research laboratories. Several current decontamination protocols for CTxs rely on procedures demonstrated to be effective on low molecular weight toxins of biological origin . Other pTo determine the effect of chemical treatment on the secondary structure of α-CTx MII , α-helical content was estimated from the measured ellipticity at 222 nm. Typical CD spectra for chemical treatments of α-CTx MII  are shown in m/v) sodium hypochlorite (80.5% decrease), and 1% (m/v) Contrex™ EZ (76.8% decrease). Chemical crosslinking with 2% (m/v) glutaraldehyde did not decrease the α-helical content, but interestingly, treatment with a combination of glutaraldehyde and formaldehyde seemed to disrupt secondary structure, with a 33.5% decrease in α-helical content. Treatment with ozone, hydrogen peroxide, and dithiothreitol (DTT) resulted in no significant change in α-helical content, and glutathione was only modestly effective at disrupting secondary structure, with a demonstrated decrease of 9.4%.The ability of chemical agents to disrupt the secondary structure of α-CTx MII  is summarized in m/z and a triply charged ion observed at 547.211 m/z . This result is consistent with the formation of imine bonds by two glutaraldehyde molecules forming intra-peptide crosslinks. The mass addition of each glutaraldehyde crosslink (C5H8) is 68 Da. The mass increase of 136 Da in the doubly charged mass spectrum of glutaraldehyde treated peptide can be attributed to the addition of C10H16 to the molecular formula of α-CTx MII . Inter-peptide crosslinking between several α-CTx MII  molecules was not observed. Sodium hypochlorite treatment entirely digested α-CTx MII , and no discernable peaks in the mass range 250–2900 Da were observed in the mass spectrum..211 m/z a. Contrerged ion d. The shm/z for the untreated α-CTx MII  , as shown by the disappearance in the band around 270 nm . The neaV, E11A) a. Intere3 did not affect the secondary structure of α-CTx MII , leading to the conclusion that ozonolysis is an ineffective decontamination method. Hydrogen peroxide treatment followed by exposure to ultraviolet light generates hydroxyl radicals with potential to cleave peptide bonds. However, hydrogen peroxide treatment did not alter the secondary or primary structure of α-CTx MII , as evidenced by the absence of either significant decrease in α-helical content by CD, or mass shift by MS (data not shown).Ozonolysis of peptides has been shown to affect amino acids with aromatic side chains and histidine . α-CTx MTreatment of α-CTx MII  with 8 M urea was effective at disrupting the secondary structure. The peptide samples treated with 8 M urea were diluted immediately prior to CD measurement in order to obtain each spectrum. Although treatment with urea did disrupt secondary structure, it did not alter the primary structure of α-CTx MII , as determined by MS. To be an effective decontamination method, additional treatment following urea induced peptide denaturation would be required. Treatment with 6 M hydrochloric acid was effective at denaturing α-CTx MII , but this is not a practical protocol for ameliorating spills due to highly hazardous properties.m/v)) treatment caused complete disruption of secondary structure and digestion of α-CTx MII . This result may be due to excess sodium hydroxide present in commercial Clorox used to stabilize the sodium hypochlorite in the product, or because of oxidative cleavage of peptide bonds by sodium hypochlorite. Sodium hypochlorite has a pungent odor and acts as a strong oxidizer that can react unfavorably with common laboratory chemicals, however, bleach is ubiquitously available, inexpensive, and frequently used as a disinfectant in laboratories. Sodium hypochlorite (6% (m/v)) appears to be an effective method to use in biosafety protocols for the decontamination of α-CTxs in research laboratories.Two chemical treatment options, sodium hypochlorite and Contrex™ EZ, were identified as the most effective and applicable laboratory decontamination methods reviewed in this investigation. Sodium hypochlorite  solution may be suitable for inclusion in biosafety protocols requiring decontamination of dilute solutions of α-CTxpeptides , laboratory spills of dilute peptides, or cleaning of equipment exposed to trace amounts of α-CTxs. In situations requiring decontamination of concentrated solution or solid peptide, or highly potent α-CTx , 6% sodium hypochlorite has been confirmed to be the recommended method to ensure inactivation. The scope of this study was limited to the evaluation of decontamination procedures for α-CTxs. The recommended decontamination procedures may not apply to all CTxs, particularly those with significant differences in disulfide framework, solubility, or peptide size.The results of this study indicate the use of 1% Contrex™ EZ (m/v) solution proved to be an effective chemical treatment for α-CTxs. Alternatively, treatment with 1% solution of Contrex™ EZ resulted in a 76.8% decrease in α-helical content and digestion of α-CTx MII  into smaller peptide fragments as demonstrated by mass spectrometry. The results of this investigation indicate that partial digestion occurs within the first 15 min of treatment. For liquid chemical waste decontamination, all liquid waste containing α-CTxs, including HPLC waste, may be stored in 4 L carboys, to which 40 g of Contrex™ EZ powder should be added to achieve ~1% solution (% w/v). A 4 lb container of Contrex™ EZ can be purchased from fishersci.com for $54.00 USD, and 121 oz of Clorox HE performance bleach can be purchased from Amazon.com for $15.75 USD. Decontamination of a 4 L bottle of waste containing 100 μg conotoxin for every 1 mL of solution will cost on the order of $1.20 USD for Contrex EZ as compared to $1.60 USD for bleach. In the studies reported herein, the decontamination methods were tested on α-CTx containing solutions of high peptide content  per 1.0 mL) and low chemical treatment concentration (1% Contrex™ EZ solution). A recommended time for decontamination of liquid waste is a minimum of one hour to ensure more than adequate time for α-CTx degradation.In laboratories where use of bleach is acceptable, sodium hypochlorite 6% (m/v), 12 M hydrochloric acid (HCl), 50% glutaraldehyde (m/v), 37% formaldehyde (m/v), dithiothreitol (DTT), and glutathione purchased from Fisher Scientific . Contrex™ EZ powdered enzymatic detergent was purchased from Decon Labs . Household Clorox bleach was used for the 6% sodium hypochlorite. High pressure liquid chromatography (HPLC) mobile phase consisted of 18 MΩ H2O and HPLC grade formic acid and acetonitrile . All α-CTxs used in this study were purchased from CS Bio  at a purity of >95%.Chemical treatments were prepared from 8 M urea, 30% hydrogen peroxide (m/v), 10% hydrogen peroxide (m/v) with UV irradiation, gaseous ozone, 6 M hydrochloric acid, 2% glutaraldehyde (m/v), 1% formaldehyde (m/v)/1% glutaraldehyde (m/v), 10 mM dithiothreitol, and 500 μM glutathione. For each of these treatments, the reaction was allowed to proceed for 15 min prior to sample dilution and analysis of secondary structure using CD spectroscopy. Prior to analysis by HPLC-MS, Contrex™ EZ, the reaction was quenched and samples were purified using a Pierce Strong Cation Exchange Spin Column , a wash buffer composed of 25 mM sodium acetate, pH 5.5, and an elution buffer of 25 mM sodium acetate, 1 M NaCl, at pH 5.5. All other reactions were quenched and samples were purified using Pierce PepClean™ C18 Spin Columns  activated with 50% methanol (v/v), and a wash buffer composed of 5% acetonitrile (v/v) and 0.5% trifluoroacetic acid (v/v), and an elution buffer of 70% acetonitrile (v/v). Ozone gas was generated with a Villa 1000 ozone generator from SD International, Inc.  operated at an output of 1800 mg ozone per hour. The reaction was performed with a 50 μM solution of α-CTx MII  exposed to flowing ozone captured in a custom-made reaction vessel for up to 24 h. α-CTx MII  treated with 10% hydrogen peroxide was exposed to UV irradiation in a biosafety hood with a UV lamp operating at an intensity of 125 μW/cm2 at a distance of ~1 m for 20 min.The following treatment conditions were used: 8 M urea, 6% sodium hypochlorite, 1% Contrex™ EZ solution (2 × dmol−1) for each spectrum was calculated from the formula θMRE = θ/(10 Crl), where θ is the measured ellipticity in millidegrees, Cr is the molar concentration, and l is the path length in centimeters. The α-helical content was estimated from the formula θMRE = −30300fH − 2340, where fΗ is the fraction of α-helical content  calculated from the θMRE at 222 nm, is a widely used proxy for helical secondary structure that is useful to assess disruption of the secondary structure, as is performed here [CD spectra were recorded on a Jasco J-810 spectropolarimeter  with a cell path length of 1 cm at room temperature in a nitrogen atmosphere. Scans were acquired from 190 to 250 nm and 250 to 300 nm for far-UV and near-UV, respectively. The bandwidth of 1 nm, a speed of 50 nm/min, and a resolution of 0.5 nm were used. A total of 5 scans per sample were averaged, baselines were subtracted, and each sample was run in triplicate. A final concentration of 50 μM and 500 μM were used for far-UV and near-UV scanning wavelengths, respectively. Analysis and processing of data were carried out with the Jasco system software and Microsoft Excel. Mean residue ellipticity : 4 or 8 L/min; drying gas temperature: 200 °C; voltage between HV capillary and HV end-plate offset: 3000 V to −500 V; mass range was set from 250 to 2900 m/z; and the quadrupole ion energy was 4.0 eV. Low concentration ESI tuning mix  was used to calibrate the system in the mass range. Samples analyzed by direct infusion with a syringe pump at a flow rate of 240 μL/h. HPLC separation was achieved using a Dionex UltiMate® 3000 uHPLC system  equipped with a Thermo Acclaim™ 120 C18 column  . The mobile phase was 0.1% formic acid in water (Buffer A) and acetonitrile (Buffer B) with a flow rate of 0.3 mL/min. A linear gradient method was used to separate the mixture starting at 5% acetonitrile and ending at 65% acetonitrile over 25 mi. The sample injection volume was 1 μL. Data were analyzed using the Compass Data Analysis software package .Untreated and treated α-CTxs were analyzed by HPLC-MS using an ultra-high resolution Quadrupole Time of Flight (QTOF) instrument  by direct injection to the MS or by HPLC-MS. The electrospray ionization (ESI) source was operated under the following conditions: positive ion mode; nebulizer pressure: 0.4 or 1.2 Bar; flow rate of drying gas (N


Viscum album L.) are used in complementary cancer treatment. These commercial extracts are water-based and do not include water-insoluble compounds such as triterpenic acids. However, both hydrophilic and hydrophobic triterpenic acids possess anti-cancer properties. In this study, a whole mistletoe extract viscumTT re-created by combining an aqueous extract (viscum) and a triterpene extract (TT) was tested for its anti-cancer potential in osteosarcoma.Osteosarcoma is the most common bone tumor and is associated with a poor prognosis. Conventional therapies, surgery and chemotherapy, are still the standard but soon reach their limits. New therapeutic approaches are therefore needed. Conventional aqueous mistletoe extracts from the European mistletoe  were detected by western blot. Apoptosis induction by co-treatment of viscum, TT and viscumTT with doxorubicin, etoposide and ifosfamide was examined by flow cytometry.In vitro as well as ex vivo, the whole mistletoe extract viscumTT led to strong inhibition of proliferation and synergistic apoptosis induction in osteosarcoma cells. In the investigations of mechanism of action, inhibitors of apoptosis such as XIAP, BIRC5 and CLSPN showed a clear down-regulation after viscumTT treatment. In addition, co-treatment with doxorubicin, etoposide and ifosfamide further enhanced apoptosis induction, also synergistically.ViscumTT treatment results in synergistic apoptosis induction in osteosarcoma cells in vitro and ex vivo. Additionally, conventional standard chemotherapeutic drugs such as doxorubicin, etoposide and ifosfamide were able to dramatically enhance apoptosis induction. These results promise a high potential of viscumTT as an additional adjuvant therapy approach for osteosarcoma.The online version of this article (doi:10.1186/s12906-016-1545-7) contains supplementary material, which is available to authorized users. Osteosarcoma (OS) is the most common bone tumor with a worldwide incidence of four to five cases annually per million . NowadayViscum album L. extracts (VAEs) are widespread in complementary cancer medicine in Europe and used in several cancer types [Viscum album L. includes a large number of chemically different substances: lectins [Viscum album L. is not fully understood. Both the European mistletoe as well as the Korean mistletoe mediated apoptosis induction by activation of the PI3K/AKT pathway [er types , 3. Visc lectins , triterp lectins , viscoto lectins , phenoli lectins , flavono lectins , oligo-  lectins , 10 and  lectins  and visc lectins , 11, and lectins , 13 and  lectins . VAE tre pathway , JNK/p38 pathway  signalli pathway . Our gro pathway , in acut pathway  as well  pathway , ALL [18 pathway .Viscum album L. contains hydrophobic compounds, pentacyclic triterpenic acids, such as oleanolic (OA) and betulinic (BA) acid. Because of their low water solubility they are not included in VAEs. OA and BA possess anti-cancer effects in different tumor entities. OA arrested the cell cycle and led to apoptosis induction in pancreatic [In addition to MLs and viscotoxins, ncreatic  and ostencreatic  while BAncreatic .To potentiate all anti-cancer effects of each mistletoe compound, a whole mistletoe extract viscumTT was created by combining mistletoe derived triterpenic acids (TT), which were solubilized by β-cyclodextrins (CD) , and a wIt is already known that adjuvant therapy with VAEs reduces side effects during chemotherapy . AdditioViscum album L. extracts were kindly provided by Birken AG . Preparation of Viscum album L. extracts from apple tree  was performed as described previously [eviously , 29. Inteviously . Both thRPMI 1640, McCoy’s 5A, penicillin, streptomycin, trypsin (0.05%) and PBS were purchased from Gibco, Lifetechnologies . Fetal Calf Serum (FCS) was obtained from Biochrom . Protein inhibitors, molecular mass standards for SDS-PAGE, sodium dodecyl sulphate (SDS), dimethyl sulfoxide (DMSO), VP16, 5, 5, 6, 6-tetrachloro-1, 1, 3, 3-tetraethylbenzimidazol-carbocyanine iodide (JC-1) and propidium iodide (PI) were purchased from Sigma Aldrich . Tween, acrylamide and dithiotreitol (DTT) were purchased from Carl Roth GmbH  and 4-hydroperoxyifosfamide  was obtained from Niomech . Doxo was kindly provided by the hospital pharmacy of the Charité.5 onto 6 well and 2*105 onto 12 well plates. For Saos-2 cells 1*106 onto 6 well and 5*105 onto 12-well plates were seeded. Cells cultured 24 h to allow cell attachment and treated with viscum , TT  and viscumTT  24 and 48 h.The human osteosarcoma cell lines 143B and Saos-2 were obtained from American Type Culture Collection . 143B cells were cultured in RPMI 1640, Saos-2 in McCoy’s 5A, both supplemented with 10% heat inactivated FCS 100 U/mL penicillin and 100 μg/mL streptomycin. For the experiments, 143B cells were seeded in 4*104 143B cells and 5*104 Saos-2 cells were seeded onto 96-well plates in triplicates and incubated 24 and 48 h with viscum, TT and viscumTT in rising concentrations  was used for quantification of cell proliferation. 2*10ions see . 10 μl WCell number was calculated using a CASY® Cell Counter from Schaerfe System GmbH . Alterations of proliferation were expressed in percent in relation to control cells . Lactate dehydrogenase (LDH) release was measured photometrically at 490 nm after 2 h using the Cytotoxicity Detection Kit  to preclude an early cytotoxic effect.2) and stained with APC-conjugated Annexin V  and 1 mg/mL PI according to manufacturer’s instructions to measure apoptosis. The cells were analyzed by flow cytometry . The results were evaluated with FlowJo Software .Both osteosarcoma cell lines were seeded onto 6 well plates and incubated with increasing concentrations of viscum, TT and viscumTT in rising concentrations see  for 24 am was measured using JC-1. After incubation, the cells were washed, re-suspended in 750 μl PBS and incubated with 2.5 μg/mL JC-1 for 30 min at 37 °C, 5% CO2. After a further PBS washing step the cells were analyzed by flow cytometry. As reference, control cells were treated with 50 μM depolarizing carbonyl cyanide 3-chlorophenylhydrazone (CCCP)  for 30 min.The ∆ΨAfter an incubation time of 24 h with viscum, TT and viscumTT in rising concentrations see , CASP3, For protein extraction 143B cells were incubated with increasing concentrations of viscum, TT and viscumTT for 24 h. The cells were washed twice and lysed using RIPA buffer  including proteinase inhibitor for 30 min. Next, the cells were centrifuged at 14,000 rpm for 30 min at 4 °C. Protein concentration was determined by Bradford reagent. After transfer of proteins to nitrocellulose membrane  primary antibody was incubated over night at 4 °C. The following primary antibodies were used: CASP3 , PARP1 , CLSPN , BIRC5 , TP53 , XIAP , BCL2 , BID , cytochrome c , ß-actin  and GAPDH . Protein signal was detected by HRP-conjugated secondary antibodies  and visualized by ECL  and ChemiDoc. For mitochondrial protein preparation, cells were lysed as described above and the cytosolic fraction was isolated as published earlier . Then, t5/well for treatment with viscum, TT or viscumTT extracts. For each tested dose, single well plate was used and experiment was performed at least in three sets of independent experiments. Then, proliferation, apoptosis induction, activation of CASP8, CASP9, CASP3 and loss of ∆Ψm were examined. Written informed consent was obtained from the patient in accordance with the Declaration of Helsinki, and the study was approved by the local ethics committee of the Charité- Universitätsmedizin Berlin.After routine surgical resection, a tumor sample from the tibia was obtained as treatment ‘residue’ from a 12-year-old boy with first-diagnosis osteosarcoma without lung metastases. This tumor material was not explicitly collected for this research. Histopathology confirmed the diagnosis. Immediately after surgical excision, the tissue was dissected into smaller pieces and cultured as a primary explant in RPMI 1640 base medium with L-glutamine supplemented with 20% heat-inactivated FCS and 1% penicillin/streptomycin solution. When cells dissociated from the explant to form a confluent monolayer culture ex vivo cells were treated in 4 trypsinized passages. For investigations, cells were seeded onto 12-well microtiter plates at 2.5×101;2exp = (E1 + E2-E1*E2), with E1;2exp = expected  effect of the combination viscumTT. E1 = observed effect of TT; E2 = observed effect of viscum; E1;2obs = observed effect of combined mixture viscumTT. Fp = E1;2obs/E1;2exp. Values Fp > 1 represent synergistic, whereas Fp = 1 additive and Fp < 1 antagonistic effects. One-way ANOVA was used for calculation of significance. All results with p ≤ 0.05 were considered significant.All in vitro experiments were performed at least in three sets of independent experiments, for which means ± standard error were calculated and plotted in bar graphs. Webb’s fractional product (*Fp) >1 was calculated for the synergistic effect of viscumTT and the chemotherapeutic agents on apoptotic induction in vitro as described earlier , 30. BriEarly cytotoxicity was analyzed by Cytotoxicity Detection Kit after 2 h whereby LDH release was investigated. Neither 143B nor Saos-2 cells showed any considerable increase of LDH in the supernatant Fig. . TherefoFor assessment of inhibition of proliferation, cells were counted using a CASY® Cell Counter and proliferation was estimated from total numbers of cells compared to control cells. Inhibition of proliferation was confirmed by WST-1 assay. Both cell lines showed strong inhibition of cell proliferation in a dose-dependent manner after treatment with viscum, TT and viscumTT after 24 h and 48 h Fig. . TT inhiNext, the potential of viscum, TT and viscumTT for apoptosis induction was determined. 143B and Saos-2 cells were analyzed by annexin V-APC/PI staining and flow cytometry after 24 and 48 h treatment. Interestingly, viscum led to only a slight apoptosis induction in both cell lines, whereas TT and viscumTT triggered apoptosis in a dose- and time-dependent manner after 24 h Fig. . This efFor investigation whether mitochondria are involved in the apoptotic process, the Δψm depolarization was evaluated by JC-1 and FACS analyses after 24 h extract treatment. ViscumTT treatment led to a significant dose-dependent loss of Δψm Fig.  and confΔψm depolarization and resulting cytochrome c release into the cytosol mediate apoptosis induction via the intrinsic pathway. Based on western blot analyses cytochrome c was detected in the cytosol fraction after treatment with viscum and viscumTT Fig.  in 143B To get a closer look at the apoptosis mechanism, we assessed the activation of CASP8 and CASP9. These caspases are upstream initiator protease caspases, which will be activated by the extrinsic (CASP8) and intrinsic (CASP9) apoptotic pathways and initiates cleavage of effector caspase CASP3. CASP3 and the presence of cytochrome c can additionally induce cleavage of CASP8 as a feedback mechanism without receptor mediation . CASP8 wBecause of previous knowledge, we wanted to know which proteins of the bcl-2 family and inhibitor of apoptosis proteins (IAPs) are altered in osteosarcoma. All three extracts led to down-regulation of anti-apoptotic protein BCL2 with simultaneous up-regulation of pro-apoptotic protein BAX in 143B cells Fig. . A reducTo investigate whether there is any apoptotic effect on primary tumor material, derived osteosarcoma cells were treated with viscum, TT and viscumTT. Figure Because of the increasing presence of resistances to conventional chemotherapy in osteosarcoma a combination treatment with complementary therapy approaches is becoming more and more important. Beside treatment with viscum, TT and viscumTT, osteosarcoma cell lines were co-treated with Doxo, VP-16 and 4OOH to examine apoptosis induction. In both cell lines apoptosis was induced even when TT and viscum alone were added to chemotherapeutic drugs. This effect was additionally enhanced in 143B after co-treatment with viscumTT Fig. . In Saoset al. proved the activation of CASP8 without any death receptor signalling [In this study we investigated the apoptotic potential of the whole mistletoe extract viscumTT in comparison to its constituents viscum and TT. ViscumTT very effectively inhibited cell proliferation and synergistically induced apoptosis in osteosarcoma cells in vitro in a time- and dose-dependent manner. Our in vitro data correlate with the assumption that whole plant extracts are often more potent in anti-cancer activity than the single ingredients , 33. Thegnalling , which sgnalling  and cytognalling  can alsognalling , 19. Howgnalling , 36, 37.gnalling , 39. Thignalling  and regugnalling , 42. p53gnalling  and humagnalling . This isgnalling . Interesgnalling . In addignalling , 47 and gnalling . It is egnalling . Our datgnalling , 51, andgnalling . Furthergnalling . An optignalling . Also, Vgnalling . In our In this study we demonstrated the high potential of viscumTT with regard to apoptosis induction in osteosarcoma. It induced apoptosis synergistically. This effect is further enhanced when it is co-administered with Doxo, VP16 and 4OOH. Because of increasing problems during chemotherapy such as resistances or negative side effects, new therapeutic approaches will become more important in the future. Hence, viscumTT may represent a promising adjuvant therapy in pediatric osteosarcoma.


CA125 is a prevalently used serum biomarker for detecting ovarian cancer over the last three decades. However, it has a significant deficiency in screening for early-stage cancer. With the purpose of exploring an effective approach to improve its performance in early diagnosis, we investigated the postprandial fluctuation pattern of cancer-derived CA125 and the underlying mechanism.In two medical centers, 551 patients sonographically diagnosed with ovarian  cysts (< 5 cm in diameter) were enrolled and divided into five disease groups . The subtle differences in 1-h postprandial serum CA125 increases were compared between disease groups. A support vector machine (SVM)-based algorithm was used for refining the performance of CA125 postprandial increment. Ovarian cancer xenograft animal and cancer cell models were used to recapitulate the clinical findings and reveal the molecular basis of postprandial blood glucose and insulin in invoking the synthesis/secretion/re-absorption of CA125.Patients with ovarian cancer presented the highest postprandial increment 13.3 ± 0.7% (mean ± standard deviation) among the five disease groups. Using a CA125 increment ≥ 10% criterion, the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) reached 83.3, 96.3, 61.1 and 98.8%, respectively, for early-stage ovarian cancer. This performance was further improved by the SVM-based CA125-increment algorithm, which exhibited 91.7% sensitivity, 99.2% specificity, 89.2% PPV and 99.4% NPV. Both modalities manifested diagnostic advantages over the traditional CA125 test . Regarding the molecular basis, the postprandial blood glucose and insulin-invoked overexpression of Mucin 16 (encoding CA125) were demonstrated in animal and cancer cell models, which were mediated by the PI3K-Akt pathway. Nevertheless, a Mesothelin-based CA125 re-absorption behavior was noted in the treated cancer cells, which contributed to the over-drop following the postprandial peak of serum CA125.Cancer-derived serum CA125 possesses a unique and distinctive postprandial pattern, that distinguishes it from the common CA125 elevation in a benign disease condition. The dynamic measurement/assessment strategy can achieve a discriminatory power superior to that of a static test.The online version of this article (10.1186/s12967-018-1489-4) contains supplementary material, which is available to authorized users. Earlier diagnosis of ovarian cancer could significantly increase survival. The general 5-year survival rate of ovarian cancer patients is approximately 90% at stage I but only 5–60% at stage II or above . LocatedEfforts to improve the efficacy of TVU and CA125-based screening include the adoption of more effective subsidiary/surrogate cancer biomarkers . Of themBoth the synthesis and secretion rates of CA125 in ovarian cancer cells are keenly influenced by extracellular signals from circulating cytokines. Konishi et al. reported that the secretory levels of CA125 in cells cultured in vitro can be increased upon administration of epithelial growth factor (EGF) . We, theFrom January 2015 through June 2017, patients were enrolled at two tertiary medical centers in Shanghai, China: The Department of Gynecological Oncology, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University and The Department of Gynecology, Obstetrics and Gynecology Hospital, Fudan University. The inclusion criteria were (1) an ovarian  tumor (cyst) ≤ 5 cm in diameter under ultrasonography; (2) a scheduled exploratory laparotomy. The exclusion criteria were (1) any elevated serum AFP, CA199, or CEA level; (2) an ultrasonography-confirmed dermoid cyst (teratoma) or other germ cell tumor; (3) computed tomography- or MRI-confirmed abdominal metastasis or metastatic gastrointestinal cancer; (4) late-stage or nonepithelial ovarian cancer demonstrated by postoperative pathology; (5) a history of diabetes mellitus.2 at 37 °C. The final concentrations of insulin , LY294002 , AktVIII (Selleck), AZD6244 (Selleck), and CA125 standard (Thermo Fisher) were 100 nM, 20, 1 mM, 5 μM and 200 U/mL, respectively. The high glucose condition referred to a glucose concentration of 2 g/L.The ovarian cancer cell lines SKOV-3, HO8910, ES-2, and OVCAR-3 were purchased from the Cell Bank, Chinese Academy of Sciences . Cells were cultured in DMEM  supplemented with 10% FBS  in 5% CO6) were subcutaneously injected into specific pathogen-free (SPF)-grade Balb/c nude mice weighting between 20 and 22 g. As tumors grew to 1 cm in diameter, mice were divided into four groups. (1) In the control group, each mouse was administered a dose of 0.2 mL saline p.o., and 0.2 mL saline i.h.; (2) in the glucose group, each mouse was administered 0.2 mL of 10% glucose p.o. and 0.2 mL saline i.h.; (3) in the insulin group, each mouse was administered 0.2 mL saline p.o. and 0.2 mL insulin (5 mU) i.h.; (4) in the glucose plus insulin group, each mouse was administered 0.2 mL of 10% glucose p.o. and 0.2 mL insulin (5 mU) i.h. The type I diabetes mouse model was built by administration of alloxan (Sigma-Aldrich) .Total RNA was isolated from the cultured cells using TRIzol (Thermo Fisher) and then reverse-transcribed into cDNAs using a Universal cDNA Synthesis Kit . Real-time PCR was performed using the primer pairs for Mucin 16 , MSLN  and β-actin . SYBRGreen I dye (Thermo Fisher) served as a quantitative indicator in the qPCR reaction. An ABI PRISM 7900 Sequence Detection System [Applied Biosystems, Carlsbad, CA, USA) was used for PCR experiments. The cycle threshold (CT) of each qPCR assay was recorded, and the results were presented as 2The primary antibodies used for recognizing the target proteins were as follows: mouse anti-human Mucin 16 monoclonal antibody , mouse anti-human Mesothelin monoclonal antibody  and mouse anti-human β-actin monoclonal antibody , which were then detected using the secondary antibody provided by the Wes Mouse Master kit . All the Western blotting experiments were performed using Wes (ProteinSimple). The intensities of the resultant bands were quantified and visualized using the “Compass for SW” software (ProteinSimple).2 test/Fisher’s exact test and Student’s t test  were used for nominal and numerical data, respectively. A support vector machine (SVM) was generated using the “kernlap” package in R software (version 3.3.3). The sensitivity of a diagnostic criterion was defined as the percentage of correctly identified cases among all pathologically confirmed ovarian cancer cases. The specificity was defined as the percentage of correctly identified cases among all pathologically confirmed non-cancer cases. The positive predictive value (PPV) was defined as the percentage of correctly identified cases among all the cases that could be judged as ovarian cancer by a given diagnostic criterion. The negative predictive value (NPV) was defined as the percentage of correctly identified cases among all the cases that could be judged as benign diseases by a given diagnostic criterion. SPSS 18.0 software  was used to complete the statistical analyses . p ≤ 0.05 was considered significant. All animal and in vitro experiments were carried out in triplicate unless otherwise indicated.Two-sided χA total of 551 patients sonographically diagnosed with cysts (tumors) of the ovary (or adnexa) were enrolled  for a 75-g oral glucose tolerance test (OGTT), among whom multipoint  serum insulin and CA125 tests were additionally performed. We observed that 1-h postprandial CA125 levels were generally higher than fasting CA125 levels in the disease groups  xenograft animal models were built. In the xenografted nude mice, a slow ascending curve of the serum CA125 level was observed after administration of 20 mg oral glucose, and the peak of the curve emerged in approximately 2 h . We noted that the addition of 2 g/L glucose to the culture medium only slightly increased CA125 concentration in the supernatant, while co-administration of glucose and insulin stimulated an active release of CA125 Fig. a. An enhTo further elucidate the molecular basis of glucose and insulin-induced CA125 synthesis/secretion, we investigated the intracellular signaling pathways that could be responsible. Western blotting and RT-qPCR experiments showed that the addition of insulin activated the expression of CA125 and its surface receptor, Mesothelin (encoded by MSLN), at both protein and mRNA levels Fig. a. Two MSIn this study, the enrolled population was a typical representation of patients encountered in a gynecological clinical condition. The gynecological diseases they suffered included four major classes of benign cysts, i.e., pelvic inflammatory cysts, adnexal retention cysts, ovarian endometrioma and benign/borderline cystadenoma, which need to be discriminated from early-stage ovarian cancer due to the high-risk manifestations, such as long-term abdominal pain (inflammatory cysts), progressive CA125 elevation , rapid tumor enlargement (retention cysts) and postmenopausal pelvic mass (retention cysts) , 23, 24.Accepted as a basic carcinogenesis theory, earlier studies have indicated that the PI3K-Akt pathway is frequently altered in cancer cells, which exhibits far stronger activity than it does in healthy cells , 26. ForOur animal and cancer cell models both revealed that, insulin, only when combined with a high-glucose condition can invoke the active release of CA125; otherwise, it promotes re-absorption of CA125 Figs.  and 4. TAlthough in this work, by including the postprandial increase in the serum CA125 test, the CA125 cut-off was successfully lowered and the efficacy of early-stage ovarian cancer detection was accordingly improved, we still noted that there were cancer cases missed, even after adopting a postprandial increment-based diagnostic criterion with the highest efficacy . Most of these cases (5/6) were characterized by CA125 > 105 U/mL , would be beneficial to gain a more stable, reliable, and informative dataset on the postprandial CA125 fluctuation patterns in cancer and non-cancer patients. Therefore, further efforts should focus on investigating the applicability of standard OGTT (or IVGTT)-associated fluctuation pattern(s) of cancer-derived serum CA125 in large-scale patient populations.In conclusion, the present study adopted the concepts of “glucose and insulin-induced CA125” and “dynamic measurement/assessment of serum cancer biomarkers”. Moreover, by exploiting the subtle differences in postprandial increases in serum CA125 among patients with endometriosis and benign and malignant ovarian tumors, an SVM-based CA125-increment algorithm was built. This algorithm exhibited improved efficacy on detecting early-stage ovarian cancer. Additionally, to a certain degree, this work provides a paradigm for future explorations of the dynamic and inductive behaviors of more serum cancer biomarkers, such as HE4, CA199, CEA, and PSA.Additional file 1: Table S1–S5. Demographic, clinical and pathological characteristics of the study population. Serum CA125 levels (random) detected in the study population. Comparisons of the differences of CA125 serum levels (random) among the five disease groups. Comparisons of the differences between fasting and postprandial CA125 serum levels for the five disease groups. Coordinates of the ROC curve for fasting CA125 levels .Additional file 2: Figure S1–S3. Comparisons of the fluctuation patterns of blood glucose, serum insulin and CA125 between the ovarian cancer and non-cancer patients. Comparisons of relative CA125 and Mesothelin protein levels between ovarian cancer cells (OVCAR-3) treated with different agents, namely DMSO, insulin, insulin with high glucose, and insulin with high glucose and PI3K-Akt/MEK-Erk inhibitors. The time-dependent re-absorption of Alexa Fluor 488-labeled CA125 by ovarian cancer cells (OVCAR-3) under the high-glucose plus insulin condition.


In the last decade, autosomal recessive interleukin-12 receptor β1 (IL-12Rβ1) deficiency, the most common cause of Mendelian susceptibility to mycobacterial disease (MSMD), has been diagnosed in a few children and adults with severe tuberculosis in Iran. Here, we report three cases referred to the Immunology, Asthma and Allergy ward at the National Research Institute of Tuberculosis and Lung Diseases (NRITLD) at Masih Daneshvari Hospital from 2012 to 2017 with Mycobacterium tuberculosis and non-tuberculous mycobacteria infections due to defects in IL-12Rβ1 but with different clinical manifestations. All three were homozygous for either an IL-12Rβ1 missense or nonsense mutation that caused the IL-12Rβ1 protein not to be expressed on the cell membrane and completely abolished the cellular response to recombinant IL-12. Our findings suggest that the presence of IL-12Rβ1 deficiency should be determined in children with mycobacterial infections at least in countries with a high prevalence of parental consanguinity and in areas endemic for TB like Iran. Mycobacterium bovis strain) vaccines, non-tuberculous mycobacteria (NTM) and non-typhoid Salmonella, and/or Mycobacterium tuberculosis (MTB), relies on the functional integrity of the interleukin (IL) 12/23-interferon (IFN)-γ axis enabling cross-talk between macrophages and T-lymphocytes/NK cells  and two X-linked genes (IKBKG and CYBB) , IFNGR1 (39%), IL12B (9%), STAT1 (5%), IFNGR2 (4%), and IKBKG (3%) . The prevalence of TB in IL-12p40- and IL-12Rβ1-deficient patients is however lower than BCG or NTM infection  or to unrelated parents (patient 2) and were referred to the Immunology, Asthma and Allergy ward at the National Research Institute of Tuberculosis and Lung Diseases (NRITLD) at Masih Daneshvari Hospitals, Shahid Beheshti University of Medical Sciences, Tehran-Iran, between January 2012 and July 2017.Mycobacterium bovis BCG sub-strain Pasteur at birth. Two months later, she developed a progressive bilateral axillary adenopathy . A few months later, AFB was isolated following bone marrow (BM) aspiration and an immunodeficiency was suspected.Two years later, the patient was hospitalized due to high blood pressure, pyrexia, myalgia, cough, and severe weight loss. Evaluation of gastric washing (GW) cultures, smear-detected Mycobacterium, acid fast bacilli (AFB), and a polymerase chain reaction (PCR) test was positive for the MTB complex and BCG. Chest radiography showed right-sided pleural effusion and adenopathy in the para-aortic, celiac, and mesenteric regions was seen. Both kidneys were normal in size although evidence of abscesses on the left side was observed by ultrasound. PCR tests for urine and pleural fluid were also positive for BCG. An anti-TB treatment course was started with isoniazid, rifampin, ethambutol, Klacid, ofloxacin, and Lanoxin. Two months later, despite a negative PCR result for the MTB complex in urine and GW, the previous treatment regimen was complemented with clarithromycin, captopril, and IFN-γ ; Hb10.6 g/dl; HCT33%; and the ESR was 39 mm/h in the first hours with detected high C-reactive protein (CRP) . Liver function tests revealed an aspartate aminotransferase (AST) of 36 u/l , alanine aminotransferase (ALT) of 10 u/l , and a decreased ALT/AST ratio. Antibody responses to tetanus and diphtheria were normal and DHR123 and nitroblue-tetrazolium (NBT) tests were within the normal range. The patient was treated again with amikacin, Klacid, cycloserine, Lanoxin, captopril, and IFN-γ (25 μg/m2). After a few months of therapy, whole blood was collected from both the patient and her parents and the release of IL-12 and IFN-γ from stimulated peripheral blood mononuclear cells (PBMCs) was assessed. In brief, PBMCs were isolated from whole blood containing EDTA using Ficoll-Paque before being stimulated with rhIL-12 , rhIFN-γ , and/or BCG for 48 h and IFN-γ and IL-12 release was measured by ELISA.Eight months later, clinical manifestations (persistent cough and dyspnea) continued to be reported despite treatment and the patient was admitted to the hospital. Various mycobacteriologic analyses , PCR, and other tests) were performed. GW PCR showed the presence of MTB complex that was isoniazid- and rifampin-resistant. Blood tests showed a WBC of 6.1 × 10IFN-γ release following stimulation with BCG + rhIL-12 was extremely low and a 100-fold lower than that observed from PBMCs from control subjects. Although the addition of rhIFN-γ to BCG-stimulated cells further increased IL-12p70 release from PBMCs 4.4-fold, this remained far less than that seen from normal healthy controls Table . These fIL12RB1 exon 5, c.512A>C, which results in the amino acid substitution p.Q171P was identified , Hb 11.9 g/dl, PLT 120 × 103/μl with a low liver function test ALT/AST ratio (310/345), and high alkaline phosphatase (ALP) 3400 u/ml . A qualitative C-reactive protein test was positive. An evaluation of the abdominal cavity by imaging and by physical examination showed moderate abdominal ascites and splenomegaly (116 mm). Liver biopsy showed chronic hepatitis, grade 3/18, stage 2/6. This condition is seen in both viral and autoimmune hepatitises. Plasma cell infiltration, lymphoid aggregates, or follicles (seen in autoimmune hepatitis) were absent and both liver biopsy and ascites were negative for AFB and mycobacteria. An MSMD functional assay was conducted in whole blood (PBMCs) and revealed a lack of response to recombinant IL-12 following BCG stimulation , Hb 6.3 g/dl, platelets 95 × 103/μl, ESR 135 mm/h, and CRP 24 mg/lit. BM aspiration was hypocellular. An evaluation of the abdominal cavity by imaging and by physical examination showed abdominal ascites, splenomegaly, and lymphadenopathy in the para-aortic, mesenteric, and celiac regions. A tuberculin skin test was negative and serum levels of IgG and IgE were elevated. GW was positive for mycobacteria on Lowenstein-Jensen medium. The lymphocyte transformation test (LTT) for BCG was 2.1  and an NBT test was normal. Based on the physical analysis, laboratory data, and clinical signs, the patient underwent MSMD functional evaluation. A year and a half later, despite treatment with rifampin, isoniazid, ethambutol, and clarithromycin, the patient was re-admitted to the hospital due to backache, abdomen distension, and ascites. Radiologic analysis showed splenomegaly (126 mm). The ascites smear test was negative for AFB.The third patient was a 7-year-old male vaccinated with BCG at birth that developed axillary lymphadenopathy at 5 months of age , Hb 12.4 g/dl, and PLT 241 × 103/μl and the CD4/CD8 ratio was decreased compared to control levels (9/40). Serological assays and PCR for HIV were both negative. Thus, levofloxacin and IFN-γ were added to the treatment regimen . However, at 5.5 years of age, the general condition of the patient deteriorated severely and the patient was again admitted to the hospital complaining of seizure and experienced respiratory distress, scrotal edema, severe backache, abdominal ascites, and distension during the hospitalization. Complete blood analysis gave the following results: WBC 6.8 × 103/μl , Hb 10.7 g/dl, and PLT 142 × 103/μl and CRP was 61 mg/l. A low liver function test ALT/AST ratio (15/20) was observed. Multiple brain abscesses and cerebritis were seen by MRI (Fig. Four months later, the blood culture was positive for Kingella species and blood tests revealed the following results: WBC 7.05 × 10IL12RB1 mutation c.517C>T in exon 5 (Fig. Progression from localized disease into disseminated infection suggested an underlying immunodeficiency and a genetic and functional analysis of the IL-12/IL-23/IFN-γ loop was performed. Functional analysis revealed possible defects in the IL-12Rβ1 (Table n 5 Fig. , and Iran 5 Fig. . The pren 5 Fig. . To our In young children, TB is often disseminated due to early, hematogenous spread of the Mycobacterium after primary pulmonary infection (Alcaïs et al. In the present study, we screened for IL-12Rβ1 mutations in suspected patients with MTB infection and reported three homozygous mutations: Q171P, R283X, and R173W. Patients 1 and 2 are the first reports of children with Mycobacterium infection due to IL-12Rβ1 deficiency in Iran. Patient 3, with an R173W mutation, is only the second child reported in Iran with this particular mutation (Boisson-Dupuis et al. However, this deficiency is probably not unique to children, as a young adult case with TB and IL-12Rβ1 deficiency has also been identified in Iran (Tabarsi et al. www.lovd.nl/IL12RB1 for IL-12Rβ1 defects, and that are also available for most other genes that cause MSMD, together with awareness of these immunodeficiencies prior to BCG vaccination will improve the health and quality of care for these patients.Mandatory screening for IL-12Rβ1 mutations that cause a defective IL-12/IL-23/IFN-γ axis would not only improve the quality of patient care but also increase the safety monitoring for potential complications including infectious diseases in these subjects. A global registry of patients with genetic defects in the IL-12/IL-23/IFN-γ axis such as that available at