Long-terminal repeat (LTR) retrotransposons are transposable genetic elements that replicate intracellularly, and can be considered progenitors of retroviruses. Ty1 and Ty3 are the most extensively characterized LTR retrotransposons whose RNA genomes provide the template for both protein translation and genomic RNA that is packaged into virus-like particles (VLPs) and reverse transcribed. Genomic RNAs are not divided into separate pools of translated and packaged RNAs, therefore their trafficking and packaging into VLPs requires an equilibrium between competing events. In this review, we focus on Ty1 and Ty3 genomic RNA trafficking and packaging as essential steps of retrotransposon propagation. We summarize the existing knowledge on genomic RNA sequences and structures essential to these processes, the role of Gag proteins in repression of genomic RNA translation, delivery to VLP assembly sites, and encapsidation. Saccharomyces cerevisiae. Ty1 and Ty3 are the most extensively characterized LTR-retrotransposons  retrotransposons are transposable genetic elements that comprise a significant fraction of many eukaryotic genomes ,2. They  reviews ,12). Ty1revisiae ,14. Ty3 proteins . In haplproteins ,17,18. Fproteins  as well proteins ,8. ThereORFs), GAG and POL, flanked by long terminal repeats (LTRs) [GAGORF encodes the Gag capsid-like protein, while the POLORF encodes enzymes required for Gag and Gag-Pol maturation (protease (PR)), retrotransposon replication (reverse transcriptase (RT)) and integration into the host genome (integrase (IN)). Organization of the Ty3 POL3 ORF is akin to that of retroviruses (PR-RT-IN) while the Ty1 POL ORF sequence order is PR-IN-RT [Ty1 and Ty3 share many important structural and functional characteristics. The elements are similar in size; integrated full-length Ty1 is 5.9 kb, and the Ty3 element is 5.4 kb in length. Both elements contain two overlapping open reading frames (s (LTRs) ,17,20,21PR-IN-RT ,23,24.S. cerevisiae cells [GAG and POL open reading frames flanked by untranslated regions (UTR) at the 5′ and 3′ termini [Retrotransposons exploit cellular machinery to transcribe their genomic copy, and both Ty1 and Ty3 transcripts synthesized by RNA polymerase II are capped and polyadenylated before nuclear export. Despite the fact that Ty transposition is a rare event, Ty1 RNA comprises up to ~10% of polyadenylated mRNA in haploid ae cells ,25,26. Tae cells  and receae cells . Ty1 and termini ,17,20,24 termini ,30,31,32 termini ,34,35,36 termini ,38,39.POL-encoded enzymes, and two copies of Ty gRNA organized in a dimeric form [GAG ORF while the 5′ and 3′ portions of the Ty3 PBS are located at opposite ends of the genome [The Ty1 and Ty3 gRNA, Gag, and Gag-Pol precursors colocalize in specific cytoplasmic foci termed retrosomes, where they assemble into VLPs ,41,42,43ric form ,45. Onceric form ,47,48. te genome ,50. The e genome ,52,53,54e genome ,56,57,58e genome .cis-acting sequences required for gRNA dimerization, packaging, and priming of reverse transcription. The cis-acting sequences were first delimited for Ty1 using a mini-Ty1 donor-helper system [trans, enabling mini-Ty1s containing cis sequences to transpose. These studies demonstrated that regions located within or adjacent to the LTRs are important while up to 5 kb of Ty1 internal sequences can be deleted without any significant effect on transposition. The minimal Ty1 element capable of retrotransposition when proteins are supplemented in trans contains 380 nt of 5′-end of the (+) RNA genome and 357 nt from its 3′-end. Using a similar assay, cis-acting sequences in Ty3 RNA were delimited at the 5′ and 3′-ends, and nt 429 to 4979 were demonstrated dispensable for retrotransposition when proteins were supplemented in trans [Retroelement gRNAs contain internal structures fundamental to propagation. Prominent among these motifs are r system . Mini-Tyr system ) lackingin trans ,36.GAG ORF and the largest region separating its PBS sequences is 28-nt, while parts of the Ty3 PBS are located at the opposite ends of the genome, adjacent to the 5′ UTR  and in the 3’ UTR (two segments) [tRNAiMet is essential for Ty1 and Ty3 retrotransposition. Using mutational analyses Ty1 gRNA sequences required for primer binding were located between nt 94 and 104  based onegments) ,48. In Tegments) . For botegments) ,63. Ty1 egments) . Howeveregments) ), Boltonegments) . In partegments)   . The 5′-egments) ,70. Inteegments) .Orthoretrovirinae subfamily also have nucleocapsid protein with only one ZF (Gamma- and Epsilonretroviruses), while others possess two motifs. Spumaretrovirinae NC-like proteins lack ZF motifs [Gag and its mature products are the major VLP structural components, serving as multifunctional regulators that orchestrate retrotransposon replication. Organization of the 290-amino acid Ty3 Gag3 is related to the counterpart proteins from simple retroviruses encoding a capsid (CA) and nucleocapsid (NC) domain, separated by short spacer (SP) . CA is cF motifs . Ty3 NC F motifs ,78. DuriF motifs . Based oF motifs ,80,81,82F motifs ,78. The F motifs .Although functionally related, Ty1 Gag lacks sequence and structural homology to Ty3 or retroviral Gag proteins . The 441Ty1 and Ty3 gRNAs are not divided into separate pools of translated and packaged RNA, thus gRNA trafficking and packaging into VLPs require an equilibrium between competing events of translation and packaging. VLP assembly starts at the specific, microscopically distinct, cytoplasmic foci known as retrosomes, where gRNA and Gag colocalize ,41,42. Bsrp68-DAmP mutation, Gag is present but rapidly turned over and retrosomes do not form. Stable Ty1 Gag is retro-translocated from the ER lumen to the cytoplasm and binds translating Ty1 gRNA. Multimerization of Gag bound to Ty1 gRNA may repress gRNA translation and induce a shift from its translation to packaging into VLPs [Little is known about Ty gRNA trafficking to retrosomes and it remains unclear where the first interaction with Gag occurs. Recent findings indicate that Ty1 gRNA is translated in association with signal recognition particle (SRP), which interacts with nascent Gag polypeptide for transport into the ER . The SRPnto VLPs .Current data raises an interesting possibility that Ty1 and Ty3 Gag proteins play a role in nuclear export of gRNAs ,97. UsinMany lines of evidence indicate that both Ty1 and Ty3 require mRNA processing body (P-body) proteins for effective retrotransposition and VLP assembly. Eukaryotic P-bodies are cytoplasmic ribonucleoprotein granules wherein mRNA deadenylation-dependent and nonsense-mediated decay factors are concentrated along with their mRNA substrates and where mRNA decay processes can occur ,104,105.POL, and 3′ UTR are dispensable for Ty1 RNA localization to retrosomes [POL sequences [GAG sequence was sufficient. Based on the mini-Ty1 donor-helper system Xu and Boeke [cis-acting sequences required for gRNA packaging reside within 580 nt at the RNA 5′ terminus. When additional nucleotides, up to position 380, were deleted from mini-Ty1 RNAs, packaging was reduced to 80%. Further deletion of nucleotides 237–380 reduced packaging to 15% and significantly impeded the amount of RNA co-purified with VLPs. Collectively, these observations argue that either major cis-acting sequences required for Ty1 RNA packaging reside within 237–380 region or its presence facilitates proper exposure of packaging element. The 237–380 fragment encompasses part of the sequence essential for folding of the Ty1 RNA pseudoknot (nt 256–270) [Although Ty gRNA is selected from a pool of excess cellular RNAs and selectively packaged into VLPs ,113, seqtrosomes  while Tyequences . For effequences  or Ty3 [equences  GAG seqund Boeke  demonstr256–270)   ). Intere256–270) . In theo256–270) ,117,118.256–270) .cis-acting sequences in gRNA by Gag. Moreover, it is not known if the same nucleotide sequences are recognized by Gag during nuclear export, trafficking to retrosomes and gRNA packaging [cis-acting signals for dimerization, packaging, and initiation of reverse transcription [Both genetic studies and limited in vitro data imply that Ty encapsidation relies on recognition of ackaging ,42,60. Tcription . A recencription  . Cluposition . Althougposition ,124, thiposition ,85,122. position ,126. Ty3position . Yeast fposition ,114,127.Based on mutational analyses, Gag residues important for Ty1 VLP assembly and structure have been mapped within aa 62–114 and 340–363 . In anotSimilar to retroviruses , the CA The NC domain of uncleaved Ty3 Gag3, but not mature NC, is required for gRNA packaging into VLPs since mutations disrupting mature NC production did not inhibit gRNA encapsidation . The conThe spacer domain (SP), located between CA and NC domains of Ty3 Gag3, is not critical for VLP assembly. However, deleting the SP domain results in formation of more compact VLPs that are defective for retrotransposition. The Ty3 SP domain is more acidic than known retroviral SP and substitutions of acidic residues to alanine led to VLP disruption and inhibition of retrotransposition . Based oThere are many mechanisms that may limit the effects of retrotransposition on the host genome. With regards to Ty RNA packaging, one such mechanism is of particular interest. Garfinkel et al. discovered Ty1 copy number control (CNC)  which isCis-acting sequences and structures important for packaging are not clearly defined and it is likely that multiple RNA sites scattered outside major packaging regions may contribute to the process. Moreover, it is not known if the same nucleotide sequences are recognized by Gag during gRNA nuclear export, trafficking to retrosomes and packaging. Many questions remain open but it is clear that RNA packaging and VLP formation is critical for Ty propagation. The VLP protects retrotransposon gRNA and concentrates all factors required for reverse transcription. VLP formation may also protect the host from high levels of free reverse transcriptase that could be potentially harmful.gRNA plays two distinct roles in the retrotransposon life cycle, namely as a template for translation and reverse transcription . gRNAs a


Enterobacter asburiae strain PDA134 genome was sequenced. This bacterial strain was isolated from the root tissue of a date palm, where it has the ability to produce 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase and indole-3-acetic acid (IAA) under salinity stress.In this report, a draft of the  Endophytic bacteria have the ability to provide host plants with their required nutrition and phytohormones . These bEnterobacter asburiae strain designated PDA134 was reported. This strain was previously described based on functional characterization and the biochemical products with relation to plant growth promotion , DNA gyrase (gyrB), initiation translation factor 2 (infB), and ATP synthase β subunit (atpD) gene sequence analyses, as previously described  was fragmented, and DNA adapters were ligated to both ends of the DNA fragments. The library was then sequenced on the Illumina HiSeq 2500 sequencer. The sequence reads were filtered and trimmed based on Phred quality scores. The analysis was carried out using the merGenie . The conmerGenie . DNA seqEnterobacter asburiae PDA134 genome consisted of 4,699,624 bp, assembled into 26 scaffolds ranging in length from 900,290 to 328 bp, with a G+C content of 56.15%. There were 4,352 putative coding regions, including 3,310 genes with a known function representing 1,549 enzymes mapped on 2,987 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The gene list also contained 69 tRNA genes and two rRNA clusters.The results showed that the d-cysteine desulfhydrase (The announced endophytic bacterial genome in this project encoded a wide-host-range VirA protein , a nitrofhydrase , severalfhydrase , multiplfhydrase , ampicilfhydrase , and a sfhydrase . The preLSQV00000000. The version described in this paper is version LSQV01000000.This whole-genome shotgun project has been deposited at DDBJ/ENA/GenBank under the accession no.


Drug resistance was extensive and care was complex; nevertheless, high rates of treatment completion were achieved albeit at considerable cost. To describe factors associated with multidrug-resistant (MDR), including extensively-drug-resistant (XDR), tuberculosis (TB) in the United States, we abstracted inpatient, laboratory, and public health clinic records of a sample of MDR TB patients reported to the Centers for Disease Control and Prevention from California, New York City, and Texas during 2005–2007. At initial diagnosis, MDR TB was detected in 94% of 130 MDR TB patients and XDR TB in 80% of 5 XDR TB patients. Mutually exclusive resistance was 4% XDR, 17% pre-XDR, 24% total first-line resistance, 43% isoniazid/rifampin/rifabutin-plus-other resistance, and 13% isoniazid/rifampin/rifabutin-only resistance. Nearly three-quarters of patients were hospitalized, 78% completed treatment, and 9% died during treatment. Direct costs, mostly covered by the public sector, averaged $134,000 per MDR TB and $430,000 per XDR TB patient; in comparison, estimated cost per non-MDR TB patient is $17,000. Drug resistance was extensive, care was complex, treatment completion rates were high, and treatment was expensive. Mycobacterium tuberculosis poses substantial obstacles to tuberculosis (TB) control. In the United States, multidrug-resistant (MDR) TB (resistant to at least isoniazid and rifampin) comprises only 1.0%–1.5% of TB cases but requires lengthy regimens of toxic drugs, imposes high costs on the health care system and society, and causes high mortality rates.Drug-resistant Studies of MDR TB in the United States have been limited by small sample sizes, limited study periods, minimal information on outcomes and costs, or reliance solely on surveillance data  by California, New York, NY, and Texas during 2005–2007. These 3 areas contribute about half of US MDR TB cases annually.CDC and local institutional review boards approved the study and granted a waiver of patient informed consent and patient authorization. We defined a 5-drug regimen  to be coEach site identified cases of MDR TB and extensively drug-resistant (XDR) TB reported to CDC during 2005–2007. The study included all XDR TB cases and a 75% simple random sample of MDR TB cases from California and New York, NY (New York City), and a 50% sample from Texas. Using standardized forms, we abstracted hospital, laboratory, and public health clinic records retrospectively for patient demographic, socioeconomic, and clinical characteristics and for treatment, case management, outcomes, and costs. Total charges for each TB-associated hospitalization were abstracted from hospital UB-04 forms. To ascertain sputum-culture conversion and drug resistance, we examined all available culture and drug-susceptibility testing (DST) results from diagnosis through treatment.To assess representativeness, we compared our sample with National TB Surveillance System data from all US sites. We identified characteristics among MDR TB patients associated with the following 3 dichotomous outcomes: drug resistance acquisition, expert consultation use, and death during TB treatment. Multivariable logistic regression was used with backward selection at p<0.05 to identify variables remaining in final models . Adjusted odds ratios (AORs) significant at 95% CIs and Schwarz Criterion statistics are reported for goodness-of-fit. Variables included in initial models were patient demographics , socioeconomic factors , medical risks , TB history, disease severity , drug-resistance pattern, receipt of TB clinic outpatient care, and additional relevant characteristics .Inpatient costs were measured; outpatient costs and productivity losses were estimated . HospitaStudy total direct costs were compared with estimated direct costs for cases of non-MDR TB . We repo2 statistics are reported to show goodness-of-model-fit.For productivity losses from hospitalization, we applied an updated 2010 dollar value of work-plus-home production of $224/day for employed patients and $40/day for home-only production of unemployed patients (The sample consisted of 135 patients (130 with MDR TB and 5 with XDRTB), representing 36% (130/364) of MDR TB and 56% (5/9) of XDR TB cases reported in the United States during 2005–2007. Among these patients, 87% were foreign born and 36% had prior TB disease –3. AmongOf the 135 patients, disease was pulmonary for 85%, extrapulmonary only for 6%, and disseminated for 9%. Of 127 patients with pulmonary or disseminated disease, 69 (54%) had extensive disease. Of 8 patients with extrapulmonary-only disease, 2 (25%) had extensive disease. Of 134 patients alive at diagnosis, 77% (103) had at least 1 AFB-smear–positive specimen, 72% from sputum.M. tuberculosis isolates were tested for susceptibility  and were resistant to several  at any time during treatment. The following mutually exclusive resistance patterns were identified: 4% XDR, 17% pre-XDR, 24% total first-line resistance, 43% isoniazid/rifampin/rifabutin-plus-other resistance, and 13% isoniazid/rifampin/rifabutin-only resistance  of the 130 MDR TB patients and XDR among 4 (80%) of the 5 XDR TB patients. DST was conducted for first-line drugs during the first month of treatment, for second-line drugs during the second month of treatment, for linezolid during the fourth month, and for clofazimine during the seventh month.At various times during treatment, sistance . InitialAmong 128 patients for whom DST was conducted multiple times on separate dates, acquired resistance to an anti-TB medication during treatment was detected for 27 (21%). According to multivariable analysis, acquisition of drug resistance during treatment was more likely for patients who were recently homeless , who had pre-XDR or XDR TB , or who were Black  . AcquisiFor 74 patients who had TB symptoms before diagnosis, a median of 1.6 months elapsed from symptom onset to initial TB diagnosis. Of 134 patients alive at diagnosis, 123 (92%) started receiving ≥2 second- or third-line medications a median of 2.4 months after initial TB diagnosis. The median duration of infectiousness was 10 months.Among 105 patients who completed treatment, treatment duration varied. Median durations were 32.3 months (interquartile range [IQR] 30.6–37.8) for those with XDR, 25.1 months (IQR 23.6–29.2) for those with pre-XDR, 25.7 months (IQR 22.4–26.9) for those with total-first-line–resistant, 24.1 months (IQR 20.1–27.0) for those with isoniazid/rifampin/rifabutin-plus–resistant, and 20.0 months (IQR 19.4–24.5) for those with isoniazid/rifampin/rifabutin-only–resistant TB.Providers changed medications for 134 patients alive at diagnosis a median of 7 times during treatment; 33% of 988 medication changes were because of adverse events and 10% because of DST results. Of 134 patients, 34% received a 5-drug regimen before sputum-culture conversion, and 61% ultimately received a 5-drug regimen. Of the 134 patients, 81% received an effective  injectable medication and 86% received an effective fluoroquinolone medication during treatment. Of 123 patients who received outpatient care, 90% received >80% of outpatient medication doses by directly observed therapy (DOT).>2 times (range 2–6). Detailed data were available for 83% of inpatients. Among multiple possible reasons, severe worsening of TB disease was the reason for 50% of hospitalizations, followed by the need to initiate or change treatment (40%), implement respiratory isolation (21%), manage adverse events (7%), manage concurrent conditions (3%), and perform surgery (1%). One patient had undergone TB-related lung lobectomy. Four XDR TB patients were hospitalized for a median of 282 days (range 14–850) and non-XDR patients for a median of 27 days (range 1–759). Home isolation was prescribed for 37% of patients; a median of 102 days (range 4–337) for non-XDR TB patients and 257 days for the 1 XDR TB patient.Most study patients were hospitalized for TB, often several times; 73% (98) were hospitalized at least 1 time and 29% were hospitalized Of the 134 patients alive at diagnosis, 81% had documentation of physician consultation with an MDR TB expert during inpatient or outpatient care. Expert consultation was more likely for patients managed primarily by a TB clinic  and less likely for those with private insurance  . OverallOf the 134 patients alive at diagnosis, ≈90% were assigned a case manager. Case management activities included home visits (68%), social worker assistance (37%), transportation assistance (32%), incentives (25%), housing assistance (17%), and other activities including legal orders for DOT or isolation (9%–15%). Only 4% of patients received none of these case management services. Interpreter use was documented for 60% of 107 patients who understood some or no English.Of 112 eligible patients, including all XDR TB patients, sputum culture converted to negative for 109 (97%). Patients considered ineligible for culture conversion included 1 patient whose TB was diagnosed after death, 6 who died during treatment, 3 who were transferred to another US jurisdiction or out of the United States, 7 who had extrapulmonary-only disease, and 6 without a positive sputum culture result. Of the 3 for whom no culture conversion was documented, 1 was lost to follow-up after 166 days and 2 completed treatment. Culture conversion occurred within a median of 2 months from starting a 5-drug-regimen but varied by resistance pattern .>65 years , smoking , or HIV infection   . When TB.1–37.7) . No HIV-<50 cells/mm3 at initiation of TB treatment and the other had a CD4 count of <100 cells/mm3. Of these 4 patients, 2 were taking >3 anti-HIV medications.Of the 4 HIV-infected patients who died of TB-related causes, 3 had a CD4 count of Among patients alive at diagnosis, a median of 1 adverse event (average 2.9) resulted in medication change or adjustment, but only 2 patients completely stopped treatment. According to multivariate linear regression, use of clofazimine was significantly associated (p<0.05) with more adverse events but was not associated with death during treatment. Of 9 patients receiving clofazimine, 6 experienced postinitiation gastrointestinal effects that resulted in their discontinuing clofazimine. Because of MDR TB or its treatment, of the 134 patients, 13% experienced hearing impairment, 13% hepatitis, 11% renal impairment, 8% difficulty ambulating, 7% visual impairment, and 1% seizures. Depression or psychosis was documented for 19% (80% of whom were taking cycloserine), and pulmonary impairment was documented for 4%. Of 103 impairments, 66% were mild, but another 7% were graded most severe and occurred for 6 patients, 3 of whom completed treatment; 2 of those patients died (lung and mobility impairments), and 1 transferred outside the country. Direct costs averaged $134,000  per MDR TB patient and $430,000  per XDR TB patient. In comparison, costs are estimated at $17,000  per non-MDR TB patient . For iso2 = 0.55). When days hospitalized were controlled for, characteristics associated with greater direct-plus-productivity-loss costs were death, XDR TB, non–injection-drug use, HIV-infection, diabetes, or being male (R2 = 0.66)  M. tuberculosis isolates were resistant to a large number of medications, care was complicated by extensive disease and by concurrent conditions, and patients were highly infectious.In this population-based sample, which comprised 36% (130/364) of MDR TB and 56% (5/9) of XDR TB cases reported in the United States during 2005–2007, MDR/XDR TB diagnosis and treatment were very complex: –Despite this complexity, for nearly all eligible patients, sputum cultures converted to negative and 78% of patients completed treatment, including all those with XDR TB. Only 1% stopped treatment because of adverse events. The mortality rate (10%) was lower than that for other countries  of patients were hospitalized; duration was 1 month for non-XDR TB patients and 9 months for XDR TB patients. Extensive DST of first-line and second-line medications was conducted within 2 months of treatment initiation for most patients. At some point during treatment, 61% of patients were receiving a 5-drug regimen. For outpatient care, DOT was used nearly universally, including during home isolation as recommended by national guidelines (www.cdc.gov/tb/topic/laboratory/guide.html).Deficiencies in practices were identified. Despite CDC recommendations, ≈20% of patients had no documentation of expert consultation. Outcome was unknown for 3% who transferred within and 6% outside the United States and for 2% who were lost to follow-up. Acquisition of any drug resistance during treatment occurred for 21% of patients and was more likely to occur in populations difficult to treat (those who had pre-XDR TB or XDR TB or were homeless), suggesting a need for more vigilant treatment monitoring. There were delays of ≈3 months before patients started a 5-drug regimen. Today, the use of more rapid molecular diagnostic techniques could shorten the time to initiation of an appropriate treatment regimen (Because MDR TB treatment lasts >2 years (vs. 6 months for drug-susceptible TB), uses expensive medications, and requires hospitalization for ≈75% of patients (vs. 50% with drug-susceptible TB), it was very costly to treat and manage; average direct cost was $134,000 per MDR TB patient and $430,000 per XDR TB patient. The estimated $17,000 per non-MDR TB patient is 8 and 25 times lower than the costs for MDR TB and XDR TB, respectively. In contrast, lifetime care per HIV-infected patient costs $380,000 (updated to 2010 dollars)  to quickly diagnose MDR/XDR TB and isolate and effectively treat the patients will be critical for preventing deaths and transmission of drug-resistant TB organisms. Investment in infection control infrastructure and the capacity to prevent TB among MDR TB patient contacts is also critical; our cost-of-illness estimates did not include these programmatic costs of preventing cases. During the 1979–1994 TB resurgence and MDR TB outbreaks in the United States, New York City renovated hospitals and the Rikers Island prison and treated 20,000 excess TB patients at a cost of ≈$1.7 billion (updated to 2010 dollars)  medication prices negotiated with pharmaceutical companies. Estimates of out-of-pocket costs were not included.In this population-based sample of MDR/XDR TB patients in the United States, despite the extensive drug resistance found at the time of diagnosis, culture conversion and treatment completion rates were high and mortality rates were low. Records of treatment practices documented near-universal use of DOT. However, these outcomes came at a high cost to the public sector, providing incentives for the United States to prevent MDR/XDR TB. Preventing MDR/XDR TB in the United States will require addressing factors associated with development of drug resistance in countries where foreign-born US patients originate, as well as rapid diagnosis, appropriate regimen selection, robust case management practices, and continued emphasis on DOT in the United States.Additional methods, cost analysis, definitions, and additional results with regard to treatment practices, outcomes, and costs of MDR TB and XDR TB in the United States, 2005–2007.


Myostatin mutant) and Ujumqin (wild type) sheep across the five fetal stages, we constructed and examined the Myostatin-Pax7 pathways in muscle. Then we validated the signals by RNAi in the proliferating and differentiating sheep myoblasts in vitro at mRNA, protein, and cell morphological levels. We reveal that Myostatin signals to Pax7 at least through Ezh2, Src, and Akt during the sheep myoblast proliferation and differentiation. Other signals such as p38MAPK, mTOR, Erk1/2, Wnt, Bmp2, Smad, Tgfb1, and p21 are most probably involved in the Myostatin-affected myogenic events. Myostatin knockdown significantly reduces the counts of nucleus and myotube, but not the fusion index of myoblasts during cell differentiation. In addition, findings also indicate that Myostatin is required for normal myogenic differentiation of the sheep myoblasts, which is different from the C2C12 myoblasts. We expand the regulatory network of Myostatin-Pax7 pathways and first illustrate that Myostatin as a global regulator participates in the epigenetic events involved in myogenesis, which contributes to understand the molecular mechanism of Myostatin in regulation of myogenesis.Myostatin and Pax7 have been well documented individually, however, the mechanism by which Myostatin regulates Pax7 is seldom reported. Here, based on muscle transcriptome analysis in Texel ( Myostatin gene lead to a hypertrophic phenotype in mice, sheep, cattle, dog, and human [Myostatin) than in Ujumqin sheep (wild type) [Myostatin mutation changes Pax7 expression in muscle during fetus development. In myogenesis, Myostatin signaling through Smad2, Smad3 and Smad4 is regulated by the inhibitory Smad7 via a negative feedback mechanism [Myostatin, a member of the transforming growth factor-β (TGF-β) family, is predominantly expressed and secreted by skeletal muscle and functions as a negative regulator of muscle growth. Mutations in nd human –6, suggend human . On the nd human –11. Pax7nd human , 11–13 and human , 14–21. ld type) , suggestechanism , 24, andechanism . Myostatechanism . In vivoechanism , 28. In echanism –33. Howeechanism . It is uMyostatin mutant while the Ujumqin serves as a wild type control [In the present study, the Texel is a  control . Texel a control . All expThe sheep-specific transcriptome-wide microarray (Agilent) contained three samples at least at each development stage for each breed. Microarray analysis was performed as we previously described . The orihttp://www.ingenuity.com) was used to construct the Myostatin-Pax7 pathways. Data for analysis were two differentially expressed gene lists for Texel and Ujumqin sheep across five fetal stages, respectively  program  with a final concentration of 4 μg/μL polybrene, 1mL Opti-MEM was added in 1.5mL centrifugal tube, with addition of corresponding volume of lentivirus and polybrene. The cells were then incubated at 37°C and 5% carbon dioxide (CO2) for 12 h.The sheep myoblasts were isolated from the longissimus muscle of adult female Ujumqin sheep in our lab , and cel our lab . For knoThe MTT method was used for proliferation assay. Briefly, the sheep myoblasts were seeded in 96-well plate  and maintained in growth medium (GM) (DME containing 10% FBS) at 37°C and 5% CO2 for 7 days. Cells were incubated for 1 h with 10 μM BrdU for examination of the absorbance at 1, 3, 5, and 7 days by a spectrophotometer. The amount of the absorbance of Brdu is proportional to the proliferation rate of cells. The RNA and protein levels of signal molecules in proliferating cells were detected at 3d by quantitative real-time PCR and Western blot. For differentiation assay, the shRNA-infected and the control myoblasts were cultured in differentiation medium (DM) (DME containing 2% horse serum) at 37°C and 5% CO2 for 7 days. Cells were collected at day 3 for examination of mRNA and protein expression, as well as differentiation by the nuclei DAPI staining. Fusion Index (FI) was calculated for evaluating the myogenic potential of myoblasts at this stage.5 cells/well) in 6-well plates. After 24 h of incubation, they were transfected with Akt-siRNA, Ezh2-siRNA, and Src-siRNA, in serum-free medium using Lipofectamine 2000 (Invitrogen) respectively. Vector (10 μg) and 20 μl Lipofectamine 2000 were mixed and incubated for 15 min at room temperature. The solution was then added to the primary myoblasts and incubated without serum or antibiotics for 6 h, after which the medium was replaced with medium containing serum, but no antibiotics.The sheep primary myoblasts were isolated from the muscle of newborn lamb as described . Cells wRPS18, ACTB, or GAPDH expression using the 2-ΔCt method. Data were analyzed using SAS 8.0.Total RNA was used to make cDNA using the PrimeScript 1st Strand cDNA Synthesis Kit (Takara). qPCR was performed on an ABI 7500 instrument using the Fast EvaGreen Master Mix (Biotium). Thermal cycling consisted of an initial step at 95°C for 10 min followed by 40 cycles at 95°C for 30s and 62°C for 30s. The qPCR measurements were performed in triplicate for each cDNA sample and gene expression was quantified relative to either Equal amounts of proteins from the cell extracts were used for Western blotting. Briefly, 20 μg of protein per sample was subjected to electrophoresis on 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels. The separated proteins were transferred onto nitrocellulose membranes, which were incubated overnight with one of the 20 primary antibodies : anti-ERFor within breeds across developmental time, we identified 3711 and 3521 differentially expressed (DE) probes in Texel and Ujumqin sheep, respectively. With annotation by BLASTN, these probes matched 2807 and 2251 human genes, respectively  Tables. Tni, Klf10, Fst, Atf2, Cdk2, Myod1, Src, Histone h3, and Akr1b1. Class II, those uniquely differentially expressed in Ujumqin sheep such as Smad4, Myod, Smad2, Dcn, Fmod, Cdkn1a, Ubc, and Bmi1. Class III, those DE genes shared in both breeds of sheep (named as “co-expressed” genes), such as Serpinh1, Myf5, Smad, Myh4, Fn1, Akt, Pparg1a, Jnk, p38MAPK, Fyn and Ezh2. Accordingly, those pathways containing the exclusive DE genes in either breed may be involved in breed-specific regulation of muscle development. Whereas the pathways containing the “co-expressed” genes mean that, whether there is mutation in Myostatin or not, these pathways are doomed to be differentially expressed and basically indispensable for Myostatin-regulated Pax7 expression in both breeds.According to the characteristics of gene expression in each breed, there were three classes of DE genes in the pathways . Class IAkt, p38MAPK, Myf5, Bmi1, Src, Histone h3, Myod, Myog, Mstn, and Pax7 in muscle in Myostatin and Pax7 in muscle at earlier stages (70d and 85d)  . We consMyostatin [Myostatin knockdown promoted the myoblast proliferation at early stage in GM  and delayed slightly the myogenic differentiation by reducing the fusion index (P>0.05), as compared with the control  for 3 days,  control . qPCR anx7 Figs.  and 6. Ayoblasts . Similaryoblasts , Cateninyoblasts . Unlike Pax7 expression. At the same time, we chemically synthesized the siRNAs for inhibition of Src, Ezh2, and Akt in the primary myoblasts, respectively. qPCR analysis illustrated that the Pax7 mRNA level was significantly downregulated by either of the siRNAs for Src, Ezh2, and Akt in the primary myoblasts, compared with their controls  viral oncogene homolog), not only is a protein-coding gene, but also is affiliated with the lncRNA class. The proto-oncogene v-src can promote the myoblast proliferation [Src suppresses the formation of myotubes and expression of myogenin [Src suppressed Pax7 mRNA expression in the sheep myoblasts , a histone methyltransferase and catalytic component of polycomb repressive complex 2 (PRC2), epigenetically regulates chromatin structure, gene expression, and recruitment of DNA methyltransferases for gene silencing in embryonic and adult stem cells , 63. Stust Figs.  substantIn addition, the observation that variations in expression of the signals examined at mRNA and protein levels under different conditions (GM or DM) Figs.  and 5 suMyostatin is required for normal myogenic differentiation of the sheep myoblasts. Myostatin signals to Pax7 at least through Ezh2, Src, and Akt in the sheep myoblasts. In addition, other signals such as Erk1/2, p38MAPK, mTOR, Wnt, Bmp2, Smad, Tgfb1, and p21 are most probably involved in the myostatin-affected proliferation and differentiation.S1 Table(XLS)Click here for additional data file.S2 Table(XLS)Click here for additional data file.S1 File(DOC)Click here for additional data file.


SK Hep-1 cells (SK cells) derived from a patient with liver adenocarcinoma have been considered a human hepatoma cell line with mesenchymal origin characteristics, however, SK cells do not express liver genes and exhibit liver function, thus, we hypothesized whether mesenchymal cells might contribute to human liver primary cancers. Here, we characterized SK cells and its tumourigenicity.We found that classical mesenchymal stem cell (MSC) markers were presented on SK cells, but endothelial marker CD31, hematopoietic markers CD34 and CD45 were negative. SK cells are capable of differentiate into adipocytes and osteoblasts as adipose-derived MSC (Ad-MSC) and bone marrow-derived MSC (BM-MSC) do. Importantly, a single SK cell exhibited a substantial tumourigenicity and metastatic capacity in immunodefficient mice. Metastasis not only occurred in circulating organs such as lung, liver, and kidneys, but also in muscle, outer abdomen, and skin. SK cells presented greater in vitro invasive capacity than those of Ad-MSC and BM-MSC. The xenograft cells from subcutaneous and metastatic tumors exhibited a similar tumourigenicity and metastatic capacity, and showed the same relatively homogenous population with MSC characteristics when compared to parental SK cells. SK cells could unlimitedly expand in vitro without losing MSC characteristics, its tumuorigenicity and metastatic capacity, indicating that SK cells are oncogenic MSC with enhanced self-renewal capacity. We believe that this is the first report that human MSC appear to be transformed into cancer stem cells (CSC), and that their derivatives also function as CSCs.Our findings demonstrate that SK cells represent a transformation mechanism of normal MSC into an enhanced self-renewal CSC with metastasis capacity, SK cells and their xenografts represent a same relative homogeneity of CSC with substantial metastatic capacity. Thus, it represents a novel mechanism of tumor initiation, development and metastasis by CSCs of non-epithelial and endothelia origin. Carcinomas are a heterogeneous population of a series of cells with different phenotypes; however, the origin of many cancers remains unknown. Human liver carcinoma is the fifth most common cancer worldwide www.atcc.org); adipose-derived mesenchymal stem cells (MSC-ad) and bone marrow-derived mesenchymal stem cells (MSC-BM) were purchased from ScienCell Research Laboratories (www.sciencellonline.com). The cell culture conditions for growing and expanding these line cells were per the manufacturers instructions.SK Hep-1 cell, a hepatoma cell line, was purchased from ATCC , CD44, CD73 , CD71 (Transferrin receptor protein 1 (TfR1)), CD105 , CD166 , CD29 , CD106 , CD49d , CD146 , CD133 , CD13 (Aminopeptidase N), CD54 ICAM-1 , CD9, CD10 , ABC (major histocompatibility complex (MHC) class I antigen), HLA DR (major histocompatibility complex (MHC) class II antigen), and Alkaline phosphatase (ALP), CD31 (endothelia cell marker), CD34 and CD45 (hematopoietic markers). The cells were stained as previously described We evaluated the potential of SK cells, MSC-ad, and MSC-BM to differentiate into adipocytes and osteocytes. The differentiation and staining with red oil R and alizarin red S was performed as previously described RNA was extracted from the SK cells, xenograft cells, as well as undifferentiated and differentiated MSC cells using the Qiagen mini RNA kit, and cDNA was generated as previously described SK cells were injected into 8–10 weeks, female NOD/SCID/IL2rg mice (Jackson Laboratory) by subcutaneous injection of 1, 10, 100, 500, 1,000, 5,000, or 10,000 cells. The re-cultured cells from primary subcutaneous tumor and metastatic tumors produced by the injection of parental SK cells were also injected into the same mouse models with 10 and 100 cells. The small number of cells (1 and 10) was mixed with 30% of Matrigel (BD Sciences) for the purpose of avoiding to lose cells during preparation and transplantation www.invitrogen.com) for 10 min in the dark to destroy the blood cells, then filtered with a 100 um cell strainer and spun again. Finally the cell pellet were re-suspended, and re-cultured with MEM .The tumors xenografts were harvested and cut into small piece under sterile conditions, and treated with dispase (1 mg/ml) and collagenase type IV (1 mg/ml), and incubated at 37C for 20 min, then the tumor tissue was homogenized with a serological pipette, and supernatants were collected. The remaining tissue was treated with the same solution for an additional 2–4 times until almost all tissue was digested. The supernatants were spun at 300 g for 5 min, the cell pellet was re-suspended with MEM medium after discarding the supernatant and was treated with fixative-free lysing solution .In vitro migration and invasion was performed using a basement membrane-based invasion assay . 100,000 SK cells or MSC-ad or MSC-BM were cultured in a 24-well size transwell containing growth medium with low serum (2% of FBS), while the bottom wells were filled with normal growth medium containing 10% of FBS. After 24 hours culturing, non-invasive cells were removed, and invasive cells that passed through the basement membrane were stained and imaged. In a parallel experiment, the invasive cells were stained and quantified by measuring OD 560 nm values. 20000, 40000, 60000, 80000, and 100000 SK cells and MSC were seeded in the bottom well, and stained and quantified as a standard curve. The procedures of the invasion assay were performed per the manufacturer's instructions.All data were summarized as means ± SEM from at least three independent measurements. An unpaired Student t test was used to analyze the data. p<0.05 was considered statistically significant.We compared surface markers of SK cells to mesenchymal stem cells isolated from adipose and bone marrow . We found that classical MSC markers, such as CD90, CD44, CD73, CD71, CD105, CD146, CD166, CD29, CD13, CD9, CD10, HLA ABC, and ALP were also present on SK cells ; howeverAfter culturing with adipogenic or osteogenic differentiation medium for 2 weeks, SK cells were differentiated into adipocytes and osteoblasts, with a similar differentiation pattern as MSC-ad and MSC-BM do, thereby confirming its MSC-like characteristics. Adipogenic differentiation was evidenced by Red oil R staining, whereas Alizarin red S staining confirmed the accumulation of calcium deposits, a characteristic of osteogenic cells, in differentiated SK cells, MSC-ad and MSC-BM . ALP, anHighly immunodeficient mice, NOD/SCID/IL2rg mice were used to evaluate the tumorigenic and metastatic potential of SK cells by us. What surprised us was that as few as 10 SK cells caused tumors in over 96% of injected mice, and even a single cell generated tumors in 10 of 28 injected mice . The timThe primary tumors produced by the subcutaneous injection, and metastatic tumors in lung and liver were collected and the cells from these tumors were isolated and re-cultured. The morphology of these cells was the same as those of parental SK cells , and floAn in vitro invasion assay showed that SK cells had greater migration and invasion capacity when compared to MSC-ad and MSC-BM, a well described invasive cell type. Large number of SK cells passed the basement membrane 24 hours under low FBS conditions, as determined by microscopy after staining . A quantWe have determined that SK Hep-1 cells expressed almost all MSC markers, such as CD9, CD10, CD13, CD29, CD49d, CD54, CD71, CD73, CD146, CD166, CD90, CD44, CD105, HLA-ABC, and alkaline phosphatase (ALP) SK cells were isolated from a patient with liver adenocarcinoma Normally MSC can maintain multipoptency and their phenotype for a short period at low passage after isolation, and they will lose this property and differentiate or transdifferentiate into other cell types after long-term culture at higher passages in vitro. SK cells appear to function as oncogenically transformed MSC with enhanced self-renewal and proliferation, because they can be unlimitedly expanded and maintained in culture without losing multipotency and changing its phenotype. Although the concept of cancer stem cells is still controversial, recent reports indicate that CSCs do exist in tumors employing mouse models The primary tumors or the xenografts produced by cancer stem cells or tumor-initiating cells represent heterogeneous population with different cell types, only a small number of cells remain cancer stem cell property. However, the subcutaneous tumors or metastatic tumors produced by a single SK cells or 10,000 SK cells exhibited a relative homogenous population with MSC phenotype which is the same with parental SK cells. Parental SK cells with MSC characteristics did not differentiate into other cell lineages such as adipose, osteoblast, or chondrocytes during the tumor formation or metastasis after injection into mice. The formation of the subcutaneous tumors and metastatic tumors is just proliferation or self-renewal of SK cells in vivo as they do in vitro. This phenomenon or mechanism is apparently different from those of epithelial cell- and endothelial cell-based tumor/cancer formation and metastasis.In summary, parental SK cells and their xenografts exhibit same oncogenic MSC characteristics with highly metastatic capacity, even a single SK cell has such tumorigenicity and metastatic capacity, moreover, parental SK cells and their xenografts present a relative homogeneity with the same phenotype. SK cells' mesenchymal origin and mesenchymal phenotype demonstrated that mesenchymal cell type contributed to the liver carcinoma of the patient which SK cells was derived from. Therefore, it represents a novel mechanism of tumor initiation, development and metastasis by cancer stem cells of non-epithelial and endothelia origins.Figure S1Characterization of surface markers of SK Hep-1 cells and mesenchymal stem cells. Flow cytometry was employed to detect and measure classical mesenchymal stem cell (MSC) markers in three cell types, SK Hep-1 cells, adipose-derived MSC (MSC-ad), and bone marrow-derived MSC (MSC-BM).(JPG)Click here for additional data file.Figure S2The tumor growth and metastasis characteristics. A, the tumor appearance before open of the skin when the mouse was sacrificed, and the tumor apparently looked small. B, the tumor appearance after open of the skin before the isolation, the tumors grew invasively. C, the tumors were isolated from the mice, and embedded holes were left after the tumors were removed. D, this tumor sizes:>0.5 cm (height) ×1 cm (width) ×1.5 cm (length). E, the average tumor sizes were ≥0.5–1.0 cm (height) ×1.0–1.5 cm (width) ×1.5–2.0 cm (length) after isolation. F, Metastatic tumors were found in multiple organs in single mouse, for example, metastatic tumors were found in lung and liver in one mouse (left), and metastatic tumors were also found in kidney and up-ureter in the same mouse. Arrow indicates normal ureter (right). G, lungs, liver, and kidneys with metastatic tumors were isolated from this single mouse shown in F. H and I: metastatic tumors were found in livers (H) and lungs (I) of all treated mice.(JPG)Click here for additional data file.Figure S3Histological features of primary tumors and metastatic tumors. A–C, H and E staining reveals that the histological and pathological features of primary subcutaneous tumor cells (A), metastatic tumor cells (arrow) in liver (B), and lung (C). Magnifications: 40 x.(JPG)Click here for additional data file.Figure S4Histological features of metastatic tumors. A–C, H and E staining reveals that the histological and pathological features of metastatic tumor cells (arrow) in pancreas (A), kidney (B), and spleen (C). Magnifications: 40 x.(JPG)Click here for additional data file.Figure S5Histological features of metastatic tumors. A–C, H and E staining reveals that the histological and pathological features of metastatic tumor cells (arrow) in colon (A), uterus (B), and ovary (C). Magnifications: 40 x.(JPG)Click here for additional data file.Figure S6Characterization of surface markers of primary tumor cells and metastatic tumor cells. Flow cytometry was employed to detect and measure classical mesenchymal stem cell (MSC) markers in three isolates from the primary tumors (Skin-isolate) produced by the subcutaneous injection, and metastatic tumors in liver (Liver-isolate) and lung (Lung-isolate).(JPG)Click here for additional data file.Table S1List of antibodies used. Abbreviations: ALP, Alkaline phosphatase; PE, phycoerthrin; HLA ABC, human major histocompatibility complex (MHC) class I, human leukocyte antigens, A, B, C; HLA DR, MHC class II cell surface receptor; FC, flow cytometry.(DOC)Click here for additional data file.Table S2Information of primers and probes used for qPCR. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; AFP, alpha fetoprotein; α-SMA, alpha smooth muscle actin; FABP4, fatty acid binding protein 4; RGC32, response gene to complement 32 protein; COL1A1, Collagen, type I, alpha 1; MYF5, Myogenic factor 5; MYOG, Myogenin or myogenic factor 4; GFAP, Glial fibrillary acidic protein; E-SELE, E-selectin; vWF, Von Willebrand factor; C/EBP, CCAAT-enhancer-binding protein; PPAR, Peroxisome proliferator-activated receptor.(DOC)Click here for additional data file.


VHL gene (von Hippel-Lindau), RET gene (Multiple Endocrine Neoplasia type 2), and NF1 gene (Neurofibromatosis type 1), 10 novel genes have so far been implicated in the occurrence of paragangliomas/pheochromocytomas: SDHA, SDHB, SDHC, SDHD, SDHAF2, TMEM127, MAX, EGLN1, HIF2A, and KIF1B. It is currently accepted that about 35% of the paragangliomas cases are due to germline mutations in one of these genes. Furthermore, somatic mutations of RET, VHL, NF1, MAX, HIF2A, and H-RAS can also be detected. The identification of the mutation responsible for the paraganglioma/pheochromocytoma phenotype in a patient may be crucial in determining the treatment and allowing specific follow-up guidelines, ultimately leading to a better prognosis. Herein, we summarize the most relevant aspects regarding the genetics and clinical aspects of the syndromic and nonsyndromic forms of pheochromocytoma/paraganglioma aiming to provide an algorithm for genetic testing.Paragangliomas are rare neuroendocrine tumors that arise in the sympathetic or parasympathetic nervous system. Sympathetic paragangliomas are mainly found in the adrenal medulla (designated pheochromocytomas) but may also have a thoracic, abdominal, or pelvic localization. Parasympathetic paragangliomas are generally located at the head or neck. Knowledge concerning the familial forms of paragangliomas has greatly improved in recent years. Additionally to the genes involved in the classical syndromic forms:  Paragangliomas are neuroendocrine tumors that can originate in either the parasympathetic or sympathetic nervous system. Most parasympathetic paragangliomas are chromaffin-negative (meaning that they do not stain brown when exposed to potassium dichromate) and do not secrete catecholamines. Sympathetic paragangliomas  are chromaffin-positive tumors that generally secrete catecholamines .The designation of pheochromocytoma appears in the literature associated with different meanings. The World Health Organization (WHO) Tumor Classification defines pheochromocytoma as a paraganglioma derived from the adrenal medulla , whilst Sympathetic paraganglia are mainly found in the adrenal medulla but also in the axial regions of the trunk along the prevertebral and paravertebral sympathetic chains and in the connective tissue within/near pelvic organs. In contrast, parasympathetic paraganglia are almost exclusively confined to the head and neck in the vicinity of major arteries and nerves . ParaganThe clinical presentation of these patients is highly variable, with most symptoms being nonspecific and mimicking other clinical conditions. Headaches, hypertension, tachycardia, diaphoresis, pallor, anxiety, and panic attacks are the most frequent signs and symptoms at presentation . The claParagangliomas are rare tumors occurring with an overall estimated incidence of 1/300 000, with an average age at diagnosis of around 40 years and no gender differences , 11. How SHDB gene (discussed in detail later), which have been associated with up to 72% of malignant tumors [Although most tumors are benign, about 10% of pheochromocytomas and 15% to 35% of extra-adrenal paragangliomas are malignant . Prior tt tumors . VHL gene in von Hippel-Lindau disease, RET gene in Multiple Endocrine Neoplasia type 2 (MEN 2), and NF1 gene Neurofibromatosis type 1, 10 novel genes have so far shown to be implicated in the occurrence of paragangliomas/pheochromocytomas [SDHA, SDHB, SDHC, and SDHD) and one complex cofactor, SDHAF2, mainly involved in head and neck and abdominal paragangliomas and initially discovered by Baysal et al. [ TMEM127, MAX, HIF2A, EGLN1, KIF1B, and H-RAS complete the list of susceptibility genes implicated in the development of paragangliomas/pheochromocytomas [ H-RAS mutations have been identified only at a somatic level.Paragangliomas can be classified into either sporadic or familial. In the last years, our knowledge concerning the familial forms of paragangliomas has greatly improved. Additionally to the genes involved in the classical syndromic forms:ocytomas –29. Amonl et al. –34. Moreocytomas –40. So f VHL, RET, SDHD, and SDHB genes. Younger age at presentation (24.9 versus 43.9 years of age), multiple tumors (32% versus 2%), and presence of extra-adrenal tumors (28% versus 8%) were significantly associated with the presence of a mutation [ VHL, RET, NF1, SDHB, and SDHD [Pheochromocytoma (here meaning catecholamine-secreting paraganglioma) was known as the 10% tumor, meaning that 10% of cases were familial, 10% bilateral, 10% malignant, and 10% extra-adrenal . In thismutation . In 2006and SDHD . So, it and SDHD .In this review, we summarize the clinical and genetic aspects of the syndromic and nonsyndromic forms of pheochromocytoma/paraganglioma. The risk of developing pheochromocytoma/paraganglioma will be addressed for each gene. A clinically oriented strategy for genetic testing will be discussed.von Hippel-Lindau (VHL) disease is an autosomal dominant syndrome characterized by a variety of benign and malignant tumors including retinal and central nervous system hemangioblastomas, clear renal cell carcinoma and renal cysts, pheochromocytomas, pancreatic islet cell tumors and pancreatic cysts, epididymal cystadenomas, and endolymphatic sac tumors .This disease affects about 1 in 36 000 live births and is divided into 2 clinical categories according to absence (type 1) or presence (type 2) of pheochromocytomas, respectively , 46. VHLα) and several other proteins involved in tumorigenesis [α subunits of HIF1 and 2, targeting it for ubiquitination and proteasomal degradation. Conversely, in hypoxic conditions or when VHL gene is mutated, HIFα is able to interact with HIFβ, inducing the transcription of hypoxia-inducible genes, leading to an increased expression of angiogenic growth and mitogenic factors [von Hippel-Lindau protein (pVHL) is a tumor suppressor protein that regulates the activity of hypoxia-inducible factor alpha , it comprises 3 exons that encode for the 2 isoforms of the pVHL protein [ VHL germline mutations have been associated to the VHL disease. These mutations are missense, deletion, nonsense, or frameshift mutations and are distributed throughout the coding sequence [ de novo mutation occurs [ VHL deletions or nonsense mutations, whereas families at risk for developing pheochromocytoma (type 2 families) almost invariably present with VHL missense mutations [ VHL mutations associated with the phenotype 2A or 2B have been shown to affect the proteasomal degradation of HIF1, whereas type 2C mutations do not disrupt the ability of pVHL to downregulate HIF1, suggesting that pheochromocytoma formation is not related with HIF1 expression levels [ protein . More thsequence , 58. Altn occurs . Genotyputations , 58, 60.n levels , 62. It n levels , 63, 64.Multiple endocrine neoplasia type 2 (MEN 2) is an autosomal dominant cancer syndrome characterized by the association of medullary thyroid carcinoma (MTC), pheochromocytoma, and hyperparathyroidism . Dependi RET (REarranged during Transfection) protooncogene is located. It comprises 21 exons that encode for a tyrosine transmembrane receptor with three domains: extracellular, transmembrane, and intracellular. When a ligand of the glial-derived neurotropic factor (GDNF) family binds to RET protein, it triggers RET dimerization and autophosphorylation, inducing a signaling phosphatidylinositol 3′-kinase- (PI3K-) mediated cascade that regulates cell proliferation and apoptosis. This requires the presence coreceptors of the GDNF family receptor-α1–4 (GFR-α1–4) at the cell surface [The genetic basis for MEN 2 syndrome lies within the long arm of chromosome 10 (10q11.2), where the surface . RET mutations. More than 98% of MEN 2A families present with missense mutations in one of five codons: 609, 611, 618, 620 (exon 10), or 634 (exon 11). Codon 634 mutations represent almost 90% of MEN 2A cases and a cysteine to arginine substitution at this codon (p.Cys634Arg) is found in more than 50% of cases [MEN 2 subtypes have been associated with specificof cases –73. All of cases –76. Abouof cases –73. Inteof cases . Generalof cases , 71–73. of cases .Pheochromocytomas in MEN 2A and B syndromes are generally benign tumors and bilateral in >50% of the patients . Extra-aBased on a large study enrolling 323 MEN 2A patients, Quayle et al. reported an overall penetrance of pheochromocytoma of 32%, with a median age at diagnosis of 34 years; the earliest pheochromocytoma was observed at 15 years; bilateral pheochromocytomas were observed in 66% of patients; the following codon-specific expression of pheochromocytoma was observed: codon 634 was expressed in 50%, codon 618 was expressed in 22%, codon 620 was expressed in 9%, and codon 609 was expressed in 4%. The mean age at diagnosis did not differ amongst these codon-grouped patients . RET mutations [ RET mutations associated with MEN 2B and mutated RET codons 634 and 630 and by the age 20 years in carriers of other MEN 2A RET mutations. Patients with RET mutations associated only with FMTC should be screened at least periodically from the age of 20 years [Childhood pheochromocytoma is rare in MEN 2, but reports at 12 years of age have occurred for both the 918 and 634utations , 81. The20 years . cafe au lait macules; two or more cutaneous/subcutaneous neurofibromas or a single plexiform neurofibroma; axillary or inguinal freckling; optic nerve glioma; two or more Lisch nodules (iris hamartomas); dysplasia of long bones or pseudarthrosis; and a first degree relative with NF1 [Neurofibromatosis type 1 (NF 1), or von Recklinghausen's disease, is an autosomal dominant disorder clinically diagnosed by six or morewith NF1 , 82. Patwith NF1 , 84. Wor NF1 gene [ NF1 gene result in constitutive activation of RAS activity triggering a kinase cascade and the activation of mitogen-activated protein kinases (MAPK), mammalian target of rapamycin (mTOR), and PI3 K pathways, therefore regulating the transcription of genes associated with cell proliferation, cell death, differentiation, and migration [ NF1 gene, the presence of 36 pseudogenes, and the fact that nearly half of NF 1 cases present de novo mutations [NF 1 is caused by loss of function mutations in the tumor-suppressorNF1 gene . This geigration . Mutatioutations , 88.Pheochromocytomas are a rare feature in NF 1, affecting approximately 0.1% to 6% of all patients , 89. A p NF1 mutational genotype and the clinical features of pheochromocytoma [Unlike mutations in VHL or MEN 2 disorders, NF 1 mutations that offer an increased risk in pheochromocytoma remain to be identified. A study carried out by Bausch et al. in NF 1 patients with associated pheochromocytoma showed that the cysteine-serine rich domain was affected in 35% of the cases whereas the Ras GTPase activating protein domain in only 13%, suggesting that the cysteine-serine rich could play a role in the formation of NF1-associated pheochromocytoma. Moreover, in accordance with the Knudson's two-hit theory that states that pheochromocytoma development requires biallelic inactivation, loss of heterozygosity (LOH) was shown in NF 1-related pheochromocytoma. No association was found betweenmocytoma . SDHA, SDHB, SDHC, and SDHD. Complex subunits A (flavoprotein) and B (iron-sulfur protein) constitute the catalytic core of the enzyme, while subunits C and D anchor the complex to the inner mitochondrial membrane. In general, inactivating mutations in one of the SDHx genes leads to accumulation of succinate and formation of reactive oxygen species, stabilizing HIFα and activating hypoxia-dependent pathways [ SDHD, SDHAF2 (responsible for the flavination of subunit A), SDHC, and SDHB, respectively [ SDHB mutation, whereas a weak diffuse staining often occurs with SDHD mutation [ SDHx paragangliomas sometimes release dopamine and/or norepinephrine, originating raised plasma levels of methoxytyramine, contributing to distinguish SDHx patients from those with VHL, RET, or NF1 mutations [Familial paraganglioma syndromes (PGLs) are a group of autosomal dominant disorders responsible for the development of paragangliomas/pheochromocytomas caused by mutations in the genes encoding for the succinate dehydrogenase (SDH) mitochondrial complex. SDH or respiratory complex II is an enzyme complex that catalyses the oxidation of succinate to fumarate in the Krebs cycle and participates in the electron transport chain . SDH is pathways . Four PGectively –33. Immumutation , 93. Funutations , 94. Howutations . SDHD gene, which are inherited in an autosomal dominant fashion with a predominant paternal transmission, suggesting a maternal imprinting of this gene [ SDHD mutation, loss or mutation of the wild-type SDHD allele, and loss of a further imprinted  tumor suppressor gene from chromosome 11 [ SDHD gene, 79% had head and neck paraganglioma, 53% had pheochromocytoma, and 39% thoracic/abdominal paraganglioma, whereas 74% of the patients presented with multiple tumors [ SDHD-derived sympathetic or parasympathetic paragangliomas [ SDHD, mainly nonsense, missense, and frameshift, but its relation with the phenotypic expression of the disease is still unclear [PGL 1 syndrome is caused by mutations inhis gene , 44, 96.his gene –99. A thosome 11 . PGL 1 pe tumors , 102. Ringliomas –105. Sev unclear , 102. SDHAF2 gene  that encodes for a succinate dehydrogenase complex assembly factor 2 (SDHAF2), which is responsible for the flavination of SDHA enabling SDH complex activity [Familial PGL 2 syndrome is a very rare condition characterized by multiple head and neck paragangliomas, of which only few cases have been reported , 107. Itactivity . To our activity , 107. Bo SDHAF2 mutations was reinforced by the failure to document mutations in this gene among 315 patients with paraganglioma and without mutations in the SDHD, SDHC, or SDHB genes. Nonetheless, it is justified to screen for SDHAF2 mutations in young patients with isolated head and neck paragangliomas or in individuals with familial antecedents who are negative for other risk genes [The scarcity ofsk genes . SDHC gene are causative for familial PGL syndrome 3, which has an autosomal dominant mode of transmission without a parent-of-origin effect [Mutations in then effect . This isn effect , 108–111n effect  and Schin effect , the mea SDHC gene [ SDHC gene have been found [ SDHC gene is extremely rare with only two cases described so far, with distinct causal mutations [About 4% of paraganglioma patients carry mutations in theDHC gene , 112. Dien found , 112. Mautations , 114.Familial PGL 4 syndrome is characterized by abdominal and pelvic catecholamine-secreting paragangliomas, which can also be present in adrenal medulla and head and neck , 115. Sy SDHB gene, which encodes for the iron sulfur subunit of the SDH complex (subunit B), are responsible for PGL 4 familial syndrome [α, causing overexpression of hypoxia-induced angiogenic pathway genes, such as VEGF  and EPAS1 , providing therefore support for tumor growth [ SDHB mutations [ SDHB gene have also been associated with an increased susceptibility to develop other neoplasms, namely, renal cell carcinoma, gastrointestinal stromal tumors, papillary thyroid cancer, and neuroblastoma [Germline mutations insyndrome . Functior growth –118. Losutations , 116. Ofblastoma , 119. SDHB mutations have been found associated with PGL 4, namely, missense, frameshift, splicing, nonsense, and large deletions. However, several studies have failed to unveil genotype-phenotype correlations, particularly in what concerns tumor location, age of presentation, and aggressiveness of the tumor [ SDHB gene are the most frequent cause of metastatic paraganglioma tumors, it has consistently been proposed that all patients presenting with malignant paraganglioma/pheochromocytoma should be tested for SDHB gene mutations.A wide spectrum ofhe tumor , 120. Si SDHA gene and paraganglioma development was only unveiled in 2010, when a patient with an extra-adrenal paraganglioma was found to have an SDHA missense mutation [ SDHA, like other SDHx genes, operates as tumor suppressor gene and activates the pseudohypoxic pathway leading to tumorigenesis. Furthermore, in accordance with Knudson's two-hit hypothesis, it was shown that the SDHA-mutated tumors have lost the wild type allele [ SDHA-affected individuals described so far have presented with distinct phenotypic characteristics: pheochromocytoma, sympathetic , and parasympathetic head and neck paragangliomas [ SDHA gene mutations have also been implicated in the development of gastrointestinal stromal tumors [The long-sought link betweenmutation . Functioe allele , 121. Thngliomas , 121–123ngliomas , 121–123l tumors , 125.TMEM127 is a tumor suppressor gene initially identified as a pheochromocytoma susceptibility gene [ TMEM127 gene encodes a highly conserved transmembrane protein, transmembrane protein 127, which is associated with several cellular organelles and thought to limit mTORC1 activation thus controlling protein synthesis and cell survival [ RET and NF1 genes [ TMEM127 mutations in patients with paraganglioma/pheochromocytoma varies between 0.9 and 2%. Different missense, frameshift, or nonsense TMEM127 mutations have been found across the three exons of the gene [ity gene  and lateity gene –130. TMEsurvival . MutatioF1 genes . Patientthe gene , 129. MAX gene as responsible for the development of bilateral pheochromocytoma in eight index patients [ MAX germline mutations in 23 nonrelated patients, all with adrenal tumors; among the 19 patients considered for phenotypic associations, 13 (68%) presented with bilateral or multifocal pheochromocytoma and 16% developed additional thoracoabdominal paragangliomas [ MAX germline mutations were found in 1.12% of patients without other mutations [ MAX mutations [ MAX tumors have an intermediate biochemical phenotype with a predominant normetanephrine release [Comino-Méndez et al. identified mutations inpatients . This aspatients . The latngliomas . Median utations . Both stutations , 131. MA release .MAX (myc-associated factor X) gene is a tumor suppressor gene that encodes for MAX protein, which is a component of the MYC-MAX-MXD1 complex that regulates cell proliferation, differentiation, and apoptosis [ MAX gene have a paternal mode of transmission and are responsible for the loss of the wild type allele with consequent abrogation of protein expression. Consequently, inhibition of MYC-dependent cell transformation by MAX protein is disrupted, causing tumor development [poptosis , 133. Muelopment .α proteins  are transcription factors that respond to oxygen concentrations in tissues. Under hypoxic conditions, stabilization of HIFα proteins occurs, allowing transcription of genes involved in angiogenesis, glycolysis, erythropoiesis, apoptosis, proliferation and growth [ VHL and SDHx genes have been shown to induce pseudohypoxic states that induce the development of paragangliomas/pheochromocytomas. In 2012, Zhuang et al. described two somatic mutations in the gene encoding of the hypoxia-inducible factor 2α (HIF2A) in two patients with polycythemia and multiple paragangliomas . Functional assays show that both mutations affected pVHL hydroxylation, impairing HIF2α degradation leading to an intact/increased transcriptional activity of genes downstream of HIF2α, such as VEGFA and erythropoietin [ HIF2A gain-of-function mutations as causative for the development of polycythemia and multiple paragangliomas/pheochromocytomas and somatostatinomas in patients, corresponding to a novel syndrome [ de novo postzygotic event early in the embryogenesis [ HIF2A have also been identified in sporadic pheochromocytomas/paragangliomas in the absence of erythrocytosis [As stated before in the context of VHL disease, HIFd growth . Mutatioopoietin . These fsyndrome –142. Theogenesis , 138. Soocytosis .α under normoxic conditions, allowing pVHL binding and proteosomal degradation of HIFα proteins [ EGLN1 gene (formerly known as PHD2) [ EGLN1 allele suggests that EGLN1 may act as tumor-suppressor gene. Functional studies indicate stabilization of HIF2α in the presence of EGLN1 mutant protein [ EGLN1 mutations in paragangliomas.EGLN (egg-laying-defective nine) family of proteins  are responsible for hydroxylation of prolyl residues of HIFproteins . The assas PHD2) . Loss of protein . Additio VHL/SDHx/EGLN1 genes) and a kinase receptor-signaling cluster (associated with RET/NF1/TMEM127/MAX/KIF1B mutations) [ H-RAS gene in four male patients presenting with pheochromocytoma (3 patients) and paraganglioma (1 patient) [ H-RAS somatic mutation in a patient with unilateral pheochromocytoma [The RAS-ERK pathway has long been associated with the development of cancer . Regarditations) . Evidencpatient) . Very remocytoma .KIF1B) gene expression results in two protein isoforms, KIF1Bα and KIF1Bβ, which are motor proteins involved in the anterograde transport of mitochondria and synaptic vesicle precursors, respectively [ KIF1B missense mutations as causative of pheochromocytoma in two tumor samples [β acts downstream from oxygen-dependent prolyl hidroxylase EGLN3 (or PHD3) to induce apoptosis. These loss of function mutations in KIF1Bβ could therefore protect neuroblasts from apoptosis, leading to tumor development [ KIF1B mutation. These individuals presented unilateral or bilateral pheochromocytoma and other nonneural crest-derived malignancies, such as ganglioneuroma, leiomyosarcoma, and lung adenocarcinoma [β mutant pheochromocytomas showed that these tumors are transcriptionally related to RET and NF1-associated tumors.Kinesin family member 1B . Absence of a known family history is not enough to exclude this hypothesis. However, there are subgroups of patients with a very low risk as it is the case for those with apparently sporadic paraganglioma/pheochromocytoma after age 50 , 151. VHL, RET, and NF1 genes are associated with other malignant tumors. Thus, an early diagnosis of patients will allow an improved lifelong surveillance and forehand treatment with a consequent improved prognosis [There are several meaningful motives in favor of offering the genetic testing to paraganglioma patients. The inherited syndromic forms caused by mutations inrognosis –29. On trognosis . MoleculAdvanced techniques like whole genome sequencing or next-generation sequencing appear to be promising genetic strategies for testing paraganglioma patients –155. Nev VHL, RET, SDHB, SDHD, NF1, or HIF2A [ VHL or RET gene, respectively [ RET gene [ SDHD or, more rarely, SDHAF2 [ VHL and RET. Thus, SDHB, SDHD, TMEM127, or MAX genes should be considered only in a second step [ SDHB, SDHD, and VHL mutations [ SDHD  and SDHB gene mutations and less often by VHL and SDHC gene mutations [ SDHAF2 might be considered for analysis [ SDHB, so initial analysis should address this gene [ VHL, NF1, SDHD, or MAX genes can be considered for investigation [Patients presenting specific syndromic features or a positive familial history should be considered for the analysis of the specific genes:or HIF2A , 33, 39.ectively , 29. TheRET gene . Express, SDHAF2 , 106. Adond step , 131. Exutations , 77, 112utations , 50, 96.tigation , 96, 131 VHL gene appears as a first target, since missense mutations in this codon are strongly associated with pheochromocytoma development [ SDHx genes in different countries such as the Netherlands, Poland, Italy, Spain, and Portugal in order to develop effective screening protocols [A few and quick ways are likely to improve cost-effectiveness of molecular genetic testing. For instance, in VHL disease-associated pheochromocytoma, codon 167 ofelopment . In addirotocols –162. SDHx-related paragangliomas [Partial or large deletions may respond for false-negative results, when using conventional PCR followed by automatic sequencing techniques. Large deletions account for about 10% of the cases ofngliomas . Ideally RET-associated pheochromocytoma, young relatives carriers of RET mutations may undergo prophylactic thyroidectomy to prevent the development of medullary thyroid carcinoma [ SDHB gene mutations, a closer biochemical and imaging follow-up might be provided in order to prevent the development of metastatic disease [Identification of a mutation allows tailoring treatment and follow-up therefore contributing to a better prognosis. The same holds true for the patients' relatives. In the specific cases ofarcinoma . On the  disease . RET gene in multiple endocrine neoplasia type 2, VHL in von Hippel-Lindau disease; and NF1 in neurofibromatosis type 1. The discovery of the succinate dehydrogenase genes associated with the development of familial paraganglioma syndromes, in particular the SDHB gene, frequently associated with malignant tumors, brought new insights into the management and prognosis of paragangliomas.A great deal of knowledge has been added to the genetics of paragangliomas since the beginning of the millennium. Until then, the genes responsible for inheritable forms of paragangliomas were restricted to those underlying the syndromic forms of the disease;RET, VHL, NF1, SDHA, SDHB, SDHC, SDHD, SDHAF2, TMEM127, MAX, EGLN1, HIF2A, H-RAS, and KIF1B) have been associated with the development of paragangliomas. These genes have been divided into two tumorigenesis clusters: a pseudohypoxic cluster (associated with mutations in VHL/SDHx/EGLN1/HIF2A genes) and a kinase receptor-signaling cluster (associated with RET/NF1/TMEM127/MAX/KIF1B gene mutations). Functional studies involving these genes and paraganglioma-associated mutations as well as gene expression profiles of tumor samples have greatly contributed to our understanding of tumorigenic pathways of paragangliomas. Progresses in genetic knowledge and the evidence for genotype-phenotype correlations have largely influenced the care of patients with positive impact.So far, at least 14 genes (In this review, we summarized the most relevant aspects regarding the genetics and clinical aspects of the syndromic and nonsyndromic forms of pheochromocytoma/paraganglioma aiming to provide an algorithm for genetic testing. Recent comprehension of the molecular pathways involved in the tumorigenesis of paragangliomas is likely to be improved by further functional assays, possibly hinting novel molecular-targeted therapy approaches.


Portulaca oleracea L., belonging to the Portulacaceae family, is commonly knownas purslane in English and Ma-Chi-Xian in Chinese. It is a warm-climate, herbaceous succulentannual plant with a cosmopolitan distribution. It is eaten extensively as a potherb and added insoups and salads around the Mediterranean and tropical Asian countries and has been used as afolk medicine in many countries. Diverse compounds have been isolated from Portulaca oleracea, suchas flavonoids, alkaloids, polysaccharides, fatty acids, terpenoids, sterols, proteins vitamins and minerals.Portulaca oleracea possesses a wide spectrum of pharmacological properties such asneuroprotective, antimicrobial, antidiabetic, antioxidant, anti-inflammatory, antiulcerogenic, and anticanceractivities. However, few molecular mechanisms of action are known.This review provides a summary of phytochemistry and pharmacological effects of this plant. Portulaca oleracea L. is a warm-climate, herbaceous succulent annual plant with a cosmopolitan distribution belonging to the Portulacaceae family. It is commonly known as purslane , rigla (Egypt), pigweed (England), pourpier (France), and Ma-Chi-Xian (China) [ Portulaca oleracea also provides a source of nutritional benefits owing to its rich omega-3 fatty acids and antioxidant properties [ (China) . It is d (China) . America (China) . Portulaoperties .Portulaca oleracea has been used as a folk medicine in many countries, acting as a febrifuge, antiseptic, vermifuge, and so forth [so forth . It exhiso forth , antiulcso forth , anti-inso forth , antioxiso forth , and wouso forth  propertiso forth . The Chiso forth , 13. It so forth –16.Portulaca oleracea has a high potential to be used as human and animal food and to be utilized as a pharmacological agent in medicine. In this paper, phytochemistry and pharmacological activities of this plant are reviewed and its potential for further investigation, exploitation, and utilization are discussed. Portulaca oleracea have been isolated, including flavonoids, alkaloids, fatty acids, terpenoids, polysaccharides, vitamins, sterols, proteins, and minerals; these are listed in Many constituents of Portulaca oleracea plant, the flavonoids levels vary according to the part of the plant; the highest levels are present in the root followed by stem and the leaf; and seven different flavonoids are present in this plant, including kaempferol, myricetin, luteolin, apigenin, quercetin, genistein, and genistin [One of the most effective constituents present in Chinese Herbal Medicines are flavonoids which are biologically active and possess a wide range of pharmacological properties such as antibacterial, antivirus, anti-inflammation, and antioxidation properties. In thegenistin . Howevergenistin . Portulagenistin . Flavonogenistin .In addition to flavonoids, another important chemical found in this plant is alkaloids including dopa, dopamine, and noradrenalin. The content of dopamine and noradrenalin is higher in leaves compared to stem and seeds. The amount of dopamine and noradrenalin obtained from leaves varies according to the solvents used in the extraction process, suggesting that the levels of these compounds are dependent on the solvents used during the extraction process . OleracePortulaca oleracea is also an excellent source of omega-3 fatty acids, which is usually present in oil and fat of fishes but not normally found in plants. Omega-3 fatty acids play an important role in the enhancement of immune function [α-linolenic acid and linoleic acid, which are essential for normal growth, health promotion, and disease prevention in humans. Polysaccharides found in Portulaca oleracea are potential therapeutic agents for the treatment of diabetes mellitus owing to their modulation of blood lipids, metabolism, and decrease of blood glucose. Portulaca oleracea contains monoterpenes such as portulosides A and B, diterpenes such as portulene, and β-amyrin type triterpenoids [α-tocopherol, and B-complex vitamins, for example, niacin, pyridoxine, and riboflavin [β-carotene, glutathione, melatonin, portulacerebroside A, catechol, and bergapten.function  and prevfunction . It inclrpenoids , 25; in boflavin . Further Portulaca oleracea. This review provides a comprehensive summary of the main pharmacological properties which are presented below.Over the past decades, numerous researchers have investigated the pharmacological activities of Portulaca oleracea can scavenge free radicals and antagonize rotenone-induced neurons apoptosis, dopamine depletion, and complex-I inhibition in striatum of rats, suggesting that Portulaca oleracea may be a potential neuroprotective candidate against Parkinson's disease [ Portulaca oleracea (EP) protects nerve tissue/cells from hypoxic damage probably by elevation of glycolysis, EPO, and hypoxia inducible factor-1 expression levels [α expression is associated with the neuroprotective effects of EP against hypoxia injury by eliciting endogenous erythropoietin expression [β-Cyanin evidently inhibits D-galactose-induced neurotoxicity in mice, which at the doses of 50 and 100 mg/kg upregulates the activities of superoxide dismutases, catalase, glutathione reductase, and glutathione peroxidase, whilst reducing the level of the lipid peroxidation product malondialdehyde in the brain of D-galactose-treated mice. When compared to vitamin C, β-cyanin play a more pronounced effect on alleviating cognition deficits in mice [ Portulaca oleracea significantly inhibited AChE activity at a final concentration of 100 μg/mL with the IC50 value of 29.4 μg/mL. The use of AChE inhibitors has been a promising treatment strategy for Alzheimer's disease (AD); therefore, Portulaca oleracea may be an effective agent for the prophylaxis and treatment of AD [Administration of disease . The extn levels . The ethpression . β-Cyani in mice . The totnt of AD .Portulaca oleracea attenuates body weight, serum free fatty acids, and hyperinsulinemia. It also increases insulin sensitivity and ameliorates impaired glucose tolerance and lipid metabolism in rats with type 2 diabetes mellitus induced by injection of streptozotocin (25 mg/kg) and feeding of high calorie forage, suggesting that Portulaca oleracea alleviates insulin resistance [ Portulaca oleracea seeds are valuable and effective as an adjunctive and alternative therapy for the treatment of type 2 diabetes mellitus [sistance . Adminismellitus . Portulaca oleracea also prevents diabetic vascular inflammation, hyperglycemia, and diabetic endothelial dysfunction in type 2 diabetic db/db mice, suggesting its protective role against diabetes and related vascular complications [The aqueous extract ofications . The cruications , whilst ications . Portulaca oleracea is attributed to its constituents, such as gallotannins, omega-3 fatty acids, ascorbic acid, α-tocopherols, kaempferol, quercetin, and apigenin [β-actin and liver PPAR a/β-actin and inhibiting the protein expression of p-PERK and the FAS mRNA expression of liver and spleen in mice [μg/mL and the ethanolic extract at a range of 1200 and 1800 μg/mL, respectively, exerted cytoprotective effects on 2,2′-azobis hydrochloride-induced hemolytic damages of RBCs in a concentration-dependent manner [The antioxidant property ofapigenin , 16, 17.apigenin . The aqu in mice . In anott manner . Portulaca oleracea display several biological activities, such as anticancer, antioxidation, anti-inflammation, and immunity enhancing properties [ Portulaca oleracea, have a suppressive effect on the growth of HeLa and HepG2 cells in vitro, suggesting that the sulfation of POP increases the cytotoxicity in tumor cells [ in vitro cytotoxic activities against human cancer cell lines. Portulacerebroside A stimulates human liver cancer HCCLM3 cell apoptosis via the activation of the p38 MAPK- and JNK-triggered mitochondrial death pathway [50 value of 1.6 ug/mL than mitomycin C which has an IC50 value of 13.0 ug/mL. Portulacanones B is active against SGC-7901 cell lines with an IC50 value of 16.2 ug/mL, which is very close to the value obtained with mitomycin C. 2,2′-Dihydroxy-4′,6′-dimethoxychalcone is moderately active against K-562 cells with an IC50 value of 24.6 ug/mL and portulacanones B–D show selective cytotoxic activity against SF-268 and/or NCI-H460 cells with IC50 values of 14.3–20.1 ug/mL [trans-Feruloyltyramine, (7′R)-N-feruloylnormetanephrine, 1,5-dimethyl-6-phenyl-1,2-dihydro-1,2,4-triazin-3(2H)-one, and (3R)-3,5-bis(3-methoxy-4-hydroxyphenyl)-2,3-dihydro-2(1H)-pyridinone have weak bioactivities against K562 with IC50 values of 222.77, 66.94, 90.09, and 41.52 umol/L, respectively, and moderate bioactivities against A549 with IC50 values of 28.80, 21.76, 24.54, and 37.20 umol/L, respectively [ Portulaca oleracea has a potential application in the treatment of cancer.Polysaccharides fromoperties –40. Polyoperties . Sulfateor cells . In addi pathway  and 2,2′.1 ug/mL . N-transPortulaca oleracea possesses antibacterial, antifungal, and antiviral activities as revealed by its antifungal effect against dermatophytes of the genera Trichophyton [ Portulaca oleracea shows antibacterial activity against the Gram-negative stains: Escherichia coli, Pseudomonas aeruginosa, and Neisseria gonorrhea with inhibition zones of 14, 15, and 15 mm, respectively, and the Gram-positive strains: Staphylococcus aureus, Bacillus subtilis, and Streptococcus faecalis with inhibition zones of 13, 14, and 15 mm, respectively, as well as antifungal activity against Candida albicans with inhibition zone of 12 mm [hophyton . A pectihophyton . A 70% m Portulaca oleracea inhibits tumor necrosis factor- (TNF-) α-induced production of intracellular reactive oxygen species (ROS) and overexpression of intercellular adhesion molecule- (ICAM-) 1, vascular cell adhesion molecule (VCAM)-1, and E-selectin in human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner. This extract also suppresses the translocation of nuclear factor κB (NF-κB) p65 to the nucleus, TNF-α-induced NF-κB binding, and the degradation of inhibitor molecule (IκB)α. Furthermore, an inhibition in the adhesion of HL-60 cells to TNF-α-induced HUVECs and TNF-α-induced mRNA expression of interleukin- (IL-) 8 and monocyte chemoattractant protein- (MCP-) 1 is also observed. The aqueous extract of Portulaca oleracea may also play an important role in the suppression of the vascular inflammatory process related to the development of atherosclerosis [Pretreatment with the aqueous extract ofclerosis . Portulaca oleracea at 0.8 g/kg and 1.4 g/kg, respectively, can reduce the severity of HCl-induced gastric ulcers in a dose-dependent manner; this is comparable to the effect observed with sucralfate 0.1 g/kg. In addition, the aqueous extract (0.56 and 0.8 g/kg) and the ethanolic extract (0.8 and 1.4 g/kg) display suppression of lesions induced by absolute ethanol. The oral and intraperitoneal doses of both extracts dose-dependently increase the pH of gastric juice in mice with pylorus ligation. Thus, Portulaca oleracea holds great promise as an effective therapeutic agent for gastrointestinal diseases due to its gastroprotective activity [Aqueous and ethanolic extracts ofactivity .4 elicits liver injury in rats, which notably upregulates the levels of total bilirubin and serum hepatic marker enzymes, including glutamate pyruvate transaminase (GPT) and glutamate oxaloacetate transaminase (GOT). A 70% alcohol extract of Portulaca oleracea significantly reverses the increase in hepatic marker enzymes and total bilirubin levels, confirming the hepatoprotective activity of this plant [Intraperitoneal administration of CClis plant . Portulaca oleracea at a concentration range of 100, 200, and 400 mg/kg, respectively, displays a dose-dependent effect in prolonging the survival time of mice in hypoxic models, including closed normobaric hypoxia and potassium cyanide or sodium nitrite toxicosis. This extract also enhances the activities of phosphofructokinase, pyruvate kinase, and lactate dehydrogenase in glycolysis and the level of adenosine triphosphate of mouse cortices in hypoxia models [ Portulaca oleracea has been appraised in Mus musculus JVI-1 and it has been shown that a fresh crude extract significantly accelerates the wound healing course by the stimulation of wound contraction and downregulation of the surface area of the excision wound [ Portulaca oleracea also has the ability to accumulate Se even at the shortest time span of 42 days, and hence it can perform the dual functions of preventing the occurrence of Se deficiency linked diseases such as Keshan and Kashin-Beck diseases [The ethanol extract froma models . The prediseases .Portulaca oleracea is of considerable importance to the food industry and also possesses a wide spectrum of pharmacological properties such as neuroprotective, antimicrobial, antidiabetic, antioxidant, anti-inflammatory, antiulcerogenic, and anticancer activities, which are associated with its diverse chemical constituents, including flavonoids, alkaloids, polysaccharides, fatty acids, terpenoids, sterols, proteins, vitamins, and minerals. Portulaca oleracea are substantiated by using in vitro and in vivo studies including animal models and cell culture studies, the mechanisms of action have not been addressed. Hence, more mechanistic studies are required before Portulaca oleracea can be considered for further clinical use. This review concludes that Portulaca oleracea is an edible and a medicinal plant which is important to the food industry and may also have a significant role to play in health care provided that adequate studies are conducted.Although bioactivities of extracts or compounds isolated from


TP53, PIK3CA, CCND1, MYC, HER2 (ERBB2), MCL1, PTEN, FGFR1, GATA3, NF1, PIK3R1, BRCA2, EGFR, IRS2, CDH1, CDKN2A, FGF19, FGF3 and FGF4. Aberrations included mutations (46%), amplifications (45%), deletions (5%), splices (2%), truncations (1%), fusions (0.5%) and rearrangements (0.5%), with multiple distinct variants within the same gene. Many of these aberrations represent druggable targets, either through direct pathway inhibition or through an associated pathway . The ‘molecular individuality’ of these tumors suggests that a customized strategy, using an “N-of-One” model of precision medicine, may represent an optimal approach for the treatment of patients with advanced tumors.Our analysis of the tumors of 57 women with metastatic breast cancer with next generation sequencing (NGS) demonstrates that each patient's tumor is unique in its molecular fingerprint. We observed 216 somatic aberrations in 70 different genes, including 131 distinct aberrations. The most common gene alterations (in order of decreasing frequency) included:  Genomic profiling demonstrates a multitude of aberrations across a broad spectrum of cancer-related genes , 2 and rHerein, we demonstrate that, amongst 57 patients with metastatic breast cancer, no two patients had the same molecular portfolio. The number of distinct aberrations driving advanced tumors may therefore be responsible for the remarkable resistance of metastatic malignancies to prolonged remission or cure, especially with our current therapeutic strategies . These dWe used next generation sequencing (NGS) of 182 to 236 genes to interrogate tumors from 57 women with metastatic breast cancer (26 with hormone receptor (HR)-positive and HER2-negative tumors; 7 with HR-positive/HER2-positive tumors; 4 with HR-negative/HER2-positive tumors; and, 20 with triple negative breast cancer (TNBC)  [4]. ImpTP53, PIK3CA, CCND1, MYC, HER2 (ERBB2), MCL1, PTEN, FGFR1, GATA3, NF1, PIK3R1, BRCA2, EGFR, IRS2, CDH1, CDKN2A, FGF19, FGF3 and FGF4  were: and FGF4 . The mosCCND1 (12 patients), MYC (12 patients), MCL1 (7 patients), HER2 , and FGFR1 (6 patients). All amplification had been previously reported in breast cancer.A total of 131 distinct somatic aberrations  were identified: 80 mutations (including mutation variants in the same gene); 37 amplifications; five deletions; five splices; two truncations; one fusion; and, one rearrangement. Of the 80 distinct mutation variants we observed , 49 havePIK3CA mutations with eight different variants were noted ; PIK3CA amplifications (3 patients); PTEN deletions (6 patients); PIK3R1 mutation (5 patients with 4 different mutation variants); AKT3 amplifications (2 patients); and PTEN or AKT1 mutation (1 patient each). Other druggable aberrations include, but are not limited to, EGFR, HER2, CDKN2A, FGFR/FGF family, KRAS, MDM2, BRCA2, and more.Many, if not most, of the genomic aberrations represent actionable targets, either by approved drugs or by agents in clinical trials , 9-14. APIK3CA is the oncogene showing the highest frequency of gain-of-function mutations in breast cancer [PIK3CA was the second most common aberrant gene, and was involved in 12% of abnormalities , the subset of patients expands. Of therapeutic interest, the BOLERO-2 study of the hormone modulator exemestane and the mTOR inhibitor everolimus showed that patients whose tumors had 0 or 1 genomic aberrations in PIK3CA or FGFR pathways or in CCND1 had better outcomes [PIK3CA-mutant breast and gynecologic tumors have higher than expected response rates  when mTOR inhibitor-based combination regimens are administered [Our observations are consistent with the hypothesis that metastatic tumors, even when originating in the same organ , are mostly distinct at the molecular level  . Of spect cancer , 10-12 , 95% (54 of 57) of our patients still had distinct genomic portfolios. The reality that NGS interrogation of these tumors has revealed is that, since most patients with metastatic breast malignancies have unique genomic landscapes, the standard methods deployed for therapeutic prosecution of molecular aberrations may need to be revised.ALK-rearranged advanced lung tumors respond to the ALK inhibitor crizotinib [Classic clinical trial design mandates grouping patients by tumor site and histology  and evaluating the efficacy of a drug or combination of agents. In the era of molecular therapeutics, the first generation of newer clinical trial designs stratify patients, often on the basis of a single molecular aberration as identified by a companion diagnostic, and test the efficacy of a cognate inhibitor to improve outcomes. The latter model represents a scientifically rational advancement that has been successful to an extent. Indeed, patients with HER2-positive breast cancer respond to trastuzumab, lapatinib, and pertuzumab , and ALKizotinib . HoweverPreviously, we have shown that, using genomic aberrations to match patients with advanced, heavily-pretreated malignancies (including breast cancer) to drugs, in the context of phase I studies, resulted in improved outcomes as assessed by response rate, progression-free survival and overall survival, as well as progression-free survival on matched Phase I therapy as compared to last conventional therapy; in the case of melanoma, progression-free survival on matched phase I therapy was improved as compared to first-line treatment , 18, 19.In summary, NGS may represent a disruptive technology that mandates a new approach to clinical research and cancer treatment. Even if there is some convergence of pathways with functional impact -23, intrμm of FFPE tissue (minimum 20% tumor cells) using the Maxwell 16 FFPE Plus LEV DNA Purification kit (Promega) and quantified using a standardized PicoGreen fluorescence assay (Invitrogen). Library Construction was performed using 50-200 ng of DNA sheared by sonication to ~100-400 bp before end-repair, dA addition and ligation of indexed, Illumina sequencing adaptors. Enrichment of target sequences  was achieved by solution-based hybrid capture with custom biotinylated oligonucleotide baits. Enriched libraries were sequenced to an average median depth of >500X with 99% of bases covered >100X (Illumina HiSeq 2000 platform using 49 × 49 paired-end reads) and mapped to the reference human genome (hg19) using the Burrows-Wheeler Aligner and the publicly available SAMtools, Picard and Genome Analysis Toolkit. Point mutations were identified by a Bayesian algorithm; short insertions and deletions, determined by local assembly; gene copy number alterations (amplifications), by comparison to process matched normal controls; and gene fusions/rearrangements, by clustering chimeric reads mapped to targeted introns. Local site permissions to use clinical samples were also obtained.Fifty-seven sequential consented patients with advanced, pathologically confirmed breast cancer who had next generation sequencing (182 to 236 genes) were analyzed. Tumor samples were evaluated for genomic alterations including base substitutions, short insertions and deletions, amplifications, homozygous deletions, gene fusions, truncations and rearrangements . DNA was extracted from 40