PO) or maternal ) and 40,XX controls. Aortic valve morphology was normal (tricuspid) in all of the 39,XPO and 40,XX mice studied, but abnormal (bicuspid or quadricuspid) in 15% of 39,XMO mice. Smooth muscle cell orientation in the ascending aorta was abnormal in all 39,XPO and 39,XMO mice examined, but smooth muscle actin was decreased in 39,XMO mice only. Aortic dilation was present with reduced penetrance in 39,XO mice. The 39,XO mouse demonstrates aortopathy and an X-linked parent-of-origin effect on aortic valve malformation, and the candidate gene FAM9B is polymorphically expressed in control and diseased human aortic valves. The 39,XO mouse model may be valuable for examining the mechanisms underlying the cardiovascular findings in TS, and suggest there are important genetic modifiers on the X chromosome that modulate risk for nonsyndromic BAV and aortopathy.Turner syndrome (TS), most frequently caused by X-monosomy , is characterized in part by cardiovascular abnormalities, including aortopathy and bicuspid aortic valve (BAV). There is a need for animal models that recapitulate the cardiovascular manifestations of TS. Extracellular matrix (ECM) organization and morphometrics of the aortic valve and proximal aorta were examined in adult 39,XO mice (where the parental origin of the single X was paternal (39,X Turner syndrome (TS), most frequently caused by X-monosomy 45,X), is characterized by diverse developmental, endocrine and cardiovascular abnormalities, and affects approximately 1 in 2500 live born females . Aortic 5,X, is cM) whereas in the remainder it is inherited from the father . X-linked parent-of-origin effects (POE), i.e. where the appearance of 45,XM and 45XP subjects differs significantly, have been described on diverse phenotypes including: brain morphology and cognitive function, neck webbing, sensorineural hearing loss, renal and ocular abnormalities [M subjects relative to 45,XP subjects [MY) relative to females  [M and 45,XP individuals [i.e., genes that are solely, or predominantly, expressed from either the paternally or maternally inherited allele) [M individuals only) [Two distinct genetic mechanisms may contribute to the phenotype of individuals with a 45,X constitution: first, haploinsufficiency for one or more of products of genes that escape X-inactivation  . Howeverividuals ,13. X-li allele) ; to datePO or 39,XMO) partially recapitulates several aspects of the Turner syndrome phenotype, including X-linked POE on cognition [MO mice would exhibit higher rates of aortopathy and aortic valve malformation than 39,XPO  mice.The 39,XO mouse model, where the parental origin of the X chromosome can be varied , or quadricuspid according to established definitions [vs. 39,XO), and X-linked POE  on the measures described. Morphometric data were analyzed by One Way ANOVA with a between-group factor of KARYOTYPE. Data regarding frequency of aortic valve malformations were analyzed by a one-tailed Freeman Halton extension of Fisher’s Exact Test. Human FAM9B expression was examined in human aortic valve control and disease samples by reverse transcriptase polymerase chain reaction using the following primers: 5′-CGCCTCTAGCTTCCCAGGACAA-3′ and 5′-GGCTTCTTGGTATTAGCCCCCGT-3′. FAM9B cDNA was used as the positive control .40,XX, 39,Xeviously . Mice aginitions . Morphominitions . By earlinitions , therefoinitions ,21. Extrinitions . The aorPO (n = 18) and 40,XX (n = 21) mice studied, four mice (15%) from the 39,XMO group (n = 27) exhibited valve abnormalities (p = 0.03); three 39,XMO mice displayed BAV and one a quadricuspid aortic valve . While aortic valve morphology was normal (tricuspid) in all of the 39,Xic valve D–G. Two 0,XX n = 1 mice stn = 5), 39,XPO (n = 5) and 39,XMO (n = 9) groups with regard to aortic root dimensions , aortic dimensions , or aortic valve annulus size , but there appeared to be a trend whereby all aortic measurements were greater in 39,XO mice than 40,XX mice with 39,XMO mice showing the greatest deviation from 40,XX subjects; a proportion of 39,XO mice showed aorta measures greater than two standard deviations from the 40,XX mean , 39,XPO (n = 5) and 39,XMO (n = 9) was 12.5%, 72.7% and 82.6%, respectively  with 39,Xogenesis . The funuggested ,28. Theri.e., 46,XX and 45,XMO cells in some individuals), by the relatively low frequency of abnormalities in the 45,XM group, and/or by using scanning techniques to examine cardiac morphology rather than post mortem histology. An obvious candidate gene for any X-linked POE on aortic valve development in humans is FAM9B, the closest orthologue of Xlr3b [FAM9B is located on the short arm of the X chromosome at Xp22.32 within a region recently shown to be important in aortic valve and aorta development [FAM9B is polymorphically expressed in human aortic valves , aortic valve morphology (tricuspid or bicuspid), or the presence of aortic valve disease (AVD) (pediatric or adult). Whether FAM9B is imprinted in human heart tissue, why it is polymorphically expressed in the aortic valve, and how it influences cardiac biology remains to be investigated.In terms of relevance to Turner syndrome, our data indicate two possibilities: (i) there is an X-linked POE on aortic valve morphology in mice but not in humans due to species’ different physiology; or (ii) there is an X-linked POE in both species, in the same direction, and of a similar magnitude. It is possible that, in some studies, an X-linked POE in humans could have been obscured by the presence of mosaicism (of Xlr3b . FAM9B ielopment . In an ac valves . In thisFAM9B as a potential novel X-linked genetic modulator of CVM risk.In summary, our findings suggest the 39,XO mouse as a much-needed animal model for examination of the biological mechanisms underlying the cardiovascular findings in Turner syndrome, and indicate that there may be important genetic modifiers on the X chromosome in nonsyndromic BAV, aortic valve disease and aortopathy. Specifically, our work suggests


Escherichia coli (aEPEC) strains are unable to produce the bundle-forming pilus (BFP), which is responsible for the localized adherence pattern, a characteristic of the pathogenicity of typical EPEC strains. The lack of BFP in aEPEC strains suggests that other fimbrial or non-fimbrial adhesins are involved in their adhesion to the host cells. The aim of this study was to investigate the distribution of major subunit fimbrial genes known to be important adherence factors produced by several E. coli pathotypes in a collection of 72 aEPEC strains. Our results demonstrate that a high percentage (94–100%) of aEPEC strains harbored ecpA, fimA, hcpA, and lpfA fimbrial genes. Other fimbrial genes including pilS, pilV, sfpA, daaC, papA, and sfa were detected at lower frequencies (1–8%). Genes encoding fimbrial subunits, which are characteristic of enteroaggregative E. coli or enterotoxigenic E. coli were not found. No correlation was found between fimbrial gene profiles and adherence phenotypes. Since all aEPEC strains contained ecpA, the major pilin gene of the E. coli common pilus (ECP), a subset of ecpA+ strains was analyzed for transcription of ecpRABCDE and production of ECP upon growth in three different culture conditions at 37°C. Transcription of ecpRABCDE occurred in all conditions; however, ECP production was medium dependent. In all, the data suggest that aEPEC strains are highly heterogeneous in terms of their fimbrial gene profiles. Despite lacking BFP production, other mechanisms of cell adherence exist in aEPEC strains to ensure host colonization, e.g., mediated by other prevalent pili such as ECP. Moreover, the production of ECP by aEPEC strains might be influenced by yet unknown post-transcriptional factors.Atypical enteropathogenic  Escherichia coli typically harbor between 12 and 16 different pili operons but the function of the majority of these pili systems and whether they are expressed or not remain uncharacterized , colonization factors (CFs), and aggregative adherence fimbriae (AAF) are found in enteropathogenic (EPEC), enterotoxigenic (ETEC), and enteroaggregative (EAEC) E. coli strains, respectively (E. coli common pilus (ECP) are among the ubiquitous fimbrial adhesins of E. coli pathotypes. The ECP that has been detected in enterohemorrhagic E. coli (EHEC), EPEC, EAEC, ETEC, uropathogenic and avian pathogenic E. coli , 40 pmol of each primer, and 2.0 μL of DNA template, obtained from a colony from culture on Luria-Bertani (LB) agar boiled in 300 μL of water for 10 min. The PCR was carried out at 94°C for 5 min, 30 cycles of 94°C for 1 min, annealing temperature . Twenty-two LAL aEPEC strains were selected for PCR analysis of ecpRABCDE genes as described above.The following fimbriae-encoding genes were searched among the 72 aEPEC strains by PCR: ecpRABCDE transcription and ECP production the strains were initially grown in LB at 37°C for 18 h. The bacterial cultures were then transferred to LB, Dulbecco’s modified Eagle Medium (DMEM) or preconditioned DMEM (PC-DMEM) (1:100) followed by static incubation at 37°C until a final absorbance of 0.6 at 600 nm (A600). PC-DMEM was prepared by incubating DMEM  in the presence of monolayers of HeLa cells for 48 h at 37°C. The supernatant referred to as “preconditioned medium” was collected, adjusted to pH 7.4, and filtered through a 0.2 μm pore membrane  were selected for ECP production. For analysis of membrane . tEPEC E strains  were useg for 5 min. Pellets were resuspended in 1 mL of the respective culture media and subsequently used for RNA extraction using RNA Protect and RNeasy Mini kit , according to the manufacturer’s recommendations. cDNAs were synthesized using 1 μg of RNA for each strain, by Superscript III First-Strand Synthesis Systems kit for RT-PCR , following the manufacturer’s recommendations. The presence of ecpRABCDE genes in the cDNAs obtained were assessed by PCR as described in Table 1. PCR for the constitutive gene rpoA was included as control of cDNA integrity. All reactions were performed in triplicates in three independent experiments.Strains BA2103, BA3378, BA4132 and BA4147, as well as appropriate control strains were grown in LB, DMEM and PC–DMEM and centrifuged at 4,000 × g and the pellets were resuspended in 10 μL of the respective medium and fixed on glass slides with 4% p-formaldehyde at 4°C for 18 h. The slides were washed twice with 0.01 M phosphate buffer saline pH 7.4 (PBS), blocked with 10% goat serum in PBS (GS–PBS) for 1 h at room temperature, followed by incubation at 4°C for 18 h with rabbit anti-EcpA antibody . The slides were washed twice with PBS and incubated with goat anti-rabbit IgG conjugated with FITC  diluted 1:500 in GS–PBS at room temperature for 1 h. After incubation, the slides were washed twice with PBS and mounted with Vectashield antifade mounting medium  and covered with glass coverslips. The preparations were visualized under laser scanning confocal  microscopes with original magnification of 1,000×. Triplicates of three independent experiments were performed.The production of ECP was studied by immunofluorescence microscopy (IFM) in LAL aEPEC strains BA2103, BA3378, BA4132, and BA4147 growing in the three different media described above. Briefly, the 18 h bacterial cultures were centrifuged for 5 min at 900 × We also determined ECP production in cultures of strain BA2103 obtained in LB, DMEM or PC–DMEM by transmission electron microscopy (TEM) using immunogold-labeling method. Strains E2348/69 and 1551-2 were used as controls. Briefly, preparations were blocked with a solution of 0.2% BSA in PBS and then incubated with rabbit anti-EcpA at 1:100 dilution for 2 h at room temperature. Following this period, the preparations were incubated with goat anti-rabbit antibody labeled with 10 nm colloidal gold particles  at 1:50 dilution for 2 h at room temperature. Preparations were negatively stained with 2% uranyl acetate in water, applied onto formvar-coated nickel grids and observed under TEM  at 80 kV. Triplicates of three independent experiments were performed.ecpA (100%), fimH and hcpA (97.2%), fimA (94.4%), lpfA1-2 (68%), lpfA2-1 (50%), lpfA1-1 (19.4%), O113lpfA (13.8%), lpfA1-3 (11.1%), pilS and ldaH (8.3%), pilV (4.2%), papA and daaC (2.7%) and sfpA (1.4%) . This large repertoire of fimbrial genes was distributed in different combinations among the 72 strains, demonstrating no correlation between fimbrial gene profiles and adherence pattern (Table 2). None of the aEPEC strains contained the following fimbrial genes: aggA, aafA, agg3A, agg4A, cooA, cotA, cstA, cofA, csaA, csfA, cssA, lngA, lpfA1-5, and sfa (Table 2).Fifteen fimbrial genes were detected in our collection: ecpA+ aEPEC strains contained also the ecpR, ecpB, ecpC, ecpD, and ecpE, suggesting that the ECP operon is highly conserved among aEPEC (data not shown). To better understand the correlation between the expression of ecpA and production of ECP in different culture conditions we selected four representative LAL aEPEC strains , all of which display different fimbrial profiles (Table 2). All the six-ecp genes were expressed in the four strains regardless of the growth medium . In general, a noteworthy increase in ECP production is apparent when the strains were grown in PC-DMEM in comparison to DMEM . Confirmation of ECP production by strain BA2103 was demonstrated by TEM and immunogold labeling .The 22 LAL tEPEC adherence to enterocytes is multifactorial involving the formation of localized microcolonies on host epithelial cells, which is mediated by BFP, promoting bacterium-cell and bacterium–bacterium interactions ; followeE. coli major fimbrial subunit genes known to be involved in bacterial adhesion processes. In agreement with previous reports, none of the genes encoding EAEC fimbriae or the CFs of ETEC were present in the aEPEC strains studied  genes were present in the majority (94–100%) of the strains, while pilS, pilV, sfpA, daaC, papA, and sfa genes were detected at lower frequency (1–8%) regardless of their adherence pattern. Since each strain studied displayed a specific genetic fimbrial profile, these data indicated that there was no correlation between fimbrial gene profile and adherence pattern phenotypes, as similarly elsewhere described  strain (IHE 3034) via mat promoter, either by a positive autoregulatory circuit or repressing the negative control exerted by H-NS (ecpA mRNA under some conditions as low growth temperature, acidic pH or elevated levels of acetate (ecpA transcript as shown for mRNA of the major cold-shock protein CspA (LEE4 and LEE5 transcripts playing an important role in the post-transcriptional regulation of A/E lesion formation. Genes within the GlmY and GlmZ regulon include stress-related genes, virulence factors, genes involved in osmoregulation and adhesins, which is the case of ECP. The role of these post-transcriptional regulators in ECP production by our strains is currently under investigation by our group.As production of ECP was not detected in aEPEC BA3378 in any growth condition tested, it is possible that this strain produces a variant of EcpA, which is not recognized by the anti-ECP employed, or is under strict regulation. Thus, post-transcriptional factors possibly regulate ECP production in some strains, as in BA3378 of our study. In fact, the operon regulator EcpR/MatA was shown to have dual role in the control of operon expression in a neonatal meningitis  by H-NS . EcpR/Ma acetate . The disein CspA . Small Rein CspA . Also, GThe reactivity of anti-EcpA antibody was remarkably more intense in bacterial cell grown in PC-DMEM than in DMEM. Similar to tEPEC strains, in which ECP and BFP act in combination to promote adherence, it is reasonable to suggest that ECP could act together with other adhesins of aEPEC to favor gut colonization and survival in and outside the host. Also, the high prevalence of ECP among aEPEC indicates that this adherence factor has been maintained during evolution to ensure intestinal colonization.E. coli fimbrial genes profiles are present in the aEPEC strains studied and that there is no correlation between these profiles and adherence patterns. The high prevalence of ECP, HCP, and type 1 fimbriae among aEPEC suggest that they are likely to contribute to cell adhesion. We also demonstrated that culture conditions influenced ECP production by aEPEC strains, likely attributed to post-transcriptional regulation.In summary, our data demonstrated that different DM, JN, KN, BY, FV, YM-L, FM, CA, WE, and RP: conception and design. DM, JN, NF, CM, KN, BY, FV, FM, and CA: acquisition and analysis. DM, JN, NF, CM, KN, BY, FV, YM-L, JG, FM, CA, WE, and RP: interpretation and substantial contributions. DM, CM, YM-L, JG, FM, CA, WE, and RP: drafting the work or revising it critically. DM, JG, CA, WE, and RP: final approval of the version to be published.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Tajikistan is a small country located in Central Asia. The mostly mountainous terrain with a continental, subtropical, and semiarid climate, is characterized by diverse flora. Many people in Tajikistan rely on medicinal plants as their traditional form of medicine to prevent and cure health disorders. Aromatic medicinal plants, in particular, have played an important role for the local people. In this review, we present a summary of the uses of 18 aromatic medicinal plants from Tajikistan and their compositions of secondary metabolites. Plants have been and continue to be valuable natural treasures, providing an important source of nutrients and therapeutic agents. Plants must defend themselves against herbivory and microbial infections, and over the last 400 million years they have evolved a high diversity of secondary metabolites that are toxic to animals and microorganisms. Because of this evolutionary background, most secondary metabolites are biologically active. This did not escape our ancestors who started to use plants as a means to treat infections and health problems ,2. In thThe defense chemistry of plants includes a surprisingly wide diversity of biologically active compounds, such as alkaloids, glucosinolates, cyanogenic glycosides, flavonoids, tannins, coumarins, lignans, terpenoids, saponins, organic acids, and many others. A number of secondary metabolites, especially mono- and sesquiterpenes but also phenylpropanoids, are volatile. These compounds serve not only as deterrents against herbivory and often against microbial infection, but also as signal compounds to attract pollinators or predators. Aromatic plants have attracted human attention for a long time because of their mostly pleasant fragrances. As a consequence, many of them are used as raw materials for the production of perfumes and cosmetics; others have found application in aromatherapy and phytotherapy. Many aromatic plants serve as spices because they can reduce the load of microbial pathogens in food, improve the taste, and support digestion .2 (93% of this area is mountainous) and has a population of approximately eight million people. Tajikistan is divided into four provinces (viloyat). These are the provinces of Sughd and Khatlon, the autonomous province of Gorno Badakhshan, and the Districts of Republican Subordination  juniper woodlands; (3) desert steppes; (4) high mountain deserts dominated by cushion plants; and (5) alpine.Tajikistan is a mountainous country in the southeastern part of Central Asia  [Materia Medica of Avicenna comprised more than 50 cardiac, 70 antiasthmatic, and 75 antidiabetic plants; 110 plants were described as useful for the treatment of kidney and gallstones, more than 40 plants for the treatment of vitiligo, dozens for wound healing, and others with antitoxic, antitumor, hemostatic and several other activities [Many Tajik plants have been used since ancient times in traditional medicine. The scientist Abu Ali ibn Sina (Avicenna)  described more than 750 pharmaceutical substances of vegetative, animal, and mineral origin, several of them from Central Asia, in his book Al-Qanoon fi al-Tibb (The Canon of Medicine). Many medicines (drugs) described by Avicenna have entered the pharmacopeia and are still in use . His expearlier) . The MatMedicinal plants generally contain complex mixtures of biologically active compounds. They affect multiple targets and, in general, show low toxicity. Some of the active secondary metabolites can have advantages in treating chronic diseases. In aromatic plants, part of their therapeutic effects comes from their essential oils. Most secondary metabolites in essential oils are small lipophilic natural products, which allow them to readily enter body tissues by free diffusion. The lipophilic components can interact with biomembranes and membrane proteins. They thus influence membrane fluidity and permeability. This explains why many components of essential oils exhibit antibacterial, antifungal, antiviral, and cytotoxic activities .Tajikistan has a rich flora including large numbers of herbs and aromatic plants. According to preliminary estimates, about 1500 species of Tajik plants are used in folk medicine, but only a small number of them are important in modern medicine . In thisAchillea species have been used in folk medicine in Europe and Asia [A. filipendulina has been employed since ancient times in traditional herbal medicines against a variety of ailments [A. filipendulina have been used to treat “breaking the muscles” and chronic inflammation of the sciatic nerve (sciatica). In Tajik folk medicine, a decoction from the dried flowers of A. filipendulina is applied as a children’s digestive aid, to treat stomach ache and cough [A. filipendulina is rich in santolina alcohol (1) (43%–46%), 1,8-cineole (2) (9%–11%), borneol (3) (5%–6%), isoborneol (5%) and cis-chrysanthenyl acetate (4) (7%–9%) [A.filipendulina leaf oil from Iran has shown antibacterial activity [A. filipendulina are quercetagetin and centaureidin [A number of and Asia . A. filiailments . Accordind cough ,13. Thisnd cough , and hasnd cough . The ess (7%–9%) . 1,8-Cin (7%–9%) . A.filiactivity . The majaureidin . Quercetaureidin  and HIV aureidin ), while aureidin .A. graveolens are widely applied for improving appetite, treating flatulence, stomach problems, digestive disorders, insomnia, cramps, inflammations of the respiratory tract, and for stimulating the release of milk in nursing mothers [A. graveolens essential oil from Tajikistan are carvone (5) (52%), trans-dihydrocarvone (15%), dill ether (6) (13%), α-phellandrene (7) (8%), and limonene (8) (7%) [This plant is widely used in Europe and Asia for flavoring foods and beverages due to its pleasant spicy aroma. It has been extensively utilized as a traditional herbal medicine throughout Europe, Asia, and America. In Tajik folk medicine, water extracts (tea and infusion) of  mothers ,14. The (8) (7%) .50 values were 0.093 mg/mL for HeLa, 0.216 mg/mL for Caco-2, and 0.067 mg/mL MCF-7 cell lines. A. graveolens essential oil was toxic in the brine shrimp lethality test (LC50 = 15.9 μg/mL), but showed only marginally antimicrobial activity against Escherichia coli (MIC = 625 μg/mL) [A. graveolens oil is most likely due to the major components carvone and limonene. Both (R)-(+)-limonene and (S)-(+)-carvone increase the production of reactive oxygen species (ROS) and decrease mitochondrial membrane potentials (MMP) [S)-(+)-carvone is utilized as a stomachic, carminative, and for treatment of nervous tension and several skin disorders [The cytotoxicity of the oil was assessed against human cervical cancer (HeLa), human colorectal adenocarcinoma (Caco-2) and human breast adenocarcinoma (MCF-7) cancer cell lines. IC5 μg/mL) . The biols (MMP) . Carvoneisorders .A. absinthium is an herb also used traditionally in Tajikistan. This plant is known to possess several biological properties, especially anthelmintic, digestive, antifungal, antibacterial, but also balsamic, diuretic, and emmenagogue activities. Extract of wormwood  is utilized to treat hyperacidity, gastric colic, gastritis, flatulence, and conditions of the liver and gallbladder. Chronic, large doses of wormwood have been reported to upset the nervous system [A. absinthium oil from Tajikistan are myrcene (9) (9%–23%), cis-chrysanthenyl acetate (4) (8%–18%), a dihydrochamazulene isomer (6%–12%), germacrene D (10) (2%–8%), linalool (11) (5%–7%), and β-thujone (12) (up to 7%), and is phytochemically distinct from A. absinthium from Europe, the Middle East, or Siberia [Wormwood is a traditional medicinal plant in Europe and Asia. It was widely used in the alcoholic beverage “absinthe” that was later banned in many countries because of its neurotoxicity, which is thought to be due to the presence of thujone. . A. absis system ,12,25. T Siberia .A. absinthium essential oil is used in the manufacture of alcoholic beverages and in pharmaceutical preparations as a mild sedative in the treatment of insomnia [Antifungal, antimicrobial activity, choleretic, antiseptic and balsamic properties of plant can be explained by the composition of its essential oil. Myrcene-containing insomnia .A. rutifolia from Tajikistan belongs to the thujone-rich chemotype, in contrast to the cineole/camphor chemotype found in Mongolia, and is dominated by α-thujone (12) (21%–37%), and β-thujone (13) (36%–47%), as well as 1,8-cineole (2) (3%–12%), and germacrene D (10) (2%–3%) [This plant is used as a tonic, febrifuge and anthelmintic in traditional medicine. A powder of the plant mixed with honey is useful against worms. A tea prepared from the dried and chopped herb is drunk to treat asthma, weakness of the heart, and also as anti-inflammatory, diuretic, and anthelmintic ,7,12. A. (2%–3%) .A. rutifolia is most likely due to the thujones that are present in its essential oil. However, thujone has psychotropic effects, acting on the γ-aminobutyric acid-gated chloride channel, a member of the superfamily of ligand-gated ion channel receptors [A. rutifolia is rich in guaianolide, germacranolide, and eudesmanolide sesquiterpenoids [The bioactivity of eceptors . In addirpenoids ,30,31. Irpenoids ,33.A. scoparia are used to treat kidney disorders, as well as a diaphoretic, diuretic, and anthelmintic. Decoctions of the plant are considered useful against epilepsy, rheumatism, fever, and inflammation of the respiratory tract [A. scoparia are useful as an expectorant. A. scoparia essential oils from different geographical locations exhibit a great variability, but that from Tajikistan is dominated by the diacetylenes 1-phenyl-2,4-pentadiyne (34%) and capillene (14) (5%), as well as β-pinene (15) (21%), α-pinene (16) (5%), methyl eugenol (17) (6%), myrcene (9) (5%), limonene (8) (5%), and (E)-β-ocimene (4%) [In the folk medicine of Tajikistan, decoctions and infusions from the tops of the shoots of ry tract . Accordiry tract ,25 the antadiyne % and capPolyacetylenes from plants are known to be highly toxic against fungi, bacteria, and mammalian cells, and to display neurotoxic, anti-inflammatory and anti-platelet-aggregation effects and to be responsible for allergic skin reactions . BecauseB. persicum is extensively used in traditional medicine to treat chronic diseases of the stomach (chronic gastritis), intestines (colitis), liver (jaundice), chronic cholangitis, swelling, and also against kidney stones. Tea from B. persicum seeds is considered a popular means to increase the appetite. According to Sakhobiddinov [B. persicum essential oil has demonstrated antispasmodic activity in support of its traditional therapeutic use of the plant against gastrointestinal disorders [B. persicum is composed largely of cuminaldehyde (18) (36%–37%), γ-terpinen-7-al (19) (15%–17%), α-terpinen-7-al (20) (13%), γ-terpinene (21) (10%–11%), β-pinene (16) (9%), and p-cymene (5%). The composition of wild-growing B. persicum from Tajikistan is comparable to that found in commercial B. persicum from India: cuminaldehyde (30%), γ-terpinen-7-al (17%), α-terpinen-7-al (8%), γ-terpinene (11%), β-pinene (8%), anthemol (10%), and p-cymene (13%). biddinov , the frubiddinov ,14. B. pisorders . Oil froThe carbonyl group of aldehydes is very electrophilic and can react with a variety of nucleophiles such as amino groups either from proteins and nucleic acids ,40. CumiG. fragrantissima are a series of aliphatic aldehydes and alcohols such as (2E)-dodecenal (22) (84%), (2E)-dodecenol (23) (8%), (2E)-tetradecenal (24) (3%) and dodecanal (25) (2%).This plant is distributed in Afghanistan, Kyrgyzstan, Uzbekistan and Tajikistan. The leaves and young shoots are used as a spice for soups and other dishes . The maiG. fragrantissima oil exhibits cytotoxicity in HeLa, Caco-2 and MCF-7 cancer cell lines: IC50 values were 0.206 mg/mL for HeLa, 0.074 mg/mL for Caco-2, and 0.058 mg/mL for MCF-7 cell lines [E)-dodecenal (22), (2E)-tetradecenal (24) and dodecanal (25) are correlated to physico-chemical damage to the cells, such as the disruption of the membrane and probably interference with proteins [ll lines . The actproteins  and nuclproteins . Additioproteins .H. perforatum has been linked to the presence of hypericin and pseudohypericin [H. perforatum is widely used in the treatment of gallbladder conditions and cystitis, chronic gastritis, gastric ulcer and duodenal ulcers, wounds, burns, gout and rheumatism, to strengthen the gums and alleviate halitosis, and also to treat involuntary urination in children. H. perforatum is used as a wound-healing aid in France [H. perforatum “means for the ninety-nine disease.” According to Russian national doctors, “As without flour it is impossible to bake bread, so without H. perforatum it is impossible to treat many illnesses of people and animals.” [H. perforatum stimulates appetite, improves bowel, enhances diuresis, and has a styptic and general calming effect. Its tincture has a beneficial effect against chronic gastritis [H. perforatum extract reduced colonic damage, attributable to anti-inflammatory and antioxidant effects [H. perforatum based on essential oil composition and geographical location [H. perforatum oil was characterized by germacrene D (10) (14%) as a major secondary metabolite, α-pinene (16) (5%), β-caryophyllene (26) (5%), caryophyllene oxide (27) (4%), bicyclogermacrene (28) (4%), dodecanol (5%), and spathulenol (29) (3%) [St. John’s wort is a traditional medicinal plant in Europe and Asia. In modern phytotherapy, special extracts are employed with proven efficacy against depression ,2, possiypericin , along wypericin . In folkn France  and in Tn France . Flavonon France . The peonimals.” . H. perfastritis . In a ra effects ,54. Therlocation . Tajik H29) (3%) .Aspergillus niger as well as cytotoxic activity in PC-3 cells [in vitro cytotoxicity against MCF-7, PC-3, and Hep-G2 cells [Germacrene D has shown antifungal activity against -3 cells . β-Caryo-3 cells . CaryophG2 cells .H. scabrum is used in traditional medicine to treat a variety of disorders of liver, heart, stomach, intestines, bladder, cough, etc. A “marham” (poultice made from the herb and butter) is applied externally to treat sores, ulcers, abscesses, furuncles, and mastitis. An infusion of the flowers is recommended against jaundice [H. scabrum from Tajikistan is dominated by α-pinene (16) (45%), with lesser amounts of spathulenol (29) (7%), verbenone (30) (6%), trans-verbenol (31) (4%), and γ-muurolene (32) (4%) [H. scabrum essential oil [jaundice ,11. The 32) (4%) . Both H.tial oil  and methtial oil  have shotial oil . This plant is a perennial semi-shrub that can be found growing wild in the northwestern part of Tajikistan. Hyssop has been used in Tajik folk medicine for centuries. Avicenna recommended hyssop for its antiseptic, anti-inflammatory, wound healing, analgesic, antitussive and stimulating activities . DecoctiH. seravschanicus growing wild near the high ridge mountainous regions of Varzob, Northern Dushanbe in Tajikistan at an altitude approximately 2500 m above sea level, were extracted and analyzed using gas-liquid chromatography-mass spectrometry (GLC-MS). The most abundant components were cis-pinocamphone (33) (57%–89%), β-pinene (15) (up to 6%), 1,8-cineole (2) (2%–4%), camphor (34) (up to 4%), and spathulenol (29) (up to 5%). The essential oil from H. seravschanicus showed low antibacterial activity against Bacillus cereus and Staphylococcus aureus [cis-pinocamphone [The volatile compounds from s aureus , likely camphone ,64.Lemon balm has been used in Europe and Asia to reduce stress and anxiety, promote sleep, improve appetite, and ease pain and discomfort from indigestion . AvicennMelissa officinalis from Tajikistan were geranial (35) (43%), neral (36) (31.5%), trans-anethole (37) (12%), β-caryophyllene (26) (4%) and citronellal (38) (3%). Melissa officinalis essential oil was cytotoxic to MCF-7 cells (IC50 = 0.062 mg/mL) and active in the brine shrimp lethality test (LC50 = 21.8 μg/mL), but showed only marginal antimicrobial activity against Bacillus cereus (MIC = 313 μg/mL) and Aspergillus niger (MIC = 625 μg/mL) [in vitro cytotoxic activity [The main constituents of the essential oils of 5 μg/mL) . The bioonellal 3 (3%). Meactivity . Citral activity , while gactivity . Lemon bal (36) 3.5%, tranle (37) 1%, β-caryMentha species have been in use by humans since ancient times in many parts of the world [Mentha species are commonly used in many processed foods as well as in herbal teas. In folk medicine, mint is widely utilized as a tea or as a gargle against both acute and chronic conditions of the upper respiratory tract. It is prescribed against liver disorders , intestinal spasms, biliary tract infections (acute and chronic cholecystitis), and cholangitis [M. longifolia essential oils from different geographical locations varies considerably, even within Tajikistan [M. longifolia can be dominated by cis-piperitone epoxide (39) (up to 78%), piperitenone oxide (40) (up to 49%), carvone (5) (up to 22%), menthone (41) (up to 17%), as well as pulegone (42) (1%–5%) and thymol (2%–4%) [he world . The aerlangitis ,14. Mintlangitis . Mint islangitis . Mountaijikistan . Wild po (2%–4%) . The ess (2%–4%) .etc. The plant is widely used in food and oral care products and well known in folk medicine. In Tajik folk medicine, the water extract of basil is used to treat inflammatory diseases of the upper respiratory tract , chronic gastritis, enterocolitis, and food poisoning. Additionally, hot basil tea is taken to treat nausea, flatulence, and dysentery. The volatiles of basil are known to repel flies [(11) (47%), methylchavicol (estragole) (43) (32%), and pulegone (42) (5%). The major components in basil oil, linalool and methylchavicol, have shown anti-inflammatory activities [Basil is a popular plant cultivated in Europe and Asia, but also in Tajikistan and used frequently to flavor soups, desserts, pickles, pizza, spaghetti sauce, tomato juice, salads, el flies , mosquitel flies , and othel flies . The esstivities , supporttivities  and antitivities  activitiO. tyttanthum is a common species, this plant has commercial value. Cultivation of O. tyttanthum covers a total area of over 140,000 hectares, yielding annually a total of 6490 tons of air-dried raw materials [O. tyttanthum has traditionally been used as an expectorant, carminative, diaphoretic, stimulant, stomachic, and tonic. In addition, it has been used as a folk remedy against colic, coughs, headaches, nervousness, toothaches, and irregular menstrual cycles. Teas prepared from the aerial parts of O. tyttanthum have been used to treat tuberculosis and against human intestinal parasites. They are also used for sedative purposes and widely used against flatulence and as a gargle against laryngitis, stomatitis, and angina [(44) (34%–59%), thymol (45) (11%–46%), and p-cymene (46) (1%–7%) [O. tyttanthum from Tajikistan [In Tajikistan, where aterials . As a med angina ,14,37,82 (1%–7%) . The oinjikistan  as the mjikistan . Pelargonium graveolens L. Her. ex Ait. (synonym P. roseum Willd.) was introduced into Tajikistan approximately 80 years ago, and it does not have a traditional medicine background. In Tajikistan, it is cultivated for the production essential oil for perfumery and as a decorative ornamental plant. P. graveolens oil is a recommended aromatherapy for anxiety and depression, and the oil has demonstrated anxiety reduction after inhalation [P. graveolens is dominated by citronellol (47) (38%), lower amounts of geraniol (48) (6%) and linalool (11) (3%), along with numerous citronellol and geraniol esters  [halation . The Tajctively) ,87. S. sclarea is taken to improve digestion and appetite, and also as a diuretic. S. sclarea fruits are utilized to treat dysentery and bloody diarrhea. S. sclarea is applied externally to soften the skin [(49) and linalool (11), and Tajik oil is comparable with 39% linalyl acetate and 13% linalool, as well as germacrene D (10) (11%), α-terpineol (50) (6%), geranyl acetate (4%), and β-caryophyllene (2%) [Salvia are a rich source of polyphenols  which are believed to be responsible for the many biological activities of sage and their use in traditional medicine [Clary sage is a traditional medicinal plant in Europe and Asia . This plthe skin ,14,82. Cene (2%) . Plants medicine . Small lT. parthenium have been used for the treatment of migraine, arthritis, and problems associated with menstrual cycle. Parthenolide [T. parthenium. The essential oil of T. parthenium from Tajikistan revealed only eight components. The major components were camphor (34) (70%–94%), camphene (51) (2%–12%), and bornyl acetate (52) (4%–9%). [Feverfew is a traditional medicinal plant in Europa and Asia . Aerial henolide  and tanehenolide  are the (4%–9%). . The ess(4%–9%). ,40.Z. clinopodioides is an edible medicinal plant that is widely distributed in Tajikistan. The leaves, flowers and stem of the plant are frequently used as a wild vegetable or as an additive to foods. The plant has been used since ancient times in traditional herbal medicines for the treatment of colds and cough, stomach ache, nausea, poor appetite, sexually transmitted diseases, and as an antiseptic and to promote wound healing [Z. clinopodioides have been recognized according to the composition of the essential oil, namely pulegone-rich, thymol-rich, and cineole-rich chemotypes [Z. clinopodioides oil is a pulegone-rich oil, composed mainly of pulegone (42) (73%–35%), neomenthol (53) (7%–23%), and menthone (41) (6%–13%) [Z. clinopodioides oil has lipophilic properties, targeting biomembranes, which would explain its antiseptic properties [ healing ,12,14. Temotypes . Tajik Z(6%–13%) . Pulegon(6%–13%) . Pulegonoperties . The rich and diverse flora of Tajikistan provides numerous locally available plants useful in traditional herbal medicine, among them Tajik aromatic medicinal plants with bioactive essential oils. Many of them have shown antibacterial, antiviral, antioxidant, and cytotoxic bioactivities. The essential oils play a commercial role as flavors, fragrances, cosmetics, and pesticides. In addition, they are also used to prevent and treat infections and other human health disorders, including diabetes, ulcers, inflammation, cancer, and cardiovascular diseases. Interestingly, many essential oils from Tajikistan show remarkable differences in their compositions compared to samples from other geographical locations, which can be attributed to the very different climatic and topographic characteristics of the country. Future studies on ecological, phytochemical, and therapeutic characteristics of medicinal plants from Tajikistan, in comparison to other locations outside and within the country, are encouraged. In addition, we encourage the preservation of traditional knowledge and uses of medicinal plants and their conservation.


N-terminal hairpin turn, four kringle domains, and a serine protease-like domain. Several naturally occurring alternatively spliced isoforms of HGF were also identified. The NK1 variant contains the N-terminal hairpin and the first kringle domain, and the NK2 variant extends through the second kringle domain. These alternatively spliced forms of HGF activate the same receptor, MET, but they differ from the full length protein in their cellular activities and their biological functions. Here, we review the species-specific expression of the HGF isoforms, their regulation, the signal transduction pathways they activate, and their biological activities.Hepatocyte growth factor (HGF), also known as scatter factor (SF), is a pleotropic factor required for normal organ development during embryogenesis. In the adult, basal expression of HGF maintains tissue homeostasis and is up-regulated in response to tissue injury. HGF expression is necessary for the proliferation, migration, and survival of epithelial and endothelial cells involved in tissue repair in a variety of organs, including heart, lung, kidney, liver, brain, and skin. The administration of full length HGF, either as a protein or using exogenous expression methodologies, increases tissue repair in animal models of tissue injury and increases angiogenesis. Full length HGF is comprised of an  C-terminal multifunctional docking domain that was phosphorylated in response to HGF [Hepatocyte growth factor (HGF), or scatter factor (SF), was first identified as a factor from the plasma from humans and rabbits, and also rat platelets, that could induce the proliferation of hepatocytes in culture ,2,3. Fole to HGF ,22. Likee to HGF ,26,27,28e to HGF ,30,31. Te to HGF ,26.N-terminal hairpin loop and four kringle domains in the alpha subunit, and a serine protease-like domain in the beta subunit [HGF was identified by Northern blot, with an estimated size of 1.5 kb, with a predicted translation product of ~33 kDa protein containing the N-terminal hairpin loop and the first two kringle domains of HGF (named NK2) [N-terminal hairpin loop and the first kringle domain (named NK1) [Shortly after the discovery of HGF, several isoforms of the factor were identified. Full length HGF was shown by Northern blots to correspond to an mRNA of about 6 Kb, encoding a protein of ~82,000 kDa, that is proteolytically processed to produce a ~69 kDa alpha subunit with a single disulfide bond to a ~34 kDa beta subunit . Structu subunit ,33. An amed NK2) . A seconmed NK1) . This lamed NK1) . Both thmed NK1) ,37,38,39HGF is a high-molecular-weight polypeptide growth factors whose domain structure and mechanism of activation resemble those of the blood protease plasminogen, and belongs therefore to a family of plasminogen-related growth factors . BesidesHGF and MET related gene sequences of limited length and without clear functional similarity can be found in invertebrates as well. The most common transcript variant of HGF is the full length HGF. The human orthologs are 4792 nt (XM_006715956), 2820 nt (NM_000601), and 2805 nt (NM_001010932) long transcripts that contain 18 exons [Functional orthologs of HGF and MET can be found in vertebrates from bony fishes to humans, and 18 exons . The firNK1 isoform is the shortest functionally active isoform of HGF. In humans, it is transcribed as a 2079 nt-long transcript (NM_0010934) consisting of five exons that encode a 201 aa polypeptide. NK1 sequences are predicted in several other species including the mouse , rabbit , opossum , and non-human primates . They are highly similar among different species. Primates share ~98.5% sequence identity, and the murine and human NK1 coding regions are 88.63% identical. The NK1-3'UTR of primates are ~95.7% identical, but the 3'UTR of rabbit and human share 71.57% identical sequence. The 3'UTR of the murine NK1 is only 48.85% identical to the human. The HGF gene contains an alternative splice acceptor in the intron that follows the five coding exons among these species.The HGF is the NK2. It is transcribed in humans as a 1292 nt (NM_001010933) or 1307 nt (NM_001010931) mRNA of seven exons, encoding 284 aa or 290 aa, respectively.The other functionally active isoform of NK2 similar sequences in several other species like Chrysochloris asiatica , Ceratotherium simum simum  and Canis lipus familiaris , in addition to primates (HGF sequence does not contain the splice sites required for the generation of NK2 (NK2 isoform was not expressed in mice [Pan troglodytes (chimpanzee) XM_003318558 and Nomascus leucogenys (gibbon) XM_003252220 share 97% similarity with the two NK2 isoforms (NM_001010933 and NM_001010931) of human  of its regulation in development/growth, tissue repair, fibrosis, and oncogenesis [HGF expression is positively regulated by other growth factors, such as epidermal growth factor, platelet-derived growth factor, and several members of the fibroblast growth factor family [HGF expression is also increased in response to inflammatory cytokines interleukin (IL)-1α and -1β, IL-6, IL-8, and interferon-γ [HGF expression during early phases of inflammation that precede tissue repair. Analyses of the HGF promoter revealed functional cis-elements for transcription factor binding including IL-6 response elements, activating protein-2 (AP2) elements, nuclear factor-1 (NF1) elements, nuclear factor-IL6 elements, a cyclic AMP response element, several binding sites for the SP family of transcription factors, an upstream stimulatory factor (USF) element, a chick ovalbumin upstream promoter-transcription factor element, a complex C1 element, and the cell type specific CCAAT/enhancer binding protein (C/EBP) binding element [HGF promoter, the peroxisome proliferator-activated receptor gamma (PPAR γ)-responsive element, was shown to strongly regulate HGF expression in response to either PPAR γ 1 or PPAR γ 2 ligands, but because this binding site overlaps with the NF1 and chicken ovalbumin upstream promoter-transcription factor elements, the binding of these other factors repress PPAR γ activation [HGF in response to tissue injury and/or inflammation is believed to occur through factor binding to the C/EBP element [ogenesis ,47,48. Hr family . HGF exprferon-γ ,51,52,53 element ,55,56,57HGF expression is tightly regulated, and several elements within the promoter suppress its expression. Binding of activating protein-2 (AP2) to a regulatory site −230 to −260 bp upstream of the transcriptional start site suppresses HGF expression [HGF expression, a repeat of 30 deoxyadenosines (termed “deoxyadenosine tract element” or DATE) is located about 750 bp upstream of the HGF start site [HGF dysregulation in breast cancer [pression . Anotherart site . The DATt cancer .HGF expression is potently suppressed in fibroblasts and other cell types by the pro-fibrotic cytokine transforming growth factor beta 1 (TGF-β1), by hypoxia, and by glucocorticoids [HGF occurred without affecting NK2 expression in the cells [HGF by TGF-β1 occurs through TGF-β1 up-regulation of miR-199a-3p [HGF mRNA 3'UTR leading to degradation of HGF mRNA. The NK2 mRNA has an alternative 3'UTR sequence that is not regulated by miR-199a-3p [rticoids ,60. In ahe cells . Recent -199a-3p . The miR-199a-3p .1234 and Y1235), two other phosphotyrosines in the carboxy-terminal multifunctional docking domain, Y1349 and Y1356, regulate downstream signaling [HGF is a pleiotrophic factor, inducing motogenesis, mitogenesis, survival, and in some cell types, morphogenesis ,62,63. Hignaling ,66,67,68ignaling ,75,76,77ignaling ,79. Whilignaling ,79. Morpignaling ,82,83,84Following their initial discoveries, it was demonstrated that NK1 and NK2 bound to MET and induced its phosphorylation in a competitive manner with full length HGF ,36,38,85NK1 induced motility in Madin Darby canine kidney (MDCK) cells and 32D cells expressing MET ,87,88,89in vivo, a study of melanoma cells in NK2 transgenic mice demonstrated that the tissues of these mice had reduced MET phosphorylation and reduced p42/p44 MAPK activation in contrast with HGF transgenic mice, suggesting that signaling by this isoform in vivo is compromised [Like NK1, NK2 induced motility in MDCK cells, but NK2 failed to induce motility in human umbilical vein endothelial cells ,86,87. Ipromised . The sigHGF die in utero during early stages of embryogenesis [HGF plays an essential role during development in the placenta , liver , and kidogenesis . A patteogenesis ,101.Although the expression patterns and effects of HGF have been investigated in development, the roles of the two naturally occurring isoforms of HGF, NK1 and NK2, in development are not yet understood. One study of HGF isoform expression during development in macaques found low levels of expression of both NK1 and NK2 as well as novel forms of NK1 and NK2 (dNK1 and dNK2) with a five amino acid deletion in the first kringle domain . IncreasHGF is a growth factor, survival factor, and mitogen for epithelial and endothelial cells from the lung, skin, kidney, liver, heart, and brain ,110,111.in vivo [4 treatment [HGF expression is greatly increased in response to most types of tissue injury . HGF expin vivo ,132. Addin vivo ,133,134.in vivo ,136. Alsreatment . Severelreatment . The faireatment . Wound hreatment .The administration of HGF, whether as a purified protein or through ectopic expression methods, was demonstrated to increase normal tissue repair in experimental injury models of the lung, skin, liver, kidney, heart, pancreas, and brain, and improves angiogenesis ,156,157.in vivo has been shown to have varying effects on cellular proliferation, survival and migration, cellular events that are all believed to be required for normal tissue repair.The basal expression of NK1 and NK2 has been observed in multiple tissues, including in normal fibroblasts from the lung . Interesin vivo and to reduce liver fibrosis in murine partial hepatectomy model [In cell culture, NK1 has been shown to act as a mitogen and anti-apoptotic factor for normal hepatocytes in culture . Transgemy model . Adminismy model .in vivo [Murine models of tissue injury suggest that NK2 does not induce the proliferation of normal cells and may inhibit normal tissue repair. NK2 transgenic mice did not exhibit the phenotypic consequences observed in response to HGF over-expression, including the inhibition of hyperplastic lesions of the kidney or olfactory mucosa, and abnormalities of the mammary gland and skeletal muscle . NK2 train vivo ,147. In in vivo .4-induced liver injury, but did not inhibit endogenous hepatocyte proliferation following liver injury [4-induced liver injury in mice, it was found that NK2 promoted liver fibrosis and prevented normal tissue repair [In an early investigation of a murine model of liver injury, the inducible expression of HGF both prevented the induction of liver injury and induced tissue repair . In contr injury . In thisr injury . In a lae repair .MET, originally identified as a proto-oncogene [Expression of HGF and/or its receptor MET is increased in a wide variety of cancers . The HGFoncogene ,180, wasoncogene ,185,186.oncogene ,187,188.oncogene ,193,194.oncogene ,197,198.in vitro [Early studies suggested that NK1 and NK2 did not induce cancer cell proliferation in cell culture, and that the activation of motility was also reduced in some cancer cell types. For instance, both NK1 and NK2 inhibited the proliferation and motility of A549 lung cancer cell line in vitro . In thesin vitro . Howeverin vitro ,199; thein vitro ,90,200. in vitro ,201.In vivo studies challenged the original views of NK1 and NK2 as inhibitors of cancer. Transgenic mice over-expressing NK1 had a number of similar oncogenic events as found in mice over-expressing full length HGF, including mammary, hepatocellular, and melanocytic tumors [in vivo [NK2 gene transfer was also demonstrated to inhibit proliferation of glioma cells in vivo [c tumors . In tran[in vivo . NK2 gen in vivo . However in vivo .HGF gene therapy is safe and improved wound healing, reduced the necessity for amputation, improved pain at rest, and improved hemodynamic measurement without adverse effects on the quality of life in the critical limb ischemia population. HGF is also under investigation for treatment of diabetic peripheral neuropathy [HGF is in Phase III Clinical Trials to treat severe peripheral arterial disease in Japan [HGF gene-transfer is under investigation as a potential treatment for coronary artery disease in a Phase I Clinical Trial in China [HGF has been used successfully as both protein and DNA as a therapeutic agent in preclinical animal models for ischemic heart disease, renal fibrosis, pulmonary fibrosis, and for other diseases where there is a need for increased tissues repair ,202,203.uropathy . Furtherin Japan , and adein China . Phase IN-terminal hairpin domain and the subsequent four-kringle domains, and has been shown to act as an antagonist to HGF monogenic activity [NK4 in gene therapy for the treatment of cancer, including pancreatic cancer, gastric carcinoma, hepatocellular carcinoma, breast and endometrial cancer, lung cancer, and prostate cancer [+ T cells, preclinical models have also shown that NK4 may provide a new approach for the treatment of rheumatoid arthritis [NK4, an artificial HGF isomer, contains the activity . Based oe cancer ,213,214.rthritis ,216. TheIn contrast with full length HGF and the synthetic HGF antagonist NK4, the naturally occurring isoforms of HGF are not currently in clinical trials. Although NK1 has been shown to induce all the signal transduction pathways of full length HGF, its lower level activity suggests that it may not be as beneficial as full length HGF for clinical applications. Additionally, NK2, though its ability to antagonize HGF proliferation could be useful for anti-cancer therapy, however its partial activation of MET may also preclude this application.The naturally occurring isoforms of HGF, NK1 and NK2 are expressed in human tissues during development and in normal adults. Studies in cell culture and in transgenic animals suggest that NK1 is capable of recapitulating normal HGF signaling and biological activities, while NK2 appears to be an antagonist for HGF-induced cellular proliferation. NK2 expression is increased relative to full length HGF in human fibrotic organ diseases, and it is possible that NK2 may play a role in the failure of normal repair. The normal biological roles of the HGF truncated isoforms remain to be determined. Further understanding of the normal functions of these proteins may provide insight for their use in human diseases.


To assess the prognostic roles of BAP1, PBRM1, pS6, PTEN, TGase2, PD-L1, CA9, PSMA, and Ki-67 tissue biomarkers in localized renal cell carcinoma (RCC).Patients who underwent a nephrectomy during 1992–2015 and had a primary specimen of their kidney tumor were included. The nine tissue biomarkers were immunohistochemically stained on tissue microarrays of RCC, and the semi-quantitative H-score, including intensity score, was used to grade the sample. The Cox proportional hazards model was used to evaluate tissue markers significant for overall survival (OS), cancer-specific survival (CSS), and recurrence-free survival (RFS) after adjusting for significant clinicopathological parameters.Samples from 351 RCC patients were included. The mean age of the patients was 53.9 years; the rates of pathologic T1-2/≥T3 stage, Fuhrman 1+2/3+4 grade, recurrence, and death were 269/65(80.5/19.5%), 222/107 (67.5/32.5%), 6.6%, and 10.5%, respectively. Median OS, CSS, and RFS were 220.6, 220.6, and 147.1 months, respectively. The multivariable analysis showed that pathologic T stage and Fuhrman nuclear grade were significantly associated with OS and CSS. Pathologic T stage and tumor size were associated with RFS. After adjusting for these significant prognostic clinicopathological factors, Ki-67 was significantly associated with OS , CSS , and RFS  and pS6 was associated with CSS  and RFS  in the multivariable model (p<0.05).pS6 and Ki-67 are significant prognostic factors of RCC; however, BAP1, PBRM1, TGase 2, PD-L1, CA9, PTEN loss, and PSMA markers did not show this association. Two-thirds of all newly diagnosed renal cell carcinoma (RCC) cases are localized , with 30Prognostic markers of RCC are important for clinicians to prevent recurrence following surgical therapy. Significant prognostic clinicopathological features such as TNM stage, Fuhrman nuclear grade, histologic subtype, lymphovascular invasion and sarcomatoid differentiation have been identified ; howeverTissue microarrays (TMA) combined with immunohistochemistry (IHC) permits the analysis of large cohorts, leading to a better understanding of disease pathogenesis, differentiating between disease characteristics, and determining the origin of metastatic cancers without a clear tissue of origin. It can encompass a wide spectrum of diverse tumor presentations and disease states for tumor marker analysis in order to predict clinical behavior and prognosis . Herein,Following approval of this retrospective study by the Institutional Review Board (IRB) of the National Cancer Center (IRB No. NCC 2015–0219), an exemption was granted for the need of written consent from patients. This study was conducted according to the principles expressed in the Declaration of Helsinki.All patients with RCC who underwent either a radical or partial nephrectomy between 1992 and 2015, with available primary tumor and control specimens of the kidney cancer were included in the study. All samples were reviewed retrospectively by one 15-year experienced uropathologist (WSP) in a blinded manner, according to the guidelines of WHO/International Society of Urological Pathology (ISUP) consensus conference [The IHC of the TMA was performed using a previously described method . Thirty The nine markers, BRCA1 associated protein-1  (BAP1), polybromo 1 of chromatin-histone regulator gene (PBRM1), phosphorylated S6 protein (pS6), phosphatase and tensin homolog (PTEN), tissue glutaminase, protein-glutamin γ–glutamyltransferase (TGase-2), programmed death-ligand 1 (PD-L1), carbonic anhydrase 9 (CA9), prostate-specific membrane antigen (PSMA), and Ki-67 were assessed by IHC staining of the TMA blocks, using a standard protocol and an automated immunostainer . After deparaffinization of the FFPE block, heat-induced antigen retrieval was performed in solution, according to the standard protocol , and reaThe expression score of IHC, defined as the staining intensity multiplied by the percent tumor positive area, was semi-quantitatively determined by the H-score (0–300), calculated by the multiplication of the intensity score  by the area of expression (0–100%) , 12, 13.To examine the prognostic value of these tissue biomarkers in terms of overall survival (OS), cancer-specific survival (CSS), and recurrence-free survival (RFS), previously identified clinicopathological variables associated with prognosis were analyzed using a Cox proportional hazards model. Significant clinicopathological variables in the univariable model were included into a multivariable model of clinicopathological variables, using a backward variable selection method with an elimination criterion of 0.05. After adjusting significant clinicopathological variables, the nine tissue biomarkers were respectively evaluated in the final multivariable model. The results were presented as hazard ratios (HR) with their 95% confidence intervals (CI). P-values of less than 0.05 were considered statistically significant. All statistical analyses were performed using SAS version 9.3 .The mean age of the patients was 53.9 years , and the rate of T1-2 pathological stage, Fuhrman 1+2/3+4 grade, recurrence, and death were 269 (80.5%), 222 (67.5%), 6.6% (n = 23), and 10.5% (n = 37), respectively. Median OS, CSS, and RFS were 220.6, 220.6, and 147.1 months, respectively. The remaining clinicopathological characteristics of the patients and the results of IHC staining for each tissue biomarker are summarized in According to the univariable analysis, the clinicopathological parameters associated with poorer prognosis in terms of OS were male sex , diabetes , smoker , stage ≥T3 , Fuhrman grade 3+4 , sarcomatoid differentiation , lymphovascular invasion , tumor size , and capsular invasion ; in terms of CSS were diabetes , stage ≥T3 , tumor size , Fuhrman grade , sarcomatoid differentiation , lymphovascular invasion , and capsular invasion ; and in terms of RFS were stage ≥T3 , tumor size , Fuhrman grade 3–4 , sarcomatoid differentiation , lymphovascular invasion , and capsular invasion , all with p-value less than 0.05 . The ninIn the multivariable analysis after adjusting for the significant prognostic clinicopathological variables identified above, pathologic T stage and Fuhrman grade were associated with poorer OS and CSS, and pathologic T stage and tumor size were associated with a shorter RFS (p<0.05). Ki-67 remained prognostic for OS , CSS , and RFS  p<0.05, , whereasFor several decades, clinicians have observed the limited accuracy and value of the currently used clinicopathological variables and have attempted to search for new specific molecular tissue biomarkers , 14. ThiThe expression levels of each of the tissue markers were similar to those reported in previous studies, as shown in The pS6 protein was more frequently expressed in metastases than in the primary RCC . OverexpAs for the other candidate tissue biomarkers, this study showed none to be significantly prognostic. The BAP1 loss and PBRM1 loss were recently found to be poor prognostic biomarkers for clear cell RCC, whereas Kapur et al.  demonstrThe study showed PTEN loss, CA9, TGase-2, and PD-L1 were not prognostic markers p>0.05, . PTEN loAs for clinicopathological factors, male sex (only for OS), diabetes (only for OS and CSS), smoker (only for OS), stage ≥T3, tumor size, Fuhrman grade 3+4, sarcomatoid differentiation, lymphovascular invasion, and capsular invasion were associated with prognosis in terms of OS, CSS and RFS in the univariable model p<0.05, . In the The study had several limitations, including its retrospective design, and some technical errors in TMA preparation. The expression levels of nine proteins were evaluated by IHC staining, which can be variable in experimental setting, as well as in the interpretation of the scoring. Nevertheless, this study is the first to evaluate the clinical implications of these nine biomarkers relating to the pathophysiology of RCC, including disease progression, in a large TMA-IHC series. Finally, this study showed that none of the markers, except for pS6 and Ki-67, had predictable roles in disease prognosis for primary RCC. A further study assessing differences between these prognostic factors for metastatic lesions and primary tumors using tissue biomarkers, and associations with prognostic effect in metastatic RCCs would be needed.This TMA study showed only pS6 and Ki-67 biomarkers are prognostic for survival after adjusting for clinicopathological parameters, whereas BAP1 loss, PBRM1 loss, TGase 2, PD-L1, CA9, PTEN loss, and PSMA markers did not have any prognostic role in determining OS, CSS, and RFS.S1 FigDistribution of the Ki-67 and pS6 markers in the primary renal cell carcinoma specimen; (1) Representative photomicrographs of Ki-67 immunohistochemistry . All immunohistochemical staining was scored as either positive (scores of 2 and 3) or negative (scores of 0 and 1); (2) Representative photomicrographs of pS6 (X60) immunohistochemistry; A, intensity score 0; B, intensity score 1; C, intensity score 2; D, intensity score 3.(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


DMRTC2, PAX7, BRACHYURY/T, and TERT were associated with defective mini-puberty and were responsive to GnRHa. These results indicate novel testosterone-dependent genes and provide valuable insight into the transcriptional response to both defective mini-puberty and curative GnRHa treatment, which prevents infertility in man with one or both undescended (cryptorchid) testes.Defective mini-puberty results in insufficient testosterone secretion that impairs the differentiation of gonocytes into dark-type (Ad) spermatogonia. The differentiation of gonocytes into Ad spermatogonia can be induced by administration of the gonadotropin-releasing hormone agonist, GnRHa ). Nothing is known about the mechanism that underlies successful GnRHa treatment in the germ cells. Using RNA-sequencing of testicular biopsies, we recently examined RNA profiles of testes with and without GnRHa treatment. Here, we focused on the expression patterns of known gene markers for gonocytes and spermatogonia, and found that  During mini-puberty, which occurs between 30 and 90 days of postnatal life in male infants, the substantial increase in gonadotropin releasing hormone (GnRH) secretion induces gonadotropin and testosterone production ,2,3. As Cryptorchid boys, of a median age of eight years, who were treated with a gonadotropin-releasing hormone agonist, showed post-puberty improved sperm concentrations, when compared to an untreated control group . The treThough expression patterns may differ, development in both humans and mice appear to involve a similar set of genes, which can also be used as markers to distinguish gonocytes and spermatogonia . BecALPP, NANOG, POU5F1, and TFAP2C encode transcription factors known to be important for pluripotency. The differentiation of gonocytes into spermatogonia is associated with upregulation of certain genes, including MAGEA4, DDX4 and TSPY1 [POU5F1, ALPP, TFAP2C, NANOG and KIT are downregulated [ALPP/PLAP, EPS8, KIT/c-KIT, NANOG, POU5F1 and TFAP2C/AP2γ are gonocyte markers, while nd TSPY1 ,17, whilegulated ,19,20,21DMRTC2, PAX7, BRACHYURY/T, and TERT genes to be associated with defective mini-puberty and responsive to GnRHa.In this study, we investigated the molecular events underlying human male germ cell development, focusing on the testosterone-dependent transition from gonocytes to Ad spermatogonia as well as the molecular impact of early GnRHa (Buserelin INN) treatment. Utilizing testicular gene expression profiles from testes with insufficient testosterone secretion, before and after GnRHa administration, and testes with completed mini-puberty, we identified the We selected 15 patients with isolated cryptorchidism, based on histological results, and divided them into 2 groups. Seven belonged to the Ad− (lacking Ad spermatogonia) and 8 to the Ad+ (presenting Ad spermatogonia) group. Data from Ad− bilateral cryptorchid boys treated with GnRHa (Buserelin) following the first orchidopexy (surgery) (4 patients) were retrieved from an ongoing randomized study. Initial biopsies revealed no Ad spermatogonia, indicating defective mini-puberty (Ad− group). The second testis was managed by orchidopexy and biopsied 6 months after the initial surgery. Thus, results from 19 biopsies were compared. Patients were age and ethnicity matched. RNA sequencing data from manually selected germ cell marker genes from our two previous studies ,23 were A cryptorchid testis is defined as a testis localized outside of the scrotum and incapable of being brought into a stable scrotal position. All undescended testes in this study were located in the inguinal region. Testicular biopsies were taken at the time of orchidopexy. This sample was then subdivided, with one fragment fixed in glutaraldehyde for histological processing, while the other one was immediately immersed in RNAlater  and stored at –25 °C until further processing (for RNA extraction and RNA- sequencing).Biopsies were fixed in 3% glutaraldehyde in phosphate-buffered saline  and then embedded in Epon resin. Semi-thin sections (1 μm) were cut using a Reichert Om-U3 ultramicrotome . Sections were mounted on glass slides, stained with 1% toluidine blue, and examined under a Zeiss Axioskop light microscope  with an integrated photo-camera. Biopsies were histologically examined by two of the authors (F.H. and D.D.), each with expertise in the interpretation of semi-thin sections of prepubertal testes.During histological analyses, at least 100 tubular cross sections per biopsy were evaluated, with regard to their number and absence of Ad spermatogonia. In the prepubertal testes, Ad spermatogonia were identified according to the criteria first published by Seguchi and Hadziselimovic . This tyThe workflow from RNA isolation, through to purification, library preparation, sequencing, data analyses, and expression level analyses, has been described previously ,23.Determination of differentially expressed genes, statistical analyses and model design were described previously ,23. OnlyTwo gonocyte marker genes, 19 spermatogonial marker genes and 8 putative spermatogonia genes, all of which are differentially expressed between the two groups (Ad− and Ad+), were used as input to obtain the protein–protein interaction network using STRING version 10.0 .Investigations were carried out in accordance with the Declaration of Helsinki of 1975, revised in 2008. All aspects of this study were approved by the Institutional Review Board and the Independent Ethics Committee of Vilnius University. Approval was also provided for research involving the use of material (data records or biopsy specimens) that had been collected for non-research purposes .Here, we focused on selected marker genes for gonocytes and Ad spermatogonia . Gonocytes are defined as small cells originating from the primordial germ cells and localized predominately in the center of the tubule and small typical mitochondria. They give rise to the fetal spermatogonia, which are the largest germ cells in prepubertal testis . This tyPOU5F1 and TFAP2C showed lower expression levels in Ad− testes with testosterone deficiency  as well as mitotic-to-meiotic germ cell transition and differentiation   was less expressed. Interestingly, 19 marker genes showed no difference in expression . There were no increased RNA levels observed for any of the gonocyte and spermatogonial markers in testes without Ad spermatogonia  . These r-puberty ,28 and eatogonia .EPS8 and KIT showed decreased RNA expression after GnRHa treatment , and the expression levels of ETV5, ID4 and UCHL1 were lower than in testes with Ad spermatogonia, indicating that GnRHa treatment further decreased the expression of these genes  responded with an increase in RNA expression. TSPAN8 was not differentially expressed between Ad− and Ad+ testes. PAX7, BRACHYURY/T, and TERT were both less expressed in Ad− testes and upregulated following GnRHa treatment  in the context of physical protein–protein interactions and functional interactions, by integrating our data with information available in the literature . Markerg-db.org . Especiag-db.org .POU5F1 encodes a transcription factor that plays a key role in both embryonic development and stem cell pluripotency. In human fetal gonads, POU5F1 regulation differs in male and female germ cells. While POU5F1 expression is gradually downregulated during gonocyte differentiation, in human males, in females it is downregulated much earlier in fetal life, when the oocytes enter the first meiotic prophase [POU5F1 and TFAP2C RNA, observed in Ad− testes compared to Ad+ testes, lead to the assumption that they play an important role in LH and in the testosterone-dependent gonocyte-to-Ad spermatogonia transition.prophase . TFAP2C/prophase . The redETV5 encodes a glial cell-derived neurotrophic factor (GDNF)-inducible transcription factor, regulating several genes known to be important for stem cell self-renewal, including BCL6B, LHX1, CXCR4, BRACHYURY/T, and RET [ETV5-null mice are infertile demonstrates the importance of ETV5 for spermatogenesis [Brachyury/T silencing significantly reduces the number of donor cell-derived colonies formed in recipient mouse testes, suggesting that BRACHYURY/T functions as a part of GDNF/ETV5 signaling to promote self-renewal [ETV5, CXCR4, BRACHYURY/T, and RET—which indicates disturbed GDNF-dependent self-renewal in the germ cells. and RET ,33. The ogenesis . Moreove-renewal . BRACHYU-renewal ,36. Ad− The pluripotency transcription factor, SALL4, was found to be localized to primordial germ cells and most gonocytes in the prenatal and early postnatal testes, as well as in undifferentiated spermatogonia of marmoset, human and mouse pubertal and adult testes ,38. SALLNanos3 deficiency results in the loss of primordial germ cells during migration and leads to sterility in male and female mice [Nanos2 loss results in decreased germ cell numbers and causes infertility in only male mice [Nanos2 expression is directly dependent on retinoic acid (RA) for its downregulation and fibroblast growth factor 9 (FGF9) for its upregulation [Nanos2 expression is restricted to prenatal germ cells and a small number of spermatogonia in adult mice [Nanos2, Sohlh1 and Sohlh2 all play substantial roles in male germline development in mice, and our results strongly support the notion that they fulfill the same role during mini-puberty in humans.NANOS2 and NANOS3 are RNA binding proteins from the NANOS family. In mice, a ale mice . In contale mice . Murine ale mice . Furthergulation . While Nult mice ,43, NANOult mice . Therefoult mice ,46. In sPax7 expression [PAX7 expression in Ad− testes with low testosterone levels as well as increased PAX7 expression in the testes of boys with increased testosterone levels, following GnRHa treatment.Testosterone treatment of mouse satellite cells was shown to increase pression , supportLim and colleagues observed a predominant expression of the transcription factor POU2F2 in Ad spermatogonia . AlthougTERT) reporter mice [TERT expression levels relative to normal testes, leading to the suggestion that telomerase plays a role in maintaining germ cell proliferation [TERT mRNA expression level was shown to be effective in both classifying spermatogenesis disorders in patients, and in predicting successful sperm recovery in azoospermia patients [TERT expression in human primary hematopoietic cells [TERT expression. This supports both, our finding of reduced TERT expression in lower testosterone Ad- testes, but also the observed increased TERT expression in Ad– testes with increased testosterone levels after treatment. Additionally, GnRHa treated testes showed significantly increased CYP19A1 gene expression levels (absolute fold change logFCGnRHa + 2.51) [High telomerase expression levels were found to be a hallmark of undifferentiated spermatogonia using telomerase reverse transcriptase  .BRACHYURY/T, has yet been reported. However, BRACHYURY/T was shown to bind to the promotor of the androgen receptor (AR) and regulate AR expression in prostate cancer cells [BRACHYURY/T levels after GnRHa treatment point towards its role in Ad spermatogonia formation. This is a new observation which indicates this gene to be an important marker of Ad spermatogonia.Neither testosterone, nor LH dependent gene expression of er cells . IncreasEGR3 expression is stimulated by RA and downregulated by KIT Ligand (KITLG) [EGR2 and EGR3 are transcription factors used as spermatogonial markers, and EGR3 immunoreaction was reported in A single or paired germ cells in mice . Further (KITLG) .NRG1 was upregulated, while KITLG was downregulated (logFCGnRHa − 1.10).The neuregulins, NRG1 and NRG3, are essential for the proliferation of spermatogonia and the initiation of meiosis . NRG1 anRBMY is expressed only in spermatogonia and early spermatocytes [While mouse atocytes  and its atocytes , human Ratocytes ,62. TherDMRTC2/DMRT7 and TSPY4 gene expression. DMRT7 mutant mice show meiotic arrest at the pachytene stage [TSPY4 is unknown, from sequence similarity it is assumed to be involved in sperm differentiation and proliferation . Interestingly, TSPY was reported to bind to androgen receptor and AR variants, and thereby increase the transactivation of the AR/AR-V7 target genes [We also found a treatment related increase in ne stage ,64, and et genes . WhetherHere, we report that testes with defective mini-puberty, with lower testosterone levels, and lack of Ad spermatogonia had significant lower RNA levels for selected gonocyte and spermatogonial marker genes (21 genes) relative to testes with Ad spermatogonia. We suggest that these differentially expressed genes reflect molecular functions involved in the gonocyte-to-Ad spermatogonia transition in humans during mini-puberty. Furthermore, we propose that higher expression levels of these 21 genes in testes presenting Ad spermatogonia are the result of testosterone-dependent expression, since the lack of testosterone increase during mini-puberty causes developmental arrest. The finding of four gonocyte markers and 15 spermatogonial marker genes that are not differentially expressed argues against a dilution effect and supports the importance of these findings.PAX7, BRACHYURY/T, and TERT responded positively to GnRHa treatment, and were markers with reduced expression in Ad− testes. We suggest that the genes DMRTC2, EGR2, NRG1, NRG3, RBMY1B, RBMY1E, RBMY1J and TSPY4 represent potential new markers for spermatogonia in infant testes, and that they may have key functions in the gonocyte-to-Ad spermatogonia transition. Additionally, it seems likely that they are testosterone-responsive genes, given that GnRHa treated boys were reported to show increased testosterone and LH levels.EGR2, DMRTC2, PAX7 and BRACHYURY/T, the growth factor like proteins, NRG1 and NRG3, and the RNA binding and Y chromosome encoded genes, RBMY1B, RBMY1E, RBMY1J and TSPY4.The absence of an apparent GnRHa stimulation for 11 out of 16 genes remains unexplained, but one possibility could be that they are epigenetically downregulated and therefore unresponsive to GnRHa treatment at the mRNA level. Nonetheless, the absence of GnRHa-responsive key players strengthens and supports the need for alternative pathways, for which we suggest the stimulation of the transcription factors, ETV5 and ID4), others control the differentiation process (DMRT1 and UCHL1). Similarly, genes that respond positively to GnRHa treatment are also involved in self-renewal (PAX7 and BRACHYURY/T) and differentiation . The differentially expressed genes EGR2, ETV5, ID4, TSPAN8 [T [FOXO1 [KIT [NANOS2 [NRG1 and NRG3 [PAX7 [PAX7, BRACHYURY/T, EGR2, NRG1, and NRG3) are therefore linked to both FGF/GDNF and RA signaling.A clear pattern was not observed regarding the function of GnRHa-responsive and unresponsive genes in germ cell differentiation or gene regulation. While some of the negatively regulated genes are involved in self-renewal , suggesting expression induced by GnRHa. LH-dependent testosterone secretion was reported to regulate SSC self-renewal by suppressing Wnt5a expression in mouse Sertolli cells [WNT5A expression (logFCGnRHa − 0.86), suggesting a similar testosterone-dependent regulation of SSC self-renewal, by Wnt5a suppression in humans.The balance between self-renewal and differentiation depends not only on the described intrinsic factors, but also on extrinsic factors, including GDNF, RA, WNT and testosterone signaling. Spermatogonial cell development in mice depends on testosterone-dependent secretion of GDNF by peritubular myoid cells . GDNF exli cells . GnRHa tDMRTC2, PAX7, BRACHYURY/T, and TERT, highlights their importance for the development of Ad spermatogonia with specific functionalities in self-renewal and differentiation, and following GnRHa curative treatment. We suggest that GnRHa induced testosterone and a LH increase reconstitute self-renewal properties of the Ad spermatogonial stem cells, and induce RA-responsive genes, such as NRG1, NRG3 and PAX7, to help prepare them for commitment to differentiation.Our differential gene expression profiling of gonocyte and spermatogonial markers, particularly EGR4 and PITX1 controlled by PROK2/CHD7/FGFR1/SPRY4 genes expression is responsible for LH deficiency, which in turn affects the germ cell transitional effectors, FGFR3, FGF9, NANOS2, NANOS3, SOHLH1 and SOHLH2. Upon GnRHa treatment, however, alternative pathways are activated, including the LH-secretion orchestrating factors, EGR2, EGR3, TAC1, TAC3, PROP1 and LEP, and the gonocyte-to-Ad spermatogonia transition effectors, DMRTC2, T, PAX7, TERT, NRG1, NRG3, RBMY1B, RBMY1E and RBMY1J.Together with our earlier observations on the level of the HPG-axis of differentially expressed genes in Ad– testes ,23, we s


Malignant mesotheliomas are aggressive, asbestos-related cancers with poor patient prognosis, typically arising in the mesothelial surfaces of tissues in pleural and peritoneal cavity. The relative unspecific symptoms of mesotheliomas, misdiagnoses, and lack of precise targeted therapies call for a more critical assessment of this disease. In the present review, we categorize commonly identified genomic aberrations of mesotheliomas into their canonical pathways and discuss targeting these pathways in the context of tumor hypoxia, a hallmark of cancer known to render solid tumors more resistant to radiation and most chemo-therapy. We then explore the concept that the intrinsic hypoxic microenvironment of mesotheliomas can be Achilles' heel for targeted, multimodal therapeutic intervention. Determining cancer etiology is an intricate process because evidence from genetics, cellular and molecular biology, as well as epidemiology and pathology must be integrated to gain a complete understanding of carcinogenicity . MalignaThe annual incidence of malignant mesothelioma in the United States alone is approximately 3,200 cases/year -11 and eBAP1 germline mutations [BAP1 mutations are at increased risk of mesothelioma when exposed to asbestos, including low doses that usually are not sufficient to cause cancer [Mesothelioma is often caused by asbestos exposure , 24. Howutations . Moreovee cancer , 36. In e cancer , chronice cancer , 39, ione cancer , 41, ande cancer -46. Secoe cancer , 48, bree cancer , and tese cancer .To reliably diagnose and determine pathological subtypes of the disease, diagnostic surgery and biopsies of malignant tissues are needed so they can be subjected to further histological examination , 52. MesBAP1 , NF2 [, LATS1,2 [PBRM1 [TP53 [AURKA [CDKN2A [RB1 [BRCA2 [CCND1 [SETD2 [SMARCC1  or PCNA [Although the number of genomic aberrations in mesothelioma is typically lower when compared to other cancers , genome-P1 , NF2 , LATS1,2 LATS1,2 , 72-74, 2 [PBRM1 , TP53 [7M1 [TP53 -77, AURK3 [AURKA , CDKN2A  [CDKN2A , RB1 [801 [BRCA2 , CCND1 [2 [CCND1 , SETD2 [1 [SETD2 , SMARCC1 or PCNA  are alsoIn addition to the cancer cell intrinsic mediators of tumor progression listed above, the tumor microenvironment is known to regulate a variety of genes associated with tumor progression, treatment resistance, and an aggressively metastatic tumor phenotype. The potential role of the tumor microenvironment and tumor hypoxia in driving mesothelioma progression has been under-studied despite evidence that mesotheliomas contain hypoxic tumor cells -87. In t2 < 10 mmHg, equivalent to < 1.3% O2in vitro) is created when oxygen demand by the proliferating tumor cells exceeds the supply of oxygen provided to the tumor through the bloodstream [Normal tissues exist over a range of oxygen tensions, and low levels of oxygen (hypoxia) are required for a variety of normal processes including embryogenesis, wound healing, and stem cell renewal in the bone marrow. In solid tumors, hypoxia  tracers [In vivo, F-FDG uptake in pleural mesotheliomas shows high correlations with GLUT1, HIF1, VEGF, CD34, Ki67, and MTOR upregulation [HIF-1 activation increases glucose transport (via GLUT-1) as well as glutamine and L-type amino acid transport (via LAT1) in pleural mesotheliomas [Cancer cells derive their energy from aerobic glycolytic metabolism for cellular processes based on Warburg's classical observations . Hypoxia tracers . In vivogulation , and pooheliomas , 95. Hypheliomas .In Figure Hypoxia as a unique feature of solid tumor biology provokes a need for clinically applicable gene expression signatures and poses a great opportunity for selective antitumor therapies , 34. ApaTherapeutically, hypoxia is exploited in cancer treatment by bioreductive prodrugs such as AQ4N, PR-104, TH-302 or hypoxia-responsive polymeric nanoparticles (containing chemotherapy) . These cAlong with well-known GLUT-1 regulation, carbonic anhydrases (CA), large family of zinc metalloproteases, are strongly upregulated by HIF-1 and are required for maintenance of pH, proliferation, and metastasis , 112. CAThe vasculature delivers oxygen and nutrients to all cells within the body. Hypoxic regions trigger HIF-related pathways that are key regulators of sprouting angiogenesis via modulating vascular endothelial growth factor (VEGF) . VEGF-inEGFR, MET or AXL [PI3K/AKT/MTOR is an oxygen and energy-sensing pathway essential for regulation of cell cycle progression and cell proliferation , and cloT or AXL , 140. InT or AXL . There aT or AXL , MET inhBRCA1 associated protein-1 (BAP1), a C-terminal family of deubiquitinating enzymes (DUBs) linked to DNA damage repair regulation [BAP1's function has been implicated in various other cancer types such as uveal melanoma [BAP1's crystal structure has been solved [BAP1 has not been developed. Moreover, cell cycle related genes often found mutated in pleural mesothelioma and regulated by hypoxic stress [CDKN1,2A) [A tumor suppressor gene most commonly deleted or mutated in mesotheliomas (∼60% of cases) is gulation , 143-145melanoma , clear cmelanoma , and cutmelanoma , 116. Aln solved , a therac stress , 135 areDKN1,2A) , 147. UsDKN1,2A) .NF2, with ∼ 45% incidence of aberration), a negative regulator of E3 ubiquitin ligase, and the Large Tumor Suppressor kinase 1/2 , two components of the Hippo pathway [NF2 and LATS2 can be regulated by hypoxia [LATS2 and NF2 delivered into the hypoxic tumors may prove more potent and call for thorough clinical investigations  and RAD50 are highly overexpressed, especially post-chemotherapy [CHEK1, required for checkpoint mediated cell cycle arrest in response to DNA damage, is overexpressed [CHEK1 inhibitor, LY2606369, is currently used in clinical trials for breast cancer patients with BRCA1/2 mutations and could be re-purposed for treating pleural mesothelioma malignancy. Other DNA damage repair genes deregulated in mesotheliomas include Fanconi anemia group D2 (FANCD2), RAD21 and RAN . Thus, co-targeting DNA damage genes in addition to chemotherapy may improve patient survival.Typically, as an adaptive response to hypoxia, tumors increase genetic instability by down regulating DNA repair genes such as pathways , 151. Thpathways . More sp and ATR . In pleuotherapy . Furtherxpressed  in pleurRAD52, another key gene involved in homologous recombination repair, is important for chemotherapy resistance and can be translationally repressed by miR-210 [in vivo demands closer investigations and gives countless potentially druggable targets for the development of innovative cures. miR-210 , a micro miR-210 -156, miR miR-210 , and miR miR-210 , 159 wit miR-210 . The the miR-210 , 161 in Under cellular stress conditions (e.g. low nutrient or oxygen levels), alteration/induction of proteasome and autophagic lysosome degradation pathways occurs as an adaptive response to mitigate the new cellular energy demands , 163. SpHIF induction is necessary for continued growth and survival of these tumors.Some of the outstanding questions in the field of malignant mesothelioma biology include deciphering which molecular events are involved in the genesis of mesotheliomas and would this inter and intra-heterogeneity among tumors change from onset to progression? Understanding the genomic landscape of mesotheliomas and integrating that knowledge for designing optimal and tailored therapeutic strategies is critical for improved patient outcome. Another important aspect is to understand whether and how the microenvironment of malignant mesotheliomas contributes to changes in oxygen permeability, nutrition and pH of tumors  and whetHypoxic cells within the tumor mass are distant from blood vessels, resistant to most anticancer drugs, and present a major obstacle to delivery of targeted therapies. In this review, we suggest that the hypoxic environment of solid tumor mesotheliomas can be used as the Achilles' heel for targeted drug delivery. We ask whether new therapies for pleural mesothelioma, such as those being used in clinical trials worldwide Table , prove mHow we address the challenge of individualized and direct assessment of oxygen pressure within mesotheliomas, correlate that measurement to tumor function, and incorporate it as part of a standard of care remain outstanding areas for investigations.


Listeria monocytogenes isolates linked to fatal outcomes in elderly patients with cancer. In addition, listeriosis patients with cancer present low IL-17A/IL-6 ratios and significantly reduced levels of anti-GAPDH1–22 antibodies, identified as two novel biomarkers of poor prognosis. Analysis of these biomarkers may aid in reducing the incidence of listeriosis. Moreover, GAPDH1–22-activated monocyte-derived dendritic cells of listeriosis patients with cancer seem useful tools to prepare clinical vaccines as they produce mainly Th1 cytokines.Two regions of northern Spain, Gipuzkoa, and Cantabria present high annual incidence of listeriosis . We report that the high annual incidences are a consequence of infection with highly virulent  Listeria monocytogenes genotypes show greater virulence, contributing to more human listeriosis cases , and one case of sporadic listeriosis occurred in an apparent non-risk patient (6.6%)  and Cantabria , were selected to calculate annual incidence, since these communities have voluntarily reported a high number of listeriosis cases since 2008 , 11. We L. monocytogenes from 15 clinical isolates and serotyped the strains by agglutination  and performed multilocus sequence typing (MLST) to establish L. monocytogenes sequence types (STs) using the primers and conditions described on the Pasteur Institute web page (http://bigsdb.pasteur.fr/listeria/listeria.html)  as serotype 1/2b, and 23% (3/15) as serotype 1/2a, with 12 and 3 clinical isolates of lineages I and II, respectively. MLST genotyping revealed the following distribution: 43% ST1, 14% ST219, and 7% each of ST6, ST26, ST37, ST87, ST213, and ST391 genotypes. These distributions differ from those registered on Pasteur Institute database  –18. In t reports , 18, folin vitro replication of invasive L. monocytogenes isolates was assayed in human monocyte-derived dendritic cells (MoDC)   that reached the spleens in C57BL/6 mice, a mice model highly resistant to listeriosis , ST219 (CC4), ST213 (CC213), ST6 (CC6), ST87 (CC87), and ST26 (CC26)] revealed the presence of at least two hypervirulent complex clones previously reported in France, CC4 and CC6   infectedaterial) –22. Thisytogenes , 21. Notcontrols , 20, 23. and CC6 , which,  reports , since t reports , or CC87L. monocytogenes infection in our patients is reduced immunocompetency, it is possible that specific immunological parameters contribute more significantly as risk factors. Two parameters appear relevant for the immune status of invasive L. monocytogenes-infected individuals. First, the Th1/Th17-Th2 cytokine balance , presenting the deceased patients basal levels (0.02–0.05 OD) (Table 1–22 column). Anti-listeriolysin O (LLO) antibodies were detected at extremely low levels in all listeriosis patients (≤0.15 OD), eliminating the possibility of using LLO antibodies as biomarkers (data not shown). Accordingly, we conclude that low levels of anti-GAPDH1–22 antibodies, together with low IL-17A levels, are two valid immunological biomarkers of poor prognosis in listeriosis patients with tumors under chemotherapeutic treatment.High levels of IgG antibodies (OD ≥ 2.0) against the bacterial epitope, GAPDH1–22 , 21, werin vitro using MoDC or in vivo revealed 1000-fold higher RI values and CFU than the standard bacteria , and 1 child (stillbirth). Our selection of listeriosis patients was based on those representative of the most prevalent clinical manifestations in our institutions, specifically, those with tumors, autoimmune disease, miscarriages, and renal or hepatic transplants , 4, 11. + T cell immune responses . Third, two biomarkers in the sera of patients with poor listeriosis prognosis were distinguished: (i) low IL-17A/IL-6 ratios that reflected a shift in the immune balance toward Th2 anti-inflammatory responses and (ii) low production of anti-GAPDH1–22 antibodies that suggested reduced CD4+ T cell–B cell responses. Dysfunction of Th17 immune responses in listeriosis patients with tumors under chemotherapeutic treatment appears to explain the high bacterial loads and reduced production of antibodies  with the reference number 2014.228 . All participants signed the Informed Consent documents, and these documents are in the custody of physicians in accordance with the Spanish Law . The study was carried out in accordance with the Guide of Care and Use of Laboratory Animals of the Spanish Ministry of Science and Innovation. The Ethical Committee of Animal Experiments of the University of Cantabria approved the protocol (permit number: 2012/06) that followed Spanish legislation (RD 1201/2005). Surgeries were performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering.Listeria isolates from listeriosis patients and the serotyping at Hospital.CA-D, planned, directed the study, performed the meetings with all participants to arrange the performance of the study and acted as a corresponding author. ORCID code: 0000-0002-4585-6959. RC-G, performed the study and collected all samples from both Hospitals. HT-N helped with the performance of the study. JM collected the listeriosis samples from patients: bacteria isolates, cells, sera, LCR, blood tests, and clinical data of Hospital Universitario Donostia and performed the Listeria serotyping. CG-R collected the listeriosis samples from patients: cells, sera, LCR, blood tests, and clinical data of Hospital Universitario Marqués de Valdecilla. JC-M collected the Listeria isolates of Hospital Universitario Marqués de Valdecilla and performed the Listeria serotyping. EF-C helped with the cytokine analysis and dendritic cell preparation. MA-G helped to collect the listeriosis samples from patients: bacteria isolates, cells, sera, LCR, blood tests, and clinical data of Hospital Universitario de Donostia and cited the listeriosis patients. MF directed and cited the listeriosis patients, agreement documents from patients and directed the collection of clinical samples and data from listeriosis patients at Hospital Universitario Marqués de Valdecilla. LM-M directed the collection of the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.