Leishmania infantum for diagnosis of VL in humans and dogs.Visceral leishmaniasis (VL) is the most severe form of leishmaniasis in Iran with high mortality rates in the case of inaccurate diagnosis and treatment. This study aimed to prepare and evaluate a new rk39 recombinant antigen from an Iranian strain of L. infantum antibodies. We screened 84 human sera and 87 dog sera from clinical cases in the endemic area of Meshkin-Shahr, Iran along with 176 sera from healthy controls (collected from 86 humans and 90 dogs) during 2013–2016.rK39-based enzyme-linked immunosorbent assay (ELISA) was compared with the direct agglutination test (DAT) for the detection of anti P<0.05).Using the rK39 ELISA, a sensitivity of 85.7%  and a specificity of 86.0%  were detected in human sera at a 1:800 (cut-off) titer when DAT-confirmed cases were compared with healthy controls; a sensitivity of 96.6%  and specificity of 94.4%  were found at a 1:80 (cut-off) titer compared with DAT. Kappa analysis indicated agreement between the rK39 ELISA and DAT (0.718) when using human sera at a 1:800 (cut-off) titer as well as (0.910) at a 1:80 (cut-off) titer when using dog sera (Leishmania infantum seems to be used for diagnosis of VL in humans and dogs. Further extended field studies are recommended.New rk39 recombinant antigen from an Iranian strain of  Visceral leishmaniasis (VL) is a parasitic disease with high levels of morbidity and mortality. Dogs are the main reservoir host for the Mediterranean type of VL, which is endemic in some areas of Iran ,2.Visualization of the parasite in bone marrow, liver or splenic aspirates and isolation by culture are currently available diagnostic procedures, but these are invasive or not sensitive enough for the diagnosis all forms of VL .rK39, which includes a 39-aminoacid-repetitive immune-dominant epitope of the kinesin-related antigen, has shown high sensitivity 93% to 100%), specificity (97% to 98%) and strong antigenicity even among HIV co-infected patients (sensitivity 81%) compared to other recombinant VL antigens00%, spec.Leishmania disease; however, the C-Ag assay has shown limitations in terms of specificity, sensitivity and has also shown cross-reactivity with sera from patients with trypanosomiasis, tuberculosis and toxoplasmosis  preparation has also been commonly used to diagnose this lasmosis .L. infantum (rK39 RDT) have been introduced by Iranian researchers as a rapid and non-invasive method to identify VL, especially in suspected symptomatic reservoirs.The main goal of this study was to evaluate rk39 recombinant antigen from an Iranian strain of This study was approved by the Ethical Committee of Tehran University of Medical Sciences  in accordance with the Helsinki Declaration and guidelines. Dog sera were collected in coordination with Iran Veterinary Organization after informed consent from the owners.The human sera were collected from volunteers following informed consent. Children were included in this study after consent from their legal guardians.L. infantum strain (MCAN/IR/14/M14 with GenBank Accession number KT201383) used in this project was isolated from a VL infected dog in Meshkin-Shahr. Promastigote culture and DNA extraction were performed (The erformed .L. chagasi sequence (GenBank Accession number AF131228), forward, F, 5′- GGATCCATGGCAGCCGAACTTGATGCCG -3′ containing a BamHI site and reverse, R, 5′- CTCGAGCTGGCTCGCCAGCTCCG -3′ which contained a XhoI site for k39 gene, and amplified by ABI DNA thermal cycler. The mixture for the PCR reaction included 1.5 μl (5pm) of each primer, 0.5 μl dNTPs, 0.5 μl Mgcl2 and 0.5 μl DMSO. Amplification was performed at 94 °C for 5 min, followed by 35 cycles at 94 °C for 1 min, 68 °C for 1 min, 72 °C for 1 min and 30 sec and a final extension for 20 min.To amplify the K39 gene, primers were designed based on E. coli . Plasmid DNA was extracted using the YTA Miniprep Kit  and digested using BamH1 and XhoI. The insert was subcloned into pET-32a (+).The k39 band, of a predicted length of approximately 843 bp, was ligated into pCR®-TOPO® vector by TOPO®TA Cloning® Kit  and transformed into PCR screening in the pCR®-TOPO® and pET-32a (+) recombinant plasmids was performed by M13 and T7 terminator primers. The recombinant plasmids were digested using BamHI and XhoI to confirm insertion of the rK39 gene.The recombinant pET-32a (+) with N and C -terminal His-tag fusions for affinity purification was expressed, purified and confirmed .L. infantum were washed in cold PBS for 3 times and ruptured by sonication. The crude Ag in the supernatant was stored at −20 °C after centrifugation at 4500 rpm for 20 min at 4 °C. Protein concentration was estimated by Lowry’s method  including 84 humans and 87 dogs from the endemic area of Meshkin-Shahr, Iran.All the cases showed anti-Leishmania antibodies ≥1:320.Most of the canine sera were obtained from symptomatic dogs with cutaneous lesions, alopecia, diarrhea and titers of anti-In parallel, 176 healthy control samples were obtained from geographical areas not endemic for VL (86 human and 90 dog samples) along with 10 patient sera and 8 canine sera with unrelated infectious diseases .A checkerboard titration was carried out to optimize the concentration of rK39 and CA using standard ELISA methods.ELISA plates (Nunc™ MaxiSorp™) were coated overnight with rK39 (1 μg/ml) and crude antigen (6 μg/ml) at 4 °C in carbonate/bicarbonate buffer at pH=9.6. All the samples were analyzed by ELISA .The optical density (OD) was measured at 492 nm and entered into MedCalc (ver. 17.7.2). The ROC- curve was created for each Ag separately in this software.Leishmania laboratory of the school of public health at Tehran University of Medical Sciences. All the samples were tested according to standard methods  and signs of VL were considered as being in the symptomatic stage.Based on previous studies on the diagnosis of asymptomatic stages of VL, titers equal or higher than 1:800 and 1:80 were considered as the cut-off point for human and dog sera, respectively –17. We ck) values with 95% confidence intervals were determined to show the degree of agreement between expected result  and actual result (DAT results) based on criteria scales (17.7.2). In order to determine the diagnostic efficacy.The sensitivity and specificity, positive predictive and negative predictive values were calculated in comparison with DAT. Moreover, Kappa  showed 843 bp band of interest.Amplification of K39 of PCR screening in pCR®-TOPO® plasmids was carried out by F&R M13 primers. The recombinant bands were 1044 and the nonrecombinant ones yielded a product of 201bp  while thThe both of plasmids were digested by restriction enzymes (BamHI and XhoI) and revealed two different bands.L. infantum k39 gene with N and C -terminal His-tag fusions were expressed in the pET 32a (+) vector. The recombinant protein was purified from the soluble fraction by NIIDA resin affinity chromatography. Western blotting using pooled serum of 20 VL-confirmed patients and anti-His antibodies revealed a single band of 58 kD. The yield of protein ranged from 47.8 to 55 μgr/ml after purification and dialysis.The L. infantum rK39 antigen were determined by ELISA compared to DAT and CA- ELISA , but this was not observed for dogs (P=0.078).There were no significant differences among results obtained from rK39-ELISA and DAT in humans k index using the SPSS software. The data indicated a moderate agreement between rK39-ELISA and DAT (k =0.485), but no concordance of CA-ELISA with DAT in asymptomatic humans (k =0.092); conversely the degree of agreement for symptomatic humans was very high using the rK39-ELISA (k =0.823), but weak in CA-ELISA (k =0.340). Totally, k coefficient for rK39-ELISA and CA-ELISA were 0.711 and 0.284, respectively in humans. These agreement values showed almost perfect agreement in dogs for rK39 and a significant result for CA with DAT (k=0.910 for rK39- ELISA and k =0.763 for CA-ELISA).The level of agreement of rK39-ELISA and CA-ELISA with DAT was measured by The Youden’s indexes for rK39-ELISA and CA-ELISA were 0.720, and 0.285 in human and 0.910 and 0.764 in dog reservoirs, respectively that indicate a high accuracy for rK39-ELISA in human and canine while an acceptable accuracy was seen for CA-ELISA in dog.L. infantum. The cloning of this antigen could be challenging due to high GC content in the gene (more than 67%), secondary structures and repetitive sequences. Repetitive DNA made problem in amplifying, cloning, sequencing, maintaining in bacteria, and gene synthesis  compared to symptomatic humans (94%) infected with infantum . Our datOn the contrary, low sensitivity of rK39-ELISA (62.5%) has been observed in self– healing or sub-clinical human infection. Sensitivity ranging was described from 52.9% to 64.7% to diagnose infection in asymptomatic CVL . MoreoveThe sensitivity of CVL and HVL were 93.1% and 84.6%, respectively by CA-ELISA in cases with clinical signs, while a lower positivity of approximately 59.4% was seen in sera with lower levels of antibodies  was observed for CSA-ELISA in this group . The reak =0.340). By contrast, this value is acceptable in dogs with a high titer of anti-body (k =0.763).Interestingly, in spite of a remarkable surge of sensitivity (84.6%) for CA-ELISA in HVL with clinical manifestations compared to another group, it fails to reach a good agreement in the association with DAT (L. infantum LON49(Our results indicate the lower specificity 86%) of ELISA using the rK39 in human in comparison with specificity reported from many VL-endemic countries, such as India and Nepal (97% to 98%) . This fi% of ELIStum LON49,34,35. HL. tropica in dogs  was found higher than rK39-sub (95%) prepared by Iranian researcher to detect antibody in people who have clinical signs ≥3200) (00 (2). TL. infantum seems to be used for diagnosis of VL in humans and dogs. Future attempts to design a double blind study to determine final validity of the antigen in the endemic areas of VL in Iran where L. infantum is the main causative agent for this disease.New rk39 recombinant antigen from an Iranian strain of


Background: Apicomplexan parasite Theileria annulata causes significant economic loss to the livestock industry in India and other tropical countries. In India, parasite control is mainly dependent on the live attenuated schizont vaccine and the drug buparvaquone. For effective disease control, it is essential to study the population structure and genetic diversity of the Theileria annulata field isolates and vaccine currently used in India.Methodology/Results: A total of 125 T. annulata isolates were genotyped using 10 microsatellite markers from four states belonging to different geographical locations of India. Limited genetic diversity was observed in the vaccine isolates when compared to the parasites in the field; a level of geographical substructuring was evident in India. The number of genotypes observed per infection was highest in India when compared to other endemic countries, suggesting high transmission intensity and abundance of ticks in the country. A reduced panel of four markers can be used for future studies in these for surveillance of the T. annulata parasites in India.Conclusion: High genetic variation between the parasite populations in the country suggests their successful spread in the field and could hamper the disease control programs. Our findings provide the baseline data for the diversity and population structure of T. annulata parasites from India. The low diversity in the vaccine advocates improving the current vaccine, possibly by increasing its heterozygosity. The reduced panel of the markers identified in this study will be helpful in monitoring parasite and its reintroduction after Theileria eradication. Theileria species, linked to economic loss and mortality worldwide, are Theileria annulata and Theileria parva. Theileria annulata is responsible for the majority of theileriosis cases in India, while there are no reports of T. parva from the country  of T.annulata were reported from different states of India  gene of the parasite . Approximately 3 ml of blood was collected in EDTA coated vacutainer tubes (BD) with the help of trained veterinarians. A total of 125 blood samples, including the parasite .T. annulata (N = 125) were used as a template for amplifying the 10 markers from each sample using a previously described protocol . Multilocus genotype (MLG) data for each sample were generated using the predominant peak (sR) were calculated for all the markers using the FSTAT 2.9.4 program  in the population using the MLG data. Population genetic differentiation (Fst) was calculated on the Genepop 4.2 Web Server (K = 1–12) with a burn-in of 50,000 steps and then 500,000 Bayesian Markov Chain Monte Carlo (MCMC) steps using the admixture model. The optimal number of clusters was identified using method described by The file generated from capillary electrophoresis was imported into Gene marker 2.7.0 software  for further analysis. Allele scoring for each marker was done in the previously described range for ant peak . For comant peak . The MLGant peak  as an exant peak . Mean al program . Lian 3. program  was usedb Server  using thb Server . Twenty The complete allelic profile of each sample was next used to prepare a binary data set for the presence and absence of alleles for understanding the genotypic diversity considering multiple parasite genotypes in each sample. A similarity matrix and dendrogram were created based on the allelic data by Jaccard’s similarity index using online server DendroUPGMA . InteracThe Bottleneck analysis was done using the allele frequency data for assessing the change in the population size by measuring excess or deficit in heterozygosity by Bottleneck software version 1.2.02 . The twoRecent effective population size reductions (genetic bottlenecks) were studied using allele frequency data and BOTTLENECK software , including the vaccine for assessing genotypic diversity of in India . The parand TS31 . The genand TS31 . The genand TS31 . The comand TS31 . Since mand TS31 . Out of and TS31 . Telangaand TS31 .T. annulata isolates from countries, like Oman, Portugal, Sudan, Tunisia, China, and Turkey  to 12.1 , Haryana (0.046), and Telangana . We found significant LD in parasite population of Telangana , Bihar  and in total Indian population , Turkey , Tunisia , Sudan , and India and other countries . Further bottleneck analysis was done using allele frequency data for checking the recent population size reduction in the Indian parasites under the TPM model. Statistical analysis of the data was carried out, under the assumption of mutation-drift equilibrium, by Sign rank test and the Wilcoxon test. A significant heterozygosity deficit was seen in the population, indicating nonbottlenecked parasite population in India  for the study were collected from four different geographical locations, including the T. annulata vaccine from India. The genotyping analysis identified high diversity among the microsatellite makers and the presence of multiple genotypes in all the samples. Based on the analysis, we found less genetic diversity in the T. annulata vaccine when compared to the parasite population from the field.This study is the first comprehensive analysis of the E; 0.864) was found in the parasite population of all four locations; similar diversity was reported from other endemic countries, like Turkey, Tunisia, Oman, and Sudan (T. annulata is high in India (f = 1/ne) and extent of outcrossing (>50%) for the T. annulata population in India using the mean number of clones per infection (ne = 12.64). This method is prevalidated in similar kinds of studies on T. annulata and Plasmodium falciparum . Moderate to high genetic differentiation was observed when the Indian parasite population was compared with other countries, like Oman, Turkey, Tunisia, and Sudan. The parasite population from Bihar was found to be genetically distant based on the Fst analysis when compared to the other three states. The low genetic differentiation between the parasites may be connected to the free movement of the animals in the country for commercial purposes. No correlation was found between the parasites based on the geographic and genetic distance (Fst). There was some evidence of regional distribution in the parasite population based on the clusters formed in the DAPC analysis. The current vaccine should ideally comprise of sufficient genotypic diversity for protection in all the states; however, it clustered near to the Bihar and Telangana population, signifying the presence of heterologous parasites in the field. The heterologous parasites in the field might be due to the frequent movement of animals inside and between the states, which provides an opportunity for the development of diverse parasites, making the population highly complex in India. The DAPC analysis reconfirmed the high genetic differentiation of the parasite population from India and other countries , which is evident as the animal movements between the countries are zero to none. As multiple parasites were present in every infection, an analysis based on the predominant allele might be biased. The phylogenetic analysis based on the complete allelic profiles of each sample also identified regional clustering in India. This implies that the genotype circulating in a particular region is similar and did not exactly match with other areas. This also proves that even though there is an allelic similarity among different states based on MLG, the genotypes circulating in different states are different. It might have a significant implication in parasite control as different genotypes may respond differently to control measures, and in the future, state-wise control strategies should be adopted. Therefore, diversity and genotypes circulating in a particular region have to be considered while implementing control measures.T. annulata population diversity. However, in the future, if we plan to track the parasite control measures in these states, such studies might not be feasible due to time and economics involved. Based on our genotyping data, we next checked whether it is possible to use a smaller number of markers for future genetic diversity studies. We identified that a minimum of four markers could be used to study the population structure of the parasite without missing the critical information and reducing the cost of the assay.The previous studies from other countries, have utilized 10 microsatellite markers for understanding the T. annulata parasites in India. The low genetic diversity observed in the vaccine highlights the scope for improvement in the current vaccine. The detection of multiple unique genotypes other than the vaccine, calls for increased efforts for Theileria control. We hypothesize that using a cocktail of parasites having sufficient genetic and antigenic diversity might be a good idea for the future vaccine. For a better understanding of the genotypic and antigenic composition of the parasite populations in the country, sample numbers and sites will have to be increased substantially in future studies. Our results can be used as the baseline data for future studies and will be helpful in monitoring the parasite population in the country. Our findings for the use of a smaller number of markers for genotype allocation in these states will be useful for the Theileria control programs.Our study helps to understand the population structure of The original contributions presented in the study are included in the article/PS and SR designed the experiments. SR, MB, and VBd have done the experiments and data analysis. VBo, PK, JA, and SS have helped in sample collection, designing study, data analysis, and manuscript editing. PS, SR, and VBd edited the paper. All authors have given approval to the final version of the manuscript.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.


Isoetes cangae and Isoetes serracarajensis have been recently described in the State of Pará in the Amazon forest located in northern Brazil. Isoetes L. has survived through three mass extinctions. Plants are considered small-sized, heterosporous, and can display a great diversity of physiological adaptations to different environments. Thus, the current study aimed to estimate the genetic variation of the populations of I. cangae and I. serracarajensis to generate information about their different mechanisms for survival at the same geographical location that could point to different reproductive, adaptative and dispersal strategies and should be considered for effective conservation strategies.Two endemic lycophyte species I. cangae and I. serracarajensis were investigated using Inter Simple Sequence Repeat (ISSR) molecular markers. Total genomic DNA was isolated, and the genetic diversity parameters were calculated.The genetic diversity and population structure of I. cangae and I. serracarajensis, respectively. The coefficient of genetic differentiation between population areas (GST) showed a higher value in I. serracarajensis (0.5440). Gene flow was higher in I. cangae (1.715) and lower in I. serracarajensis populations (0.419). Overall, the results further show that I. serracarajensis and I. cangae are two species with considerable genetic variation and that these differences may reflect their habitats and modes of reproduction. These results should be considered in the development of effective conservation strategies for both species.The sixteen primers produced 115 reproducible bands, 87% of which were polymorphic. A high level of polymorphic loci (81.74% and 68.48%) and a high Shannon index (Sh = 0.376 and 0.289) were observed for  Isoetes L. is characterized by approximately 200 species of vascular plants that have been catalogued with molecular dating studies, tracing its evolutionary roots to the Devonian in the Palaeozoic , a dominant marker with a multiallelic nature, high reproducibility, and large genome coverage. ISSR reveals a large number of polymorphic fragments and thus constitutes a suitable marker of intrapopulation and interpopulation genetic diversity for Isoetes .I. cangae population and four I. serracarajensis populations in the Brazilian Amazon region. We aimed to estimate (1) the genetic variation of the populations of I. cangae and I. serracarajensis and (2) the genetic structure and its correlation with geographical distribution. Our findings should be considered for effective conservation strategies.In this work, we used 16 ISSR markers to estimate the genetic variation of the only known Isoetes cangae and Isoetes serracarajensis , categorized population density, and number of samples are presented in http://www.icmbio.gov.br) number 59724-2. and S11 . The fouI. cangae was estimated to understand the processes related to the maintenance of the diversity of this species and its restricted occurrence. For density estimation, cover and number of individuals were determined by free diving at approximately 12 points randomly distributed in each of the four sampling areas. At each point, a 1 ×1 m square of plastic (PVC), subdivided into four quadrants of 0.25 m2, was arranged randomly on the substrate. In each square, the percentage coverage (percent area of the square occupied) and density estimation  were estimated. Among fifty squares sampled, counting was performed in fifteen squares. The density category was established as a plus symbol “+” corresponding to an average of 3 plants per square metre. The coordinates (latitude and longitude) for each sample point were recorded with GPS. The areas occupied by the sampled specimens (AOPEA) and respective distances between sampled areas were measured in Google Earth Pro (Google LLC 2019). For the determination of the area, a polygon comprising all the samples of each area was drawn and is represented in m2. The smallest distance between the polygons of the different sampling areas was used to establish the distance among them. The maps were made in Adobe Illustrator CS6.The density of Total genomic DNA was isolated from 1 gram of dry leaves. Leaves were macerated using liquid nitrogen and the extraction was performed with the help of the DNeasy Plant Maxi Kit (Qiagen). The DNA was quantified by Nanodrop, and 50 ng of genomic DNA was used for each PCR. Annealing temperatures varied between 48 °C and 52 °C according to the primer used. Among the twenty tested primers, sixteen were selected for further analysis . IntegriA binary matrix was constructed from the gel analysis. The presence of bands is indicated by the number 1 and the absence of bands by the number 0. Genetic dissimilarity through the weighted index was determined using this binary matrix.ST = (HT-HS)/HT] and an estimate of gene flow (Nm) from GST as Nm = 0.5 (1 - GST)/GST were determined using POP gene version 1.32 , number of polymorphic loci (NPL), and percentage of polymorphic loci per primer (PPL%) were calculated with the software GenALEx 6.5 . The occion 1.32 .(UPGMA) in the programme GenALEx 6.5 and MEGA7  software   and that, with the lowest was IS11 (18%) (= 0.245) . For I. ICW area ; Table 2ICW area , while tICW area . The pop11 (18%) . Among t11 (18%) .I. cangae presented a high Shannon diversity index value at the species level, Sh = 0.376, and high genetic flow (1.714). The ICW and ICN areas displayed the highest values of Sh = 0.318 and Sh = 0.208, respectively , a GST equal to 0.5440 and low genetic flow (0.419)  (ectively . It is iectively . The pop (0.419) . The ISV (0.419) . From th (0.419) . Compariulations . A dendr 0.0001) .The genetic diversity of a species is a result of evolutionary processes, including recent changes such as habitat reduction, which may lead to a decline in genetic diversity and decrease the survival potential for a given species.I. cangae. In genetic structure studies, I. cangae presented a high level of intrapopulation genetic diversity . I. cangae is one of the most diverse species in the world. Similarly, high diversity was found in I. malinverniana  , I. yunguiensis   and I. hydrophila     for = 0.217) . Similar 0,1848)  and the = 0,781) . The appI. cangae. The presence of long-lived individuals and overlapping generations observed may also be a contributing factor to the genetic diversity of this species. Similar results were found in species such as I. asiatica, I. taiwanensis, and several endangered Isoetes and fern species . Despite moderate genetic diversity, sexual reproduction was not observed. Asexual reproduction by division of the corm through the emergence of individuals from the nonfertile mother plant is shown  of all the remaining populations, even populations under ecological stress, probably due to the maintenance of the ancestral genetic diversity of a historic moment of interpopulation connectivity and high gene flow  and among populations   .ST = 0.544, AMOVA, P=45%), so both within-population diversity and diversity among populations contributed significantly to the total diversity of the species. Other species also showed high diversity among populations, such as Argania spinosa L. Skeels , and in the dry season (June to October), the species seems to disappear (few leaves resist air for an extended period) ( period) , the cor period) . These p period) .I. serracarajensis, IN3, N6, ISV, and IS11, were grouped by Nei’s genetic distances, and a correlation was observed between genetic and geographical distance according to the Mantel test  indicate that the geographical isolation barrier could principally affect gene flow. In fact, the Mantel test is reliable for determining the correlation between genetic and geographical distances among populations such as Ottelia acuminate var jingxiensis  found indicates that spore dispersion is reduced among populations . Populattel test . The anatel test . The lowgxiensis  and Braschreberi . Spores d plants , which cI. cangae), and the other classified as vulnerable (I. serracarajensis). Both species have high genetic diversity. The knowledge gained from the current study represents an indispensable prerequisite for effective conservation programmes (in situ and ex situ strategies) for both species since maintaining genetic diversity reduces extinction risk. Intra- and inter-region translocations are appropriate for in situ conservation strategies when conflict exists between conservation and economic interests. Translocations to the same or between management units are recommended to alleviate the impact of inbreeding and genetic drift. Interregional translocations must be performed very carefully because environmental differences can result in adaptive diversity loss. Additionally, ex situ conservation is also an important species maintenance procedure. The genetic diversity stock can be kept in active germplasm banks and spore banks. The source population(s) can be used to produce new plants to reintroduce into the natural environment if necessary. Our results provide valuable biological information for the conservation of the species and for future functional studies to uncover the physiological adaptations behind the endemic quillwort (Isoetes L.) species from Amazonian ferruginous outcrops to their specific environments. Detailed studies of ecological dynamics such as spore dispersion should be carried out to yield valuable information for further conservation of I. cangae and I. serracarajensis. Future studies on the effect of storage methods on spore viability will also be necessary.This work aimed to evaluate the genetic diversity of two species: one critically endangered  I. cangae and I. serracarajensis (from left to right). (B) Young sporophytes generation from in vitro fecundation of I. cangae. (C) Asexual sporophytes generation (white arrow) from young plant nonfertile of I. serracarajensis. (D) Young sporophyte (white arrow) near of fertile adult plant of I. cangae observed in greenhouse.Click here for additional data file.10.7717/peerj.10274/supp-2Supplemental Information 2 I. serracarajensis location on the ferruginous plateaus of the southern portion marsh ISV during rainy and (B) dry seasons. (C) Detail of I. serracarajensis leaves during dry season (white arrows).Figure S2. (A)Click here for additional data file.10.7717/peerj.10274/supp-3Supplemental Information 3Gel results of ISSR primers, Mean Population Genetic Distance Matrix for Binary Distance, Results of Analysis of Molecular Variance.Click here for additional data file.10.7717/peerj.10274/supp-4Supplemental Information 4Nei’s genetic and geographic distance matrices. Mantel test matrix.Click here for additional data file.


Genetic adaptation of maize to the increasingly unpredictable climatic conditions is an essential prerequisite for achievement of food security and sustainable development goals in sub-Saharan Africa. The landraces of maize; which have not served as sources of improved germplasm; are invaluable sources of novel genetic variability crucial for achieving this objective. The overall goal of this study was to assess the genetic diversity and population structure of a maize panel of 208 accessions; comprising landrace gene pools from Burkina Faso (58), Ghana (43), and Togo (89), together with reference populations (18) from the maize improvement program of the International Institute of Tropical Agriculture (IITA). Genotyping the maize panel with 5974 DArTseq-SNP markers revealed immense genetic diversity indicated by average expected heterozygosity (0.36), observed heterozygosity (0.5), and polymorphic information content (0.29). Model-based population structure; neighbor-joining tree; discriminant analysis of principal component; and principal coordinate analyses all separated the maize panel into three major sub-populations; each capable of providing a wide range of allelic variation. Analysis of molecular variance (AMOVA) showed that 86% of the variation was within individuals; while 14% was attributable to differences among gene pools. The Burkinabe gene pool was strongly differentiated from all the others , with no gene flow (Nm) to the reference populations (Nm = 0.98). Thus; this gene pool could be a target for novel genetic variation for maize improvement. The results of the present study confirmed the potential of this maize panel as an invaluable genetic resource for future design of association mapping studies to speed-up the introgression of this novel variation into the existing breeding pipelines. Zea mays L.) is one of the most important cereal crops consumed in sub-Saharan Africa (SSA) and an essential component of livestock feed in the developed as well as developing world. Approximately 10,000 years ago in Central Mexico, maize was domesticated from its ancestor, the wild grass-teosinte (Zea mays spp. parviglumis) [Maize (iglumis) . Mutatioiglumis) ,4. Thus iglumis) . The laniglumis) . Indeed,iglumis) . Modern iglumis) . In ordeThe high selection pressure under low input and climatically stressed maize growing environments in Africa is likely to have resulted in local adaptations with potential value for breeding stress tolerant and nutritionally enriched varieties . Indeed,Recently, we phenotypically characterized a panel of maize landraces originating from Burkina Faso, Ghana, and Togo, which cover a wide range of climatic conditions classified as Sahel and coastal West Africa (WA) . The stuThe assessment of genetic diversity by genotyping-by-sequencing (GBS) provides robust estimates of diversity and has been increasingly adopted as a fast, high-throughput, and affordable tool for whole-genome genetic diversity analysis in large germplasm sets . The divWe analyzed 208 maize accessions obtained from international and national gene banks in Africa . The maihttps://www.diversityarrays.com/orderinstructions/plant-dna-extraction-protocol-for-dart/). The quality of each DNA sample was visualized by electrophoresis on 0.8% agarose gel, and the purified DNA was further quantified using a nano-drop spectrophotometer . Certified DNA samples were then sent to the Integrated Genomic Service and Support (IGSS) genotyping platform, Nairobi, Kenya, for genotyping. High-throughput genotyping was conducted in 96 plex DArTseq protocol, and SNPs were called using the DArT’s proprietary software, DArTSoft, as previously described [Zea mays L. genome reference, version AGPV3 (B73 Ref-Gen v4 assembly) [For each accession, total genomic DNA was isolated from bulked leaf composites from 15 seedlings at two weeks old according to the DArT protocol (escribed . Reads assembly) .o), and expected heterozygosity (He) using PowerMarker v. 3.2.5 [adhoc number of clusters (k) was varied from 1 to 12, with 10,000 burn-in steps, followed by 10,000 Markov chain Monte Carlo simulations, as previously described [STF) and haploid number of migrants (Nm) between gene pools was performed using GenAlEx v6.503 [STF measures the amount of genetic variance that can be explained by population structure based on Wright’s F-statistics [NmNm value less than 1 indicates limited gene exchange among subpopulations [A total of 47,441 putative DArTseq markers were generated from the 208 maize panel. Prior to further analysis, the raw data set was filtered to remove markers with call rate <0.8, minor allele frequency (MAF) <0.05, and unmapped SNP markers. Thereafter, markers with no missing rate were retained using the TASSEL software version 5.2.12 . The retv. 3.2.5 . The popv. 3.2.5 . The adhescribed . For eacescribed  embeddedescribed . Accessiescribed . The popescribed . Membersescribed . An unroescribed  with 100escribed . The resescribed  and editescribed . Geneticescribed  to partix v6.503  with 999atistics , while Nulations .e, Ho, MAF, and PIC values estimated for the entire maize panel (208 accessions) averaged 0.36, 0.50, 0.28, and 0.29, respectively , while chromosome 10 had the least (422) . He, Ho,ectively . The aveThe model-based simulation of population structure analysis of the maize panel (208 accessions) showed that the delta K values from the mean log-likelihood probabilities plateaued at K = 3 (389.43), followed by K = 4 (276.33), and K = 2 (273.07) a. At K =Using the Bayesian information criterion (BIC) implemented in DAPC, a maximum of K = 3 was obtained, which corresponded to three groups of maize accessions in the panel . EstimatFurther investigation of the genomic structure of the maize panel using the PCoA indicated three subpopulations as per the STRUCTURE simulation and DAPC analyses . The totAs per the STRUCTURE, DAPC, and PCoA results, the NJ phylogenetic tree also showed three sub-populations with higher degrees of admixture among Ghanaian and Togolese landraces, and reference populations . The neiSTF value of the maize panel was 0.21, and the Nm value was 1.58. As shown in STF value (0.28), and the Ghanaian and Togolese gene pools had the lowest . The pairwise STF values ranged from 0.14 (Ghanaian vs. Togolese) to 0.31 (Burkinabe vs. reference populations). Similarly, Nm values between gene pools varied from 0.98 (Burkinabe vs. reference populations) to 2.83 (Ghanaian vs. reference populations). The Nm value between the Ghanaian and Togolese gene pools was 2.63.The AMOVA revealed that 14% of the total variation was found among gene pools, while the rest (86%) was within gene pools . The ovee (0.36) and Ho (0.5). The He obtained in this study was comparable to the 0.36 reported for provitamin A (PVA) quality protein maize (QPM) germplasm from IITA-MIP [e = 0.25), Western Africa (He = 0.18), and Sahel Africa (He = 0.24) [e = 0.22) [e = 0.36, Ho = 0.50) contained slightly higher diversity than the Burkinabe  and Ghanaian  landrace pools. Further, the low variation in the genetic indices identified between the landraces as a group, and the reference populations showed that the two germplasm sets possessed similar genetic diversity   as well  = 0.22)  includin = 0.22) . Charactiversity . These r. The additional smaller peaks observed at K = 4 (276.33) and K = 2 (273.07) implied the presence of subgroups within the three major groups  supported the presence of significant genetic divergence within the maize panel. Wright [STF values, the Burkinabe gene pool was the most differentiated  suggested little involvement of the original Sahelian gene pool in the development of the modern maize varieties presently grown in the sub region. This observation is biologically and historically meaningful since in West Africa, the reference maize gene pool called Composite Y [STF (0.14) and high Nm (2.63) values. This result further reflected the gene flow via seed exchanges and local preferences towards a given agrotype owing to similar climatic conditions. These may have significantly shaped the distribution of the genetic diversity within Ghanaian and Togolese maize landraces, as was previously suggested [STF and Nm values (According to Frankham et al. , an FST . Wright  reported. Wright . Accordientiated , in agreposite Y , which wposite Y . In the posite Y  showed tposite Y  was obseposite Y ,48. The uggested . The FSTm values  suggestem values . Indeed,In the present study, we explored the genetic diversity and relationships within and between a maize panel comprising landrace gene pools from Burkina Faso, Ghana, and Togo and compared each to a reference maize population. The analysis of genetic diversity parameters indicated ample genetic diversity in the maize panel. The four multivariate methods were consistent in dividing the maize panel into three distinct genetic groups, each capable of providing different sources of variation for maize genetic enhancement. The genetic divergence of the Burkinabe gene pool was particularly remarkable. It, therefore, clearly represents an invaluable genetic resource that should be exploited to address the overarching goal of improving maize for adaptation to different environments, ecosystems, and stress situations. Overall, the genetic diversity revealed in this study has provided an invaluable resource for future analyses of candidate genes for local adaptations using robust association mapping experiments.


JAK2/STAT3 pathway plays an important role in cell proliferation, differentiation and inhibition of apoptosis in number of cancers. However, the relationship and interaction between MLAA-34 and JAK2/STAT3 has never been investigated in AML.MLAA-34 is a novel leukemia-associated gene closely related to the carcinogenesis of acute monocytic leukemia (AML). MLAA-34 over expression has been observed to inhibit apoptosis in vitro and in vivo. We constructed MLAA-34 knockdown vector and transfected U937 cells to observe its apoptotic activities in relation to JAK2/STAT3 signaling pathway in vitro and then in vivo in mouse model. Levels of expression of MLAA-34 and JAK2/STAT3 and its downstream targets were also measured in AML patients and a few volunteers.This study investigates and reports a novel relationship between MLAA-34 and JAK2/STAT3 pathway in AML both in vitro and inhibited tumor growth in vivo. Components of the canonical JAK2/STAT3 pathway or its downstream targets, including c-myc, bcl-2, Bax, and caspase-3, were shown to be involved in the carcinogenesis of AML. We also found that the JAK2/STAT3 pathway positively regulated MLAA-34 expression. We additionally identified a STAT3 binding site in the MLAA-34 promoter where STAT3 binds directly and activates MLAA-34 expression. In addition, MLAA-34 was found to form a complex with JAK2 and was enhanced by JAK2 activation. Correlation of MLAA-34 and JAK2/STAT3 was further confirmed in AML patients.We found that MLAA-34 knockdown increased U937 apoptosis In conclusion, MLAA-34 is a novel regulator for JAK2/STAT3 signaling, and in turn, is regulated by this interaction in a positive feedback loop. Thus we report a novel model of interaction mechanism between MLAA-34 and JAK2/STAT3 which can be utilized as a potential target for a novel therapeutic approach in AML. Acute monocytic leukemia (AML) is a cancer of the myeloid lineage of blood cells. It is the most common acute leukemia, which results in white blood cells accumulation in the bone marrow with ultimate impaired hematopoiesis. Although the frequency of complete remission of AML has increased, its prognosis is usually poor due to its resistance to majority of the chemotherapeutic treatments In the last two decades, immunotherapeutic approaches targeting leukemia-associated antigens (LAAs), have been indicated as a promising new strategy for AML patients MLAA-34 over expression markedly inhibited apoptosis in U937 cells Thus, the objective of this study was to determine the interaction mechanism between MLAA-34 and JAK2/STAT3 pathway in AML. We functionally validated the role of MLAA-34 knockdown and its effects on JAK2/STAT3 pathway. JAK2 activator and inhibitor were used to assay the relationship between JAK2/STAT3 and MLAA-34. The interaction between MLAA-34 gene promoter and STAT3 was additionally certificated. Finally, this relationship between MLAA-34 and STAT3/JAK2 was validated in AML patients. Our findings provide new mechanistic insights into the crucial roles of MLAA-34 in regulation of JAK2/STAT3 pathway. Simultaneously, knockdown of MLAA-34 plays an important tumor-inhibiting function through JAK2/STAT3 signaling. These findings provide a novel intervention target for clinical implications in AML treatment.Antibodies, including MLAA-34, STAT3, JAK1, JAK2, c-Myc, Bax, Caspase-3, bcl-2, IgG, β-actin, GAPDH, secondary mouse and rabbit antibodies, were purchased from Cell Signaling Technology, Shanghai, China. Annexin V-FITC/PI Bradford Reagent Kit was bought from Roche, USA. AG490 was purchased from Calbiochem-Nova- biochem, Bad Soden, Germany. IL-6 was purchased from Raybiotech, Inc., Norcross, GA, USA. Quick Change Site-directed Mutagenesis Kit , ChIP and EMSA Assay Kits  were used in the experiment. The pGCSIL-GFP, pcDNA3.1, pGL3-Basic/LUC, pRL-TK vectors, were purchased from Clontech Corporation. Reagents of cell culture were purchased and authenticated from Gene Chem Inc . Bradford Protein Assay Kit, Nu Page 4-12% Bis-Tris protein gels, ECL reagent used were Bio-Rad, Invitrogen, and Amersham Biosciences, respectively. Other reagents were bought from Sigma Aldrich, USA.In this study, 41 AML patients  enrolledU937 cells  were cultured in RPMI-1640 medium, and HEK293 cells were cultured in DMEM. Additionally, U937 cells were treated with the JAK2 inhibitor AG490 (0-50 μM) for 6-24 h or the JAK2 activator IL-6 (0-40 ng/ml) for 12-48 h.MLAA-34 knockdown lentivirus was generated in the pGCSIL-GFP vector (KD), referred to as pGCSIL-GFP-MLAA-34. An empty pGCSIL-GFP vector was used as a normal control (NC). The siRNA sequence for MLAA-34 was 5′-GTACGTGGAGTTGTCAACA-3′. Following U937 cells were cultured in a 24-well plate for 24 h. The viral stock with a multiplicity of infection (MOI) of 100 was added and the transfection mixture was replaced with normal complete growth medium. The infected cells were referred to as MLAA-34-shRNA/U937 (KD) and Vec/U937 (NC) cells.7 cells/200 μl) were injected into the caudal veins of female NOD/SCID mice (10/each group). We also subcutaneously injected mice with cyclophosphamide (100 mg/kg) for 5 days continuously before cell injection. Tumor size was assessed once per week, and tumor volume was calculated as previous description NOD/SCID mice  were purchased from Vital River Laboratories . The mice were housed and had free access to food and drinking water in the SPF laboratory. All animal care and euthanasia were performed in accordance with the guidelines of our Institutional Animal Care and Use Committee (IACUC), University Laboratory Animal Research. Previous KD and NC cells  and analyzed with CELLQUEST software (BD Biosciences) For the western blot assay, infected U937 cells were cultured for 48 h and were collected. The protein concentration was measured using a Bradford protein assay kit. Fifty micrograms of protein were loaded in NuPage 4-12% Bis-Tris protein gels and were detected by ECL reagent GAATTCCGGAGAAACAGTTGGGACC-3′, R: 5′-CGGGGTACCCGTTCTCAGCTCCTCACATG-3′. Total RNA was extracted from U937 cells, and RT-PCR and PCR were used to amplify the STAT3 gene. The amplified STAT3 fragment and the pcDNA3.1 vector were digested with EcoRI and XhoI and then ligated and transformed into E. coli DH5α. After resistance selection and sequence, the recombinant plasmid was named as pcDNA3.1-STAT3, and an empty pcDNA3.1 vector was used as a control. Under the mediation of cationic liposomes, pcDNA3.1-STAT3 and the empty vector pcDNA3.1 were transfected into U937 cells for 24 h. Then, RT-PCR and western blotting were used to assay the expression of MLAA-34 and STAT3 in U937 cells.The primers for STAT3 were as follows: F: 5′-CCGTo analyze the interference of the transcription regulator of STAT3 on MLAA-34 expression, the interference region was selected. STAT3 siRNA sequences were designed as follows: F: 5′-GCAGCAGCUGAACAACAUG-3′, R: 5′-CAUGUUGUUCAGCUGCUGC-3′. Similarly, under the mediation of cationic liposomes, si-STATT3 and NC-siRNA were transfected into U937 cells for 24 h. The expression of MLAA34 and STAT3 in the transfected cells was detected by RT-PCR and western blotting.To further verify the STAT3 binding site on the MLAA34 gene core promoter, a mutant active detection vector with EGFP was constructed and named pGL3-Basic/EGFP/sSTAT3 (mut-STAT3). The core region of the MLAA-34 promoter with EGFP was constructed and named pGL3-Basic/EGFP/MLAA-34-wt (WT) and used as the positive control. The two constructed plasmids were transfected into U937 cells for 24 h, and the expression of green fluorescent protein was observed under a fluorescence microscope.The core region in the MLAA-34 promoter was predicted and amplified by PCR using the following primers: F: 5′-GGGGTACCAAACCTCCCGAGCGCAGGTGCC-3′, R: 5′-CCAAGCTTAACAGCTGCGCCACCACTCCAGT-3′. The recombinant plasmid was named pGL3-Basic/LUC/MLAA-34-wt . According to the sequence (GGAAGG) of the STAT3 binding site in the core region of the MLAA-34 promoter, primers with site-specific mutations were generated using a QuickChange site-directed mutagenesis kit with the following sequences: F: 5′-TCTACCGGGGATGAGACTCTTCGTAACCTCCGCCGG-3′, R: 5′-CCGGCGGAGGTTACGAAGAGTCTCATCCCCGGTAGA-3′. The pGL3-Basic/LUC/MLAA-34-wt plasmid was used, and PCR was performed according to the manufacturer's protocol. Then, the PCR products of the STAT3 mutation were confirmed by sequencing. The site-specific mutagenized plasmid was named pGL3-Basic/LUC/mut-STAT3 (mut-STAT3).HEK293 and U937 cells were inoculated in a 24-well culture. Then the pGL3-Basic/LUC/mut-STAT3 plasmid was cotransfected into NIH3T3 cells with 2 ng Renilla luciferase vector of pRL-TK as an internal interference. The same ratio of pGL3-Basic/LUC , pGL3-Promotor vector  and pGL3-Basic/LUC/MLAA-34-wt  vectors together with the Renilla luciferase vector of pRL-TK were also cotransfected into U937 and HEK293 cells for 24 h. The relative luciferase activity (RLA) was calculated by normalizing the fluorescent luciferase with the internal standard Renilla luciferase.7 cells for 10 min and cells were then washed with ice-cold PBS, harvested by scraping and suspended in lysis buffer containing protease inhibitors. Following incubation on ice, 500 bp DNA fragments were formed under sonication. Anti-STAT3 was incubated with the supernatants with centrifugation for 10 min at 4 °C.ChIP was performed according to the manufacturer's instructions. Briefly, 1% formaldehyde was used to crosslink 1×10IgG or no antibody was used as a control. After using salmon sperm DNA/protein A-agarose to precipitate the immune complexes, they were recovered, and eluted with the ChIP elution buffer and reverse cross-linked by heating at 65 °C for 4 h. A small chromatin-protein sample was excluded and used as an input sample for the positive control in the PCR reaction. Extracted DNA was used as a template for PCR amplification. The primers for the STAT3 binding site in the MLAA-34 gene promoter or the control region were as follows: STAT3 sense: 5′-AGCCTTCATCTCAACCACAAC-3′ and antisense: 5′-CTCCCATGTAGTGATCGGTTT-3′; control sense: 5′-AGGCTGGTCTTGAACTCCTGA-3′and antisense: 5′-GTGTGAACGCTGGTGAGAGC-3′; both at 60 °C for 30 cycles Nuclear extracts were prepared and incubated with biotin-labeled wt or mutant probes. DNA-protein complexes were separated and transferred to nylon membranes, and shifted probes were detected with HRP-conjugated streptavidin. The probe was 5′-AGCGCAGGTGCCCTCTGGCCGGGAAGTACTTCACCAT-′3. After incubation for 20 min at room temperature, 4% native polyacrylamide gel was used to separate the samples. For competition analysis, STAT3 proteins were incubated with unlabeled probes for 20 min at room temperature before the addition of labeled probes Total RNA from NC and KD cells was extracted using TRIzol reagent. Microarray analysis was performed using an Affymetrix microarray human genome U133 chip containing 40,000 genes. Microarray data were further analyzed by ingenuity pathway analysis (IPA). Isolated RNA was assay for the mRNA levels of STAT3, JAK2, c-myc, Bax, caspase-3 and bcl-2 using RT-PCR kits, as in our previous study Co-immunoprecipitation (CO-IP) was used to analyze the interaction between MLAA-34 and JAK2. Briefly, U937-MLAA-34 cells were cultured and transfected with pGCFU-MLAA-34 vectors to generate U937-MLAA-34 cells. Lysis buffer was added to the transfected cells. A protein agarose A+G of 30 μl and 0.5 µg of other antibodies of the same species were added to react for 2 h at 4 °C and centrifuged at 3000 rpm for 15 min. The antibodies were added to the supernatant and incubated for 12 h. A protein agarose A+G of 30 μl was added and incubated for 3-6 h and centrifuged at 3000 rpm for 5 min. The supernatant was discarded, and 1ml lysate was added to react for 10 min. The precipitate was collected, and 30 µl 2× sample buffer was added and placed in boiling water for 5 min. The tubes were immediately placed on ice to cool and then centrifuged at 12000 rpm for 10 min. A 30 µl sample was used for western blotting.Immunohistochemistry (IHC) was performed on 4 μm sections from frozen tumor xenografts of mice as described previously using bcl-2 (1:100), Bax (1:100), JAK2 (1:100), and STAT3(1:100) antibodies for 60 min at room temperature or overnight at 4 °C. Slides were developed as described previously P-values were generated using unpaired Student's t-test and considered significant at P ≤ 0.05, and all experiments were performed in triplicates.All data were expressed as the mean ± S.D. The Mann-Whitney U test was used to evaluate the differences between groups. Spearman's correlation analysis was used for AML patient data. Figure 1, MLAA-34 was successfully knocked down compared with the vector control . We found that MLAA-34 knockdown significantly decreased (P<0.05) cell proliferation in a time-dependent manner . In addition, apoptosis was significantly increased (P<0.001) in KD cells compared with NC cells . These results demonstrated that knockdown of MLAA-34 played an important role in inhibiting the proliferation of U937 cells.To determine the role of MLAA-34, the U937 cells were transfected with the KD and NC vectors. As shown in in vivo, we determined the tumorigenic potential of U937 cells in a NOD/SCID mouse model. We observed that tumors in mice that received KD cells grew much less than tumors that received NC cells . Tumor volumes were significantly decreased (P<0.001) in KD mice compared with those in NC mice after 21 days . Correspondingly, tumor weight was significantly decreased (P<0.001) in KD mice . Similarly, the results of MLAA-34 protein expression levels indicated that MLAA-34 was knocked down  and indicated a significant reduction (P<0.001) in mRNA expression levels in the KD group . Apoptotic cells were significantly increased (P<0.001) in KD mice compared with NC mice . Additionally, obvious reduction in bcl-2  and increment in Bax  were observed in splenic tissues of KD mice compared to NC mice. These results extend the in vitro findings of tumor-suppressive capabilities of MLAA-34 knockdown.To evaluate the tumor-promoting or proliferation effects of MLAA-34 Figure ). This signified the importance of MLAA-34 in AML progression. Additionally, MLAA-34 knockdown mediated genes that are either components of the JAK2/STAT3 pathway or its downstream targets . Important components of this pathway that were differentially regulated in KD cells compared to those in NC cells, included STAT3 (~7-fold downregulated) and JAK2 (~2.5-fold downregulated). The downstream targets of the JAK2/STAT3 pathway included proto-oncogenes, such as c-Myc (2-fold downregulation), survivin (~3.5-fold downregulation) and antiapoptotic genes of bcl-2 (~3.5-fold downregulation). Apoptotic genes of Bax and caspase-3 were upregulated (2- to 4-fold upregulation). Next, we investigated the variations of JAK2/STAT3 and these involved genes by qRT-PCR and western blotting. The results showed a significant reduction in the expression levels of JAK2, STAT3 and c-Myc in KD cells . We observed a significant increment in the expression levels of Bax and caspase-3 . JAK2, STAT3 were also confirmed by IHC analysis in tumors derived from kidney  tissues. As shown in Figure We performed microarray analysis using an Affymetrix gene chip to explore the mechanism involved in MLAA-34-knockdown-mediated tumor suppression. We found that MLAA-34 knockdown significantly affected cellular pathways involved in carcinogenesis. The relative enrichment pathway terms were JAK-STAT, Fas, NFKB and so on . When we treated U937 cells with the JAK2 activator IL-6  for 12, 24 or 48 h, marked increments in MLAA-34, JAK2 and pSTAT3 expressions were observed . The findings demonstrated that the JAK2/STAT3 pathway is positively correlated with MLAA-34 expression. This positive regulation effect was observed in a dose-dependent manner in this experiment.To investigate the correlation between the JAK2/STAT3 pathway and MLAA-34, we measured the effect of JAK2 inhibitor and activator on the expression of MLAA-34 and JAK2/STAT3. As shown in Figure ). Overexpression of STAT3 enhanced MLAA-34 expression in U937 cells, while siRNA-mediated knockdown of STAT3 reduced MLAA-34 expression . When the binding site of STAT3 in the MLAA-34 gene promoter was mutated, the expression of GFP decreased . The role of the STAT3 binding site within the MLAA-34 gene promoter was further demonstrated using a luciferase reporter assay. As shown in Figure , we found that in both U937 and HEK293 cells, the mutation of the STAT3 site (mut-STAT3) resulted in reduction of luciferase activity (P<0.05 and P<0.01). Furthermore, ChIP and EMSA were performed to verify whether STAT3 was directly bound to the STAT3 binding site in the MLAA-34 gene promoter. U937 cell lysate was immunoprecipitated with anti-STAT3 antibody, and the fragment containing the STAT3 binding site in the MLAA-34 gene promoter was amplified by PCR . As shown in Figure The interaction between MLAA-34 and STAT3 was assayed. Based on the transcription factor (TF) prediction programs Gene-regulation, Transfac6.0 and ALGGEN-PROMO, several putative TF binding sites were identified, including USF2, E2F1, SP1, MZF-1 and STAT3 at the MLAA-34 promoter. The binding site for the STAT3 transcription factor is 5′-GGAAGG-3′  but not JAK1 or STAT3 . This interaction was enhanced following treatment with the JAK2 activator IL-6 . Our results indicate that MLAA-34 interacts with JAK2 and that MLAA-34 is required for cytokine-induced JAK2 activation.Our results thus far suggest that JAK2 activation may be directly or indirectly dependent on the presence of MLAA-34. Therefore, we tested the possible interaction between endogenous JAK2 and MLAA-34 using the co-IP method. Based on theU937-MLAA-34 cells, we found that MLAA-34 formed a complex with endogenous JAK2 .To further verify the correlation of MLAA-34 and JAK2/STAT3, we analyzed the mRNA levels of MLAA-34, JAK2, and STAT3 in AML patients and healthy volunteers. In this study, 41 AML patients, including 17 incipient patients (IP), 14 nonremission patients (NRP), and 10 remission patients (RP), and 6 healthy control volunteers (CV) were analyzed. The clinical characteristics at diagnosis were shown in Figure Based on previous findings, we propose an interaction and a schematic diagram of a positive feedback regulation between MLAA-34 and JAK2/STAT3 signaling in AML carcinogenesis, as shown in in vitro and in vivo. Down regulation of MLAA-34 decreases JAK2/STAT3 signaling as well as its downstream target genes. On the other hand, we found that JAK2/STAT3 signaling positively regulated MLAA-34. We found the STAT3 binding site in the MLAA-34 promoter. A complex formed between MLAA-34 and JAK2 was demonstrated, and this was enhanced by IL-6-mediated JAK2 activation . To our knowledge, this is the first study that explores the interaction between MLAA-34 and JAK2/STAT3 signaling pathway and demonstrates its key role in AML.MLAA-34 is a novel M5-associated antigen that is highly expressed in U937 cells and M5 patients. However, little is known about the key regulatory proteins and signaling pathways for MLAA-34. In this study, we revealed a positive feedback regulation between MLAA-34 and JAK2/STAT3. On the one hand, we observed that MLAA-34 knockdown decreases proliferation, increases apoptosis and inhibits tumor growth Our previous studies revealed that MLAA-34 has a potential antiapoptosis effect and may be a novel antiapoptotic factor related closely to carcinogenesis or the progression of AML JAK2/STAT3 signaling can induce cell proliferation, differentiation and antiapoptosis. Aberrantly activated JAK2/STAT3 signaling is critical in malignant progression by promoting cell growth. Inhibited growth and induction of apoptosis through inactivation of JAK2/STAT3 signaling in many cancer cells and tumors have been demonstrated in many published studies. Cellular pathways affected by MLAA-34 knockdown are mainly correlated with cell apoptosis or proliferation processes, immune regulation and inflammation reaction, and KEGG pathways. This finding is similar to our previous findings of shotgun analysis of protein interactions with MLAA-34 Figure ).The JAK2/STAT3 signaling pathway can be activated by several cytokines, including IL-6. IL-6-mediated activation of JAK/STAT3 signaling has been shown to increase the proliferation of cancer cells 2-6 NAA) Figure ).STAT3 belongs to a family of DNA-binding proteins that not only fulfill their roles in normal cell signaling but may also contribute to oncogenesis In conclusion, we revealed that MLAA-34 knockdown is accompanied by a decrease in the JAK2/STAT3 pathway. Simultaneously, JAK2/STAT3 enhances MLAA-34 activation. A positive feedback loop was formed between MLAA-34 and JAK2/STAT3 pathways. MLAA-34/JAK2/STAT3 signals modulate its downstream target genes in AML carcinogenesis. Our results provide the first evidence that MLAA-34 is central to JAK2/STAT3 signaling. We validated that the knockdown of MLAA-34 is highly like to potentiate an antitumor response, thus highlighting the JAK2/STAT3 pathway via MLAA-34 as a potential target for new therapeutic approaches in AML.Supplementary table S1.Click here for additional data file.


Sabellaria alveolata (L.)), a reef-building polychaete that supports high biodiversity, we carried out RAD sequencing using individuals from along its complete latitudinal range. Patterns of neutral population genetic structure were compared to larval dispersal as predicted by ocean circulation modelling, and outlier analyses and genotype-environment association tests were used to attempt to identify loci under selection in relation to local temperature data.Under the threat of climate change populations can disperse, acclimatise or evolve in order to avoid fitness loss. In light of this, it is important to understand neutral gene flow patterns as a measure of dispersal potential, but also adaptive genetic variation as a measure of evolutionary potential. In order to assess genetic variation and how this relates to environment in the honeycomb worm (ST = 0.28 ± 0.10), in line with the low dispersal potential identified by the larval dispersal models. The North Atlantic reef emerged as a distinct population and this was linked to high local larval retention and the effect of the North Atlantic Current on dispersal.We genotyped 482 filtered SNPs, from 68 individuals across nine sites, 27 of which were identified as outliers using BAYESCAN and ARLEQUIN. All outlier loci were potentially under balancing selection, despite previous evidence of local adaptation in the system. Limited gene flow was observed among reef-sites (FAs an isolated population, with limited potential for natural genetic or demographic augmentation from other reefs, the North Atlantic site warrants conservation attention in order to preserve not only this species, but above all the crucial functional ecological roles that are associated with their bioconstructions. Our study highlights the utility of using seascape genomics to identify populations of conservation concern. Conservation strategies in the context of climate change rest on the premise that populations can show three responses to avoid fitness loss: evade, evolve or acclimatise . CharactHomarus americanus H. Milne Edwards, 1837), whereas sea surface temperature acted as an agent for natural selection. Ocean circulation modelling for understanding dispersing larval stages has emerged as an important tool to explain neutral population structure where isolation-by-distance measures alone are uninformative [Despite genomic advances allowing investigations of genetic diversity in non-model species, which is particularly important for species of conservation concern, understanding of gene flow and local adaptation in marine environments had, until recently, lagged behind that for terrestrial species –16. Receormative .Intertidal communities have been proposed as particularly suitable for studying the impacts of climate change, as many species already exist at the limit of their thermal tolerance ranges , 22. EcoSabellaria alveolata ), is an ecosystem engineer that constructs biogenic reefs along temperate coastlines of North Africa and Europe [Sabellaria, are listed under the EC Habitats Directive (92/43/EEC) Annex 1 and are thus of conservation interest [S. alveolata. Neutral population structuring is expected because (i) adults are sessile, and (ii) during the dispersive planktotrophic larval stage of this species, individuals have been shown to demonstrate positive taxis towards the cement of sand grains and bioclasts of S. alveolata tubes [The honeycomb worm  andta tubes  leading ta tubes . HoweverS. alveolata show local adaptation to temperature in terms of their membrane lipid composition [S. alveolata provide an excellent opportunity to investigate if/how adaptive population structure is related to temperature, and assess the role of neutral processes in gene flow.We have previously demonstrated that position  but the S. alveolata, and the relationship with the environment. We test: 1) if gene flow is restricted among S. alveolata reef sites and whether population structure can be predicted by ocean circulation modelling; and 2) whether S. alveolata show evidence of local adaptation and if this relates to local thermal conditions.In this study, we explore the extent of neutral and adaptive genetic variation among reef sites of the honeycomb worm, Due to high variability in sequence coverage between samples . Observed heterozygosity was low across all sites (Ho = 0.11 ± 0.02) but was significantly lower than He only in the Balearic Sea site, which had a low sample size (Table P < 0.01) and within rather than between the genetic clusters identified by STRUCTURE (see below) .All sites showed reasonable numbers of alleles per locus, nucleotide diversity and expected heterozygosity  and BAYESCAN identified 28 as outliers .Pairwise FST distances .Maps of larval densities from dispersal simulations show the range of potential dispersal pathways for a single generation released from each sample location: from the most conservative , Fig. es Table . Under tBoth ARLEQUIN and BAYESCAN analyses suggested that the 27 loci identified as outliers were under balancing selection Fig.  and onlyS. alveolata reef sites spanning the latitudinal and longitudinal range of the species revealed low gene flow between sites, equating to low effective dispersal, with 25 out of 36 pairwise FST values showing significance and an average pairwise FST of 0.28 ± 0.10 across the system. The DAPC supported the differentiation of the Tyrrhenian Sea site from the other sites, as also identified by high FST values between this site and all other sites (FST = 0.32 ± 0.12), and the STRUCTURE analysis revealed the North Atlantic site to be a separate population from all the others (FST = 0.42 ± 0.10). Genetic divergence in terms of FST was not significantly correlated with either straight-line distance or shortest ocean distance. Presence of low gene flow between reef sites is in line with a growing number of studies that have identified population structuring in marine species which, as with S. alveolata in our study, have a dispersing larval stage and no hard geographical barriers to dispersal [ST = 0.0019) in the American lobster (H. americanus) along a sea surface temperature gradient using RAD genotyping despite a dispersing planktonic larval stage with a duration of 4–6 weeks. Similarly, the bat star Patiria miniate  showed genetic population structure along a latitudinal gradient (average pairwise FST = 0.061) despite a longer larval dispersal period of 6–10 weeks [S. alveolata at 5–12 weeks, dependant on temperature and food availability [Population genetic analysis using genome-wide SNPs identified from nine ispersal , 48–50. 10 weeks , which i FST = 0. ± 0.10. lability . Indeed,lability  and our ST = 0.42 ± 0.10 and STRUCTURE), Tyrrhenian Sea (average pairwise FST = 0.32 ± 0.12 and DAPC) and Bay of Biscay (average pairwise FST = 0.25 ± 0.05). The observed differences in the results produced by the two cluster assignment methods can be explained by how they use data: STRUCTURE assigns cluster groupings in order to minimise Hardy–Weinberg and linkage disequilibria within clusters by looking at allele frequencies [ST) and English Channel (in terms of FST and the DAPC) sites were also all differentiated from each other . Observed heterozygosity was very low at all sites and could be due to inbreeding [High population sub-structuring could be partially explained by two behavioural barriers to connectivity: first, spawning in polychaetes is triggered by the action of waves, especially during spring tides, which tends to push larvae toward the coast rather than offshore  and secoing-tide . This ‘ting-tide , 57). Yebreeding , as a rebreeding . Heterozbreeding . Howeverbreeding , 60. FurS. alveolata, as in-depth local ecological knowledge, such as population size and breeding behaviour, is lacking in the majority of sites . showed a private Irish Sea haplotype linking two ancestral haplotypes and they suggest this could have evolved in a small ice-free area along the southwest coast of Ireland. Therefore, one potential explanation is that this population was isolated in a different glacial refugium to the rest of the sites, with subsequent admixture, but further study is needed to test this hypothesis.Despite some evidence of low levels of gene flow between the majority of the study sites within the system, both Flaciated  and genelaciated , 69 and laciated –72. In placiated  found thS. alveolata sites, is supported by predictions of larval dispersal as seen in the ocean circulation modelling. In even the most optimistic scenario for larval dispersal, the North Atlantic site was not predicted to have interchange of individuals with any of the other sampled sites and had by far the highest predicted proportion of larval retention . This can be explained by the hydrodynamic modelling in terms of current patterns within Galway Bay limiting dispersal. The presence of the North Atlantic Current, which moves eastwardly towards Ireland and then continues northwards, is also an isolating factor for the North Atlantic reef. Any larvae that do move beyond their spawning reef at the North Atlantic site are drawn northwards, beyond the current northern range limit for the species . This oScotland , 74), isS. alveolata in particular, as identified by our data in regards to the isolated North Atlantic site, our ocean circulation models of larval dispersal did not show a significant correlation with observed genetic distance between sites. This is surprising as both FST values and larval dispersal models suggest low connectivity between sites. This lack of correlation between our larval dispersal modelling and genetic distance values is in contrast to studies on a number of species with a dispersing larval stage including the Mediterranean shore crab  [P. miniata) [H. americanus) [Pandalus borealis Krøyer, 1838). Galindo et al. (2010) [Balanus glandula Darwin, 1854) but it also did not match an observed genetic cline in the species. They discovered that their model fit was improved by including natural selection, larval retention, and input values from an additional source population [Despite the importance of ocean circulation to larval dispersal in general , 19, 75 miniata)  and the ricanus) ; these sricanus)  found th. (2010)  modifiedpulation .S. alveolata reefs are not well documented, particularly in the southern range of the species , and the prediction of our model that genetic differentiation between our sample sites would be high could be due to the absence of intermediate reef sites within the model that would create higher levels of admixture within the system as a whole [S. alveolata reefs and ongoing monitoring of reef sites is required to generate a clearer picture of the role of connectivity in meta-population maintenance in this species and to further inform the ocean circulation model. Our study highlights the importance of comparing bio-physical models with observed population structure of a species in order to create accurate dispersal predictions.There are several factors likely to reduce the similarity between modelled larval dispersal and observed gene flow. Gene flow is representative of multigenerational mixing, while the model used in this study is representative of the dispersal patterns of only one generation. It is possible to simulate multigenerational gene flow based on dispersal models , 77, 78  a whole , 79. Thi a whole . TherefoNematostella vectensis Stephenson, 1935, covering a broad geographical range, Reitzel et al. (2013) [Twenty-seven SNPs were identified as outlier loci, all of which were putatively under balancing selection. Identification of a higher number of loci potentially under balancing rather than divergent selection is common in genome scan studies, including those on marine invertebrates , 80–82. . (2013)  identifi. (2013) .S. alveolata individuals from sites along the latitudinal range of this species  showed different responses to thermal regime changes in terms of their membrane lipid composition dependant on their site of origin [The lack of identification of loci under divergent selection using either outlier analyses or environmental association tests is surprising given that local adaptation to temperature is known to be present in this system . We prevf origin . This isf origin ) and whof origin .S. alveolata across their range, which was in part supported by the genomic data. In particular, population genetic analyses identified the North Atlantic reef is an isolated population, due to the effect on dispersal of the North Atlantic Current. These findings have implications for the management of S. alveolata engineered habitats in a changing climate and have highlighted the North Atlantic reef as at increased risk and in need of direct conservation action in order to preserve not only this species, but above all the crucial functional ecological roles  that are associated with these bioconstructions. Our study highlights the utility of using seascape genomics to identify populations of conservation concern.Modelling of larval dispersal predicted low gene flow between sites for In total, 180 mature individual worms were haphazardly collected from nine sites (20 per site) across the global geographic range between August 2013 and March 2014, from North Africa at the species’ southern boundary, along the Atlantic Coast, to the Northern Irish Sea in the north, as well as from two sites on the Mediterranean coast , visualised on 1% agarose gels, and quantified using the Quant-IT PicoGreen dsDNA Assay Kit . Single-end Restriction-site Associated DNA (RAD) libraries were prepared by digesting DNA from each individual using the EcoRI and MseI restriction enzymes. Samples were individually barcoded and sequenced  using an Illumina NextSeq 500 .Genomic DNA was extracted using chaotropic “Chaos” buffer aminomethane (Tris), pH 8.0; 0.1 M 2-mercaptoethanol, 0.5% N-lauroyl sarcosine) . Worms galaxy.sb-roscoff.fr [All analyses were carried out on the Analyses and Bioinformatics for Marine Science (ABiMS) Galaxy Platform at the Station Biologique de Roscoff . Samplscoff.fr . STACKS scoff.fr  was usedscoff.fr  namely, scoff.fr . Furtherscoff.fr . The numscoff.fr ;).The repopulations from the STACKS suite, was used to process the SNP data [populations was transformed in PGD Spider version 2.1.0.3 [The programme SNP data . RADtagsSNP data ;). LumpeSNP data , but 10xSNP data  and thusSNP data , 94 and SNP data . In orde 2.1.0.3  for use  2.1.0.3  and sign 2.1.0.3 .e) and observed heterozygosity (Ho). Separate analyses of molecular variance (AMOVA) were carried out based on site of origin and genetic clusters identified in STRUCTURE (see below) using 16,000 permutations.Genomic summary statistics were calculated in ARLEQUIN version 3.5.2.2  as mean ST than expected under a neutral model of selection . Outliers were estimated in two ways: firstly, using BAYESCAN version 2.1 [P-value of ≤0.05. Only SNPs identified as outliers by both BAYESCAN and ARLEQUIN were conservatively selected as candidate loci under balancing or directional selection. Subsequent analyses of population structure and seascape genomics were conducted separately for neutral and outlier loci.An outlier approach was used to identify loci that had a higher or lower Fsion 2.1 , which usion 2.1 , 17, 98.sion 2.1 . OutlierST were calculated in ARLEQUIN version 3.5.2.2 [marmap [ape 5.3 [ST and shortest ocean distances for each pairwise (site) combination. Divergence between sites was also assessed using a Discriminant Analysis of Principal Components (DAPC) with the R package adegenet 2.1.2 [Pairwise F 3.5.2.2  and sign 3.5.2.2  using th [marmap , where d [marmap . Isolati [marmap  with 10,[ape 5.3 , followi[ape 5.3 , and weret 2.1.2 , with 30Genetic clusters (populations) were inferred using Bayesian analyses carried out in STRUCTURE version 2.3.4 . The ana2s− 1, vertical = 0.05 m2s− 1; after Kough et al. 2016 [Hydrodynamic models are increasingly used as predictors of larval dispersal (gene flow) in marine systems . HYCOM al. 2016 . Simulatal. 2016 .S. alveolata larva, were released in a Lagrangian framework , from each of nine locations  an optimistic scenario which modelled a daily release on every day of the year. The optimistic scenario was designed to simulate all the potential pathways of dispersal if reproduction occurs throughout the year, which has been suggested in S. alveolata [GBIF.org) in which larvae could also settle if encountered. Larval exchange counts (i.e. the source-sink contribution) were generated after 5, 8, 10, and 12 weeks PLD. Model outputs were processed in Matlab . Pairwise matrices of larval exchange counts, standardised using proportions, were generated from larval fates averaged across the three focus years to compare against genetic pairwise FST matrices. Daily positions of larvae throughout the simulation were used to generate cumulative 2-D maps of larval density to highlight the predicted pathways of dispersal and areas of potential settlement.In the simulations, 100 propagules, each representing an lveolata , thus calveolata , 51. LarST  and dispersal. A Bonferroni correction was applied to assess the significance of the Mantel tests [The proportion of settlers reaching each study site from each release site under the four larval duration scenarios  in the ocean circulation modelling simulations were inverted and used to form a matrix of pairwise dispersal resistances (i.e. the proportion of larvae that did not reach each site). Mantel tests  were carel tests .S. alveolata experience at a spatial and temporal scale that cannot be achieved using satellite data [Locally collected temperature data provides insight into the conditions that ite data  and can ite data . Temperaite data  who usedite data , 118. AtS. alveolata experience tidally-driven fluctuations in water and air temperatures. Sixteen metrics were calculated to describe variation in temperature at each site as follows: mean temperature, maximum temperature, minimum temperature, temperature range (maximum-minimum), standard deviation of the mean, 95th – 5th percentile (to exclude outlier temperatures), average daily temperature range (maximum-minimum per day), and average daily standard distribution (standard deviation of the mean), per site. High-tide (water) and low-tide (air) temperature values were also used separately to calculate mean temperature, maximum temperature, minimum temperature, and temperature range (maximum-minimum) for water and air temperatures, respectively (Table S. alveolata can be found in the water column all year round [ly Table . Larvae ar round , suggestWe utilised a gene-environment association software, BAYENV2 , to testAdditional file 1. SeascapeGenomics_Muiretal.


Target specific small molecule inhibitors has driven signaling pathway discovery and are used as common positive controls in drug discovery screens. During a biophysical screen, using surface plasmon resonance spectroscopy, of a novel small molecule library for the Signal Transducer and Activator of Transcription 3 Src Homology 2 (STAT3-SH2) low molecular weight interactors we evaluated commercial inhibitors S3I-201 and 5,15-diphenylporphyrin  as positive controls.Here, we show using surface plasmon resonance spectroscopy that a common STAT3-SH2 inhibitor, 5,15-diphenylporphyrin , does not bind STAT3 core amino acid residues 127 to 722 relative to another commercially available SH2 inhibitor, S3I-201. This finding should provide caution in data interpretation when using 5,15-DPP in in vitro and in vivo laboratory investigations. It is not uncommon to probe signaling pathways using specific small molecule inhibitors in order to elucidate key cascading events . The decDiscovered through in silico screening, S3I-201 is a cell-permeable STAT3 inhibitor that binds to the STAT3-SH2 domain, prevents phosphorylation/activation, dimerization, and DNA-binding . Similar2. The sensor chips were initialized, primed, and preconditioned in horizontal and vertical parallel flow channels 0.5% (w/v) sodium dodecyl sulfate, 100 mM HCl and 0.85% (v/v) phosphoric acid at 30 μL/min. Recombinant mouse STAT3βtc  was expressed, purified [Briefly, surface plasmon resonance (SPR) spectroscopy was performed using the ProteOn™XPR36 Interaction Array System (Bio-Rad) in 40 mM HEPES, pH 7.4, 150 mM KCl and 5 mM MgClpurified ; STAT3 wStructural analysis of purified recombinant STAT3 was performed using FTIR spectroscopy Fig.  in the pD of 880 nM.The sensorgrams shown in Fig. One caveat to the work shown here may be that 5,15 DPP may indeed bind STAT3 but not at the SH2 domain; the construct used lacks the first 126 amino acids of the N-terminal cooperative DNA binding domain required for STAT3 concatemerization at DNA binding sites and may play a role in transcriptional regulation . Taken tAdditional file 1. Representative exported dataset of controls S3I-201 and 5,15-DPP from surface plasmon resonance spectroscopy screening experiment used to generate Fig.