Posttransplantation diabetes mellitus (PTDM) is a known complication of transplantation that affects the prognosis. Tacrolimus (Tac or FK506) is a widely used immunosuppressant that has been reported to be a risk factor for PTDM and to further induce complications in heart and skeletal muscles, but the mechanism is still largely unknown. In our preliminary experiments, we found that after Tac treatment, blood glucose increased, and the weight of skeletal muscle declined. Here, we hypothesize that tacrolimus can induce PTDM and influence the atrophy of skeletal muscle.  We designed preliminary experiments to establish a tacrolimus-induced PTDM model. Gene expression profiles in quadriceps muscle from this rat model were characterized by oligonucleotide microarrays. Then, differences in gene expression profiles in muscle from PTDM rats that received tacrolimus and control subjects were analyzed by using GeneSpring GX 11.0 software (Agilent). Functional annotation and enrichment analysis of differentially expressed genes (DEGs) helped us identify clues for the side effects of tacrolimus.  Our experiments found that the quadriceps in tacrolimus-induced PTDM group were smaller than those in the control group. The study identified 275 DEGs that may be responsible for insulin resistance and the progression of PTDM, including 86 upregulated genes and 199 downregulated genes. GO and KEGG functional analysis of the DEGs showed a significant correlation between PTDM and muscle development. PPI network analysis screened eight hub genes and found that they were related to troponin and tropomyosin.  This study explored the molecular mechanism of muscle atrophy in a tacrolimus-induced PTDM model by bioinformatics analyses. We identified 275 DEGs and identified significant biomarkers for predicting the development and progression of tacrolimus-induced PTDM. Posttransplantation diabetes mellitus (PTDM) is a known complication of transplantation . It has Tacrolimus Tac or FK506) has been reported as a risk factor for PTDM because it can cause insulin hyposecretion 06 has be, 12. A pGene expression profiles measure the expression of thousands of genes simultaneously to build a global picture of cell function, and gene expression profiling can be used as an important diagnostic test , 19. Thiγ which was related to insulin resistance in Tac-induced PTDM expressed in quadriceps [Therefore, we hypothesize that tacrolimus induces PTDM and affects skeletal muscle function such as atrophy. It has been reported that quadriceps are kinds of oxidative muscle which were closely related to insulin resistance  while thadriceps . So, quaTacrolimus capsules, approval number: Zhunzi J20090142, were purchased from Astellas Pharma Co., Ltd. (China); saline was purchased from Haili Chen Biotechnology Co., Ltd.; chloral hydrate was purchased from Shanghai to Beijing Biological Technology Co., Ltd. Cable; a blood glucose meter and blood glucose test strips were purchased from Changsha Sannuo Biosensing Co.; and TRIzol reagent was purchased from Gibco BRL.We designed preliminary experiments to establish the maximum dose of tacrolimus needed to control the clinical conditions of most of the patients to determine the target trough plasma levels of tacrolimus (≤20 ng/mL). We treated three groups of animals with i.p. tacrolimus . Animals were treated for 7 days, and plasma levels of the drug were serially measured. The target trough level of tacrolimus was achieved with a dose of 1 mg/kg/day.Male Sprague-Dawley rats (body weight: 200-250 g) were obtained from Vital River Laboratory Animal Technology Co. Ltd, housed in cages at 22°C under a 12 h light-dark cycle, and fed commercial pellet chow ad libitum. All animal care, use, and experimental protocols were submitted and approved by The Tab of Animal Experimental Ethical Inspection of Laboratory Animal Centre, Wenzhou Medical University. Groups of rats were injected once a day with saline (control) or FK506 in saline (1 mg/kg BW). Body weight and glucose, which was measured in the tail blood with an Accu-chek Sensor instrument, were regularly recorded every two days at the end of the light period. Once the treatment finished, animals were euthanized and their quadriceps and gastrocnemius were dissected out.μm sections and then deparaffinized and rehydrated. The muscles were stained with hematoxylin-eosin  and observed with a light microscope at 200x. The cross-section area was determined for at least 100 fibers per tissue.The quadriceps and gastrocnemius of both groups were collected and weighed and then fixed in paraformaldehyde for 36 h. The tissues were dehydrated in graded ethanol solutions, vitrified with dimethylbenzene, and embedded in paraffin. Paraffin-embedded muscles were cut into 4.5 p value < 0.05 and minimum fold change > 1.5  using GeneSpring GX 11.0 software (Agilent) and (3) a difference of intensity between the two groups ≥ 1000 when the log2 ratios = NA.This dataset included 6 samples of quadriceps muscle from PTDM rats that received tacrolimus and normal rats with three biological replicates. RNA was extracted from the samples with TRIzol reagent according to the manufacturer's protocol. RNA was reverse transcribed by a RevertAid First Strand cDNA Synthesis Kit. Further labeling and microarray hybridization experiments and washes were performed at GeneChem . Microarray analysis was performed on an Agilent platform . Differentially expressed genes in PTDM rats were defined as follows: (1) normalized intensity: after normalizing with median scaling normalization method, take the average of repeated data from the same sample. The software of Rosetta Resolver was used to list normalized data (2) https://string-db.org) is a system for determining interactions between genes or proteins and was used to guide the construction of the PPI network of downregulated top 50 DEGs to explore functional interactions [GO and KEGG enrichment analysis of differentially expressed genes in skeletal muscle from PTDM rats was performed using WebGestalt. Web Gene Ontology Annotation Plot (WEGO 2.0) was used to visualize the GO classification results. The STRING database  using a machine of 7500 Fast machine (Applied Biosystems). GAPDH was used as an internal standard, and the results were analyzed by the 2p values < 0.05 were considered statistically significant.All data are presented as mean values ± SD and performed using one-way ANOVA. p > 0.05), before the injection. After two weeks of treatment, the blood glucose concentrations of the two groups were 19.51 ± 4.22 and 6.87 ± 2.05 mmol/L, respectively, and the concentration of the PTDM group was significantly higher than that of the control group (p < 0.01)  (p < 0.0001) (p < 0.05). Those gaps between muscle fibers were narrow in the control group. Fibers of quadriceps obtained from Tac rats exhibited atrophy and disordered arrangement  . The wet 0.0001) . The cro Figures . The wei Figures .p value cutoff of 0.05 and a fold change cutoff of 1.5 (upregulated or downregulated). According to the threshold in this study, we identified 275 differentially expressed genes, 86 upregulated genes and 199 downregulated genes, between these two groups. The majority of differentially expressed genes in muscle from PTDM rats exhibited expression levels with a fold change lower than 4.0 (up or down) when compared to those in muscle from control subjects, and a few genes showed a fold change ≥ 4.0. The principal component analysis (PCA)   score plTo assess the function of the differentially expressed genes in skeletal muscle, we first assigned the genes to different GO levels. The GO classification analysis showed a relatively high abundance of genes involved in binding and cellular processes . Then, wAccording to GO enrichment analysis, downregulated differentially expressed genes between control and PTDM rats were significantly enriched in multicellular organismal movement, muscle structure development, and circulatory system processes in the biological process category, contractile fibers in the cell component category, and actin binding and cell adhesion molecule binding in the molecular function category. These findings indicated that the GO categories of these differentially expressed probes were predominantly related to muscle function and movement.The KEGG classification analysis of upregulated and downregulated DEGs was shown in Functional enrichment KEGG analysis of downregulated DEGs, as shown in The upregulated DEGs PP2A and ECM as well as downregulated DEGs such as GF, cytokine R, ITGA, and p21 were shown to be involved in the PI3K-Akt signaling pathway . Akt hasAs seen from the above analysis results, significant functional enrichment was found only in downregulated genes. We further explored whether there was a close significant relationship between the downregulated genes. The PPI network of 41 candidate rat genes obtained by screening for downregulated genes that were significantly enriched in GO and KEGG functions is shown in p value < 1.0e‐16 means that those proteins have more interactions among themselves than what would be expected for a random set of proteins of similar size, drawn from the rat genome. Such an enrichment indicates that the proteins are at least partially biologically connected, as a group. There were 24 nodes and 31 edges in the PPI network based on the top 50 DEGs between two groups, and the average node degree was 2.58. The PPI network contained 8 hub genes according to our criterion, which were Tpm3, Tnnc1, Tnnt1, Tnni3, Hrh3, Apln, S1pr3, and Cxcl12, and all of these genes are related to troponin, myosin, and muscle  which is one of the most severe complications of transplantation and influences the survival . ResearcA study showed that diabetes mellitus influences the structure and function of skeletal muscle , 29. SkeCardiac muscle contraction is also regulated by troponin and tropomyosin. Troponin is made up of troponin C, troponin I, and troponin T. Under low calcium conditions, troponin combines with tropomyosin and actin through the inhibitory protein troponin I, which leads to the diastole of the myocardium. On the other hand, the activation of myofilament and binding to troponin C are attributed to calcium. The conformational change of troponin inhibits the binding of troponin I and actin, which results in systole .In this study, the microarray method was used to investigate gene expression profiles in Tac-induced PTDM and control subjects. A list of 275 differentially expressed genes (86 upregulated and 199 downregulated) was identified in Tac-treated rats. GO classification analysis showed downregulated expression of DEGs related to muscle structure development. The DEGs were also associated with contractile fibers as a cellular component, actin binding as a molecular function, and so on.We further built a PPI network of 50 top DEGs, which is shown in Tacrolimus-induced PTDM is a severe complication of posttransplantation with poor prognosis. This study attempted to identify the association between tacrolimus and PTDM by using bioinformatics analysis. Functional analysis of 275 DEGs between the tacrolimus-induced PTDM group and the control group illustrated that muscle atrophy of PTDM is induced by tacrolimus through inhibition of the expression of Tpm3, Tnnc1, Tnnt1, Tnni3, Hrh3, Apln, S1pr3, and Cxcl12. The target genes we found may have a guiding significance for predicting or even reducing the risk to patients. Moreover, the next study will focus on experiments such as western blotting and PCR in clinical cases.


Mangrove forests are widespread along the subtropical and tropical coasts. They provide a habitat for a wide variety of plants, animals and microorganisms, and act as a buffer zone between the ocean and land. Along with other coastal environments, mangrove ecosystems are under increasing pressure from human activities, such as excessive input of nutrients and toxic pollutants. Despite efforts to understand the diversity of microbes in mangrove sediments, their metabolic capability in pristine and contaminated mangrove sediments remains largely unknown. By using metagenomic approach, we investigated the metabolic capacity of microorganisms in contaminated (CMS) and pristine (PMS) mangrove sediments at subtropical and tropical coastal sites. When comparing the CMS with PMS, we found that the former had a reduced diazotroph abundance and nitrogen fixing capability, but an enhanced metabolism that is related to the generation of microbial greenhouse gases via increased methanogenesis and sulfate reduction. In addition, a high concentration of heavy metals  and abundance of metal/antibiotic resistance encoding genes were found in CMS. Together, these data provide evidence that contamination in mangrove sediment can markedly change microbial community and metabolism; however, no significant differences in gene distribution were found between the subtropical and tropical mangrove sediments. In summary, contamination in mangrove sediments might weaken the microbial metabolisms that enable the mangrove ecosystems to act as a buffer zone for terrestrial nutrients deposition, and induce bioremediation processes accompanied with an increase in greenhouse gas emission. In recent years, the increasing level of contamination in mangrove sediments has started to pose a serious threat to mangrove forests, with plants disappearing at a rate estimated to be ~1–2% globally per year3. Although mangroves act as a natural sewage treatment plant, exhibiting a robust ecosystem restoration capacity5, the ever increasing levels of contaminants are now overloading mangrove sediments, and this influences their restoration and nutrient cycling capacity by affecting the sediment-inhabiting microorganisms9.Mangrove ecosystems are widely distributed in the transition between land and sea, particularly along the estuaries and coastlines in subtropical and tropical regions. The microorganisms that inhabit mangrove sediments play a critical role in the biogeochemical cycling of carbon, nitrogen, phosphorus, as well as the deposition of heavy metals from adjacent land and fluvial imports, through metabolic processes12. It has been demonstrated that contamination of the mangrove sediment can significantly promote the diversity of the microbial community as a whole, but at the same time it has a negative impact on some specific groups of microorganisms14. For example, acetoclastic/methylotrophic methanogens exhibited a decreased level of diversity in a contaminated mangrove in Singapore6. In addition, metals are retained in mangrove sediments rather than moving to adjacent environments, but this also markedly influences the autochthonic microbial community15. For example, both metals and metalloids have been shown to contribute to the abundance and distribution of ammonia-oxidizing archaea and beta-proteobacteria, both of which are crucial to the nitrogen cycle processes in mangrove sediments17. The fact that polycyclic aromatic hydrocarbon- (PAH-) and phenanthrene polluted mangrove sediments can also increase growth of specific microbial degraders, which illustrates the strong adaptation and bioremediation ability of mangrove sediment-residing microorganisms21. “To further confirm the distribution of microbial genes in mangrove sediment, researchers had applied metagenomic approach in many different regions recently. For instance, Andreote et al. had investigated the microbial participants in sulfur and nitrogen cycle in Brazilian mangrove sediment22; Imchen et al. studied the variation of microbial taxon and metabolic genes across diverse mangrove sediment system23; the prevalent distribution of heavy metals and antibiotic resistome in mangrove sediments at India, Brazil and Saudi Arabia was also reported24. However, the lack of a fundamental level of understanding of the microorganism-related metabolic capability in pristine and contaminated mangrove sediments hinders our determination of the carrying capacity of the ecosystem, as well as the capability of the microbiome in responding to a further increase in pollution in mangrove sediments.The effects of anthropogenic nutrients and pollutants on the microflora in mangrove sediments have been widely studied26. Located in the northern part of Hainan Island, Dongzhai harbor in Haikou city has one of the largest natural mangrove forests in China and it is the first to have been made into a nature reserve; however, the local aquaculture industry has resulted in severe eutrophication in the offshore part of reserve, while the inshore part is relatively pristine. In contrast, the mangroves in Sanya city, located at the southern tip of Hainan Island, have been partially contaminated by both pre-treated and untreated domestic sewage as well as by industrial wastewater in inshore part, while the offshore part is relatively pristine. Thus, the mangroves in Dongzhai and Sanya are ideal sites for us to investigate the metabolic capacity of microorganisms in both contaminated and uncontaminated mangrove sediments under different climatic conditions. By applying metagenomic approach, researchers had already investigatedMangroves located in the northern and southern parts of Hainan Island (China) are representative of subtropical and tropical habitats, respectivelyIn this study, we explored the community structure and metabolism capacity of the microorganisms in the heavily polluted and relatively pristine mangrove sediments in both tropical Sanya and subtropical Haikou using a metagenomic approach.As shown in supplementary Table −2 h−1) than in the PMS (−0.143~−0.017 mg·m−2 h−1), and a higher methane flux was measured in the subtropical mangrove sediment than in the tropical sites  and 2687.74 mg/kg in pristine mangrove sediment of Haikou (HKP)) and tropical  and 205.53 mg/kg in pristine mangrove sediment of Sanya (SYP)) samples. In addition, the concentration of Zn in the subtropical sites was an order magnitude higher than that in the tropical sites. Similarly, the amounts of Cd  and Pb  were also all higher in the CMS than in the PMS Table . In addies Table .After assembly of the quality controlled short reads, a total of 810,027 contigs (>400 bp) were obtained, ranging from 172,238 to 231,903 contigs in the four samples , were widely distributed among the samples. The most abundant, i.e., Gammaproteobacteria, accounted for more than 25% of all the phyla across the four samples, but they showed a marked decrease in the CMS  and SYP (12.3%). In addition, the relative abundance of Alphaproteobacteria ranged from 4.5% to 8.3% across all the samples, with the highest and lowest values found in the SYC and HKP, respectively, and this class was markedly enriched in the tropical mangrove sediment with a log10 odds ratio of −0.1  in the tropical mangrove sediment, whereas Peptococcaceae, Halomonadaceae, Desulfurococcaceae, Methanobacteriaceae, Methanococcaceae, Methanomicrobiaceae were all more abundant (with an odds ratio >0.6) in the subtropical mangrove sediment  in the CMS, while Desulfohalobiaceae, Oceanospirillaceae, and Puniceicoccaceae were more abundant  in the PMS  and at ent Fig. . A compaPMS Fig. .Figure 2nifH) was found in the subtropical region (HKC and HKP), whereas key genes in ammonia oxidation (amoA), assimilatory nitrate reduction (nirA), dissimilatory nitrate reduction (nirD), and denitrification (nosZ) were more abundant in the tropical region (SYC and SYP)  exhibited a higher abundance in the pristine samples; whereas nirA and nosZ were more abundant in the contaminated samples  Fig. . In addiles Fig. . The orgles Fig. . In addiles Fig. .Figure 3Gammaproteobacteria (affiliated with nirD and nosZ), and Alphaproteobacteria (affiliated with nirB and nirD) were found in the subtropical and tropical sediments, respectively. In addition, we also found that within the nosZ-affiliated microbial taxa, Pseudomonadales, which is a typical heterotrophic denitrifying bacteria, was abundant in the CMS  or methanol , were more abundant in the CMS (HKC and SYC). In contrast, formylmethanofuran dehydrogenase (fwdA), which is a key gene in autotrophic methanogens, was more abundant in the PMS (HKP and SYP). Essential genes within the metabolic pathway of methanotrophic bacteria (for example pmoA and mdh), were more abundant in the CMS (HKC and SYC). It was also noticeable that within the methanotrophic pathway, the gene functioning to generate carbon dioxide was more abundant in the CMS.The abundance of essential microbial methane generation and utilization genes was investigated Fig. . A compa29. This might therefore explain why less abundant diazotrophs, such as Rhodospirillacene within the Alphaproteobacteria class30, and Clostridiaceae within the Firmicutes phylum31  and SRB bacteria (Desulfovibrionaceae and Desulfobacteraceae) in the CMS, reflects the impact of contamination on the microbial communities  and acetyl-CoA decarbonylase/synthase (cdhc), whereas autotrophic methanogens can process the reduction of CO2 to methane via formylmethanofuran dehydrogenase (fwdA)48. Within the PMS, the consumption of atmospheric methane  and Sanya  in Hainan Island, China, in August 2016  and an average quality score <20, or a length <140 bp were removed. The resulting clear paired-end reads were assembled using four different assemblers: IDBA_UD60; Velvet61; MEGAHIT62; and SPAdes63, each using their default setting. After comparing the assembly results obtained from the different assemblers, those from the SPAdes software were selected for further analyses.Raw reads were treated using the FASTX-Toolkit package64. Following the analysis pipe-line previously described65, the extracted SSU rRNAs were analyzed using QIIME 1.9.1 against the Silva database (version 123). A filtered OTUs table at 0.1% abundance of each sample was generated with QIIME 1.9.1, and the community structure was visualized with the R software using the ‘vegan’ package66.Screening of SSU rRNAs from the assembled contigs was conducted using Metaxa267, with a minimum length of 200 bp. The extracted ORFs were then annotated with the Kyoto Encyclopedia of Genes and Genomes (KEGG) database68, the SEED database, and the non-redundant protein (NR) database via DIAMOND69 using the following parameters, blastp, k = 1, and an e-value = 10−7 to select the best annotation result. For each sample, clean short reads were mapped back to ORFs using Bowtie 2.2.970 to reveal the coverage information for individual functional genes. The web-based analysis tool, KAAS (KEGG Automatic Annotation Server), was used to validate the annotation results from a BLAST  search. In addition, KEGGMAPPER (http://www.genome.jp/kegg/mapper.html) was used to reconstruct the metabolic pathways.The open reading frames (ORFs) of assembled contigs were predicted using the Prodigal software3−, NH4+, and NO2−, 30 ml KCl (2 mol/L) was added to the wet sediment, after which the liquid was vibrated for 1 h and then allowed to settle for 30 min before being filtered through a 0.2 μm elutriation membrane. This filtrate was then also analyzed with the FUTURA-II analyzer71.For the measurement of nutrients and metals, mangrove sediment samples were collected in acrylic tubes and analyzed in the laboratory on land. To measure the total nitrogen (TN), total phosphate (TP) and total organic carbon (TOC), 1 g dried chips was taken out from the 150 μm mesh filtered mangrove sediment, and 2 gram of catalyst : 10 ) was then added into the powder grinded from the chips. Then, 2 ml distilled water and 5 ml concentrated sulfuric acid were added to the powder before microwave digestion of the mixture (30 seconds). After digestion, the sample was filtered through a 0.45 μm elutriation membrane, after which the filtrate was analyzed with a FUTURA-II continuously flowing analyzer . To determine the salinity, NO72. The sediment was then oven dried for 2 days at 100 °C, after which it was ground up, homogenized and sieved through a 2 mm mesh. Then 7 mL HNO3 (72% v/v) and 1 mL H2O2 (30% v/v) (Fisher Scientific) were added to 0.5 g of each dried sediment sample, and the samples were heated using a closed microwave digestion system  for 90 min until the solutions were clear. The solutions were filtered and then analyzed in triplicate for total concentrations of Zn, Fe, Mn, Cd, and Pb using a 7500 series Inductively Coupled Plasma Mass Spectrometry (ICP-MS) system (Agilent Technologies)73. All the measurements for major elements were carried out in triplicates and the standard error was <1%.The pH was measured by mixing 5 g sediment with 5 ml water, as reported previously2) was as previously reported74. In brief, at each location, a Perspex chamber was inserted 5 cm into the sediment for gas collection. The gas in the chamber was extracted with a 10 ml gas-tight syringe every 30 min over a period of 120 min . The sampled gas was then transferred to 12 ml pre-evacuated exetainers , and preserved in a small, cold and dry refrigerator . Within 24 h, the samples were analyzed with gas chromatography , USA; 3800, Varian, Netherlands), and the methane flux of sampled mangrove sediment was then calculated based on mathematic equations75. Measurements were conducted in duplicate for each location, and the final value was averaged.The flux of methane across the mangrove sediment-atmosphere interface was measured using the Closed Chamber method; the operation and construction of the Perspex chamber (length 50 cm, volume 10 L, enclosing 1000 cmSupplementary material


Taraxacum kok-saghyz (TKS) carries great potential as alternative natural rubber source. To better inform future breeding efforts with TKS and gain a deeper understanding of its genetic diversity, we utilized de novo sequencing to generate novel genomic simple sequence repeats markers (gSSRs). We utilized 25 gSSRs on a collection of genomic DNA (gDNA) samples from germplasm bank, and two gDNA samples from historical herbarium specimens. PCR coupled with capillary electrophoresis and an array of population genetics tools were employed to analyze the dataset of our study as well as a dataset of the recently published genic SSRs (eSSRs) generated on the same germplasm. Our results using both gSSRs and eSSRs revealed that TKS has low- to- moderate genetic diversity with most of it partitioned to the individuals and individuals within populations, whereas the species lacked population structure. Nineteen of the 25 gSSR markers cross-amplified to other Taraxacum spp. collected from Southeastern United States and identified as T. officinale by ITS sequencing. We used a subset of 14 gSSRs to estimate the genetic diversity of the T. officinale gDNA collection. In contrast to the obligatory outcrossing TKS, T. officinale presented evidence for population structure and clonal reproduction, which agreed with the species biology. We mapped the molecular markers sequences from this study and several others to the well-annotated sunflower genome. Our gSSRs present a functional tool for the biodiversity analyses in Taraxacum, but also in the related genera, as well as in the closely related tribes of the Asteraceae. This important crop is threatened by the South American leaf blight pathogen Microcyclus ulei (Henn.) Arx (syn. Pseudocercospora ulei ) and is losing competition for the land and the manpower against the economically favored African oil palm Elaeis guineensis Jacq4.The growing human population has generated an increased demand for resources, including rubber, a substrate used for over 40,000 commercial products1. Several thousand plant species from across the world were screened for laticiferous properties, especially at times of increased rubber demand, e.g., WWI or WWII5 , rubber ficus (Ficus elastica Roxb. ex Hornem.), and Russian dandelion . The molecular properties of rubber from each of these plants differ from those of the Hevea product6 and point toward specialty uses on species basis. For instance, the guayule rubber could be used for medical products because of the lower content of allergenic proteins2. The TKS rubber is of particular interest to the tire industry due to its high molecular weight (polymer index) and fast generation time (six months in TKS vs. seven years in Hevea), albeit with a comparatively higher content of allergenic proteins3. Moreover, each of these species could be grown in areas complementary to the Hevea palm (24°S through 23°N7) with latitudes reaching as high as temperate zones .Natural rubber from plants outperforms that from petroleum in several aspects: the polymer of the natural rubber has much higher molecular weight compared with the synthetic rubber and the sustainable and renewable production of the plant  rubber is considered superior to processing the non-renewable petroleum8. The tire industry reported in their very first uses of TKS rubber that the tires “differed but little, according to their mechanical characteristics, from those made from imported natural rubber9” (citing10). In addition, it offers an accessory gain of inulin used in the manufacturing of numerous commercial products14. Both biosynthetic pathways are linked interchangeably within the TKS metabolism15. Several establishments devoted to TKS rubber production were founded in the United States (US) and Europe  emanating from the major research projects .TKS is of particular interest for the industry due to the proven success in production of tires17 and Western Xinjiang, China18, and is currently grown in Western Europe and North America alike . The plant was a major crop and model plant for rubber studies during the times of the Soviet Union of Socialist Republics (USSR). As hypothesized in other studies, likely due to the governmental pressure on performance, the TKS germplasm was profusely confused with the common dandelion species . As a result, the world’s germplasm and botanical gardens collections were annotated as TKS for over 50 years despite being the common dandelions23. Recent United States Department of Agriculture - Agricultural Research Service (USDA-ARS) and European expeditions helped remedy this issue and provided new properly identified germplasm24. TKS is obligatory out-crossing, self-incompatible, diploid herbaceous plant (2n = 16), and morphologically resembles common dandelions, which exhibit mostly clonal reproduction due to polyploid genomic architecture25.TKS is native to Kazakhstan21, molecular26, biochemical14, physiological15, and breeding26 approaches. The outcrossing nature of TKS was regarded when devising the plant genome linkage map27, followed by its genome sequence assembly to contigs4 and transcriptome sequencing28. All of this helped elucidate the TKS latex biosynthesis pathways29. In addition, a number of physiological and developmental studies on currently available germplasm provided data that was helpful in maximizing the rubber/inulin yield in both years the plants were grown19. TKS also proved amenable to genetic transformation and tissue culture26, indicating the potential for its breeding engineering and increases in yield of rubber31 or inulin15.A recent spike in the TKS research confirmed the USDA-ARS collected species identity using the morphologicalet al.32 utilized the USDA-ARS TKS germplasm24 to estimate the genetic diversity of the species using 17 expressed-sequence tags/simple sequence repeats markers  mined from the available GenBank EST libraries, across 17 TKS populations from the species native area24. They concluded that the majority of the species diversity is captured within each population32. Similar conclusions were drawn from a study of Russian, American (USDA-ARS), and wild Chinese accessions of TKS using 23 eSSRs33. None of these studies utilized nuclear genomic short-sequence repeats markers (gSSRs) to infer the population structure and genetic diversity of this economically important plant species. Moreover, the available TKS genome assembled to the contigs level only4 is lacking an extensive annotation or higher-level organization, despite providing insights into the TKS rubber/inulin biosynthetic pathways.Although some progress has been made regarding TKS biology, physiology, and genetics, until recently only limited information was available regarding the species diversity and inheritance/interplay of traits of interest. McAssey de novo sequencing to generate novel TKS gSSRs and to estimate the genetic diversity and spatial structure of the USDA-ARS TKS germplasm. We hypothesized that the majority of the species diversity would be captured in each examined population, as found in prior studies that utilized eSSRs33. The specific research objectives included the following: (1) identifying and characterizing polymorphic gSSR loci using de novo sequence of the TKS genome and mapping of the useful polymorphic gSSRs and other marker sequences onto the well-annotated genome of the related species Helianthus annuus32; (2) estimating the genetic diversity and inferring the population structure of the USDA-ARS TKS germplasm24 and two available historical herbarium TKS samples using gSSRs; and (3) comparing the gSSR data with the published eSSR data of McAssey et al.32 and Yushuang et al.33 to reach better-informed conclusions on TKS genetic diversity. We then deployed those gSSRs in a cross-amplification study with the local US dandelion samples , including their molecular identification. Information provided here will be useful in advancing future TKS studies, in the current and future breeding efforts of this potential crop for renewable rubber, and in augmenting the currently available resources for analyses of Taraxacum spp. and related plants.The goal of our study was to infer the TKS population structure, information of high value for breeding of this potential industrial crop. We utilized de novo genome sequencing yielded 45,804,966 paired-end reads of 275 bp. After trimming, 42,367,598 reads with a mean length of 265 bp were masked and used for de novo assembly on ABySS. The resulting de novo assembly contained 8,077,494 unitigs, from which 99,429 SSRs were identified on 95,692 sequences. From these, 11,259 were compound SSRs, meaning that two SSRs were separated by at most 15 bp. Primers were computed for 22,764 perfect SSRs. The number of SSRs with primers were 15,760 for the di-, 4,893 for the tri-, and 2,111 for the tetranucleotides, respectively.The TKS et al.4 containing the markers used for the construction of TKS linkage map27 and those of the gSSR and eSSR32 population genetics studies were mapped to the sunflower genome. The markers analyzed were located across all eight TKS linkage groups based on the mapping back to the sunflower genome  networks, and population structure: Our results suggest no significant deviations from the Hardy-Weinberg equilibrium (HWE) across the 25 gSSR markers used to analyze the TKS populations . Collectively, the data indicated rather low genetic differentiation among the TKS datasets analyzed, despite high allelic diversity of the obligatory outcrossing TKS. In agreement with the spatial fixation indices accrued, AMOVA for the gSSR dataset indicated the majority of the molecular diversity partitioned among the individuals and not among populations .The amplified gSSR markers yielded from 3 to 13 alleles per locus, averaging about 6 across the TKS germplasm pool Table . The 25 ST ranged from 0.018 to 0.355 for the gSSR, and from −0.024 to 0.261 for the eSSR datasets, respectively (data not shown). The pairwise population FST distance matrices  for the gSSR and eSSR datasets provided similar results , we generated pairwise matrices of population genetic distances for both. Values of the population pairwise distance matrix of FST population-wise trees  supported the F-statistics conclusions with no evidence of population structure in the TKS germplasm analyzed with gSSRs Fig. . The lacees Fig. , with th32, the significant deviations from HWE presented a locus-wise pattern and were much more common in occurrence than in the gSSR dataset , and an inferred Nm = 2.02. Partitioning of the molecular variance with AMOVA for the eSSR dataset yielded results similar to the gSSR dataset . Differences occurred in the variance partitioned among individuals within populations, and the gSSR dataset showed higher value of this parameter than the eSSR dataset. The FST distance matrix for the eSSR dataset of TKS showed different population-wise clustering from the gSSR dataset  networks, and population structure: In the re-analyzed eSSR TKS dataset32 of TKS lacking well-defined population structure. Analysis of networks from both gSSR and eSSR datasets resulted in similar Bruvo’s genetic distance ranges, and congruently implied lack of TKS population structure. Similar to the gSSR dataset, the DAPC analysis of the eSSR dataset , Hypochaeris spp. , Krigia spp. , Lactuca , Pyrrhopappus , and Erigeron .Species genotyping and assignment and ITS phylogeny of the plant materials: Species identity of the samples collected from Tennessee, Georgia, Alabama, and Mississippi Tables  and S1 wces Fig.  and coulTaraxacum officinale spatial fixation genetics indices, Multi-locus genotype (MLG) networks, and population structure: The majority of the TKS-derived gSSRs cross-amplified the gDNA of the related US native dandelions  and of the outgroup specimens . Overall, the cross-amplification was broad and proved effective even in the specimens of related genera and tribe  developed for TKS. Our results indicated violations of HWE in both locus- and population-manner . Linkage disequilibrium range for this dataset was similar to that of the gSSR study of TKS . To gain access to that data, we coded the whole dataset as tetraploid with occasionally missing alleles and corrected the ploidy with the R package polysat before analyses.The analyses of the species diversity and population structure included 74 samples of T. officinale dataset displayed between 5 and 16 alleles per locus . Several analyses indicated the presence of population structure in this dataset. The MSN analyses populations are more diverged from one another than in TKS. Bruvo’s distance-based tree of individuals  based on our de novo sequencing of TKS and utilized them for evaluating the genetic diversity of TKS germplasm. We then carried-out an array of comparative population genetics analyses, re-analyzing the recently published genic SSR (eSSR) dataset generated on the same TKS germplasm32, and an expanded cross-amplification study with the local US dandelions using those gSSRs.In this study, we aimed to gain a deeper understanding of the genetic diversity of TKS, a potential alternative, sustainable rubber cropde novo gSSRs were distributed across the TKS genome, based on the linkage disequilibrium data, as were the eSSRs32. We mapped both types of SSRs (gSSRs and eSSRs) along with the TKS markers used for the linkage map, back to the related and well-annotated H. annuus genome, based on the TKS contigs4. This is very likely to be helpful for the future breeding efforts. We chose not to use the TKS genome assembly4 or the closely related Lactuca sativa genome assembly35 because both are more fragmented and have fewer scaffolds anchored to chromosome locations in comparison to the H. annuus genome. To further underscore the need for improved TKS genome resources, the gSSR Tara003 sequence could not be found in the TKS contigs published4. Moreover, only 15 markers out of the 65 that constructed the TKS linkage map27 were mapped back together (in pairs or in threes) to six TKS scaffolds of Lin et al.4. Also, only one of the SSRs analyzed (gSSR Tara026) co-localized with two other map markers of Arias et al.27 within a single TKS contig of Lin et al.4. Several studies independently reported the TKS genome size as ~1,420 Mb based on flow cytometry (1.45 pg/1C31). Other studies estimated the diploid plant genome size at 2,400 Mb28. Comparatively, the draft TKS genome estimates at 1,040 Mb by 19 mer, 1,140 to 1,210 Mb by flow cytometry, or the 1,290 Mb assembly  represent an underestimation, which signifies room for improvement in the TKS genome completeness and assembly. As H. annuus is related, but somewhat distant to TKS, we expected mis-localizations and/or ambiguities in the marker placement due to genome rearrangements and/or sequence diversity. It is noteworthy though, that many chromosome regions in the map , which could stem from targeting the parts of genome different in character, duplications of the non-coding regions targeted by gSSRs37, or differences in the genomes of TKS (2n = 16) and H. annuus (2n = 34).Our 27. Seedling growth characteristics were also studied38. The first attempt at estimating the species genetic diversity using molecular methods was focused on a wide collection of TKS materials and allowed for a genetic distinction of the Russian/Kazakh and Chinese TKS germplasms33. A milestone in the TKS molecular diversity research was the study of the Kazakhstan-originating USDA-ARS germplasm using a set of eSSRs32 with which we compare the statistics of our gSSR dataset. Despite our sampling scheme being lower in number than in the previous eSSRs study of TKS, our study yielded very similar results and provided significant correlation of the population distances/indices. This result was possibly accrued by employing ~50% more gSSRs at lower population sampling, yet, ensured reliability of our results. This also confirmed the general observation on the TKS diversity formulated before32 that the overall low species diversity resides mainly within populations. This observation is in agreement with our research hypothesis for this outcrossing, self-incompatible dandelion species. Comparison of the HWE violations in the gSSR and eSSR datasets shows much lower occurrence in the former dataset. This could be intrinsically related to the sequences targeted by either SSR type, or variable mutational frequency of the targeted loci40. This is further substantiated by the patterns of HWE violations detected. The (sub-) population violations in gSSR dataset could stem from the limited sampling, whereas locus-wise HWE violations in the eSSR dataset suggest a different underlying reason, with abundant (sub-) population TKS sampling32.Several studies addressed the TKS diversity at various levels; agronomic performance and rubber/inulin production was of primary concern due to the industrial potential of the plant40. Owing to the fact of differences in parts of the genome targeted, in their conserved character, and in cross-amplification rates, both types of SSRs provide slightly different but complementary information42. Thus, inferences made from both types of SSRs together will provide more substantiated conclusions on the species diversity (or other studies for which they were used). Diversity of several economically important crops was analyzed using both types of SSRs, and in almost all cases led to similar results, which could also be taken as a confirmation study. For instance, deployment of both types of SSRs on the cucumber germplasm provided consistent positioning of most of the accessions analyzed on dendrograms and detected higher polymorphism rates using the gSSRs43. Similarly, high similarity was found between the gSSR and eSSR dendrograms among the tomato germplasms with higher polymorphism rate for the gSSRs, albeit slightly lower polymorphic information content44. The authors of that study postulated that combining both marker types in tomato would be effective for the species genetic diversity analyses. In contrast, studies of soybean indicated comparatively lower agreement between the gSSRs and eSSRs46. Authors argued for use of the eSSRs in soybean diversity studies for direct access to the population diversity in genes of agronomic interest but concluded that the species diversity was effectively estimated by both types of SSRs46. Analyses of the genetic diversity in wheat repeatedly indicated higher polymorphism of the gSSRs over eSSRs, but the authors of the studies argued that use of the eSSRs allowed for a more accurate delineation of the genetic relationships49. Studies in other cereal species observed the highest proportion of trimeric eSSRs, especially those encoding for neutral bulky amino acids50. Both studies also stated that the lower level of polymorphism detected by eSSRs compared to gSSRs might be due to the more conserved character of the targeted regions with selection acting against variation, a feature that could drive the relatively higher transferability of the eSSRs and a comparatively superior genotypic identification. Another conclusion emerged from the studies of the Prunus species. Although both types of SSRs resulted in similar dendrograms, combination of both datasets increased the genotypic discrimination44 and indicated a higher polymorphism and more effective resolution by the gSSRs than by the eSSRs51. The emerging conclusion from those and other studies is that similar levels of genetic diversity between populations or species may be recorded by using either SSR type with eSSRs often detecting lower variation, but performing more reliably at species differentiation54.Developing eSSRs is generally achieved faster and easier than the gSSRs due to comparatively more conserved character of the transcriptomeTaraxacum genus, the 14 gSSRs tested extensively in this study also cross-amplified to four independent gDNA samples of T. brevicorniculatum , but the Erigeron specimens belong to a distant tribe Asterae. This indicates a possible broad application of our gSSRs in the Asteraceae crops analyses. The TKS eSSRs also cross-amplified with four gDNA samples of local dandelions32. Thus, our gSSRs present additional resources to the classical (GA/CT)n gSSRs identified by restriction digest, hybridization, and Sanger sequencing55.Cross-amplification with the TKS gSSRs proved very successful and our markers transferred to other genera of the Asteraceae Fig. . Within 56. In contrast, results of the US dandelions are in agreement with the previous studies58 that provided evidence of both sexual and asexual modes of reproduction present in T. officinale with a broad cross-amplification to related species. The retrieved ITS sequences remained largely indiscriminate as to the species identity of the local US dandelions, co-localizing with the T. officinale ITS sequence consensus and the historical Herbarium specimen. Yet, previous research indicated predominance of only three Taraxacum species in North America . Including in the phylogenetic analyses the respective ITS consensus sequences of those three species, of the historical T. officinale specimen, and of T. officinale used for previous research26 (and data not shown) suggested the bulk of the US local dandelions could belong to T. officinale, if the microspecies of Taraxacum are disregarded60. Notably, the obligatory sexual diploid TKS was segregated with high confidence from the bulk of the US dandelions, as was the Central Asia-frequent T. brevicorniculatum.Both eSSR and gSSR datasets of TKS confirmed its sexual reproduction as observed in nature25. The majority of our dataset was tri- or tetra-ploid, and it is possible that we used too few markers to capture the higher levels of ploidy of the remaining several local dandelions samples classified as diploid based on the allele counts alone. In contrast to TKS, the US T. officinale presented evidence of population structure. This is in agreement with the biology of both species, especially considering the postulated clonal reproduction of the alloploid apomictic T. officinale in North America60. The higher frequency of sampling the outgroup specimens belonging to distant genera in the Southeastern US may be worth investigating in regard to the species range.The results of our gSSR analyses of this collection of US dandelions are in agreement with the recent ploidy analyses of the North America common dandelionsTaraxacum are notorious for hybridization, which often results in genome rearrangements, regional gDNA duplications, and/or polyploidization57. Cross-amplification of the TKS gSSRs (this study) and eSSRs (confirmed on four samples32), could help invigorate the molecular and genomic analyses of the more demanding polyploid dandelions57. Our study distinguishes the local US populations of T. officinale from TKS in several aspects. First, higher frequency of HWE violations indicated a difference in the US dandelions dataset. Second, the higher ploidy in this dataset inferred from the number of alleles detected indicated the possibility of clonal/asexual reproduction, which was further supported by the Index of association (IA). Third, several analyses indicated presence of population structure in this dataset contrary to the outcrossing diploid TKS. Overall, our gSSRs present a useful analytical tool for Taraxacum spp., due to cross-amplification in related species, even in distant genera.Species of Results on the genetic diversity of TKS accrued in the course of this study may help current and future breeding efforts of this potential crop for renewable rubber. Complementary and congruent data obtained from both gSSR and eSSR study on the same germplasm provided thorough insights into the species biology. Although the TKS well-annotated genome is still to come, the combined marker map located on the related sunflower genome may help advance future TKS studies. Furthermore, cross-amplification of our gSSRs into related species of dandelions and even other genera augments the currently available resources to analyze their biodiversity and provides a platform for their further research.24 was obtained from USDA-ARS and identified in a previous study15  extraction. We extracted three to five independent plant specimens per population for population diversity study (Tables TKS germplasm (seeds) collected in Kazakhstan15 Table . Plants y Tables . In addiT. officinale Weber (n = 74) accessions from the Southeastern US and plants morphologically very similar were collected across different geographical regions  and from eight distinct populations, as well as from historical herbarium specimens (Table Taraxacum spp. (n = 23) were set as a multiple outgroup. Leaf samples were collected in January and February of 2017, before the majority of the plants set bloom. No specific permissions were required for these locations/activities, as the materials are considered common weeds and regarded as neither endangered nor protected. Collected plant tissue was placed in ziplock bags containing silica gel . gDNA was isolated from the freshly collected tissues with the DNeasy Plant Mini Kit  as per the manufacturer’s protocol. Samples of the historical T. officinale were provided to us by the University of Washington Herbarium  and Oregon State University  for destructive sampling and analyses . The optimized thermal profile used included an initial denaturation at 94 °C for 2 min, 40 cycles of 95 °C for 30 s, 60 °C for 1 min, 72 °C for 90 s, and the final extension at 72 °C for 7 min. For each PCR, 5 µl of products were electrophoresed in 2% agarose-TAE buffered gels stained with ethidium bromide to confirm the amplification, and the rest was purified with ExoSAP-IT (Thermo Fisher Scientific) according to the kit manual. Analytical sequencing was done at McLab  or University of Tennessee – Knoxville Genomics Core . Sequences were assembled using LaserGene SeqMan version 7.0.0 , manually inspected and corrected, and identified using BLAST at NCBI. The obtained sequence matrix was enriched for published TKS ITS data34 (Genbank: KF437406 and KF 437407) and the ITS consensus sequences of T. ceratophorum (n = 3), T. erythrospermum (n = 12), and T. officinale (n = 53) from NCBI, respectively  Gblocks function with all the ‘less stringent selection’ options65. This sequence matrix was then submitted for phylogenetic analyses using RAxML GUI version 1.566 for Maximum Likelihood using 100 runs, with thorough bootstrap of 10,000, bootstrap branch lengths activated, and General Time Reversible (GTR) substitution model67. Multiple outgroup was set by selecting the 23 samples identified as not Taraxacum spp.  and ITS4 (Rv: 5′-TCCTCCGCTTATTGATATGC-3′)24; the detailed lineage is a proprietary information of ESKUSA GmbH, Parkstetten, Germany; chosen owing to abundant plant growth and thus availability of fresh leaf material) was isolated with the method described by Stein et al.69 and submitted to the UT Genomics Core for Illumina MiSeq sequencing at 275 bp, paired-end, on a v3,600 cycle flow cell. The gDNA library was prepared using the Nextera XT kit  following the manufacturer’s protocol with minor modifications, that included doubled incubation times and omission of the Normalizing step.Genomic DNA from the leaf sample E55/12 ; optimum annealing temperature of 60.0 °C (in the range of 55 to 65 °C); primer GC content in the range of 40 to 60%.Illumina sequencing adapters, low quality bases , and short reads (<30 bases) were trimmed off with Skewer version 0.2.24 was used in combination with the TKS linkage map information27 to infer the genomic locations of the SSR markers in this study. We used the marker sequences published therein, those obtained from our de novo sequencing gSSR search, as well the marker information and/or primer sequences of the published TKS eSSRs32 for comparison. The marker sequences were compared to the TKS genome contigs assembly of Lin et al.4 using gmap with default scoring settings . For each best sequence match to the TKS genome, a ~1 kb region containing the marker (500 bp on either side) was selected. The resultant contig fragments were used to BLAST the genome of related species, sunflower Helianthus annuus L., HA412-HO bronze assembly75. Best-hit sequences were then drawn on a map, respective to their physical locations on the sunflower chromosomes. If multiple best-hits had the same e-value, all were retained.The TKS genome sequencede novo sequencing was used for an initial genotyping screen with 50 primer pairs (25 di- and 25 tri-nucleotide repeats) with the PCR procedure described below. The results were visualized by capillary electrophoresis using Qiaxcel (Qiagen) and analyzed by using 25 to 500 bp DNA size marker and internal 15/600 bp alignment marker. We screened the results of genotyping with the 50 gSSRs for specificity on this gDNA sample, and the best-performing 25 gSSRs were selected for the analysis of the TKS gDNA collection  DMSO, 4 ng gDNA, 1 µM of each primer, and 1 U of AmpliTaq Gold DNA Polymerase (Fisher Scientific). The experimentally optimized thermal profile used included an initial denaturation at 94 °C for 3 min, 15 touch-down cycles of 95 °C for 40 s, 63-0.5 °C/cycle for 40 s, 72 °C for 30 s, 25 cycles of 95 °C for 40 s, 55 °C for 40 s, 72 °C for 30 s, and the final extension at 72 °C for 4 min.PCR reactions of 10 µl were composed of 1 × PCR buffer, 2 mM MgCl76). In addition, the published dataset of TKS-eSSR study was retrieved32 and binned to allow comparison of the datasets. Lastly, the dataset of T. officinale collected in the US (n = 74) and genotyped using 14 gSSRs was also binned, following the same procedure as the two datasets mentioned above. The binned datasets were analyzed separately for an array of population genetics parameters. To estimate the fixation and differentiation indices , we used packages: poppr79, hierfstat81, and polysat83 in R version 3.4.384. Due to the detected variation in ploidy levels in the US dandelions dataset, the data was corrected for ploidy in R version 3.4.3 using the package polysat and then recoded as tetraploid with occasionally missing alleles when samples were actually di- or tri-ploid. The mixed ploidy of that dataset limited the scope of the indices accrued, notably the differentiation index F’ST77; we resorted to GenoType/GenoDive85 to calculate the respective T. officinale dataset-wide FST and D’ST indices. As per convention, the FST bins considered were low (FST < 0.05); moderate (0.05 < FST < 0.15), and high (FST > 0.15). Deviations of Hardy-Weinberg equilibrium (HWE) were calculated using package pegas version 0.1086 in R version 3.4.3, using the exact test based on Monte Carlo permutations of alleles  and α = 0.05. The results were depicted as a probabilistic heatmap for HWE deviation in a locus- and subpopulation-manner. The multi-locus genotype (MLG) networks were constructed using the Bruvo distances, using the minimum-spanning networks (MSN) reticulation algorithm in the package poppr in R version 3.4.3. POPTREE287 was used to calculate the population-wise distance matrices using either FST or DST indices (both standardized and unstandardized). Mantel tests were performed in R version 3.4.3 using the package MASS88. Analysis of the molecular variance (AMOVA) was performed in R version 3.4.3 using the package poppr, and the resulting Φ indices are reported as [%] values, after 1,000 permutations, at. the three levels of each dataset hierarchy: within individuals ΦIT, within individuals between subpopulations ΦIS, and among subpopulation and ΦST. The mixed-ploidy T. officinale dataset did not lend itself to the ΦIT calculations using AMOVA. Discriminant Analysis of Principal Components (DAPC) was performed in R version 3.4.3 using the package adegenet version 2.1.190.A total of 62 TKS gDNA samples were genotyped using 25 gSSRs and binned using FlexiBin (an MS Excel macroResearch involving Human Participants and/or Animals: This article does not contain any studies with human participants or animals performed by any of the authors.Supplementary InformationSupplementary Table S1


RET proto-oncogene underlie the development of hereditary medullary thyroid carcinoma (MTC). The aims of this study were to evaluate the prevalence of germline RET mutations in a large series of MTC, collected over the last 25 years, and to reappraise their clinical significance. Methods: We performed RET genetic screening in 2031 Italian subjects: patients who presented with sporadic (n = 1264) or hereditary (n = 117) MTC, plus 650 relatives. Results: A RET germline mutation was found in 115/117 (98.3%) hereditary and in 78/1264 (6.2%) apparently sporadic cases: in total, 42 distinct germline variants were found. The V804M mutation was the most prevalent in our cohort, especially in cases that presented as sporadic, while mutations affecting cysteine residues were the most frequent in the group of clinically hereditary cases. All M918T mutations were “de novo” and exclusively associated with MEN2B. Several variants of unknown significance (VUS) were also found. Conclusions: a) RET genetic screening is informative in both hereditary and sporadic MTC; b) the prevalence of different mutations varies with V804M being the most frequent; c) the association genotype–phenotype is confirmed; d) by RET screening, some VUS can be found but their pathogenic role must be demonstrated before screening the family.Background: Pathogenic germline mutations affecting the  RET mutations in hereditary medullary thyroid carcinoma (MTC) has been reported 25 years ago [RET mutations have been discovered mainly in FMTC. These mutations occur mostly in cysteines, but can also affect non-cysteine codons, mainly at codons E768 [RET mutations, of uncertain impact in disease pathogenesis , have also been reported often in a single subject or family [Medullary Thyroid Carcinoma (MTC) can be either sporadic or hereditary. The hereditary form gives origin to the Multiple Endocrine Neoplasia (MEN) disorders in which thyroid carcinoma can be associated with additional endocrine neoplasia such as pheochromocytoma and/or hyperparathyroidism MEN2A) and with non endocrine diseases such as mucosal neuromas, megacolon, marfanoid habitus and skeletal abnormalities (MEN2B). A third form of hereditary MTC is represented by familial MTC (FMTC), which is characterized by MTC alone, but in several relatives of the same family ,2. The RA and witears ago . Followiears ago ,7. In adears ago  and the ears ago , over thons E768 , L790 anons E768 , codon Vons E768  and codoons E768  and S891ons E768 . Very rar family ,15,16.RET germline mutations have been found in almost all MEN2 families, with only about 2% of families affected by hereditary MTC having no detectable germline mutations [RET germline mutations, mainly affecting non-cysteine codons, have been reported in about 5–10% of apparently sporadic MTC cases [RET mutations, commonly point mutations but also deletions/insertions, have been described in about 50% of the sporadic MTC, according to data published in the COSMIC database [RET mutation, particularly M918T, has been found to correlate with more aggressive tumor features, a more advanced stage at diagnosis and a worse prognosis [utations ,17. RET TC cases ,19,20. Adatabase . In sporrognosis ,23.Although codon 634 mutations are the most prevalent mutations in both European and non-European families ,25, mutaRET genetic screening in clinical practice for hereditary MTC favorably modified the clinical management of MTC patients. According to the recent American [RET genetic screening to identify both hereditary cases erroneously diagnosed as sporadic and RET mutation carriers in MEN2 families. Relatives who are RET negative will not develop MTC, whereas RET-positive subjects are at risk to develop the cancer and must be followed and treated [The introduction of American  and EuroAmerican , all pat treated ,31.RET mutations in our large series of sporadic and hereditary MTC after 25 years of genetic screening. A reappraisal of the clinical meaning of the RET mutations is also included.The aim of this study was to provide an update on the prevalence of germline RET genetic screening in a total of 2031 Italian subjects: 117 were clinically affected by a hereditary disease at diagnosis, 1264 were affected by a sporadic MTC form , 650 subjects were relatives of RET positive MTC patients , we have performed patients . InformeRET sequencing has been modified: in the first years only exons 10, 11 and 16 were analyzed. Following the identification of additional RET mutation spread in a larger portion of the gene the analysis of exons 5, 8, 13, 14 and 15 has been introduced. With the exception of relatives of RET positive index cases, who have been analyzed only for the presence of the mutation identified in their family, all patients included in the study have been screened for the presence of RET mutations in eight exons . In familial cases with no evidence of RET mutations in these commonly analyzed exons, the entire coding sequence of the RET gene was analyzed. The biological rationale to focus on this region of the RET gene is that these mutations cause ligand-independent constitutive activation of the tyrosine kinase receptor. Cysteine mutations induce the formation of disulphide-bonded homodimers while mutations in the tyrosine kinase domain of the receptor, in particular the M918T mutation, cause a constitutive high level of autophosphorylation. Both mechanisms lead to activation of downstream signaling pathways, independent of ligand binding to the receptor.Blood has been collected over this 25-years period and stored at −20 °C. Over the years, the procedure of DNA extraction and sequencing analysis has been modified; until 2006, genomic DNA was purified from peripheral blood lymphocytes using the QIAMP blood kit . From 2006 until today an automated method  for genomic DNA extraction has been introduced. During this time interval also the Currently, genomic DNA is amplified using KAPA2G Fast HotStart PCR Kit  in a final volume of 25 μL . Amplification cycle is performed with an initial step of 95 °C for 2 min, followed by 35 cycles of 95 °C for 15 s, 60 °C for 15 s and 72 °C for 15 s. A final extension at 72 °C for 7 min was performed at the end of the amplification protocol. Sequence analysis was performed and has been reported on previously . PrimersRET genetic screening in the period 1993-2018 (25 yrs), 117 were clinically affected by a hereditary disease at diagnosis: 115/117 (98.3%) were found to be carriers of a RET germline mutation, while two families (1.7%) had no detectable RET mutations. A germline RET mutation was also found in 78/1264 (6.2%) MTC patients who were diagnosed as sporadic according to their negative familial history and absence of other endocrine neoplasms. Considering the cases found to be familial at diagnosis  and familial cases erroneously diagnosed as sporadic (apparently sporadic cases) a total of 195 families with hereditary MTC were followed at our hospital. The remaining 1186 sporadic MTC patients were found to be negative for germline RET mutations and their sporadic nature has been confirmed.Among the 2031 subjects submitted to RET mutation in the index cases, 650 relatives have been analyzed and 256 (40%) subjects were found to carry the same RET mutation as their affected family members. The remaining 394 (60%) individuals were RET negative  and when considering clinically familial  or apparently sporadic  cases. When taken together, mutations affecting the cysteine at codon 634 (C634R/Y/G/F/W/S) were found in 45/195 cases (23%). Cys634 mutations, were the most prevalent germline mutations in the group of clinically familial cases  and were significantly (p = 0.0005) more frequent in this group than in the group of apparently sporadic cases . When considering all cysteine mutations together a significantly higher prevalence was observed in clinically familial cases than in apparently sporadic cases (54/117 (46.1%) vs 23/78 (29.5%), respectively) (p = 0.02). Conversely, germline mutations affecting non-cysteine residues were more frequent in apparently sporadic than in clinically familial cases (55/78 (69.2%) vs 61/117, (52%), respectively) (p = 0.01). The M918T mutation, found in 15/117 (12.8%) cases, was always a de novo mutation and the patients were all affected by MEN2B syndrome.All together 42 different germline missense mutations have been identified . The V80RET mutation but only the index case presented with MTC so far  previously reported but of unknown biological significance (VUS) . The VUSC so far . MoreoveRET germline mutation in exon 10 .As shown in RET mutations in the pathogenesis of MTC [RET genetic screening in patients with hereditary and sporadic MTC has been introduced in the clinical practice to identify RET positive subjects and their relatives who are at high risk of developing MTC during their life.Soon after the discovery of the driving role of s of MTC ,8 RET geRET gene in a series of 2031 Italian subjects including 1381 MTC patients and 650 relatives. As a standard routine procedure, only RET exons reported to be causative of the inherited MTC have been investigated . Furthermore, in patients with familial MTC who were negative on the first screen, the whole RET coding sequence has been analyzed. In agreement with previously reported data we have found that about 98.5% of hereditary MTC are affected by a RET germline mutation but a few cases are still RET negative despite the screening covered all RET exons. To explain the hereditary nature of RET cases, the presence of a mutation in genes other than RET could be hypothesized or the presence of a RET intronic variant with the ability to modify RET expression levels could be considered [Over the last 25 years, we have screened the nsidered .RET mutation in more than 6% of apparently sporadic MTC patients, thus confirming the important role of RET genetic screening for the identification of unsuspected MEN2 families [RET genetic screening should be performed in all first-degree family members, allowing the identification of gene carriers who can thus benefit from appropriate screening, an early diagnosis and timely or prophylactic treatment measures.In addition, and in keeping with other reports ,34, we dfamilies . Once anRET Consortium (IRC) [RET germline mutation affecting one of the following codons: 609, 611, 618, 620, 634, 768, 804 and 918. In the present series, the percentage of RET positive families  was higher than that reported in the IRC study. This finding could be explained by the inclusion of a wider portion of the RET gene in the screen , and perhaps to improvements in sequencing technologies. In fact, over the past 25 years, 197 different RET variants, (some pathogenic and others VUS), have been associated with MEN2 [RET screening approach identified 42 different point mutations. In our series, the most frequent RET mutation was the V804M in exon 14. Our findings support previously reported data [RET variant associated with hereditary MTC in Italy, while mutations at codon 634 have been found to be the most frequent in Germany [In 1994 a study performed by the International um (IRC)  analyzedith MEN2  and mostted data ,36 indic Germany  and in o Germany ,25. The RET germline mutations between the group of the clinically hereditary cases and the cohort of apparently sporadic cases. Although the V804M mutation is the most frequent single germline mutation observed in the two groups, mutations involving the cysteine at codon 634, and overall, mutations involving the cysteine residues in exons 10 and 11, are significantly more frequent in the group of clinically hereditary cases than in apparently sporadic cases. Conversely, mutations affecting non-cysteine residues are more frequent in apparently sporadic cases than in the clinically familial cases. This difference can be explained by the higher oncogenic potential and penetrance of the cysteine mutations, with particular regard to C634R, when compared to non-cysteine RET variants [The present study describes a different profile of variants . The difvariants ,38.RET germline mutations of our series, the Y791F mutation is still reported although recent studies indicate that this mutation does not increase the risk for MTC [RET screening.Among all mutations, we found some rare variants whose biological significance is still unknown (VUS). The accurate characterization of the pathogenic role of these variants would be of great relevance for patients. The aggressiveness and the biological role of these new variants can be predicted by “in silico” on line tools ,15 even  for MTC . In agreRET gene that it is known to account for 95% of MEN2B cases [RET mutations in MEN2B were all de novo. In addition to the MEN2B cases, seven additional RET positive patients have been documented to be de novo and in these cases RET mutations were discovered in exon 10 or 11. The finding of de novo mutations in MEN2A is of interest and scarcely reported and discussed. Proving the de novo origin of these mutations would require the analysis of both parents and this is not always possible, especially in adult index cases whose parents may have already died. Moreover, since is not ethically acceptable to test for paternity, doubts about the progenitorship cannot be ruled out. However, it is conceivable that, as it happens for MEN2B [RET mutation happened as “somatic” event in one of the gametes or in the early phases of embryonic development.In our series the MEN2B patients were all affected by the M918T mutations in exon 16 of the 2B cases . As repo2B cases , also inor MEN2B , also inRET mutations strengthens the idea that alterations in the RET gene are enough to induce tumoral transformation as indeed demonstrated by in vitro studies [RET mutations. In our series only three families with a non-cysteine RET mutation had members with PHEO or hyperPTH and were all harboring a V804M mutation. This evidence is relevant for clinicians because the follow up of patients with non-cysteine RET mutations could be simplified by eliminating the annual evaluations of adrenal gland and parathyroid function which are still recommended in the clinical practice guidelines. Of course, it remains mandatory in cases with cysteine mutations, particularly when a C634R is present.In agreement with previous series ,43, a st studies . PHEO anRET mutations but, in contrast to those associated with MTC, they are inactivating mutations [RET C620, C618, C611 and C609, act as “Janus” mutations [RET “Janus” mutations. However, we have identified other two families with non-cysteine RET mutations, (K821E and Y791F) with one single member with Hirschsprung’s disease and, so far, no one with MTC. It is interesting that in both cases the mutations are in the intracellular domain of the receptor, in particular in exon 13 and 14, that are commonly screened for MTC but never demonstrated to be Janus mutation. Only the long-term follow up of these young patients and their RET positive relatives will clarify if they will ever develop MTC.Hirschsprung’s disease is also associated with utations ,46. In sutations : that arRET genetic screening should be performed in all MTC cases independently from their clinical presentation; (b) V804M is confirmed to be the most frequent RET mutation in the Italian population; (c) cysteine mutations are more frequent in cases with a clinically manifested hereditary form; (d) the non-cysteine mutations are very rarely associated with PHEO and hyperPTH suggesting that the follow up of these patients could be simplified; (e) de novo mutations are frequent in MEN2B syndrome but they can be found also in both MEN2A and FMTC; (f) germline RET VUS may be found with genetic screening but their pathogenic role should be defined before testing relatives of the index case.In this study, that to our knowledge is the largest series of screened MTC at a single center, we showed that: (a)


Experimental sessions involved the ingestion of either an HGI or LGI meal, followed by the completion of the modified Loughborough Intermittent Shuttle Test (mLIST). There was no significant difference between HGI or LGI meals on sprint times (p = 0.62) and distance to exhaustion (p = 0.54) in the mLIST. Exercise heart rate, blood lactate and ratings of perceived exertion were also similar between the two meal trials throughout the mLIST . Subjective ratings of hunger, fullness, satiety and satisfaction were also not significantly different between the two meals. In conclusion, consuming either an HGI or LGI meal after a prolonged 12 h fast and ingesting the meal 45 min prior to exercise did not differ in either physiological, subjective and intermittent sprint and endurance performance outcomes.The purpose of this study is to investigate the effects of ingesting either a high glycaemic index (HGI) or low glycaemic index (LGI) carbohydrate meal  on intermittent sprint and endurance exercise performance. Ten male varsity athletes from intermittent sports (age 23.6 ± 1.7 years, VO The glycaemic index (GI) was established to enable the comparison of human physiological responses towards varying types of food and to develop food items which, when ingested, would lead to a relatively slower or more gradual increase in blood glucose concentration levels, especially for people with diabetes or glucose intolerance . CalculaInterestingly, the knowledge of the GI of different foods has since been used to examine the influence of these varying foods on substrate metabolism during exercise. A plethora of research has been conducted to investigate the effects of ingesting LGI and HGI food within 60 min to between 1 to 3 h prior to exercise performance, especially in endurance exercise performance using time-trials and time-to-exhaustion exercise protocols (see reviews) ,5,6. AltThere have been only very few studies that examined the effects of consuming HGI and LGI food prior to intermittent-type exercise performance ,10,11,12iftar meal  before commencing their exercise a short time later. Therefore, the purpose of this study is to compare the effects of ingesting either an HGI or LGI meal  on intermittent sprint and endurance exercise performance. Based on previous studies [It is common for athletes, or even among the general population, to commence exercise after being in a fasted state for a prolonged period of 10–12 h. For example, many athletes prefer to exercise very early in the morning. Hence, upon waking after an overnight fast, they may subsequently consume a small, light meal just before the commencement of early morning exercise. Another good example is in the month of Ramadan, many Muslim athletes would observe their religious fasting during the daylight hours which typically last between 10–16 h duration, hence their sporting activities may be postponed until after the breaking of the day’s fast ; in this studies ,9,14,15,peak 16 ± 0.7 km·h−1; maximal oxygen consumption or VO2max 51.9 ± 4.7 mL·kg−1·min−1) volunteered for the study. Participants were involved in their respective intermittent sport training sessions but were asked to rest completely 24 h before each experimental session. Participants were amateur-level athletes who were training between 6 to 8 sessions per week, which consisted of sport-specific technical skills, aerobic and anaerobic (speed) conditioning as well as resistance training. Ten healthy male varsity athletes from intermittent sports . The second and third visits were the two experimental sessions, i.e., either the HGI or LGI meal trials. These two sessions were randomised across the tested individuals comparing the participants’ subjective and physiological responses, and intermittent sprint and endurance exercise performance.The present study adopted a single-blind, controlled, randomized, cross-over trial experimental design. Participants made a total of three visits to the laboratory, between 3 to 7 days apart. The first visit was a preliminary test and familiarisation session. The preliminary test consisted of the V−1 body mass. The GI values of the LGI and HGI meal in the present study were determined based on the food items reported in the study of Sun et al. [The HGI meal was glutinous chicken-flavoured rice, marketed locally as Lor Mai Kai . The LGI meal provided was wholemeal bread with coconut jam and butter spread, known locally as Kaya Butter Toast. The LGI was physically prepared by the primary investigator to match the caloric and macronutrient profile of the Lor Mai Kai. Both the LGI and HGI meals were of the same caloric values of ~425 kcal , equivaln et al. . The stun et al.  which wan et al. .Participants were not informed of the true aim of the study. They were duly informed that the rationale of this research was to test two types of pre-exercise meals commonly found in Singapore on intermittent exercise performance, i.e., bread vs. rice. This helped ensure that the true intention of the study was concealed, thus minimising potential placebo effects . ParticiParticipants underwent two experimental sessions, separated by a ~3–7 day period. They were instructed to abstain from strenuous physical activity 24 h prior to each experimental trial and abstain from alcohol, caffeine and any ergogenic supplements. To limit any influence of nutritional and physical activity habits on exercise performance, subjects kept a 24 h diet and physical activity record on the day before the first experimental trial. They were instructed to follow the same dietary and physical activity pattern for a 24 h period before the second experimental session. All trials were also scheduled at the same time of the day to avoid any circadian rhythm influence on exercise metabolism and exercise performance. Participants were instructed to cease any ingestion of food and liquid (except water) the evening (by 22:00 h) before the day of the exercise until their reporting time the next morning (by 10:00 h). This is to ensure that they reported to the laboratory in a 12 h fasted state. Upon arrival to the laboratory, participants ingested either an LGI meal or HGI meal together with 500 mL of plain water within 10–15 min. Participants then remain rested for 30 min before the commencement of the warm-up to begin the intermittent sprint and endurance performance test.peak test to determine their individual running velocities for the modified Loughborough Intermittent Shuttle Test (mLIST) exercise protocol. The Vpeak test was conducted on a motorised treadmill, which commenced at 8.0 km·h−1; at a gradient of 1% for 1 min, after which the speed was increased by 1.0 km·h−1; in 1 min increments until volitional exhaustion was attained [peak and maximum heart rate (HRmax) was the highest treadmill velocity maintained for 60-s and the highest 5-s average data, respectively. During the Vpeak test, the individual’s VO2max was also measured with indirect calorimetry using a calibrated metabolic cart . Measurements of heart rate  and ratings of perceived exertion  [peak test, participants rested for 15 min and this was followed by a 15 min familiarisation trial of the mLIST.Before the experimental trials, participants completed a familiarisation session during which all the experimental procedures were taken and questionnaires were explained to them. Participants then completed the Vattained . The ind0 scale)  were monpeak determined from the Vpeak test, except for the 15 m maximal sprint effort. Each of the 15 min exercise blocks of part A consisted of ~9–10 cycles of 3 × 20 m walk (at 1.3 m·s−1), followed by 1 × 15 m maximal sprint, 3 × 20 m jog (at 55% Vpeak) and 3 × 20 m run (at 95% Vpeak). Part B consisted of 20 m shuttles alternating between running speeds corresponding to 55% and 95% Vpeak until volitional exhaustion (defined as the inability to maintain pace for three consecutive shuttles at 95% Vpeak). During the test, participants were instructed to follow the pattern of movements , except for the sprinting component where the player sprinted the 15 m distance as fast as possible i.e., all-out maximal effort for every sprint. In the mLIST, sprints were initiated from a standing position, 20 cm behind the start timing gate. Sprint times were recorded with the Speed Light Sports Timing system  to an accuracy of 1/100th of a second. The light gates were at a standardised height of 1.2 m above the ground, placed at the start and at the 15 m mark. The coefficient of variation for part A and part B in the original LIST was estimated to be ~5% and ~30% (based on duration to exhaustion), respectively [The present study used a modification of the original LIST , i.e., mHeart rate via short-range telemetry  was taken throughout exercise. Capillary blood samples to assess blood glucose concentration via finger-prick were taken before consumption of the test meal, 5-, 15-, 30- and 45-min after ingestion of the test meal, using a portable glucose meter . Capillary blood samples were additionally taken at the end of each block of exercise in part A and upon completion of part B of the mLIST to assess blood lactate concentration . The players rated their subjective RPE using Borg’s categorical 6–20 scale at pre-exercise and at the end of each exercise block of part A and at the end of part B of the mLIST.Before and at the end the mLIST, participants completed the Brunel University Mood State (BRUMS). The BRUMS comprised of 32 items of descriptors for six different mood subscales of anger, confusion, depression, fatigue, tension and vigour . Raw scot-tests with Bonferroni adjustments were used to detect the occurrences of the differences. Paired sample t-test was employed to compare the variable distance to exhaustion between the two meal conditions. Significance level was set at p < 0.05. Supplementary analysis using magnitude-based differences were also undertaken for all performance variables  [All data were analysed using statistical software . Shapiro-Wilks was conducted to assess the normality of the data. Sprint times; blood glucose; blood lactate; HR; RPE; subjective ratings of hunger, satisfaction, fullness and satiety; and the various mood states were all analysed by two-factor trial x time repeated measures of analysis of variance (ANOVAs). Mauchly’s test was consulted and Green-Geisser correction was applied if sphericity was violated. If a significant main effect was observed, post-hoc paired sample austion) . Changesp = 0.05), but no significant difference was found between the two meal trials at any time point throughout the mLIST . There was no interaction effect between trial X time for mean sprint times across three exercise blocks . For the distance to exhaustion performance, paired sample t-test showed no significant difference in the distance covered between the two meal trials (p = 0.54), although the distance to exhaustion in the HGI trial was 82 ± 28 m greater  than in the LGI trial. The qualitative outcome across the sprints and distance to exhaustion were both deemed as unclear.p = 0.002, ηp2 = 0.46) in blood glucose concentration during the post-prandial non-exercise period. Post-hoc test showed a significant difference in blood glucose concentration between HGI and LGI trials after post-meal ingestion at the 15- and 30-min mark  in blood glucose concentration throughout the mLIST exercise, but no significant interaction effect of trial X time was found . Similarly, blood lactate concentration showed a progressive increase due to exercise .A significant trial X time interaction effect was found . There were significant main effects for time factors for both HR and RPE . Similarly, there were significant main effects on time for fatigue, and tension subscales , except for the mood subscale of vigour, , but not in the LGI meal trial .There were significant main effects for time for all ratings of hunger, satisfaction, fullness and satiety  on intermittent sprint and endurance exercise performance. The results indicated that there were no significant differences between LGI and HGI meal trials for sprint times (part A) and distance to exhaustion (part B) of the mLIST, and thus, contrary to our initial hypothesis, ingestion of a pre-exercise LGI meal relative to a HGI meal did not lead to any ergogenic effects on intermittent sprint and endurance performance.The present study finding is supported by previous studies that have shown no differences in exercise performance capacity between either ingesting LGI and HGI meals on intermittent exercise performance ,10,11,12−1 body mass) and the participants’ characteristics . With regards to intermittent exercise performance protocol that is similar to the present study, only one study  has showDuring prolonged moderate- to high-intensity continuous exercise protocols, there is an increased reliance on free fatty acid oxidation and economical utilisation and sparing of limited endogenous glycogen stores, which may accrue an advantage in consuming an LGI vs. HGI meal during exercise ,14,15. HNegative mood state is usually associated with low blood glucose concentration levels . Thus, aThere are several limitations of the present study which should be highlighted. This research study involved a small number of participants, albeit similar to that previously of many other GI related studies ,12. As sThis study compared the effects of pre-exercise HGI and LGI meals  on an intermittent sprint and endurance exercise protocol. Results showed that ingesting either HGI or LGI meals did not significantly influence intermittent sprint and endurance performance or any other physiological and subjective variables measured. Thus, intermittent sport athletes may ingest either an LGI or HGI meal if they are planning to engage in exercise within an hour post-prandial.