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Arsenic mobilization in a high arsenic groundwater revealed by metagenomic and Geochip analyses

Microbial metabolisms of arsenic, iron, sulfur, nitrogen and organic matter play important roles in arsenic mobilization in aquifer. In this study, microbial community composition and functional potentials in a high arsenic groundwater were investigated using integrated techniques of RNA- and DNA-based 16S rRNA gene sequencing, metagenomic sequencing and functional gene arrays. 16S rRNA gene sequencing showed the sample was dominated by members of Proteobacteria (62.3–75.2%), such as genera of Simplicispira (5.7–6.7%), Pseudomonas (3.3–5.7%), Ferribacterium (1.6–4.4%), Solimonas (1.8–3.2%), Geobacter (0.8–2.2%) and Sediminibacterium (0.6–2.4%). Functional potential analyses indicated that organics degradation, assimilatory sulfate reduction, As-resistant pathway, iron reduction, ammonification, nitrogen fixation, denitrification and dissimilatory nitrate reduction to ammonia were prevalent. The composition and function of microbial community and reconstructed genome bins suggest that high level of arsenite in the groundwater may be attributed to arsenate release from iron oxides reductive dissolution by the iron-reducing bacteria, and subsequent arsenate reduction by ammonia-producing bacteria featuring ars operon. This study highlights the relationship between biogeochemical cycling of arsenic and nitrogen in groundwater, which potentially occur in other aquifers with high levels of ammonia and arsenic.

operon are much more prevalent among prokaryotes and well known to contribute to arsenate reduction [23][24][25] . In addition, it was reported that microbial oxidation and methylation of arsenite occur in some groundwater 26,27 . Therefore, to comprehensively understand As transformation processes in groundwater systems, those functional genes of As reduction, oxidation and methylation should be evaluated together using some high-throughput molecular technologies, such as metagenome and Geochip 28 . Besides, the integrated metabolic potential of metagenome and Geochip enables As metabolism to link to other corresponding elements cycle, such as C, N and S, and further elucidate their relationship and role in As mobilization. With the complementary beneficial characteristics including new genes discovery, low detection limits, high reproducibility, and potential for quantification, metagenome and Geochip could be combined to reveal microbial functional potentials in specific ecosystems of interest.
High levels of As in groundwater systems is often accompanied by high ammonium concentrations, such as Hetao basin, China 29 , Bengal delta plain and Ganges River floodplain, Bangladesh 30,31 , Red River floodplain, Vietnam 32 , and Mekong Delta, Cambodia 33 . It was reported that ammonium in groundwaters reflects degradation of organic matter and/or inputs of anthropogenic activities, such as fertilizers and manure, and only act on the formation of reducing conditions to favor the reductive dissolution of As-bearing Fe oxides 32,34,35 . Recently, isotopic evidence of nitrogen sources in As-contaminated groundwater suggested the prevalence of nitrification, denitrification, ammonification and dissimilatory nitrate reduction to ammonia (DNRA) 34,36,37 . These results infer that some nitrogen metabolic processes possibly link to As mobilization in groundwater systems. After all, none of the above iron-reducing, sulfate-reducing and arsenate-reducing bacteria could reasonably explain the positive correlation between As and ammonium concentrations in groundwater.
The objective of this study is therefore to further understand the microbial mobilization and transformation processes of As in groundwater, especially the role of nitrogen metabolism on As mobilization. To this end, we used the integrated techniques of RNA-and DNA-based 16S rRNA gene sequencing, metagenomic sequencing and functional gene array (Geochip 4.0) to assess microbial community composition and functional potential in a high As groundwater. This groundwater is located in Hetao Basin, Inner Mongolia, China, a representative endemic arsenicosis area with an affected population of more than two hundred thousand 38 . Our results indicated that some ammonium-producing bacteria with ars operon is likely to mediate the transformation from arsenate to arsenite in groundwater after it is released from the reductive dissolution of As-bearing iron oxides by iron-reducing bacteria.

Material and Methods
field sampling. The hand-pumped tube well (N40.96683°, E107.00517°) from resident Dingshan Li in Shahai village, Hangjinhouqi County, western Hetao Basin, was selected for this study because of high levels of As detected and arseniasis family history (two members died of long-term drinking groundwater). The sample herein was abbreviated LDS. Groundwater biomass were collected using both normal filtration, with a 0.22 μm × 142 mm (pore size × diameter) nitrocellulose membrane filter (Merck Millipore, Billerica, MA, USA), and tangential flow filtration (TFF; Prep/Scale Spiral Cartridge with 30 kDa molecular weight cutoff, Millipore, Billerica, MA, USA) 39 . The filtered water was collected into 100 mL acid-washed polypropylene bottle (to measure As species), brown glass bottle (to measure dissolved organic carbon, DOC) and serum vial with butyl rubber septum stoppered and aluminum seal (to measure methane). The filtered membrane was cut into two equal parts and placed into 15 mL sterile polypropylene tubes with/without prefilled Ambion RNA later Solution (ThermoFisher Scientific, Waltham, WA, USA) (RNA and DNA extraction for 16S rRNA gene sequencing) respectively. Concentrated cell suspension by TFF was packed into 500 mL sterile nuclease-free plastic bottles (DNA extraction for metagenomic sequencing and Geochip analyses). The groundwater volumes of normal filtration and TFF is approximately 10 L and 2000 L respectively. All samples were transported to the laboratory on dry ice and then stored at −80 °C until further analyses.
Geochemical measurements. Geochemical parameters including temperature, pH, electrical conductivity and oxidation-reduction potential (ORP) were measured in situ using a portable multi meter (HQ40D, Hach, Loveland, CO, USA). Concentrations of sulfide, sulfate, ammonium, nitrite, nitrate, phosphate, ferrous iron and total iron in groundwater were determined at the sampling with a portable colorimeter (DR890, Hach, Loveland, CO, USA) according to the manufacture's procedures. In the laboratory, four As species of arsenite, arsenate, monomethylarsenate (MMA) and dimethylarsonate (DMA) were performed using liquid chromatography combined to hydride generation atomic fluorescence spectrometry (AFS-9780, Haiguang, Beijing, China). Dissolved organic carbon (DOC) concentrations were determined using a TOC analyzer (Vario MICRO cube, Elementar, Langenselbold, Germany). Soluble methane concentrations were analyzed by gas chromatography (Thermo Trace Ultra) at the Third Institute of Oceanography, State Oceanic Administration of China. All samples were run in triplicate and then averaged. DNA and RNA extraction and cDNA synthesis. Cells in TFF concentrates were pelleted by centrifugation at 6000 rcf for 10 min after defrosting. Genomic DNA was extracted from the pellet and filter using MoBio PowerSoil DNA Isolation Kit (Qiagen, Valencia, CA, USA) according to manufacturer's instructions. Total RNA was extracted from the filter preserved in Ambion RNAlater Solution using RNeasy Mini Kit (Qiagen, Valencia, CA, USA). An Ambion Tubro DNA-free Kit (ThermoFisher Scientific, Waltham, WA, USA) was used to digest residual DNA. To verify complete removal of genomic DNA in total RNA, we utilized total RNA as a template to amplify 16S rRNA gene with general primer 515F (5′-GTGCCAGCMGCCGCGGTAA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) and performed electrophoresis detection. DNA and RNA concentrations were measured by NanoDrop One (Thermo Fisher Scientific, Waltham, MA, USA).
Reverse transcription-PCR (RT-PCR) amplification of 16S rRNA was achieved using Invitrogen SuperScript III One-Step RT-PCR System with Platinum Taq DNA Polymerase (ThermoFisher Scientific, Waltham, WA, USA). Specifically, it was carried out in a 50 μL reaction containing 25 μL 2 × Reaction buffer, 0.2 μM of both sense and anti-sense primers (515F and 806R), 10-15 ng template and 2 μL SuperScript III RT/Platinum Taq Mix using the following program: cDNA synthesis at 55  16S rRNA gene amplification, sequencing and preprocessing. The V4 region of 16S rRNA gene was amplified from both DNA and synthesized cDNA using the primer 515F and 806R combined with Illumina adapter sequences, a pad and a linker, as well as barcodes on the reverse primers. PCR amplification was carried out in a 25 μL reaction buffer containing 12.5 μL GoTaq colorless Master Mix (Promega, Madison, WI, USA), 0.5 μM of both forward and reverse primers, 10-15 ng template using the following program: initial denaturation at 94 °C for 3 min, followed by 35 cycles of 94 °C for 45 s, 50 °C for 60 s, and 72 °C for 90 s, and then a final extension at 72 °C for 10 min. Reactions were performed in triplicate and pooled.
Amplicons were verified by agarose gel electrophoresis and quantified with PicoGreen using a FLUOstar Optima microplate reader (BMG Labtech, Jena, Thuringia, Germany). 500 ng of amplicon from each sample were combined together and purified using MoBio UltraClean PCR Clean-Up Kit (Qiagen, Valencia, CA, USA) and then re-quantified with PicoGreen. Sample library was prepared according to the MiSeq Reagent Kit Preparation Guide (Illumina, San Diego, CA, USA). Briefly, the sample was denatured by mixing 10 μL 2 nM combined amplicon and 10 μL 0.2 mol NaOH and incubated for 5 min at room temperature. Denatured sample was diluted to 7 pM using HT1 buffer and mixed with 7 pM PhiX control (final PhiX control concentration, 10%). A total of 600 μL sample mixture, together with customized sequencing primers for forward, reverse, and index reads, were loaded into the corresponding wells on the cartridge of a 300-cycle MiSeq Reagent Kit v2 and run on an Illumina MiSeq system (Illumina, San Diego, CA, USA). RNA-and DNA-based 16S rRNA gene sequencing raw data were deposited to the Short Read Archive database at NCBI (Accession number: PRJNA493661).
Raw sequencing data with perfect matches to barcodes were split to sample libraries. Forward and reverse reads with at least 30 bp overlap and less than 25% mismatches were joined using Fast Length Adjustment of SHort reads (FLASH) 40 . The joined sequences were trimmed using Btrim with a QC threshold of greater than 30 over a 5 bp window size and a minimum length of 140 bp 41 . After deleting sequence containing N base and trimming by sequence length of <251 and >255 bp, the sequences were clustered to operational taxonomic units (OTUs) by Uparse at a similarity level of 97% 42 . Singletons in generated OTU tables were removed for downstream analyses. The Ribosomal Database Project (RDP) classifier was used to assign the taxonomy of representative sequences at a minimal 50% confidence 43 . Samples were rarefied at 55841 sequences per sample. The above steps were performed through a Galaxy-based pipeline compiled by Ye Deng's lab in Research Center for Eco-Environmental Sciences, China Academy of Sciences, China (http://mem.rcees.ac.cn:8080).
Metagenomic sequencing, preprocessing and genome binning. DNA was randomly sheared into small pieces of ~350 bp using Covaris M220 Focused-ultrasonicator (ThermoFisher Scientific, Waltham, WA, USA). Sample library was prepared according to the instruction manual of NEB Next Ultra DNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA). Briefly, it consists of fragmented DNA end-repaired, adaptor ligation, size selection and cleanup of adaptor-ligated DNA, PCR enrichment of adaptor-ligated DNA (5 cycles) and cleanup of PCR reaction. The library concentration was determined using Qubit 3.0 Fluorometer (ThermoFisher Scientific, Waltham, WA, USA) and sequenced on an Illumina HiSeq X Ten platform at Guangdong Magigene Biotechnology Co. Ltd (Guangdong, China). ~13 Gbp (2 × 150 bp) reads were generated. Metagenomic sequencing raw data were deposited to the Short Read Archive database at NCBI (Accession number: PRJNA493663).
The raw sequencing data was trimmed to produce clean data using Trimmomatic (Version 0.36) 44 and de novo assembled using MEGAHIT(Version 1.0.6) 45 . The assembled scaffolds were interrupted from N base site and the <500 bp scafftigs were removed. MetaGeneMark(Version 3.38) was used with default parameters to predict the open reading frame (ORF) of scafftigs and those ORFs with a length of <90 bp were omitted 46 . Redundant gene catalogue(Unigenes) was obtained using CD-HIT (Version:4.7) by grouping sequences with an identity of >95% and coverage of >90% into clusters and assigning the longest sequence as representative of the cluster 47 . The clean data was aligned to the Unigenes using BBMap (http://jgi.doe.gov/data-and-tools/bbtools/) to obtain the number of mapped reads of each gene 48 . Based on the number of mapped reads and length of gene, each gene abundance was computed and utilized for subsequent statistical analysis. DIAMOND software was used to blast the Unigenes to the sequences of bacteria, fungi, archaea and viruses extracted from NCBI non-redundant (NCBI-nr) database with the e value threshold of 1e-10 49 . The best hit was chosen to be the taxonomic unit of Unigenes based on the results of lowest common ancestor (LCA) algorithm compiled in MEGAN software 50 . Functional annotation was performed by blasting Unigenes to the functional database including NCBI-nr database, KEGG database (http://www.kegg.jp/kegg/) and eggNOG database (http://eggnogdb.embl.de/) using DIAMOND software with the e value threshold of 1e-10. For each gene's blast result, the best hit with lowest e value and Blast coverage ratio between query and reference sequences of >40 was chosen for subsequent analysis. In addition, the coverage information of the scaffolds with lengths of >5 kb and tetranucleotide frequency (TNF) were used to perform the genome binning by using MetaBAT with a parameter of verysensitive 51 . The completeness, contamination and strain heterogeneity of genomic bins were then estimated by using CheckM 52 . Finally, three genome bins belonging to Pseudomonas and Acidovorax were retained for further analysis.  53 for details. Briefly, the purified DNA was labeled with Cy-3 using random primers and the Klenow fragment of DNA polymerase I and then mixed with sample tracking control (NimbleGen, Madison, WI, USA). With an addition of hybridization buffer, the sample was hybridized at 42 °C for approximately 16 h on a MAUI hybridization station (BioMicro, Salt Lake City, UT, USA). After the hybridization, the array was scanned with a NimbleGen MS200 Microarray Scanner (Roche NimbleGen, Madison, WI, USA) at full laser power. Signal intensities were measured based on scanned images. Poor quality spots with a signal-to-noise ratio of less than 2.0 were removed and the positive signals were normalized before statistical analysis. The sample was run in triplicate and then averaged. GeoChip 4.0 hybridization data process was performed through a pipeline compiled by the Institute for Environmental Genomics, University of Oklahoma, USA (http:// ieg.ou.edu/microarray).

Results and Discussion
Geochemistry. Sample LDS is under a slightly alkaline reducing condition with a total As concentration of 1.28 mg/L (Table S1). No MMA and DMA were detected and the ratio of arsenite to total As was 0.77, suggesting that inorganic As reduction prevails. Given high concentrations of DOC (8.58 mg/L) and ferrous iron (0.15 mg/L) and total iron (0.66 mg/L), the reductive dissolution of As-bearing iron oxides seems to contribute to As release in the groundwater, similar to previously reported results 54,55 . Sample LDS has extremely low concentrations of sulfide and sulfate (below detection limit), generally excluding the role of sulfur on As mobilization or sequestration 19,56,57 . High concentrations of ammonium (1.02 mg/L) and nitrate (0.79 mg/L) in sample LDS suggest the presence of some nitrogen metabolic processes, such as denitrification or DNRA 34,36 . Taxonomic profiling of microbial community. The As mobilization and transformation in groundwater systems are mediated by a variety of microorganisms 58 . An integrated analysis of 16S rRNA gene sequencing based on both RNA and DNA and metagenomic taxonomic annotations was used to investigate the composition and structure of microbial community in sample LDS (Figure 1 and Table S2). As the representative of active , Sediminibacterium (0.6-2.4%), Geobacter (0.8-2.2%) and numerous rare genera. In contrast to the high sensitivity of 16S rRNA genes, metagenomics sequencing without deep coverage usually detect principal species in complex microbial communities 59,60 . As we found in this study, dominant phylum Proteobacteria has a proportion of up to 93.2% in metagenomic taxonomic annotation, in which major genera are mainly composed of Pseudomonas (50.9%), Sphingobium (16.7%), Acidovorax (8.6%), Hydrogenophaga (2.6%) and Novosphingobium (2.2%).
Typical iron-reducing bacteria detected by 16S rRNA gene sequencing based on both RNA and DNA, such as Geobacter and Ferribacterium, suggest that iron reduction probably occurs in sample LDS. These iron-reducing bacteria may utilize organic matter as electron donors to reduce As-bearing iron oxides and release absorbed As 5,61,62 . High concentrations of As, iron and DOC detected in sample LDS are consistent with the suggested roles of iron-reducing bacteria (Table S1). Except for reductive dissolution, As-bearing iron oxides might dissociate a small fraction of As into solution due to the competitive adsorption of phosphate in the groundwater (Table S1) 5,8,9 . As the most abundant genus in the sample LDS, Simplicispira likely involve nitrate reduction as the strains of the genus Simplicispira isolated from freshwater, soil, and activated sludge were reported to be capable of denitrification 63 . Many members of Pseudomonas and Sphingobium have demonstrated to be able to degrade aromatic compounds, and thus those derived intermediates may further serve as electron donors for themselves or other microorganisms to reduce iron, nitrate and arsenate 64,65 . functional potential of microbial community. Metagenomic functional annotation and Geochip 4.0 were used to assess microbial functional potentials in sample LDS. Close to 11 Gbp clean data from metagenomic were assembled de novo to scaftigs and further derived 240574 protein-coding genes (Table S3). Among them, 104064 genes were successfully annotated to orthologue numbers in KEGG database. The results from metagenomic and Geochip 4.0 are similar regarding the relative abundances of As, carbon, nitrogen and sulfur-metabolizing genes ( Figure 2 and Table S4). High abundances of genes in ars cluster (arsRABCH) and absence of arrA infer that arsenate reduction in sample LDS may be mediated by As-resistant organisms harboring ars operon 66 . Arsenite oxidation and methylation are much less prevalent, revealed by near absent aioA and arsM. These results coincide with high arsenite concentration found and no detectable MMA and DMA in sample LDS. Carbon compounds degradation genes are significantly higher than carbon fixation genes, suggesting the consumption of DOC as energy and electron sources. Detection of mcrA, mmoX and pmoA by Geochip 4.0 indicate that some methanogens and methanotrophs inhabit sample LDS and regulate the methane concentration together (204.8 µg/L) (Table S1). Methanogens detected by sequence-and group-specific probes in Geochip 4.0 are composed of uncultured archaea (60.7%), Methanoculleus marisnigri JR1 (10.1%), Methanocorpusculum labreanum (8.4%) and Methanococcus aeolicus Nankai-3 (6.7%) (Table S5), which is similar to our previous study on mcrA libraries in groundwater samples from Hetao basin, Inner Mongolia 67,68 . Regarding nitrogen cycle, the dominant processes in sample LDS mainly include denitrification (presence of a whole set of narG, nirK, nirS, norB and nosZ), nitrogen fixation (nifH) and ammonification (gdh and ureC). Detections of napA, nrfA, nasA and nirAB imply that DNRA and assimilatory nitrate www.nature.com/scientificreports www.nature.com/scientificreports/ reductions to ammonia may occur. The near absence of amo, hao and hzo suggest inert nitrification and anammox activities. This is also supported by no detection of these characteristic anammox bacteria (genera of Candidatus Kuenenia, Candidatus Brocadia, Candidatus Jettenia, Candidatus Anammoxoglobus and Candidatus Scalindua) in RNA-and DNA-based 16S rRNA gene sequencing 69 . High levels of ammonium in sample LDS is thus attributed to multiple production pathways of ammonium as well as lack of appreciable ammonium-consuming process. In contrast to low levels of aprA, dsrA and dsrB, high abundances of cysI and sqr indicate that assimilatory sulfate reduction rather than dissimilatory sulfate reduction dominate in sulfur-deficient sample LDS. The transformation from thiosulfate to sulfate probably occurs, revealed by the presence of sox.
Metagenomic and Geochip 4.0 analyses display different results in terms of what microorganism possess above functional genes (Table S5). For example, metagenomic analyses indicate that the majority of arsC-harboring bacteria are affiliated with Pseudomonas, followed by Sphingobium, Sphingopyxis and Acidovorax. Differently, Geochip 4.0 analyses show the presence of arsC gene in Delftia acidovorans SPH-1, Maricaulis maris MCS10, Aspergillus fumigatus Af293, Shewanella putrefaciens CN-32, Shewanella sp. ANA-3 and Pseudomonas putida GB-1. This disparity should be attributed to the different nature of those technologies 70 . It is known that Geochip requires a priori sequence information to synthesize probes before hybridization and these probes enable Geochip to identify both abundant and rare species by theirs sensitivity and specificity 70,71 . On the contrary, metagenome without a high sequencing depth only detect those dominant species in complex microbial community, as we found in taxonomic profiling results in this study. Therefore, As-, N-and S-metabolizing functional genes in metagenome are similar to the members from the dominant genera Pseudomonas, Sphingobium and Acidovorax, whereas them in Geochip is more taxonomically diverse (Table S5). It should be noted that the majority of metagenomic ORFs in sample LDS is functionally unknown ( Figure S1 and Table S3), suggesting the novelty of sample LDS and highlighting that the relative abundance of functional genes may underestimated in metagenome. Geochip results cannot go beyond the detection capability of the probes fabricated on the array and thus may overestimate relative abundance of corresponding genes. Exactly for those reasons, the relative abundance of functional genes found in metagenomic analysis is much lower than that in Geochip 4.0 ( Figure 2).

Binning of metagenomic.
Three high-quality genomic bins belonging to Pseudomonas stutzeri, Pseudomonas and Acidovorax were derived from metagenomic data in sample LDS (Table S6). CheckM result showed their completeness of 96.5-99.3% and contamination of <1.1%. The genome sizes of bins range from 4.5 Mbp to 5.5 Mbp. The number of protein-coding genes is 4238, 5084 and 5092 respectively. These three bins encode all genes involved in glycolysis and citrate cycle, but lack key genes in reverse citrate cycle (rTCA cycle), reductive acetyl-CoA pathway and reductive pentose phosphate pathway (Table 1 and Figure 3), supporting the idea that these microorganisms www.nature.com/scientificreports www.nature.com/scientificreports/ utilize organic carbon as energy and electron sources. To cope with As toxicity and sulfur deficiency in sample LDS, they all possess the pathway of As-resistant, assimilatory sulfate reduction and thiosulfate metabolism. Bin 1, Pseudomonas stutzeri, participate in other metabolic processes of nitrogen cycle except for anammox and the first step of nitrification. Bin 2, Pseudomonas, only perform ammonification and partial DNRA. Bin 3, Acidovorax, has capabilities of denitrification, DNRA, ammonification and the second step of nitrification. Dissimilatory arsenate reduction, arsenite oxidation, arsenite methylation, methane oxidation, methanogenesis, anammox, dissimilatory sulfate reduction, and sulfur oxidation were all absent in these bins, which is basically consistent with metagenomic and Geochip 4.0 functional analysis results (Figures 2 and 3).
Given that Pseudomonas dominate in microbial community and As-resistant bacteria in sample LDS (Table S5 and Figure 1), metabolic potentials of Pseudomonas stutzeri bin were further analyzed ( Figure 3). The results show that Pseudomonas stutzeri could intake a variety of substrates, such as nitrate, nitrite, urea, phosphate, arsenate, sulfate, glucose and some amino acids, as well as iron, molybdate and zinc, some general cofactors of enzymes. These transporters of substrates provide a precipitate for intracellular metabolisms. The pathways of DNRA, denitrification, nitrogen fixation, ammonification, assimilatory sulfate reduction, glycolysis, citrate cycle and As-resistance are all found in the genome of Pseudomonas stutzeri bin. It was reported that Pseudomonas stutzeri is a well-known heterotrophic denitrifying bacterium, and some strains simultaneously possess the capacities of denitrification, nitrogen fixation and DNRA 64,72 . We thus propose that Pseudomonas stutzeri utilizes organic matter as energy and electron source to produce ammonium as well as reduce cytoplastic arsenate and pump-out arsenite to alleviate the stress of As, which leads to the accumulation of arsenite and ammonium in sample LDS. The co-existence of carbon-, nitrogen-and As-related corresponding genes in the genome of bin 2 and bin 3 supports this assumption  Table S4 for details. Nitrogen-, arsenic-and sulfur-related genes marked by the superscripts of 2 and 3 indicate their presence in the genome of bin 2 and bin 3. Due to the same detection of genes from carbon metabolism and membrane transporters in all bins, the superscripts of 2 and 3 in these genes are omitted. ANRA: Assimilatory nitrate reduction to ammonia; DNRA: Dissimilatory nitrate reduction to ammonia; ASR: Assimilatory sulfate reduction; DSR: Dissimilatory sulfate reduction.
Mechanism of As mobilization. The reductive dissolution of iron oxides is the main mechanism of As release in young groundwater systems with reducing condition 3,73 . Based on the microbial compositional and functional results from this study, we propose the pathways for arsenate release and reduction in sample LDS (Fig. 4). In this study, the iron-reducing bacteria, such as Geobacter and Ferribacterium, possibly initiate arsenate release by reductive dissolution of As-bearing iron oxides 2,13 . The competitive adsorption of phosphate may also contribute to a small fraction of dissociated arsenate 5 . The desorbed arsenate is subsequently reduced to highly mobile arsenite by some nitrogen-metabolizing bacteria featuring ars operon, such as Pseudomonas stutzeri 58,64 . Organic matter provide carbon and electron sources to fuel above biogeochemical processes 11,74 . As a consequence, high levels of ammonium, ferrous iron and arsenite were present in groundwater sample LDS (Table S1).

Figure 4.
A schematic description of As mobilization mediated by microorganisms in groundwater sample LDS. OM refers to organic matter. Iron-reducing bacteria refer to Geobacter, Ferribacterium and other microorganism which are able to utilize iron as electron acceptor. N-metabolizing bacteria mainly refer to N-transforming bacteria featuring ars operon, such as Pseudomonas stutzeri.


Nanobiotechnology in Agricultural Sector: Overview and Novel Applications

The agricultural sector has many problems achieving the desired yield in products because of pathogens, viruses, pesticides, herbicides, temperature, soil moisture, among others. Nanotechnology, along with other sciences, has emerged in industry and society due to the interest in solving problems such as those mentioned above, in order to serve the needs of the world population. The present review discusses the principal topics about Nanobiotechnology, such as generalities, applications, aspects that can be improved and perspectives, beside its influence in the agriculture industry.

Introduction
Nanotechnology is present in almost all areas of our daily life and is recognized by the European Commission as one of the six "Key Enabling Technologies" that contribute to sustainable competitiveness and growth in many areas of industrial application [1]. There are a variety of concepts about nanotechnology in the literature, however, it can be defined as a novel, innovative and interdisciplinary science field that involves the designing, development and application of materials and devices at molecular level in nanometric scale [2]. In other words, it is a field of applied science and technology which deals/involves in manipulating matter and its atoms and molecules with the purpose of fabricating materials, devices and systems with new characteristics that can improve their applications [3]. The principle of Nanotechnology is to work atom by atom, with the purpose of obtaining structures with new molecular organization and through this, to

History
The concept of "Nanoscience" has been attributed to Laureate Richard Feynman in 1959 and the term "Nanotechnology" was used for the first time in 1974 by Norio Taniguchi of the Tokyo Science University [7]. After that, the development of nanotechnologies has been attributed to two inventions that have revolutionized imaging and manipulation of nanoscale objects: The Scanning Tunneling Microscope (STM) invented in 1981 and the Atomic Force Microscope (AFM) in 1986. These instruments practically opened the door for scientists to the "nanoworld" and since these discoveries the studies in nanotechnology don't cease to increase in the innovations in electronics and also in the use of nanoparticles in many areas [9].
On the other hand, the studies about nanotechnology in agriculture started growing for industrial applications nearly half a century ago, nevertheless, the impulse to other scientists for its use is attributed to recently reports published, which are focused on identifying the research areas that should be found and also on set the agenda for nanotechnology research in agricultural applications, which became the principal guiding force for many nations that have agriculture as primary occupation of the population [10].

Applications in Agriculture
Nowadays, the world's population is growing very rapidly, and it is expected to reach 8.5 billion by 2030 [11], which means that the demand for food, natural resources like water, land to establish cities and cultivate, will also increase [12]. In the same matter, the climate change due to the pollution has caused a very negative impact on the crop production [12]. In that sense, the food industry needs to come up with quick and novel alternatives in order to fulfill the heavy demand for food and the shortage of land and water.
Currently, some of the solutions to cope with those problems have been the use of fertilizers, pesticides and genetically modified organisms (GMO) [13]. Nevertheless, the indiscriminate use of chemical compounds to increase the crop production or efficiency, has caused serious issues like heavy soil, air and water contamination, human and animal diseases and destabilization of the ecosystems [12] [14]. For example, related to the overuse of pesticides, many of the pests that affect the crops have developed resistance, analogous to the bacterial antibiotic resistance, which causes that the farmers tend to use more quantity and stronger pesticides, some of them being quite toxic [15].
With that being said, bio and nanotechnology now comes into play, as innovative technologies that may have the solutions to many of the issues that the humans and agricultural industry have been facing [1] [6] [13]. As an illustration, Table 1 shows some of the main materials used in agriculture that have been developed using nanometric technologies [15] [16] [17].  [15]. However, in order to achieve the enormous required amount of food, farmers have resorted to the use of conventional fertilizers at high rates and long periods of time, which is the main anthropogenic factor that has caused the eutrophication of coastal and freshwater ecosystems [18].
In that way, different approaches have been developed to deal with this issue.
One of them is the creation of nanofertilizers, that are either nanomaterials that can supply nutrients to the plant, or those that act as enhancers of the traditional fertilizers, but do not directly provide the nutrients [18]. For example, nanoparticles made out of hydroxyapatite, TiO 2 , SiO 2 , or even carbon nanotubes [13].
In regards of the uptake mechanism by the plants, there are two main mechanisms [18]. The first consists on the nanoparticle entering directly into the plant cells through pores on the cell wall, in this case, the size of the nanoparticle needs to be smaller than the pore size (5 to 20 nm) [18]. The seconds involves the dissolution of the nanoparticles, which are absorbed through the roots of the plant and release the nutrients as soluble ions [18]. Also, the advantage of using nanoparticles is that the dissolution of these in water or soil is higher compared to the conventional fertilizers, because of its smaller particle size and higher specific surface area [18].
Another strategy has been the development of biofertilizers, in which are used beneficial microorganisms that are able to convert organic matter into compounds that are nutritional for the plant and, at the same time, maintain the soil fertility and its natural habitat [15]. Nevertheless, this approach has a few drawbacks, like short shelf life, lack of suitable carrier materials, high temperature and desiccation sensitivity [15]. Therefore, in order to overcome those issues, Journal of Biomaterials and Nanobiotechnology polymeric nanoparticles for coating to resist desiccation, might be a great application [15]. For example, scientists have investigated the interaction between gold nanoparticles (GNPs) and plant growth promoting rhizobacteria (PGPR) [17]. The results showed a significant increase on growth for some of the strains tested, like P. fluorescens, P. elgii and B. subtilis, hence, GNPs have great potential as nano-biofertilizers [17].

Nanopesticides
Some materials contain elements in the nanometer size that claims novel properties associated with these small size, in this way, nanopesticides are defined as any formulation that includes this type of modified materials to generate more efficient products. Since materials vary their properties depending on their size, there is a wide range of nanopesticides that cannot be considered as a single category. For example, some of them are made of organic ingredients like polymers and, in the other hand, some nanopesticides may have inorganic ingredients like metal oxides. It should be noted that the nanomaterials were introduced into pesticides in various forms (particles and micelles); and this nanometric materials are introduced into pesticides to release the active ingredient more slowly and in a selectively way to increase the solubility of the poorly soluble active ingredient o thereby protect the ingredient against premature degradation [19].
In these days, resist environmental conditions such as sun, heat, rain, to be transported through the target and penetrate efficiently the organism (fungus, insect, etc) or to resist defense of the pest/pathogen, are some of the more important characteristics of an ideal pesticide. Even so, pesticides do not have such good chemical characteristics, that is why, now nanomaterials are implemented in this products. Likewise, it has been observed that in economic terms, introducing nanoparticules into pesticides provide monetary, social and environmental benefits by formulating more controlled modes of action. To achieve this, nanomaterials have potential for developing safer chemical pesticide formulations for applications in the field.
Another point to take into account is that nanotechnology has been incorporated into pesticides through the use of nanosuspensions, nanoparticles and polymeric nanoparticles. Nowadays, these forms of introduction into the nanopesticides have been tested to observe their efficiency and effectiveness [15].
Among the main nanomaterials used to improve the properties of pesticides are found the iron oxide nanoparticles and gold nanoparticles, they are mostly used because their easily way to be synthesized. According to the size, the pharmacokinetic parameters can be altered shape and functionalization of the surface of the nanomaterials [20]. That is why, nanotechnology can take an important part in the development of sustainable agriculture, since this technology can control the delivery of nutrients and monitoring the water quality used in this field [5]. As a consequence, it is important to encourage the use and spread of nanomaterials in this aspect. This allows the manufacture of advanced objects such as Zeolites for the controlled release of active agents such as pesticides [21].
Reducing waste and labor costs are some of the benefits that nanotechnology promises by streamlining agricultural management. Therefore, nanopesticides increased average crop yield by 20%, sunflower by 50%; rice by 35% and cucumber by 25%. Nowadays, some researches shows that nano-iron oxide pesticides increased rice yields by 25% and soybean yields increased 48% [22].

Antifungal Properties of Nanomaterials
The efficiency of nanoparticles on the control of the growth of some bacteria and fungi on plants has been proven. New Silver nanoparticles (SNPs) have been studied as pesticides for fungal and bacterial control; this is because by having such a small size they are able to penetrate the wall and cell membrane in large scale in biological processes. The antibacterial activity of silver ions has been well established. These SNPs have fungistatic, bacteriostatic and plasmonic properties that are eco-friendly inhibitors against plant-pathogens compared with synthetic fungicides [24]. SNPs can attack membranes and structures denaturing cell. This nanomaterial disrupts transport systems, such as ion efflux, causing a rapid accumulation of silver ions, interrupting cellular processes such as respiration and metabolism by reacting with the molecules [24] [25]. Journal of Biomaterials and Nanobiotechnology In the same way, in terms of nanotechnology, studies have been published on copper nanoparticles and copper oxide nanoparticles for the phytopathological and phytosanitary application [26]. Another nanoparticle used to control fungal pathogens is the chitosan, a natural compound that has several applications in biology, because of its capacity of being biodegradable, safe and non-toxic, environmentally friendly and low-cost production. Some investigations found that chitosan has antifungal properties because the interaction between amino groups of chitosan and negative charges of the macromolecules at the outside of the fungal cell wall [27].

Nanosensors
Agricultural production continues to be constrained by biotic and abiotic factors. For instance, insect pests, diseases and weeds cause considerable damage to agricultural production [6]. Regarding this, Nanotechnology has allowed the use of nanosensors, which are sophisticated instruments that can respond to physical, chemical or biological aspects and transfer that response into a signal that can be used by humans, allowing to detect many factors due to drought, temperature, insect or pathogen pressure, or lack of nutrients [5]. fungi) via the quantitative measurement of differential oxygen consumption in the respiration of good microbes and bad microbes in the soil. Also biosensors are developed, using photosystem II, known to bind several groups of herbicides and chemicals, among others [7].
Through the development of new devices, farmers and population will be benefited in aspects such as economic gain, reduced environmental pollution and lower labor costs [30].

Nanobiosensors
Physical tissues, microorganisms (pathogens), nucleic acids, among others, which have the function of receive signals from the sample and transmit it to a transducer.
The transducer measures physical changes occurring at the biological receptor. After that, the device transforms that energy into measurable electrical emission. The detector, catches the signal from the transducer and then it is passed to a microprocessor where are amplified and analyzed. Finally, the data is transferred to a display and the user can observe the results [31].
Due to investigations that have been carried out in the last years, nanobiosensors can be classified into many types such as mechanical, optical, electronic, viral, probes encapsulated by biologically localized embedding (PEBBLE), nanoshell and enzyme-based nanobiosensors ( Table 2). The use of each type of nanobiosensor depends on the analyte, for example, biosensors are used for the detection of phenols, polyaromatic compounds, halogenated pesticides and volatile organic compounds (VOCs) while electrochemical sensors can be used as chemical gas sensors [32].
Their applications include detection of analyte like urea, glucose, pesticides, also the monitoring of metabolites and detection of various microorganisms/pathogens [7]. Regarding the latter, one example is a novel microbial detection technology based on silica nanoparticles has been developed in the last years. The NPs (60 nm) are filled with a fluorescent dye and conjugated to an antibody specific to a surface antigen of the microbe of interest [15]; this method

Disinfectants
Elimination of contaminants (such as bacteria, fungi, and viruses) is the first step of any successful investigation on in vitro plant tissue culture. One of the major problems of in vitro plant culture techniques is chronic contamination by microorganisms [35]. Frequent application of conventional antibiotics and chemotherapeutic agents may be phytotoxic or may retard plant tissue growth [35]. In

Genetic Material Delivery
Genetic transformation is a major branch of plant biotechnology [35]. Delivery of genetic material such as DNA and small interfering RNA is important for the development of pest, pathogen and stress resistant strains of crop plants by alteration of gene expression [15]. Gene delivery systems for plant transformation face obstacles such as targeting of delivery system, transportation through the cell membrane, uptake and degradation in end lysosomes and intracellular trafficking of DNA to the nucleus [15].
Nanobiotechnology offers a new set of tools to manipulate the genes using nanoparticles, nanofibers and nanocapsules. Properly functionalized nanomaterials serve as transportation and could carry a larger number of genes as well as compounds able to trigger gene expression or to control the release of genetic material throughout time in plants [7].
Particle bombardment and Agrobacterium mediated transformation are two after cultivation with conventional antibiotic is challengeable [35].
On the other hand, gene gun or particle bombardment is one of the popular tools to deliver DNA into intact plant cells. Particles used for bombardment are typically made of gold since they readily adsorb DNA and are non-toxic to cells [37]. Experiments showed that the plasmid DNA transferred by gene gun method using gold capped nanoparticles was successfully expressed in intact tobacco and maize tissues [7].
However, gold NPs (5 -25 nm) embedded carbon matrices were employed for the delivery of DNA during transformation of plant cells that carried higher amount of genetic material as compared to the microparticles. NPs had lower plasmid and gold requirement as compared to the commercial micrometer-sized gold particles. Moreover, plant-cell damage with the NPs was minimal with increased plant regeneration [15].
The major advantage is the simultaneous delivery of both DNA and effect or molecules to the specific sites that results in site targeted delivery and expression of chemicals and genes respectively [7].

Controlled biochemical manipulations in cells have been achieved through the
integration of carbon nanofibers which are surface modified with plasmid DNA [7].
Carbon nanotubes (CNTs) could also be regarded as a potential gene delivery vector to plant cells. Single-stranded DNA wrapped around single wrap carbon nanotubes was able to access the cytoplasm of walled plant cells. This could be a promising observation to introduce short dsRNA pieces into the plant cell nucleus for small RNA-induced gene activation. Protoplasts could also be a target for gene delivery where larger CNTs with adsorbed plasmids would be employed to introduce genes-of-interest into the plant genome [38].
In addition to these vectors, nano-fibers/nanoparticles/nanovesicles with cationic nature have ability to precipitate negative charged DNA molecules and bring into cell through nonporous of cell membrane. This process has similarity with microinjection method of gene delivery [39]. Nanoparticulate cationic liposomes and cationic polymers are pursued as alternatives. Due to their permanent cationic charges interact electrostatically with negatively charged DNA to form complexes (lipo-or polyplexes) [15].
Although liposomes formed from cationic phospholipids offer several advantages as vectors for gene delivery into plant cells such as ease of preparation, enhanced delivery of encapsulated DNA by membrane fusion, protection of nucleic acids from nuclease activity, targeting to specific cells, delivery into a variety of cell types besides protoplasts by entry through plasmodesmata [3] [15]. Their Journal of Biomaterials and Nanobiotechnology toxicity and relative low transfection efficiencies are disadvantageous as gene delivery vectors [15].
Polymer/DNA complexes are more stable than those involving cationic lipids, and they protect DNA against nuclease degradation. Cationic polymers such as chitosan are used to condense and deliver DNA both in vitro and in vivo. Biocompatibility, low immunogenicity and minimal cytotoxicity render chitosan NPs (100 -250 nm) a good alternative to viral or lipid mediated transfection [15].
The ability of surface functionalized mesoporous silica nanoparticles (MSNs) to penetrate plant cell walls also opens up new ways to precisely manipulate gene expression at single cell level by delivering DNA and its activators in a controlled fashion [37].
Plasmid DNA molecules carrying a reporter gene can be encapsulated in micrometer-sized calcium alginate beads referred to as "bio-beads". These bio-beads have a possibility for efficient transformation in plants [3].
The major advantage of this technology is the simultaneous delivery of both DNA and effectors molecules to the specific sites that results in site targeted delivery and expression of chemicals and genes respectively. This is how the nanoparticle mediated plant transformation differs from the conventional genetic engineering methods (like electroporation, microinjection, etc.) [37].

Bar Coding
Barcodes are referred as a small symbol which is used for fast and easy identification. It contains a portable device which is powered by batteries and interfaced with laptop or computers. It is multiplexed with various pathogen detectors and has the potential to detect different pathogens simultaneously [39].
The applications of nanobarcodes include: DNA nano barcoding for pathogen detection (such as Ebola and SARS); monitoring of bacterial systems; can be used as a "nano-sensor" to detect water levels, soil nutrient information, and chemical levels; tagging food packages (Pathogens, Temperature changes, Leaks); biomolecular tracing; maximizes productivity; monitoring the quality of agricultural products [39] [40].
Moreover, DNA barcoding methods are a simple, rapid, and low-cost method for simultaneously detecting the presence of multiple bacteria and pathogen targets in the environment and foods [41]. Novel genes are being incorporated into seeds and sold in the market which can be traced with the help of nanobarcodes that are encodable, machine-readable, durable and sub-microsized taggants.
Disease spread through seeds and many times stored seeds are killed by pathogens [7].
South Korean researchers have developed a nano-DNA barcode system, which have a specific nano particles adhere products. The small triangle of color through nanotechnology, not only be visible signs of physical security, but also be embedded in the barcode with nano materials ink. And the newly developed

Nanolaminates
The largest postharvest losses in fruit and vegetable crops are due to deterioration caused by microorganisms after harvest and during cold storage. Fruits, due to their low pH, higher moisture content and nutrient composition are very susceptible to the attack of fungi, while vegetables are generally less acidic, and their spoilage is usually by bacteria. Although it is very difficult to determine the full extent of postharvest losses due to decay (i.e., attack by microorganisms and physical damages), it is well known that these losses are significant [43].
Therefore, food packaging is one of the main focus of polymer nanotechnology and thus one of the major applications of nano-coatings once it is believed that this technology can implement and improve the principal functions of existing packaging. Depending on the type of food, packaging materials need to satisfy different requirements in terms of light, moisture, water vapor and gas (i.e. oxygen and carbon dioxide) barriers in order to avoid color or taste deterioration, loss of nutritional value and eventually growth of potential pathogens [44].
Nanolamination is a technique for protecting the food from moisture, lipids and gases. Moreover, they can improve the texture and preserve color and odor of the food [45]. Therefore, the application of nano-coatings can be of great usefulness for food packaging once their significantly reduced thickness may improve barrier properties while exchanging their resistance, flexibility and tension, as well as the food appearance. Moreover, nano-coating materials may provide other active functionalities such as antimicrobial activity [44]. The resulting properties of nano-laminate coatings such as mechanical properties, gas permeability and swelling and wetting characteristics are influenced by the kind of adsorbing materials utilized and also by the sequence, the total number of layers and the conditions used for preparation (e.g., temperature, pH and ionic strength) [43].
Nanolaminates are made up of two or more layers of nano-sized (1 -100 nm) thin foodgrade films which are used on a wide variety of foods: fruits, vegetables, meats, etc. These are prepared from edible polysaccharides, proteins, and lipids [45]. Studies show that polysaccharide-and protein-based nanolaminates are good barriers against oxygen and carbon dioxide, but poor in protecting against moisture. Whereas, lipid-based nanolaminates are good at protecting food from moisture [4].
Other materials used for the nano-coatings, applied in a continuous process as a thin amorphous film of 50 nm or less, include titanium dioxide. Another example is that of nano-selenium, which is being marketed as an additive to a tea product in China for a number of (proclaimed) health benefits [46].
Previous studies have described the design and potential for use of emulsion-based nano-laminated biopolymer coatings produced using electrostatic Journal of Biomaterials and Nanobiotechnology deposition to produce novel encapsulation and delivery systems to control the bioavailability of bioactive lipids. They also described the use of in vitro and in vivo experiments necessary to study the efficacy of such coating systems. Findings included the utility of a chitosan coating to physically or chemically protect an encapsulated bioactive component within a food product and achieve release and bioaccessibility in the digestive tract after ingestion [47].
Current developments include safety and quality indicators that can be applied as labels or coatings to add an intelligent function to food packaging. For example, to monitor the integrity of the packages sealed under vacuum or inert atmosphere by detecting leaks, freeze-thaw-refreeze scenarios by detecting alterations in time-temperature, or microbial safety by detecting the deterioration of foodstuffs. This area of application is likely to see a rapid growth in the future [46].
In this matter, whey protein nano-coatings can also act as sensors mainly intended to monitor the condition of packaged food or the environment surrounding the food. This technology can provide real-time status of food freshness by using a visible indicator (i.e. thousands of nanocoatings may be designed to fluoresce in different colors upon contact with food pathogens, chemical contaminants, or toxins in response to changes in environmental conditions e.g. pH, temperature and moisture) [44].
It is suggested that barrier properties of nano-laminate coatings are improved due to their nano-structure, which has an increased tortuosity resulting from the electrostatic interactions between the nano-laminate's components and also from the interpenetration of the successively deposited layers that hampers gas molecules migration through the structure [43].
Furthermore, nanoparticles can have a dual purpose: besides acting as carriers of additives, they may also provide improvements in the mechanical and barrier properties of the structures where they can be incorporated. However, the efficiency of nanolayer systems with a variety of features (e.g., antioxidant, antimicrobial and reduction in gas exchange) still remains little studied [43].

Nanoremediation
As previously mentioned, pesticides are an essential key part of agriculture, since they allow the growth of crops by protecting them from pests that causes major loses on yields [48]. Unfortunately, most of the pesticides being used nowadays doesn't completely reach the target and the leftovers end up contaminating the surrounding soil, water and air resources and, finally, affect other living beings like animals, plants and humans [49] [50]. For example, arsenic, chlorpyrifos and benfuracarb are some of those pesticides being used that represent a huge health risk on humans, since they are neurotoxic, cytotoxic and carcinogenic [48] [49] [50].
With that being said, one major challenge that humans face these days is the restoration of contaminated soil, water and air to reach a quality level in which Journal of Biomaterials and Nanobiotechnology they no longer represent an environmental and health harm [51] [52]. Nevertheless, traditional remediation techniques are not so effective, quite costly, laborious and may also produce its own contamination [50] [51] [52]. As a promising alternative, nanoremediation has been investigated and might be a less costly and more effective solution to conventional remediation, as it relies on certain nanomaterials, like nanoparticles, which have the capacity to react with the pollutant, enable the transformation into less toxic compounds or immobilize it by adsorption [52]. Also, nanoremediation, compared to traditional remediation, offers some advantages, like less time to complete the cleanup process, since the NPs used have higher surface area and, hence, are more reactive [50] [51] [52] [53]. Also, the use of nanomaterials provides more technologies for in situ remediation, which is less expensive, and avoids the necessity to excavate into the ground, the disposal of contaminated soil or "pump and treat" for groundwater [28] [50].
For instance, nanoscale titanium (IV) oxide is being investigated for the photocatalytic degradation of contaminants in air and water, like VOCs or NOx [52]. Also, zero valent iron nanoparticles, also known as nZVI, have been widely investigated in order to develop novel nanoremediation techniques [28] [49] [50] [51] [52] [53]. These NPs are good electron donors and have shown capacity to remediate or degrade pesticides such as atrazine, lindane, chloroacetanilide, molinate, malathion, chlorpyrifos, dinoseb, organochlorine pesticides and heavy metals like arsenic, lead, mercury and zinc [49] [52]. Nevertheless, there are also concerns because scientists haven't fully investigated the behavior, toxicity and impact on biological systems of the nZVI once they're applied [28] [52].

Impact in Agriculture: Pros and Cons
As described above, nano biotechnology is characterized for designing and developing innovation materials at nanometer level for application in diverse areas. Following this aspects, Nanotechnology presents great advantages for the development of new technologies according to agricultural requirements ( Figure 2) [1].
The possibilities of innovation have increased after the incorporation of emerging technologies such as nano and biotechnology, which impulse the search of new market opportunities after the discovery of tools that improve areas of global and commercial importance such as agriculture [54].
The use of this technology implies, in turn, the efficient use of resources and time in the generation of research within agriculture, such as reproductive science and technology, conversion of agricultural and food waste to generate energy and other valuable byproducts, disease prevention and treatment of common diseases and reducing the negative impacts of pollution due to the excess use of pesticides and fertilizers [32].
Through technology, nanoscience has shown great potential in improving food safety, quality, product traceability, nutrient delivery enhancing, packaging performance, and improving agricultural and food processing [36]. These aspects are areas of application and development of agriculture in current times, Journal of Biomaterials and Nanobiotechnology As detailed above, applications of nanomaterials in this areas includes the germination and faster production of the plant, effective protection of plants with a reduced environmental impact, as opposed to traditional approaches. In addition, intelligent nanosensors are a viable option to detect pesticide residues in the field [28].
In the other hand, studies have shown that, using nanomaterials is not intrinsically risky; but the use of certain nanometric materials in agriculture, water and food have risks for human use and consumption, for the environment or for both since their long-term impact has not been proven, such as the bioaccumulation of these materials. Likewise, is important to consider that biomagnification and biotransformation of nanoparticles genetically modified in food crops are not yet well known. In addition, a good regulation of the entry of these materials into the food chain must be taken into account, or else, the manufactured nanomaterial may represent a potential risk to humans, sand and animals [7].
The use of nanotechnology in agriculture offers great potential for significantly higher productivity and agricultural efficiency at a lower cost, less use of pesticides and less waste of resources. Even so, is important to highlight that the appearance of these applications in agriculture and other sectors has also raised concerns about the impact of the use of these technologies on the safety of environmental and human health. Nanotoxicology, defined as the study of the toxi-Journal of Biomaterials and Nanobiotechnology cology of the main components of nanometric materials, has developed an effort to answer critical questions about the hazard, the exposure and the ultimate risk [8] [55].
In the same way, in the use of nanoparticles, diffusion in nature and a possible toxicity must be controlled, size impact of the use of these components within the area of agriculture for the innovative development of technologies in this area.

Future Perspectives
Currently, the main objective of nanotechnology research focuses on applications in the field of electronics, energy, medicine and life sciences, areas that are commonly in constant investigation for their impact at the social and commercial level. On the other hand, agriculture is not considered a powerful industry, so the research directed to these issues is considerably smaller. While nanochemical pesticides are already in use, other applications are still in their early stages, and it may be many years before they are commercialized or reach the public [5]. Due to this there is a broad path for the study of new research areas within agriculture.
Since nanobiotechnology is use to develop advanced tools in agriculture, the toxicity of nanoparticles is a recurrent theme in many research studies. Integrating "sustainable development" and considering the "environmental impact" are the challenges that nanotechnology has for the use of new resources within agriculture [9].
Studies have shown that the majority of companies dedicated to capital for research in nanotechnology focus on the search of devices with high technological demand and therefore monetary. Consequently, concerns in agriculture are still not a priority, so studies should be conducted on the importance and economic impact of nanotechnology within this area.
So far, ethical and social issues about nanobiotechnology have not been an important issue for many researchers in this area. Therefore, it is proposed to generate initiatives to anticipate the ethical and social problems that may arise during the development of nanobiotechnology, in order to respond proactively and responsibly to the probable public, media and political problems [56].
Likewise, highly detailed information of how plants respond to exposure and the molecular-level effects when nanomaterials are introducing into the plant and the correlations between physiological and biochemical parameters should be provided to bring more complete understanding effects of toxicity. Between the information that must be provided includes aspects such as the study of low dose exposures with sensitive endpoints, transgenerational studies, impacts on nutritional quality, co-contaminant effects and the impacts of exudation and microbial activity on particle fate and dynamics [7]. As shown in Figure 3, nanoparticles must have specific characteristics to be used in agriculture so they must be guaranteed for their consequent use within this industry [57]. Journal of Biomaterials and Nanobiotechnology

Conclusions
Nanobiotechnology is characterized as a science with innovative features that has the possibility to generate high technology tools for the development of sustainable fields such as agriculture. Among these features are the development of materials in nanometric scale (1 -100) nm that have versatile characteristics according to their size and manufacturing form such as hydrophobicity, permeability, pH, etc.
For the agriculture sector, the properties and possibilities of nanotechnology in this area are high reactivity, enhanced bioavailability and bioactivity, adherence effects and surface effects of nanoparticles. Nanotechnology comes as an innovative technology that may have the solutions to many of the issues that the humans and agricultural industry have been facing.
Among the predominant areas in which nanotechnology has developed in agriculture in recent years are nanofertilizers, nanopesticides, disinfectants, genetic material delivery, nanolaminates, nanosensors, irrigation, bar coding, etc. Even so, it is important to highlight that the appearance of these applications in agriculture and other sectors has also raised concerns about the impact of the use of these technologies on the safety of environmental and human health. Therefore, it is important to develop adequate studies about the possible toxicity of nanomaterials on agriculture so that its proper use is made possible in the improvement of the processes that this field requires in the future. Likewise, it should be made aware of researchers and investors about the importance of developing technological tools within agriculture to reduce the use of natural resources and the environmental impact caused by the excessive use of chemicals.


extfieldsofstudy: Medicine Geography

text:
American Tegumentary Leishmaniasis in the Brazilian Amazon from 2010 to 2014

ABSTRACT American tegumentary leishmaniasis (ATL) is one of the most important but neglected infectious diseases, and can cause severe deformities. ATL incidence remains high in forest regions, such as in the Amazonas State, Brazil. However, differences within the State and over time have been observed, since infection patterns are not homogeneous, and these aspects need to be clarified. This study aimed to identify the epidemiological profile of ATL and its spatial and temporal distribution in the Brazilian Amazon, from 2010 to 2014. Data were extracted from the Information System for Notifiable Diseases, which descriptively evaluates the incidence rate, as well as the temporal and spatial distribution of the disease. The highest prevalence of ATL was found in men and in the age group of 20-40 years. Approximately 95% of the cases were of cutaneous ATL and they were identified through direct examination. The spatial analysis has shown that ATL was widely distributed, both in rural and urban areas, and more concentrated in the Southern part of the State. Moreover, although there was an expansion in the spatial distribution and an increasing incidence of ATL in Amazonas, the epidemiological profile remained unchanged, suggesting that other factors must be responsible for its widespread distribution and increasing incidence.

INTRODUCTION
American tegumentary leishmaniasis (ATL) is a zoonotic disease caused by the protozoan Leishmania, which affects both humans and animals. It is an infectious disease with chronic evolution and is classified in several clinical forms: cutaneous, mucosal and mucocutaneous 1 .
ATL, a neglected disease 2 , is a public health problem that requires attention and is classified as one of the six infectious diseases of great importance owing to its high detection coefficient and characteristics that cause deformities. Due to the occurrence of lesions, ATL can compromise the patient's psychological state, besides affecting social and economic status, as it is also considered an occupational disease 3 .
Brazil is one of the five main countries with the highest incidences of ATL worldwide 4 . The epidemiology of ATL has been continuously changing in recent years, since it was primarily characterized as a zoonosis transmitted to humans when they came into contact with the vectors in forests. However, ATL has also started to emerge in deforested rural and peri-urban areas 1 . This situation may be explained by the presence of various vectors, reservoirs and etiological agents, together with socioeconomic and environmental conditions, such as unplanned urbanization, deforestation and population concentration, which can contribute to changes in the disease cycle 4 .
Amazonas is the Brazilian State with the highest incidence of ATL, with Leishmania (Viannia) guyanensis as the etiologic agent. In addition, ATL has recently become widespread with an increased incidence in the State, especially in municipalities previously free of the disease 5 . However, there have been few attempts at scientific evaluation in the scientific world, media and general society and as it is a severe disease that can cause suffering and increase economic burden, it calls for the attention of health services 6 .
It is important to provide information that could be used to manage and control the disease in endemic areas. This is the focus of the epidemiological surveillance, because there is no objective method, such as a vaccine, for preventing this disease. Health education is required to create awareness among populations in order to promote the avoidance of areas with epidemic outbreaks, and for those in areas at risk, to promote the use of preventive strategies, such as repellents, mosquito nets, window and door screens and environmental management to avoid vector contact. Therefore, this study aimed to identify the epidemiological profile of ATL and its spatial and temporal distributions in Amazonas, from 2010 to 2014.

Study design
This was an epidemiological, observational and ecological study, performed using the ATL data annually reported by the Information System of Notifiable Diseases (SINAN), focusing on epidemiological, spatial and temporal distribution.

Study area
The study area was the entire Amazonas State, the largest State in Brazil, with an area of 1,559,146.876 km 2 7 but low population density (3,873,743 inhabitants) 8 , corresponding to 2.5 inhabitants/km 2 in 2014. However, this State has the highest incidence of ATL, with an average of 50 cases/100,000 inhabitants compared to the 10.4 cases/100,000 inhabitants recorded in the entire country in 2014 9 .

Study population and data source
The study population consisted of all newly diagnosed cases of ATL reported to SINAN in Amazonas (Brazilian Amazon), from January 1 st , 2010, to December 31 st , 2014.
Annual ATL data from SINAN were obtained from the Ministry of Health, without individual identification (updated on September 21 st , 2015). Therefore, these were secondary data, and approval from the research ethics committee for studies involving human participants was thus not required, according to the National Health Council, Resolution 510/2016 10 . Each row in the database corresponds to a case; the cases were grouped according to municipality for the analysis, as well as in appended databases according to the year of notification.
Annual population estimates were recorded in the Department of Information of Unified Health System (Datasus) and were obtained at the municipal level 11 .

Variables
The data were analyzed by: 1) demographic profile (sex, age, years of education and ethnicity). The classification of ethnicity or skin color followed the guidelines of the Brazilian Institute of Geography and Statistics (IBGE). People are asked to classify themselves based on their skin color; hence, we adopted the terms Black, Yellow, White, Mixed Ethnicity (Black and White) and Indigenous 12 . In this analysis we grouped individuals into mixed ethnicity and not mixed ethnicity groups, since the majority of the population, and consequently, the cases, belonged to this group; 2) clinical aspect, diagnosis, and follow-up (ATL clinical form, HIV-ATL coinfection, and diagnostic exams, such as direct parasitology [biopsy of the lesion whose material is used to make a smear on a slide, which is then stained with Giemsa] 3 , Montenegro intradermic reaction, and histopathology); 3) geographical aspects (municipality, urban/rural housing area and epidemiological classification [autochthonous/imported]); and 4) ATL burden: annual incidence rate, obtained using the following formula:

Data analysis
Descriptive statistics were used to summarize the data, presenting the percentage of the annualized distribution of the variables. The incidence rate was stratified into five categories, according to the mean rate from 2010 to 2014 for all municipalities: up to 25; >25 to <=50; >50 to <=100; >100 to <=200; and >200. These ATL cutoff points followed the exploratory analysis in which a similar interquartile distribution of the rate, in most years, was found. For each category of ATL rate, we obtained the median and quartile in order to understand the evolution of the disease; we have Page 3 of 8 also obtained the median for each year. The median was used since the distribution of the annual rates of ATL was not normal according to the Shapiro-Wilk test. The spatial distribution was calculated by mapping the crude annual incidence rates of ATL in Amazonas (Brazilian Amazon). The spatial results are presented in thematic maps created using Q-Gis ® free software, version 2.16.

RESULTS
ATL was predominantly found in men, comprising approximately 80% of all reported cases. It is important to note that the Demographic Census of the IBGE in 2010 registered 1,822,607 (50.6%) 8 men in the Amazonas State, which reinforces the magnitude of the higher burden of infection in males. It was also mainly observed in adults between 20 and 40 years old, with mixed ethnicity (black and white) and low educational level. In several cases, schooling data were missing or incomplete (Table 1).
Regarding the clinical aspects, for all the analyzed years, the proportion of cutaneous ATL was greater than 95%. No records of mixed forms (mucocutaneous) were found. The occurrence of HIV-ATL coinfection was low (<1% throughout the study period), but HIV tests were not performed in more than half of the cases, except in 2010. Direct parasitological examination was performed in approximately 90% of the cases. Regarding geographical aspects, ATL was found in both rural and urban areas. This clearly indicated that the transmission pattern of the disease was not only wild, but also autochthonous, corresponding to approximately 75-80% of all cases ( Table 2).

Temporal distribution
The temporal distribution showed that ATL incidence increased from 2010 to 2011, remained constant in 2012, decreased during 2012 and 2013, and increased in 2013 and 2014 ( Figure 1). In the category > 200, the median incidence rate has shown oscillations, with an increase in 2011 and reduction in subsequent years, increasing again in 2014. The opposite behavior was observed in the stratum > 100 to 200, with a pronounced increase in the year 2013. In the other strata, the median incidence rate was similar across all years (Table 3).

Spatial distribution
In the spatial distribution analysis, the incidence rate was higher in the municipalities neighboring the town of the State capital, Manaus, as well as in the Southern areas of Amazonas. In 2011 and 2012, the disease was widespread in several municipalities ( Figure 2).
Next to the capital, the Municipality of Rio Preto da Eva, had the highest incidence rates (>1,000 cases per 100,000 inhabitants throughout the whole period), followed by Presidente Figueiredo (Figure 2). Of these, only the Barcelos municipality is not located in the South of Amazonas. Therefore, areas with the highest incidence rates of ATL were the municipalities bordering other states in the South of Amazonas, such as Rondonia, Acre, and Mato Grosso, in which ATL is endemic.

DISCUSSION
The incidence rate of ATL in Amazonas is surprisingly higher than that in other Brazilian States 9 . The epidemiological profile of the cases is almost similar to that from studies conducted in Amazonas and other States 13-15 . However, the age group with the highest prevalence in this study was the one between 20 and 40 years old, which is the most economically productive age group, especially for those who live outside the capital. The occupational profile of ATL is primarily associated with a high incidence of heavy labor activities, such as mining or gold prospecting, farming 13,16 , deforestation for livestock production 17 and environmental transformations related to increased rural migration 18 .
The following are the possible explanations for the increase in reported ATL cases in the capital of Amazonas, Manaus City, as well as in some municipalities in the metropolitan region of Manaus, such as Rio Preto da Eva and Presidente Figueiredo: access to forest and periurban areas, especially for leisure activities; haphazard occupations 19 in regions where recent deforestation has occurred; tourism activities and the search for access to health services in the capital, where patients often provide the address of their relatives and acquaintances, and conceal their original addresses, which makes it difficult to report the origin of the cases. Therefore, some individuals may not feel vulnerable to ATL because they live in urban areas or because they lack information about the clinical manifestations, mode of transmission and outcome of the disease, and seek care only when the wound has not healed for some time.  Another aspect responsible for neglected ATL, especially among affected people, is that when an individual has an open wound on the skin, he/she may believe that it will heal by itself in time, and usually skin problems are not a priority and are perceived as aesthetic concerns only 20 . This may be an explanation for the persistence of the endemic disease, since the individual may remain infected for a long time without any treatment.
Data from the World Health Organization estimate that Brazil is one of the 10 countries accounting for approximately 87% of cutaneous leishmaniasis cases worldwide (in 2015) 2 , and our data confirmed the high incidence in Amazonas, with no trend for a decline. This situation calls attention for its potential to lead to severe deformities 1 , which cannot be neglected. In contrast, it is possible to infer that cutaneous leishmaniasis has been efficiently treated, since the cutaneous form occurred in more than 90% of the reported cases. It is known that the mucosal form only occurs as an ATL complication and further develops into the mucocutaneous form when untreated or neglected 20 . Mucosal ATL is more severe than other forms, responds slowly to treatment, and is more susceptible to coinfections. Therefore, early diagnosis through parasitological examination to look for protozoa in supposedly infected tissues is required 21,22 . One can observe, based on this study's results, that the parasitological examination was not performed in all most cases. The lack of parasitological evaluation may prevent early diagnosis and, consequently, compromise proper treatment.
The low proportion of HIV coinfection should also be carefully considered, since the underreporting or lack of specific diagnosis, as evidenced by the high percentage of patients that did not undergo HIV testing may compromise the analysis. ATL progression may be facilitated by HIV coinfection because of decreased immunity. In addition, coinfection demands special ATL treatment; hence, performing an HIV diagnostic test is important, but has been neglected 1 .
In the past, ATL was considered a rural disease, but is currently evident in urban-rural areas 16 , which is consistent with this study's finding that no differences were found between urban and rural areas. This may be due to environmental changes related to migration, urbanization and socioeconomic aspects, leading to large endemic areas and the emergence of outbreaks. Thus, the vectors of ATL also adapt to new environments, approaching the peridomicile, i.e., around dwellings, facilitating disease transmission even in urban areas 18 .
The municipality of residence as the probable infection site, in most situation may suggest the population's migration to periurban areas or even deforestation within the urban areas, which favors contact with vectors; however, the question arises whether the epidemiological investigation, by the surveillance service, is accurate enough to identify the patient's trips to probable sites of infection in an area distant from his or her home. In turn, autochthony facilitates tracking down and effectively diagnosing ATL cases, even in more isolated municipalities.
Although there is expansion in the spatial distribution and an increase in the incidence of ATL in Amazonas, the epidemiological profile has remained similar across the years, suggesting that other factors must be responsible for the increasing spatial and temporal distribution of the disease. Therefore, strategic actions should be implemented in municipalities that have been identified as endemic, in areas that have experienced epidemic outbreaks over the years and in individuals living in risk areas. Therefore, the results presented in this study may guide health institutions or personnel in the formulation of priorities regarding more vulnerable areas and populations. Finally, continuous research should be conducted to detect possible changes in the epidemiological pattern of ATL to support prevention and control strategies.


extfieldsofstudy: Medicine Chemistry

s2fieldsofstudy: Biology Medicine

text:
18F-FIMP: a LAT1-specific PET probe for discrimination between tumor tissue and inflammation

Positron emission tomography (PET) imaging can assist in the early-phase diagnostic and therapeutic evaluation of tumors. Here, we report the radiosynthesis, small animal PET imaging, and biological evaluation of a L-type amino acid transporter 1 (LAT1)-specific PET probe, 18F-FIMP. This probe demonstrates increased tumor specificity, compared to existing tumor-specific PET probes (18F-FET, 11C-MET, and 18F-FDG). Evaluation of probes by in vivo PET imaging, 18F-FIMP showed intense accumulation in LAT1-positive tumor tissues, but not in inflamed lesions, whereas intense accumulation of 18F-FDG was observed in both tumor tissues and in inflamed lesions. Metabolite analysis showed that 18F-FIMP was stable in liver microsomes, and mice tissues (plasma, urine, liver, pancreas, and tumor). Investigation of the protein incorporation of 18F-FIMP showed that it was not incorporated into protein. Furthermore, the expected mean absorbed dose of 18F-FIMP in humans was comparable or slightly higher than that of 18F-FDG and indicated that 18F-FIMP may be a safe PET probe for use in humans. 18F-FIMP may provide improved specificity for tumor diagnosis, compared to 18F-FDG, 18F-FET, and 11C-MET. This probe may be suitable for PET imaging for glioblastoma and the early-phase monitoring of cancer therapy outcomes.

Results
As a result of screening our α-methyl amino acid chemical library using hLAT1 and hLAT2 overexpression cell lines, FIMP was found to have higher affinity for LAT1 than 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH), a classical inhibitor of L-type amino acid transporters, which is also transported into cells as a substrate of LAT1. The half-maximal (50%) inhibitory concentration (IC50) value of FIMP was significantly lower than that of BCH (Mean ± SD, 88.5 ± 13.5 µM and 231.5 ± 10.0 µM, respectively).

Expression of human LAT1 and CD98 in cell lines and tumors. High expression levels of LAT1 and
CD98 were observed for T24 and LNZ308 tumor cells. Conversely, WI-38 normal human fetal lung fibroblast showed low to moderate expression of both proteins (Fig. 1a).
We also evaluated LAT1 and CD98 expression in tumor xenografts and normal muscle tissue. Moderate to high LAT1 and CD98 expressions were observed for T24 and LNZ308 xenografts (Fig. 1b). Conversely, LAT1 and CD98 expression was not detected for normal muscle tissue. Expression of sodium/potassium ATPase, plasma membrane markers, was comparable in all cells and tissues (Methods in Supplementary Information).
PET probe accumulation in tumor and inflamed tissue. PET probe accumulations were evaluated by small animal PET imaging using a mouse model which had both acute inflammation and a LAT1-positive tumor. After probe injection we observed time-dependent change of 18 F-FIMP accumulation by PET imaging in tumor, muscle and inflamed tissues. Time-activity curve of 18 F-FIMP accumulation in all tissues plateaued by 90 min after probe injection (Fig. 2). On the basis of this result, the biodistribution in all animals was evaluated at 90 min after probe injection. As a result of visual assessment, 18 F-FIMP showed high accumulation in the tumor region and low accumulation in the inflamed lesion. 18 F-FDG showed higher accumulation in tumor regions compared to 18 F-FIMP, 18 F-FET and 11 C-MET; however, comparatively high 18 F-FDG accumulation was also observed in inflamed lesions compared to 18 F-FIMP, 18 F-FET and 11 C-MET. 18 F-FET showed moderate accumulation in tumor regions and low accumulation in inflamed regions. 11 C-MET showed low accumulation in both tumor regions and inflamed regions (Fig. 3a). Furthermore, quantitative analyses of PET images found that accumulation levels of 18 F-FIMP in tumors (SUV 2.32 ± 0.09) were significantly higher than that of 18 F-FET (SUV 1.14 ± 0.20) and 11 C-MET (SUV 0.79 ± 0.14) (P < 0.01, respectively), comparable to that of 18 F-FDG (SUV 2.55 ± 0.59) without significant difference (P = 0.29). However, accumulation of 18 F-FIMP in inflamed lesions (SUV 0.96 ± 0.04) was comparable and significantly higher than that of 18 F-FET (SUV 1.14 ± 0.20) and 11 C-MET (SUV 0.79 ± 0.14) (P = 0.19 and P < 0.01, respectively), significantly lower than that of 18 F-FDG (SUV 1.73 ± 0.36) (P < 0.01) (Fig. 3b).
Tissue radioactivity was measured to validate the PET imaging data. Probe biodistribution data showed highest 18 F-FIMP uptake in the pancreas (33.99 ± 2.39%ID/g). Uptake of 18 F-FIMP was higher in tumor (7.78 ± 1.11%ID/g) than inflamed tissue, muscle tissue, and blood (3.89 ± 0.17, 2.36 ± 0.14, and   11 C-MET, and 18 F-FDG PET. LNZ308 cells were inoculated in right paws (arrow), and inflammation induced by injection of turpentine oil in left paws (arrow head). PET data were acquired 90 min after injection of 18 F-FIMP, 18 F-FET, and 11 C-MET, 18 F-FDG-PET data was acquired from 55 to 100 min after injection. Quantitative analysis of PET imaging data represented as (b) SUV and (c) tumor-to-muscle and inflamed lesion-to-normal muscle ratios (TMR and INR, respectively). Data are presented as mean ± SD (n = 4-9). *P < 0.05, **P < 0.01, compared with 18 F-FIMP groups.

18
F-FDG is the PET probe most commonly used for cancer diagnosis, staging, restaging, and assessment of therapy responses. However, the accumulation of this probe in inflamed lesions can lead to false positive diagnoses 1-3 . Therefore, efforts to further develop tumor-specific PET probes are important for increasing the effectiveness of cancer screening and treatment programs. In the present study, we demonstrated that the PET probe 18 F-FIMP The ratio of radioactivity in the unmetabolized fraction to that in total radioactivity was determined using a phosphoimaging plate at 90 min after injection into tumor-bearing mice. Data are presented as mean ± SD (n = 4). www.nature.com/scientificreports www.nature.com/scientificreports/ is highly specific for LAT1, with high accumulation in tumor tissue but not in inflamed lesions. T24 has higher expression of LAT1 than LNZ308 in tumor tissue (Fig. 1b). However, our final goal is to image brain tumors with 18 F-FIMP. So, we selected LNZ308 brain tumor cell line in this study, and used T24 as a positive control when examining LAT1 expression analyses. We first examined PET imaging in a subcutaneous tumor model implanted with LNZ308, and are currently considering PET imaging in a brain tumor model.
We confirmed that 18 F-FIMP is stable in aqueous 3.5% ascorbic acid. Starting with a radiochemical purity of 99.1%, this value decreased to 97.6% within 24 h after synthesis. For practical clinical and research applications, this high stability is advantageous as it allows for possible transportation of the synthesized 18 F-FIMP to hospitals and laboratories that are remote from the place of synthesis.
Inflammation is an inseparable by-product in the pathophysiology of cancer. Inflammation is not only a side-effect of cancer treatments, such as radio-and chemotherapy, but also contributes to the development and progression of cancer. At the earliest stages of neoplastic progression, inflammation can promote the progression of incipient neoplasia into invasive cancers 13 . Furthermore, inflammation can significantly hinder the efficacy of diagnostic tests. The accumulation of 18 F-FIMP in inflamed lesions was low in both animal models of inflammation (turpentine oil induced myositis model and collagen induced arthritis model); hence, suggesting that this probe may provide a more accurate approach to discriminate tumor tissues from inflamed tissues.
The efflux of radiolabeled amino acids and their metabolites from cells has been negatively correlated with their accumulation in tumors 14 . Most natural amino acid-derived PET probes, such as 11 C-MET, are incorporated into the protein fraction, resulting in an increase in nonspecific accumulation in non-tumor tissues 15 . From our results, 18 F-FIMP showed very high metabolic stability both under in vitro and in vivo conditions and is not incorporated into protein. These findings suggest that the high tumor accumulation value obtained using this tumor-specific PET probe maybe more reliable compared to that of other radiolabeled amino acids such as L-3-18 F-fluoro-α-methyl tyrosine (FAMT) 9 , O-(2-18 F-fluoroethyl)-L-tyrosine (FET) 16 , and anti-1-amino-3-1 8 F-flurocyclobutane-1-carboxylic acid (FACBC) 8 . In order to demonstrate whether 18 F-FIMP has superiority in tumor imaging, standard PET probes, such as 18 F-FDG and 11 C-MET, and 18 F-FIMP need to be directly compared in the same cancer inoculation models by PET imaging studies.
An accurate estimation of radiation exposure is indispensable for defining a safe clinical PET study protocol. According to the biodistribution data from our PET imaging studies, we were able to estimate the expected mean absorbed dose of 18 F-FIMP in humans. The effective doses of 18 F-FIMP were determined to be 25.1 ± 5.3 and 30.8 ± 6.7 μSv/MBq for males and females, respectively. These doses are comparable or slightly higher than that  17 but still indicate that 18 F-FIMP is a safe PET probe for use in humans.
However, additional studies are required to evaluate further applications of 18 F-FIMP in humans. For example, additional comparisons between 18 F-FIMP and other LAT1-specific PET imaging probes would provide a valuable assessment of its potential as a cancer diagnosis tool.
In this study, we developed a tumor imaging PET probe with a high affinity for LAT1. PET imaging studies revealed that 18 F-FIMP accumulated in LAT1-positive tumor tissue, but not in inflamed lesions. This markedly high discrimination between tumors and inflamed lesions is important for effective diagnosis and treatment. Hence, 18 F-FIMP may have advantages over existing PET imaging probes, such as 18 F-FDG and 11 C-MET.

Subcutaneous tumor xenograft and inflammation models.
All animal experimental protocols were approved by the Animal Care and Use Committee of RIKEN and performed in accordance with the National Institutes of Health Principles of Laboratory Animal Care (Approved No. MAH28-02). All applicable institutional and/or national guidelines for the care and use of animals were followed.
LAT1-positive human glioblastoma (LNZ308) cells were inoculated into the right forepaws of female BALB/ cAJcl-nu/nu nude mice (CLEA Japan, Inc., Tokyo, Japan) via subcutaneous injection of 5 × 10 6 cells in 100 μL phosphate buffered saline (PBS). Tumor growth was monitored twice weekly using a caliper. Acute-phase inflammation was induced by subcutaneous injection of 50 μL turpentine oil into the left forepaw of tumor-bearing mice 72 h before PET imaging 19 . We also used collagen-induced arthritis (CIA) model mice for evaluation of developed radiolabeled probes (see Supplementary Methods).
PET data acquisition. Mice were fasted for 14 h before 18 F-FDG administration. 18 F-FIMP and 11 C-MET-PET were administered to unfasted mice. All mice were anesthetized with 1.5% isoflurane and placed on the bed of a microPET Focus 220 scanner (Siemens Medical Solutions USA, Inc., Knoxville, TN). Radiolabeled probes were dissolved in saline (0.1 mL) and administered via a cannula inserted into the tail vein. Emission data were acquired for 90 min after administration using a 3-dimensional (3D) list-mode method for 18 F-FIMP and 11 C-MET, and for 45 min from 55 min after administration using a 3D list-mode method for 18 F-FDG. Data were reconstructed using 2-dimensional filtered back projection (FBP) for quantification and a 2-dimensional ordered subset expectation maximization (OS-EM) algorithm for region of interest (ROI) definition. For ROI definition and further analysis, summed images (0-90 or 55-100 min post injection) were reconstructed. ROIs were drawn on several areas of tumor, muscle, and inflamed tissues. Regional uptake of radioactivity in organs were decay-corrected based on injection times and expressed as the standardized uptake value (SUV), where SUV = tissue radioactivity concentration (MBq/cm 3 )/injected radioactivity (MBq) × body weight (g). Quantitative analysis of PET imaging data also represented as tumor-to-muscle and inflamed lesion-to-normal muscle ratios (TMR and INR, respectively). PET imaging was also performed with CIA mice using 18 F-FIMP, 11 C-MET and 18 F-FDG.
After PET imaging, mice were sacrificed and their organs quickly removed and washed with saline. Blood samples were obtained immediately before dissection by cardiac puncture. Excised organs and blood samples were weighed and their radioactivity determined using a Wallac Wizard 1480 scintillation counter (PerkinElmer, Waltham, MA). Results were expressed as %injected dose per gram of tissue, TMR, tumor-to-blood ratio (TBR), and INR. Dosimetry analysis. Mean absorbed doses of 18 F-FIMP (μSv/MBq) in humans were estimated on the basis of PET imaging data from mice (n = 4). The mean %ID/g values for mouse livers, kidneys, pancreases, urinary bladders, and remainder of the body were extrapolated to estimate expected uptake in organs for a 73 kg human adult male. The organ fractions of total body mass for mice (25 g), human males (73 kg), and human females (53 kg) required for this extrapolation were obtained from Hui et al. 20 for mice and ICRP Publication 60 17 for humans, respectively. Dosimetry estimations were calculated using the OLINDA/EXM version 1.1 software (Hermes Medical Solutions, Stockholm, Sweden) based on standard human male and female models 21 .
Statistical analysis. Data are presented as mean ± standard deviation (SD). All statistical analyses were performed using Microsoft Excel 2010 version 14.0 (Microsoft, Redmond, WA) using Student's t test. P-values less than 0.05 were considered significant.