All cells need iron. Both too much and too little iron lead to diseases and unwanted outcomes. Prevotellaceae and Porphyromonadaceae families and Bacteroidales order are highly sensitive to low-iron conditions, while other seemingly insensitive OTUs recover. These results provide new insights into the iron requirements of gut microbiome members and add to the growing understanding of mammalian iron cycling.Iron is an essential micronutrient for life. In mammals, dietary iron is absorbed primarily in the small intestine. Currently, the impacts of dietary iron on the taxonomic structure and function of the gut microbiome and reciprocal effects on the animal host are not well understood. Here, we establish a mouse model of low-iron challenge in which intestinal biomarkers and reduced fecal iron reveal iron stress while serum iron and mouse behavioral markers indicate maintenance of iron homeostasis. We show that the diversity of the gut microbiome in conventional C57BL/6 mice changes dramatically during 2 weeks on a low-iron diet. We also show the effects of a low-iron diet on microbiome diversity are long lasting and not easily recovered when iron is returned to the diet. Finally, after optimizing taxon association methods, we show that some bacteria are unable to fully recover after the low-iron challenge and appear to be extirpated from the gut entirely. In particular, operational taxonomic units (OTUs) from the IMPORTANCE All cells need iron. Both too much and too little iron lead to diseases and unwanted outcomes. Although the impact of dietary iron on human cells and tissues has been well studied, there is currently a lack of understanding about how different levels of iron influence the abundant and diverse members of the human microbiome. This study develops a well-characterized mouse model for studying low-iron levels and identifies key groups of bacteria that are most affected. We found that the microbiome undergoes large changes when iron is removed from the diet but that many individual bacteria are able to rebound when iron levels are changed back to normal. That said, a select few members, referred to as iron-sensitive bacteria, seem to be lost. This study begins to identify individual members of the mammalian microbiome most affected by changes in dietary iron levels. While satalase) , how iroTo address this knowledge gap, we conducted a series of experiments using conventional C57BL/6 mice to identify the most iron-sensitive microbiome members. We first established a model for low-iron (LI) challenge that includes acclimatization to a chemically defined diet, itself a major perturbance to the microbiome, prior to evaluating the impact of LI challenge on both α- and β-diversity using 16S rRNA sequence analysis. Finally, we evaluated bioinformatic selection procedures for identifying iron-sensitive taxa and quantified their ability to recover after LI challenge when dietary iron was restored. The model described here incorporates an acclimatization period into a murine model of microbiome-iron interaction and, therefore, serves as a benchmark for understanding the influence of this essential mineral on microbiome structure and function.2+) carbonate. Purified chow containing iron at 2.6 ppm  was purchased for use as an LI challenge. Since other nutritional components of the LI diet were markedly different from typical mouse chow, we first conducted a pilot study to establish a new baseline (B) of microbiome diversity for comparison. Namely, we switched the diet of conventional mice to the LI challenge chow and added ferrous iron [iron(II) sulfate heptahydrate] to drinking water at a level that maintained a similar level of iron in the gut  diet on gut microbiome diversity, we developed a feeding protocol in conventional, specific-pathogen-free mice where stool samples and blood were taken throughout dietary transitions. The typical chow used in our vivarium contained 208 mg total iron/kg of body weight (quantified by inductively coupled plasma-mass spectrometry [ICP-MS]), primarily as ferrous (Fe the gut .0] to baseline day 14 [B14]) maintained the same level of iron in their stool as they did when eating normal chow, without statistical differences (P = 0.844 by paired Wilcoxon rank sum test) (P = 0.006 by paired Wilcoxon rank sum test)  occurred and was comparable for the cohort  . This inum test) . A signi4 from the drinking water resulted in a significant decrease in total iron in stool samples over a 2-week time frame (P = 0.047 by Wilcoxon rank sum test) (14 to LI14), indicating that our iron challenge protocol was sufficient for lowering the amount of iron in the gut. Concomitant with low iron conditions in the stool, we saw a further decrease in population richness in α-diversity , where relatively little change was observed after the second week (LI14). Collectively, the results from this initial experimental setup suggest that (i) our protocol established a reasonable baseline following the dietary shift and (ii) the microbiome responded to the LI challenge.The removal of FeSOiversity  and anotiversity , indicat7) to better assess microbiome stability during baseline establishment. Consistent with results from the pilot experiment, total iron in stool did not change significantly between T0 and baseline time point B7 or B14 but decreased significantly in response to the LI challenge   . Stool i7 and B14) or LI challenge time points (LI7 and LI14), suggestive of microbiome stability during both of these intervals. Surprisingly, we found that the microbiome of these two groups of mice were markedly different at the outset of each experiment (T0), potentially reflecting vivarium-specific differences in microbiome diversity even when exposed to similar housing and feeding conditions . Regardless of the initial starting differences, similar changes in ordination space occurred throughout the LI challenge phase . However, total serum iron decreased from baseline in mice during the second week of LI challenge (B7 and B14 versus LI14), suggesting host iron stores were being drawn down. Over the same time intervals, stool iron decreased, indicating that a host metabolic response led to recycling and retention rather than excretion of systemic iron. Duodenal tissue collected at the end of the LI challenge and assayed for iron-responsive gene expression supported the proposed explanation of iron recycling and retention, as all three genes of interest were significantly upregulated , no change in host serum iron was evident either during LI challenge or repletion  as well as for two iron “repletion” experiments (repletion groups 1 and 2) that received iron repletion for 2 weeks following LI challenge  and was comparable throughout the second week of repletion (R14). Similarly, decreased α-diversity was observed in all groups between baseline and LI time points but was significantly reversed during the first week of repletion (7 to R14). Not all components of microbiome diversity were affected equally during this 2-week repletion period. For example, there was no difference in the total number of observed operational taxonomic units (OTUs)  during time points R7 to R14 (14 was significantly different from baseline (B7) in all groups . Incomplete rescue of both α- and β-diversity estimates suggests that LI challenge had long-lasting effects on microbiome diversity even after stool iron levels returned to normal.As mentioned above, we conducted three independent experiments  to understand whether alteration of microbiome diversity as a result of LI challenge would last when iron was reintroduced as opposed to simply shifting back to baseline . In these experiments, a decrease in stool iron was observed between baseline and LI challenge time points , similarepletion . Interes7 to R14 , but OTUt testing with Bonferroni correction) based on straightforward OTU selection criteria.Few comparisons of the performance of OTU-picking methods have been conducted, meaning there is currently little consensus in the field about which approach is most appropriate for identifying OTUs most affected by a given treatment. Here, we compared and evaluated four different approaches  were compared  tract. The taxonomic structure of the gut microbiome is known to be sensitive to the chemical speciation and overall amount of iron in the host’s diet. For example, feeding mice a heme-based diet was shown to result in an increased proportion of in stool , 6. Rece, HIF-2α . Such stHere, we quantified the response of a complex, native microbiome in C57BL/6 mice to a subclinical period of iron deprivation. Our approach was unique compared to other studies with respect to carefully monitoring host-microbiome adaptation to a chemically defined diet (acclimatization) prior to a 2-week period of LI challenge. With this approach, we were able to partition significant changes in microbiome diversity due to dietary change versus iron removal, which turned out to be very important, as simply administering a new dietary regimen with or without normal iron levels potentially confounds these two influences. The murine host exhibited generally stable serum iron levels throughout LI challenge but showed markedly reduced stool iron excretion, which supports host systemic iron withholding. Consistent with these observations, several known iron-regulated transporters were upregulated in the small intestine of iron-deprived mice, yet they remained outwardly healthy with no signs of anemia.Firmicutes  and Bacteroidetes . Moreover, three bacteria, each belonging to the Bacteroidales order, appeared to be hypersensitive to LI challenge, having gone below our detection limit over the relatively short (2-week) period of iron deprivation. This suggests that these Bacteroidales members are highly sensitive to low dietary iron.Over the course of the same LI challenge, the microbiome underwent dramatic changes, and members of six common bacterial families were completely lost from >50% of the mice. We expect these species have the greatest dependence on dietary iron for survival, although it is possible that these observations were indirect effects, such as the loss or metabolic change of cross-feeding members in the community. Interestingly, all six of the most affected bacteria were anaerobes belonging to the most common phyla in the mammalian gut, Pseudomonas, Staphylococcus, and some Enterobacteriaceae) are often facultative anaerobes with robust, iron-utilizing metabolic capabilities that permit somewhat unchecked growth in host tissue  during LI challenge and their continued absence even after iron was reintroduced suggests that the cooperative link by which these anaerobes fill their iron requirement is irrevocably broken. This could have occurred if members of low-abundance bacterial species capable of directly supplying heme  also underwent extinction, keeping in mind that 16S sequencing, as we and others have shown, has significant detection bias for lower-abundance OTUs . Mice were subsequently bred and maintained at the American Association for the Accreditation of Laboratory Animal Care-accredited Animal Resource Center (ARC) at Montana State University and housed under specific-pathogen-free conditions (including murine norovirus) in individually ventilated cages with sterilized bedding. Female and male mice were separated at weaning and housed separately until the start of each experiment. Mouse sex and age at the outset of each experiment is given in Table S1 in the supplemental material, and an overview of time points and experimental transitions is given in Fig. S1. Mice were reared on standard chow  and switched to an iron-deficient but otherwise nutritionally sufficient chow  with iron supplementation as iron(II) sulfate heptahydrate  dissolved in distilled, purified water . Water was made fresh in sterilized plastic bottles and changed every 3 days to account for the precipitation of iron. For low-iron challenge, dietary and living conditions were held constant, with the only difference being that iron-supplemented drinking water was replaced with regular vivarium drinking water . Vivarium water is sourced using a reverse osmosis system, and iron levels were tested and confirmed to be less than the U.S. Environmental Protection Agency standard of 0.3 mg/liter or ppm. Longitudinal serum samples were prepared from blood sampled by the submandibular bleeding method . Stool samples were collected during the same visit as blood sampling. All animal experiments were approved by the Montana State University Institutional Animal Care and Use Committee. No statistical methods were used to select Five different experiments were conducted to evaluate the effect of dietary iron on gut microbiome diversity, consisting of at least two technical replicate cages, as indicated in Table S1. The timeline of each experimental group refers to separate, nonoverlapping experiments conducted on different dates and indicative of biological replicates. Cages were composed of 5 mice each. Unfortunately, data were not available for all mice at each time point due to problems with DNA extraction, 16S rRNA amplification, and/or problems arising in the Illumina DNA sequencing pipeline.n = 14 days), the water was replaced with metal-free purified water for another 2 weeks. Stool samples were collected throughout the experiment.To establish methodology and efficacy of iron limitation, two cages of 9-week-old, female mice were switched from standard mouse chow to the low-iron chow and with iron sulfate in their drinking water as described above. After 2 weeks (n = 5) were transitioned to iron-replete water for the 2 weeks following the iron challenge. Duodenal and cecal tissues were harvested following euthanasia for evaluation of host iron responses.To evaluate iron limitation on host expression of iron metabolic genes, two experimental groups were designed to have specific endpoints as indicated. Blood and stool were sampled throughout the experiment as indicated, and half of the mice in group 3 (T0).Two additional groups were used to increase biological replication and to consider a group of male mice (repletion group 2). Both groups were treated identically to validation group 2, with the only experimental difference being that no samples were considered at the outset of the experiments (g to separate the serum. A known volume (∼20 μl) of serum was removed from the top of each sample and incubated in 1 ml of 50% nitric acid  at 55°C for 30 to 60 min or until dissolved. Samples were diluted with 9 ml of metal-free water  and centrifuged at 5,700 × g for 10 min. After centrifugation, samples were filtered to remove particulates . Stool pellets were dried overnight under vacuum and weighed before being incubated with 1 ml 50% nitric acid at 55°C for 1 h. Samples were diluted with 9 ml of metal-free water and centrifuged at 5,700 × g for 10 min before filtration to remove particulates. All samples were stored in the dark at room temperature until analysis by ICP-MS. Total iron in serum and stool was quantified using an Agilent 7500ce ICP-MS with a collision cell and a certified environmental calibration standard . Total iron per sample was calculated based on volume (serum) or dry mass (stool).Weekly blood samples (<60 μl) were collected and frozen (–20°C) until prepared for total iron quantification using inductively coupled plasma-mass spectrometry (ICP-MS). Blood was thawed at room temperature and centrifuged for 10 min at 1,677 × b (Dcytb), divalent metal transporter 1 (Dmt1), and ferroportin were evaluated relative to β-actin using primer sequences and sources listed in TC) method  and shredding . Total RNA was extracted using a kit , quantified using a spectrophotometer , and stored at –80°C until further use. cDNA was prepared using iScript polymerase master mix  and thermocycling . Quantitative PCR was performed on a Roche 96 light cycler using a SYBR green fluorescence assay . Transcription of genes encoding duodenal cytochrome Eukaryota or that remained unclassified at the domain level were removed. For normalization, each sample was rarefied to 10,000 reads.DNA was extracted using a DNeasy Powersoil kit  from stool pellets by following the manufacturer’s instructions. DNA was shipped in 96-well plates to the University of Michigan Center for Microbial Systems for Illumina paired-end (2 × 250 bp) sequencing of the variable region 4 (V4) of the bacterial 16S rRNA gene. Raw sequencing reads were processed using mothur v.1.39.5. Low-quality reads were removed by following the mothur standard operating procedure  accesse and remot test for normal data and nonparametric Wilcoxon rank sum test for nonnormal data. Changes in microbiome diversity between experimental treatments were tested for significance using PERMANOVA (adonis2 in vegan). OTUs most responsible for these changes were identified as described in Results using indicator value , and ordinations  were performed to visualize patterns of microbiome similarity (β-diversity). Both analyses are based on the presence-absence and relative abundance of taxa (OTUs) resolved by 16S sequencing, but where α-diversity estimates represent diversity within a single community, β-diversity represents diversity between several different communities. These analyses as well as bar charts representing taxon abundance were generated in R version 3.6.0  using ve .PRJNA562734.Sequencing reads were deposited in the National Center for Biotechnology Information (NCBI) BioProject database with accession number


Rice is staple food for over two billion people. Planthoppers like BPH and WBPH occur together in most of rice growing regions across Asia and cause extensive yield loss by feeding and transmission of disease-causing viruses. Chemical control of the pest is expensive and ecologically disastrous; breeding resistant varieties is an acceptable option. But most of such efforts are focused on BPH with an assumption that these varieties will also be effective against WBPH. No critical studies are available to understand rice resistance, common or otherwise, against these two planthoppers.Our studies aimed to understand the defense mechanisms in rice line RP2068 against BPH and WBPH through RNA sequencing analysis of a RIL line TR3RR derived from the cross TN1 (susceptible) and RP2068 (resistant) after infestation with BPH or WBPH. Results revealed higher number of differentially expressed genes (DEGs) in BPH infested plants than in WBPH infested plants when compared with the uninfested plants. These DEGs could be grouped into UPUP, DNDN, UPDN and DNUP groups based on whether the DEGs were up (UP) or down (DN) regulated against BPH and WBPH, respectively. Gene ontology analysis, specially of members of the last two groups, revealed differences in plant response to the two planthoppers. Abundance of miRNAs and detection of their target genes also indicated that separate sets of genes were suppressed or induced against BPH and WBPH. These results were validated through the analysis of expression of 27 genes through semi-quantitative and quantitative real-time RT-PCR using a set of five RILs that were genetically identical but with different reaction against the two planthoppers. Coupled with data obtained through pathway analysis involving these 27 genes, expression studies revealed common and differential response of rice RP2068 against BPH and WBPH. Trehalose biosynthesis, proline transport, methylation were key pathways commonly upregulated; glucosinolate biosynthesis, response to oxidative stress, proteolysis, cytokinesis pathways were commonly down regulated; photosynthesis, regulation of transcription, expression and transport of peptides and defense related pathways were exclusively upregulated against WBPH; MYB transcription factor mediated defense induction was exclusive to BPH.Rice defense against the two sympatric planthoppers: BPH and WBPH has distinct features in RP2068. Hence, a conscious combination of resistance to these two pests is essential for effective field management.The online version contains supplementary material available at 10.1186/s12284-021-00470-3. Oryza sativa L.) is one of the most important staple food crops of the world. Significant amount of the rice production is lost annually due to biotic stresses, of which about 25% is attributed to the insect pests ] and whitebacked planthopper (WBPH) [Sogatella furcifera (Horváth)] have again attained peak pest status in Asia since the beginning of this century  represented lectin receptor kinases; bph29 has a B3 DNA binding domain and Bph32 is novel with an unknown SCR domain , having shifted its host from Leersia about 0.25 million years ago , and TN1 (susceptible to both the planthoppers). Using an RNA-seq approach, specific transcripts displaying differential induction were identified in the RILs and subsequently, gene expression of 27 selected target genes was validated to understand and obtain useful insights into the molecular process of rice resistance against two of its major pests i.e. BPH and WBPH. The results of the current study indicated that rice defense against WBPH is of lower order with emphasis on tolerance as against BPH where the emphasis is on antibiosis.Omics tools have been successfully employed to investigate molecular pathways triggered or suppressed in rice genotypes when challenged by BPH. Zhang et al.  studied i et al.  adopted g et al.  studied g et al. . Li et ag et al.  made a c2 = 0.343; P = 0.558), resistance to WBPH among RILs segregated in 37R:143S suggesting two gene involvement . Other phenotypic tests such as nymphal survival (antibiosis component of resistance), days to wilt (tolerance component) and nymphal preference (antixenosis component) also revealed similar trend of segregation of resistance among RILs against BPH and WBPH. Based on the overall performance  and the gene expression level was estimated. The variability among the biological replicates indicated R2 value to be 0.759 for BPH, 0.785 for WBPH and when both BPH and WBPH data were pooled R2 was 0.345  was observed in the BPH infested plant tissue in comparison with the uninfested plant tissue than in the WBPH infested plant tissue in comparison with the uninfested plant tissue (BPH = 9361 vs WBPH = 8498). Among these, 3821 transcripts showed upregulation while 5540 showed downregulation against BPH in comparison with uninfested control tissue samples (Supplementary Data File No. Gene Ontology (GO) enrichment was performed for all the DEGs in leaf sheath tissues of TR3RR plants when challenged by either BPH or WBPH. The DEGs were first grouped into UPUP, DNDN, UPDN and DNUP categories based on their expression (whether up or down regulated) against infestation by BPH or WBPH. The DEGs were further categorized into three major categories viz. biological process (BP), cellular component (CC) and molecular function (MF). However, several of the DEGs were annotated in two or all the three categories. Among the DEGs classified under biological process, 16 clusters were represented in both UPUP and DNDN groups; 6 clusters were exclusive to UPUP while 14 clusters were represented only in DNDN group Fig. a. Five oTo identify the miRNAs, present in the RNA-seq data, all the transcript sequences in the size range of 20 to 24 nt were blasted against the miRbase. In all, 180,887 transcripts could be aligned with known rice miRNAs representing 713 families. Based on relative abundance of miRNA transcripts in plants subjected to infestation with either BPH or WBPH, 27 miRNAs were short-listed. These have been recognized to have 121 target genes. Of these, expression of 37 genes was observed to be modulated in the plants following planthopper feeding and these were identified to be modulated by 14 miRNAs Table , five geIn all, 27 DEGs were validated in five of the RILs following infestation with either BPH or WBPH Table . Five ofOf the 20 genes of the first set, 8 genes were selected from the UPUP group of DEGs  of induction in four of the RILs against both BPH and WBPH. Interestingly, two of the genes  in the susceptible TR24SS in 3 of 4 instances against both BPH and WBPH suggesting their possible role in susceptibility against the planthoppers. Five genes representing the DNUP group showed upregulation (≥ 2-fold) only against WBPH in three of the resistant test RILs , but not in the susceptible TR145RS and TR24SS. These genes displayed either down regulation or mild induction (< 2-fold) in all the RILs when infested with BPH. Only two genes (growth regulating factor protein and centromere protein coding genes) displayed threshold level of upregulation only against BPH in three of the resistant RILs . Thus, it was evident that despite 13 of the genes showing common response against BPH and WBPH with either induction or suppression, seven of the genes displayed differential response against BPH as versus WBPH.Fourteen of the DEGs, as revealed by the RNA-seq analysis, were re-validated through quantitative real time RT-PCR. Three genes from UPUP group were re-analysed. Two of the genes (B3 DNA binding domain and heat shock protein DnaJ) showed similar expression as that observed in the first set Fig. , panel 3Further, pathway analysis of these key 27 genes under four groups revealed major processes involving two or more of them ; rice green leafhopper and green rice leafhoppers  which are parapatric, with discrete geographic distribution. Among the sympatric species, members differ in their host range adaptation. Monophagous yellow stem borer (Scirpophaga incertulas) coexists with oligophagous striped stem borer  and polyphagous pink stem borer (Sesamia inferens). Monophagous BPH shares space with oligophagous WBPH and polyphagous small brown planthopper- . Many studies have been conducted to understand interspecific interactions that results in mutual survival of the sympatric species  this trend was reversed. This difference is attributed to switching off of primary metabolism related activities in the plant to switch on defense related secondary metabolism and energy partitioning between the two , derived from the cross between an elite cultivar Swarnadhan and the land race Velluthachera, is resistant to gall midge, BPH and WBPH like the parent land race  by OsmiR396 resulting in reduced expression of a key gene, OsF3H, in the flavonoid pathway led to BPH susceptibility in rice ZH11  in modulation of BPH resistance in rice has become the focus of recent studies , codes for a B3 domain protein  of expression of 27 shortlisted genes under four groups  three genes were identified that were upregulated against both the hoppers Table  includinBPH15 gene  and they speculated that this gene may be involved in functions other than volatile emission. MAP 3kinase gene family, of which STE_MEKK_ste11_MAP3K.7 - STE kinase is a member, is reported to be involved in rice resistance to BPH with down-regulation of one of the members being reported by Wang et al. (ASR) gene is a member of the transcription factor family which altered expression of OsASR2, another member of the family, and influenced rice resistance to bacterial blight and sheath blight  with diverse regulatory role  genes on chromosome 1 and 10 were dramatically induced in resistant RILs only against WBPH and not against BPH. IRLs of rice are not true isoflavone reductases like those of legumes and do not produce natural isoflavonoid products by acting on the substrate 2-hydroxy isoflavonoids  and two members of MYB transcription factor family against one of the planthoppers. This, to the best of our knowledge, is the first study to demonstrate the selective expression of rice host genes upon attack from two major insect pests of rice using genetically similar host material. Such studies are not only important to dissect the plant responses to different insect pests but information derived from such studies are urgently required to consciously combine relevant resistance strategies against both the planthoppers for their effective management.Adults of both BPH and WBPH were collected from farmers’ field in Nalgonda district of Telangana state, India, during 2014–2015 and separate colonies were established on TN1 rice in greenhouses at ABF, Hyderabad. Care was taken to prevent population admixture. Nymphs or adults, arising from these populations reared in the greenhouse, of specified age/stage were used for the experiments.14; 180 lines) . In this method the parents and test lines were infested with 2nd instar BPH/WBPH nymphs, on an average of 8–10 nymphs per seedling 10–12 days after sowing  or MO1 (WBPH). The plants were raised in 500 ml plastic pots with puddled soil from rice field which were randomized (RCBD) and then infested with ten 1st or 2nd instar nymphs per plant covered with a mylar film tube cage. Survival of the insects on the plants was observed daily and number of surviving nymphs was recorded until all surviving nymphs metamorphosed into adults. Three replications were maintained. Nymphal survival was expressed as percentage and means. Entries with ≤60% survival against BPH and WBPH were considered as resistant while those entries with > 60% survival were described as susceptible. For statistical comparison of means, values were transformed into arc-sine values .Antixenosis or non-preference (of nymphs for settling on seedlings) type of resistance mechanism was assessed while conducting the standard seed box screening test , RS (resistant to BPH only), SR (resistant to WBPH only) and SS (susceptible to both BPH and WBPH) were identified Table . From thIn order to identify the pathway genes responsible for conferring resistance to BPH and WBPH in RP2068, an NGS protocol was used. RNA sequencing (RNA-seq), was conducted with RIL TR3RR (resistant to both BPH & WBPH). TR3RR seedlings were raised in nine 3 L plastic bucket pots . Fifteen days after sowing, the designated pots were infested with 1st-2nd instar nymphs (5 nymphs for BPH and 10 nymphs for WBPH/plant). Lower leaf sheath samples  were collected for 3 biological replications at two different time points: 6 and 12 h after infestation (hai). Total RNA was isolated from these nine samples, including uninfested control plants, using RNeasy plant mini kit  as per the manufacturer’s guidelines. The RNA samples collected for each of the two time points (6 hai and 12 hai) for each replication were pooled prior to sequencing. RNA sequencing was carried out by M/s Genotypic Technology Pvt. Ltd., Bengaluru, India.The concentration and purity of the RNA was evaluated using the Nanodrop Spectrophotometer (Thermo Scientific 2000). The integrity of the extracted RNA was analyzed on the Agilent Bioanalyzer 2200  using the manufacturer’s protocols.For validation studies using qRT-PCR, similar experiment setup was repeated and in addition to TR3RR, four additional RILs  were included. Seedlings were raised in 3 L plastic bucket pots and 15 days after sowing they were infested with 1st-2nd instar nymphs of BPH or WBPH. Leaf sheath samples were collected from 3 replications of control and infested plants at two different time points: 6 and 12 hai. RNA isolation and cDNA synthesis were carried out following standard protocols . Twenty-seven genes were selected and validated; first 20 genes using semi-quantitative RT-PCR and 14 genes (including seven of the earlier set) were selected for real time quantitative RT-PCR  at M/s Genotypic Technology Pvt. Ltd., Bengaluru, India.One μg of total RNA was taken for mRNA isolation, fragmentation and priming. Fragmented and primed mRNA was further subjected to first strand cDNA synthesis in the presence of Actinomycin D  followed by second strand synthesis. The double stranded cDNA was purified using HighPrep magnetic beads . Purified double-stranded cDNA was end-repaired, adenylated and ligated to Illumina multiplex barcode adapters as per manufacturer’s protocol.Illumina Universal Adapters were used in the study. Adapter-ligated cDNA was purified using HighPrep magnetic beads and was subjected to 14 cycles of Indexing-PCR  and 65 °C for 5mins) to enrich the adapter-ligated fragments. The final PCR product (sequencing library) was purified with HighPrep magnetic beads, followed by library quality control check. Illumina-compatible sequencing libraries were quantified by Qubit fluorometer  and its fragment size distribution were analyzed on Agilent 2200 Tape station. The libraries were sequenced by using Illumina HiSeq 4000 sequencer  for 2 X 150 paired-end chemistry following manufacturer’s procedure.Analysis pipeline and the different software used for analyzing the raw sequencing data obtained from the HiSeq sequencer were as follows:The raw data generated was quality-checked using FastQC. Reads were preprocessed to remove the adapter sequences and removal of the low-quality bases (<q30). Pre-processing of the data was done with Cutadapt Martin . HISAT-2Cufflinks4 was used to estimate and calculate transcript abundance  files were viewed and inspected in standard genome viewer IGV browser . These GO were mapped to the differentially expressed transcripts/genes (DEG). Next, pathways for each gene were obtained from multiple database such as KEGG pathways and biomart and the compiled pathways for each gene were mapped to the DEGs.For each transcript, gene ontologies were downloaded from ensembl biomart database  to predict plant small RNA targets using default parameters. The miRNA precursors/miRNAs sequence of rice in the miRBase 21.0 (http://www.mirbase.org/search.shtml) database was used to identify the mature sequences and the families of miRNAs to which it belongs. The miRNA sequences were used as input in RNA fold database (http://rna.tbi.univie.ac.at/) to predict their secondary structure.The raw sequence data obtained for both control and infested samples (BPH and WBPH) were used as input for psRNATarget software (http://www.mirbase.org/cgi-bin/mirna_entry.pl) and the sequence and BLAST analyses (Ref) was performed against indica rice genome (http://plants.ensembl.org/Oryza_indica/Tools/Blast/). Genes listed as overlapping/homologous with the reference sequence were considered as the target genes. Net, expression profiles of these genes in our mRNA library were obtained and MSU japonica locus Ids and the putative nature and function of the genes were identified (http://rice.hzau.edu.cn/cgi-bin/rice2/id_mapping_rs2).Mature rice miRNA sequences for these families was downloaded from miRbase  (Supplementay Table https://www.youtube.com/watch?v=N7mkI8_xxc4&feature=emb_logo). Negative values were expressed as decimal fraction for analysis. Results are expressed as a graph constructed based on the log (2) values of the fold change using MS Excel  with two types of samples: (i) the same RNA samples that were used for RNA-seq analysis and (ii) newly isolated RNA samples from independent infestation. About 3 μg of RNA was used for first-strand cDNA synthesis using the iScript cDNA synthesis kit  following the manufacturer’s guidelines. Gene-specific primers for the RT-PCR were designed using Primer3 Software (–∆∆Ct method (Livak and Schmittgen https://www.youtube.com/watch?v=N7mkI8_xxc4&feature=emb_logo). Negative values were expressed as decimal fraction for analysis.  according to the manufacturer’s instructions. Rice ubiquitin gene, OsUbq (GenBank accession no. AK059694), was used as the endogenous control. Real Time PCR reaction volume of 10 μl contained 5 μl SYBR Green PCR Master Mix , 500 nM each of forward and reverse primers and 30 ng of the cDNA samples. To calculate mean relative expression levels, cDNAs from three independent biological samples in two technical replications each were used. PCR was initiated with denaturation at 95 °C for 5 min followed by 40 cycles of denaturation at 95 °C for 10s and annealing and extension at 60 °C for 30s. After 40 cycles, a melt curve analysis was carried out to determine the specificity of the reaction. After normalization, quantity of each mRNA was calculated from the threshold points (CT) located in the log-linear range. The data from different PCR runs or cDNA samples were compared by using the mean of CT values of the three biological replicates that was normalized to the mean of CT values of the endogenous gene. The relative standard curve method was used for the quantification of mRNA levels and displayed as Relative Expression Values (REV). Expression ratios were calculated using the 2www.ncbi.nlm.nih.gov/sra). The Bioproject accession number for Molecular basis of differential resistance in rice line RP2068-18-3-5 against BPH and WBPH is PRJNA577384, and SRA experimental accession numbers are SRR10394603, SRR10394602, SRR10394598, SRR10394601, SRR10394600, SRR10394597, SRR10394599, SRR10394596 and SRR10394595.The sequence data (raw data) generated in this study has been deposited at NCBI Sequence Read Archive (SRA) database Additional file 5: Supplementary Table S5. Mean fold values and Tukey’s HSD for separation of means for 14 genes validated with qRT-PCR (quantitative)Additional file 6: Supplementary Table S6. List of significant miRNA and their reported target genes in RILs induced by infestation either by BPH or WBPHAdditional file 7: Supplementary Fig S1. Scatter plot representing correlation coefficient between samplesAdditional file 8: Supplementary Fig S2. Pathway analysis for key genes listed in Table 2


The purpose of this study was to report the technique for intraprocedural guidance of endovascular Venous Sinus Stenting procedures using 3-Dimensional (3D) Magnetic Resonance Venography (MRV) as an overlay on live biplanar fluoroscopy.Venous sinus stenting procedures performed between April and December, 2017 with 3D MRV fusion for live guidance were reviewed in this study. A thin-slice, contrast-enhanced MR Venogram was used to create 2 3D models – vessels and skull – for procedural guidance via augmented fluoroscopy . The skull model was used in the registration of the 3D overlay on both the frontal and lateral planes, which required 1–2 min of procedural time. The vessel model was used to mark landmarks such as the cortical vein ostia and stenosis on the 3D overlay fused with biplanar fluoroscopy.The retrospective imaging review was conducted by 3 neurointerventionalists and relied on a consensus confidence ranking on a 3-point Likert scale from 1- low confidence to 3- high confidence. The neurointerventionalists first reviewed the conventional 2-dimensional pre-stent deployment fluoroscopy images and then reviewed the corresponding images with the 3D MRV overlay. They ranked their confidence in their understanding of cortical venous anatomy for each group. Statistical analysis was performed using a Paired T Test at a 99% confidence interval.p = .001).Ten cases were included in the retrospective image review. Operator confidence regarding the location of cortical veins was significantly increased using 3D MRV fusion during venous sinus stenting procedures (1.9 vs 2.9, 3-Dimensional MRV fusion is feasible and helpful in understanding the venous sinus anatomy and location of important cortical veins during venous sinus stenting procedures. Idiopathic Intracranial Hypertension (IIH) is a condition characterized by increased intracranial pressure manifesting with headaches, pulse-synchronous tinnitus, diplopia, and visual field loss from papilledema wires, (micro)catheters, and stents in venous sinuses in a retrograde fashion, there is a risk of inadvertent placement of a wire in a cortical vein resulting in catastrophic hemorrhage  to report the technique for intraprocedural 3D MRV guidance, and 2) to report our initial experience and clinical potential of this technique.Patients who underwent venous sinus stenting procedures with a 3D MRV-fusion overlay between April and December of 2017 were included in this study, which was approved by our Institutional Review Board with a waiver of informed written consent. Cases with poor quality pre-procedural MRVs  were excluded. 10 consecutive cases fit these criteria during this time period and were included in the study.As part of the standard pre-procedural assessment, all patients from the study group had a thin-slice, contrast-enhanced MR Venogram. All institutional MRV exams were performed on 3 Tesla units  with coronal 2D time of flight, sagittal 3D phase contrast (INHANCE/NATIVE), and 3D thin section T1 pre and postcontrast sequences (SPGR or MPRAGE). Contrast enhanced sequences were acquired following weight-based intravenous administration of gadobutrol (Gadavist). The fusion images used for MRV-guidance relied on two 3D models: a vessel model and a skull model. The vessel model was created using vessel segmentation software  for 3D extraction of the venous sinuses on a preprocedural MR venography series. The segmentation required the selection of two end points, one in the superior sagittal sinus and one in the sigmoid sinus, on the multi-planar reformatted MRV images  and Minitab® 17 statistical software (2010) .For comparing conventional images and MRV-guided images from the same procedures, 10 venous sinus stenosis cases with saved images of the 3D overlay on the cortical veins were reviewed to assess their clinical utility. The consensus confidence levels reached by the reviewers are shown in Table p = .001). A comparison of the imaging from both methods is shown in Fig. Operator confidence was significantly increased regarding the location of major cortical veins using 3D MRV-fusion during the venous sinus stenting procedures . Although osseous landmarks could be registered with greater accuracy on the CT modality relative to MRI, there are noteworthy limitations with the use of thin section CT venography compared to MR venography. The relatively larger contrast volumes needed in performing CT venography poses a risk for patients with reduced Glomerular Filtration Rates (GFRs), noting that patients with GFR less than 60 are at risk of further contrast-induced nephropathies compared to the average population. This is in contradistinction to the low-volumes of gadolinium-based agents used in MR venographic examinations, noting the less that 0.07% chance in development of nephrogenic systemic fibrosis in patients with markedly reduced GFRs less than 30  requires further evaluation in a prospective fashion using a control group.The main limitation of this case study was its small sample population. There could be also some recall bias for operators performing image assessment. Furthermore, it is difficult to achieve objective ratings regarding the clinical utility of the software given the inherent variability present in providing an overall grade for confidence. Lastly, this study does not provide information about benefits in term of procedural time or radiation doses given the lack of a control arm.In summary, the addition of MRV bi-plane 3D fusion during venous sinus stenting procedures for management of IIH is feasible and relatively easy. By providing both consistent visualization and accurate overlay of large cortical veins despite table position alterations, 3D MRV bi-plane guidance may help optimize venous sinus stenting procedural success.


Monocarboxylate transporter 1 (MCT1) is a regulator of cell metabolism and a therapeutic target for cancer treatment. Understanding the changes in tumour function accompanying MCT1 inhibition will better characterise the anti-tumour effects of MCT1 inhibitors, potentially enabling the identification of pharmacodynamic biomarkers for the clinical development of these agents.We assessed the impact of the MCT1 inhibitor AZD3965 on tumour metabolism and immune cell infiltration as key determinants of tumour biological function in the MCT1-dependent Raji B cell lymphoma model.Treatment of Raji xenograft-bearing severe combined immunodeficiency mice with AZD3965 led to inhibition of tumour growth paralleled with a decrease in tumour choline, as detected by non-invasive in vivo proton nuclear magnetic resonance spectroscopy. This effect was attributed to inhibition of phosphocholine de novo synthesis following decreased choline kinase α protein and messenger RNA expression that correlated with the AZD3965-induced build-up in intracellular lactate. These changes were concomitant with increased tumour immune cell infiltration involving dendritic and natural killer cells.Our data provide new insights into the metabolic and cellular changes that occur in the tumour microenvironment following MCT1 blockade, which may contribute to the anti-tumour activity of AZD3965 and could have potential as pharmacodynamic biomarkers of MCT1 inhibition. MCTs also mediate the uptake of lactate into cells, which can be used as a fuel source and oxidised to produce energy in a variety of cell types, including cancer, stromal and immune cells.5One example of the many effectors of metabolic re-wiring in cancer is the monocarboxylate transporter (MCT) family , which are transmembrane proteins that mediate the bi-directional transport of lactate  in and out of cells.8 To date, several inhibitors of MCT1 have been reported that exert promising activity in experimental cancer models, including SR138009 and AZD3965,10 and progress is being made towards the development of MCT4-targeted agents (AstraZeneca).The key role that MCTs play in regulating lactate homeostasis in tumours together with the upregulation of MCT1 and MCT4 expression observed in many cancers has led to much interest in targeting these proteins for cancer treatment.www.clinicaltrials.gov) with phase I expansion cohort enrichment for Burkitt lymphoma and diffuse large B cell lymphoma cases, given the promising pre-clinical results obtained with the drug in this setting.13 Gaining an insight into the consequences of MCT1 inhibition on metabolism and tumour function as a whole is therefore necessary to (a) improve our understanding of the processes relevant to the drug’s mechanism of action and (b) enable the discovery of pharmacodynamic (PD) biomarkers of target modulation.The MCT1 inhibitor AZD3965 is in early phase clinical trials (i) build-up. Indeed, in MCT4− cells (MCT4 expression being predictive of resistance to MCT1 inhibition13) we observed effects on mitochondrial activity, shown by increased oxidative metabolism and improved bioenergetics as well as decreased phosphocholine (PCho) levels, implying effects on phospholipid metabolism and alterations in several other metabolic intermediates.13In this regard, we have previously shown that blockade of MCT1 with AZD3965 has profound effects on human cancer cell metabolism that extend beyond intracellular lactate  spectroscopy (MRS) and evaluate the accompanying changes in the tumour microenvironment.Thus, in this study we sought to characterise the effect of MCT1 inhibition on tumour choline metabolism in vivo using non-invasive proton nuclear magnetic resonance , also representative of the phase I trial expansion cohort, while HT29 and Hut78 cell lines were chosen to provide a range of baseline expression of MCT4 . Cell culture reagents were purchased from Gibco and AZD3965 was kindly provided by AstraZeneca, UK. Cells were exposed to either dimethyl sulfoxide (DMSO) (0.01%) or AZD3965 in 0.01% DMSO for the indicated duration and at the indicated concentrations in fresh media.The human cell lines Raji (B cell lymphoma) and Hut78 (T cell lymphoma) were purchased from ATCC. HT29 human colon carcinoma cells were originally obtained from ATCC and authenticated in our laboratory by STR profiling on 13 March 2017. The cell lines were grown in either RPMI  (Raji and Hut78) or DMEM (Dulbecco’s modified Eagle’s medium) (HT29) as previously described.17 Details of the primary and secondary antibodies as well as primers used are provided in Supplementary Materials and methods.Western blotting and quantitative real-time PCR (qRT-PCR) for protein and gene expression analyses, respectively, were performed using standard conditions as previously described.Prkdcscid) mice  as previously described13 maintained in pathogen-free conditions within the Biological Services Unit laboratories, ICR. Tumours were measured as previously described16 and once they reached 300–350 mm3 in volume (after ~4 weeks), mice were randomised into two groups; one group was dosed with vehicle ) and the other with 50 mg/kg AZD3965 dissolved in vehicle (n = 9) administered orally twice daily over 5 days. This schedule was based on a previously published report10 and shown in our laboratory to cause tumour lactate build-up consistent with MCT1 blockade.13 No adverse events were associated with the procedures carried out in the experiment.Raji xenograft tumours were established by subcutaneous injection of Raji cells into the flank of female severe combined immunodeficiency (SCID)  Act 1986 and UK National Cancer Research Institute (NCRI) Guidelines for the Welfare and Use of Animals in Cancer Research.1H surface coil. After shimming (linewidth on the unsuppressed water peak of ~15 Hz on average), localised 1H point resolved spectroscopy measurements of the tumours were performed prior to (day 1) and 2 h following the last dose (on day 5), as depicted in Fig. 19 After the last scan, the experiment was terminated with a schedule 1 method  and then tumours were excised, immediately snap frozen in liquid nitrogen and finally stored at −80 °C until further processing.Tumour-bearing mice were anaesthetised with a mixture of air:oxygen:2% isoflurane and placed at the isocentre of a horizontal Bruker 7 Tesla microimaging system  with the tumours positioned in the centre of a 15 cm 20 and freeze-dried samples reconstituted in D2O containing 3-(trimethylsilyl) propionic-2,2,3,3-d4 acid (TSP) as an internal 1H NMR quantitation and chemical shift reference. Data were acquired on a Bruker 500 MHz spectrometer at 25 °C and processed as previously described.16 Metabolite content was measured by peak integration relative to TSP and normalised to cell number. For tumour metabolite analysis, peak areas of the metabolites of interest were normalised to tissue wet weight and expressed as arbitrary units/mg.Cells and xenograft tumour tissue were extracted as previously described21 Briefly, tumour fragments were digested in a mixture of collagenase and DNAse and disaggregated cell suspensions were treated with a red blood cell lysis buffer followed by cell number counting using trypan blue exclusion. To determine the frequency and phenotype of immune cells within tumours, multi-colour flow cytometry analysis of cell surface marker expression was applied using ~106 live single cells and a standard protocol as previously described.21 Details of reagents used in tumour tissue digestion as well as flow cytometry, antibodies and gating strategy are provided in Supplementary Materials and methods and Supplementary Fig. Fresh tumour tissue was collected and processed as previously described.13C NMR, a method that allows NMR signal enhancement by up to 10,000-fold, thus enabling the rapid and dynamic monitoring of metabolic reactions occurring within seconds.22 To monitor HP 13C-pyruvate-lactate exchange, PBMCs were transferred to a 5 mm NMR tube, incubated at 37 °C in FBS-free medium and 13C NMR spectra acquired with 2 s intervals and a 10° pulse for 4 min immediately after the addition of 10 mM HP [1-13C]pyruvic acid and 10 mM unlabelled lactate at pH 7 . Dynamic HP 13C time-series spectra were processed and the ratio of the areas under the curve for the lactate and pyruvate peaks (lactateAUC/pyruvateAUC) determined as previously described.13Fresh blood samples (30 ml each) were donated by consented healthy volunteers (Ethics protocol number EudraCT 2010-024463-41), in accordance with the Declaration of Helsinki. Samples were collected in EDTA tubes and used to isolate peripheral blood mononuclear cells (PBMCs) using Ficoll-Hypaque density gradient centrifugation. Isolated PBMCs (2–15 million cells/sample) were incubated ex vivo in 0.1% DMSO or 25 nM AZD3965 for 1 h  RPMI medium) and then harvested for hyperpolarised (HP) t test (for in vitro comparisons) and paired t test (for in vivo tumour changes prior to and following treatment) were used with P <0.05 considered statistically significant. Data represent the mean ± SE.Single comparison two-tailed unpaired 1H MRS of Raji tumours treated with either vehicle or AZD3965 as depicted in Fig. To evaluate the impact of AZD3965 on tumour choline metabolism in vivo, we used non-invasive 1H MRS being the most commonly used method in the clinic, enabling the detection of metabolic species such as choline-related metabolites, taurine, creatine and lipids.23MRS is a clinically translatable technique for evaluating tumour metabolite profiles, with 1H MR spectra are shown in Fig. P = 0.037), but remained unchanged in the vehicle-treated group . Interestingly, the taurine/water ratio was also significantly reduced in the AZD3965-treated group  as shown in Fig. Transverse anatomical images of a representative Raji tumour pre- and post-AZD3965 treatment together with corresponding in vivo P = 0.01), the AZD3965-treated tumours showed cytostasis (P = 0.25), consistent with drug-induced tumour growth inhibition , to activate lactate uptake and force an increase in lactatei (since lactate transport follows a concentration gradient3). The data showed that increasing lactatei content under standard or low glucose conditions was sufficient to trigger a decrease in cellular PCho .PCho is produced through de novo synthesis from its precursor choline via choline kinase α (CHKA) as part of the Kennedy pathway for the synthesis of phosphatidylcholine, the most abundant cell membrane phospholipid. PCho can also be formed through release from phospholipid breakdown via phospholipases.lls Fig. . We also 26 Fig. , althougCHKA messenger RNA (mRNA) expression showed significant decreases following exposure to AZD3965  to AZD3965 ex vivo and monitored HP 13C-lactate formation following the addition of HP 13C-pyruvate. Our results show that a 1 h treatment with 25 nM AZD3965 (a concentration previously shown to cause MCT1 inhibition in Raji cells) in human PBMCs  led to ake Fig. . These dTaken together, these results indicate that AZD3965 modulates the tumour immune cell infiltrate in vivo, specifically increasing NK and DC abundance and maturation within the tumour, but not the periphery, despite exerting an inhibitory effect on MCT1 activity in immune cells.Understanding the impact of MCT1 inhibition on overall tumour function together with identifying PD biomarkers for MCT1 modulation are key requirements for advancing the development of MCT1 inhibitors, which are in early phase clinical testing.In this study, we show that at a treatment dose and schedule that led to tumour stasis, AZD3965 caused marked effects on tumour metabolic and immune phenotypes in a B cell lymphoma xenograft model representative of the clinical trial expansion patient cohort.1H MRS of Cho/water, as a result of decreased tumour PCho levels. This effect occurred prior to any measured changes in tumour volume, indicating that 1H MRS of Cho/water has the potential as an early, non-invasive PD biomarker of MCT1 blockade.MCT1 inhibition with AZD3965 reduced Raji tumour choline content, as shown by i build-up (being absent in the cell line with negligible lactate accumulation). Forced increase of cellular lactate was also sufficient to trigger a decrease in PCho, independently of AZD3965 treatment, indicating that the fall in PCho is a downstream cellular response to lactatei build-up.The fall in PCho was recapitulated in vitro, showing a strong negative correlation with the extent of lactate24 Our results indicate that the AZD3965-induced decrease in cellular PCho is likely to be due to a decline in its de novo synthesis from choline via reduced mRNA and protein expression of CHKA, which was also confirmed in Raji tumour tissue. Interestingly, AZD3965 treatment showed a trend towards inhibition of other rate-limiting lipogenic enzymes (ACC and P-ACL) in MCT4− Raji cells, potentially suggesting an overall decline in lipid biosynthesis following MCT1 inhibition in this model.PCho can be synthesised from the phosphorylation of its precursor choline via CHKA, or it can be released following phospholipid breakdown via phospholipases.26 However, the mechanism underlying the link between lactate accumulation and decline in lipid biosynthetic enzyme expression, particularly CHKA, is unclear. Lactate has been shown to mediate gene expression regulation, including the activation of wound healing pathway genes and downregulation of lipid metabolism-related genes,29 with changes in cellular redox state invoked as one underlying mechanism.30 Whether similar or alternative mechanisms are at play here requires further investigation, particularly as previous reports have shown changes in redox state following MCT1 inhibitor treatment.9The inhibition of lipid biosynthesis observed with AZD3965 in the Raji model may in fact contribute to the anti-proliferative effects of the drug observed in our study given the role of lipogenesis in promoting cancer cell growth and proliferation.32 If validated in other MCT1-dependent models, our data support exploring the use of the clinically applicable 1H NMR spectroscopy of choline/water as a biomarker for monitoring the action of AZD3965 and potentially other MCT1 inhibitors in patient tumours.Our in vivo investigation identifies new pathways that are modulated in the tumour following treatment with the MCT1 AZD3965 and shows the potential of non-invasive metabolic studies to provide biomarkers for reporting on accompanying downstream metabolic consequences of MCT1 inhibition. Non-invasive metabolic studies are a powerful tool for longitudinal assessment of metabolism at the tumour site, with methods such MRS (single voxel and spectroscopic imaging methods) enabling the steady state as well as dynamic assessment of several key metabolic pathways in pre-clinical models and patients.33 Thus, increased tumour abundance of DC and NK cells could contribute to the growth inhibitory effects of AZD3965. A limitation of our findings is the fact that they were obtained in an immunodeficient mouse model (SCID mice lacking T and B cells), primarily due to current unavailability of suitable mouse models of MCT4−lymphoma. Nevertheless, our findings suggest a role for AZD3965 in stimulating anti-tumour immune responses involving both the innate and adaptive arms, which are likely to be involved in tumour growth inhibition following MCT1 blockade and which may be even more profound if reproduced in an immunocompetent setting.One of the aims of our study was to assess the effects of AZD3965-induced alteration in metabolism on tumour biological phenotype. Our results show that MCT1 inhibition with AZD3965 increases tumour immune cell infiltration, involving NK cells and DCs, in the responsive MCT4− Raji human lymphoma xenograft model. NK cells exert direct cytolytic action on tumour cells and are a key component of the innate anti-tumour defence system while DCs enable antigen presentation to T cells and form a key part of adaptive anti-tumour immunity.36 Interestingly, our human PBMC data also show that AZD3965 inhibits MCT1 in immune cells themselves potentially modulating their metabolism and also their activity.37 In agreement with this observation, previous work has shown that MCT1 inhibition has an immunosuppressive effect on T cells, preventing their expansion upon activation.38Tumour microenvironment conditioning through reduced lactate export is likely to be the main mechanism for the increased tumour immune cell infiltration given the known inhibitory effects of lactate on anti-tumour immune cell activity and expansion, and the lack of effect on spleen immune cell phenotype.Our data provide important new insights into the processes taking place in the tumour microenvironment, suggesting that the AZD3965-induced metabolic changes  are able to promote local anti-tumour immune cell expansion. Further studies in an immunocompetent setting are required to examine the effects of MCT1 inhibition on the function and phenotype of different immune cell subtypes involved in anti-tumour immunity including T cells.39 Our data provide preliminary evidence for investigating the impact of AZD3965 on overall immune profiles and the potential for combination therapy with immune-modulating agents.It is noteworthy that AZD3965 treatment in our model also led to the upregulation of the immune checkpoint molecule PD-L1 on NK cells, previously shown to have a regulatory function in NK cells with regards to DC maturation and activation.In summary, findings from our study provide valuable new insights into the pathways and mechanisms modulated in the tumour following MCT1 blockade, which could contribute to the anti-tumour activity of AZD3965. This knowledge may be exploited for furthering the development of AZD3965 and potentially other MCT1-targeted agents through the evaluation of non-invasive, clinically applicable response biomarkers, and exploring rational combination treatment to increase drug efficacy.Supplementary data


Alzheimer’s disease (AD) is one of the most prevalent neurodegenerative diseases, characterized by impaired cognitive function due to progressive loss of neurons in the brain. Under the microscope, neuronal accumulation of abnormal tau proteins and amyloid plaques are two pathological hallmarks in affected brain regions. Although the detailed mechanism of the pathogenesis of AD is still elusive, a large body of evidence suggests that damaged mitochondria likely play fundamental roles in the pathogenesis of AD. It is believed that a healthy pool of mitochondria not only supports neuronal activity by providing enough energy supply and other related mitochondrial functions to neurons, but also guards neurons by minimizing mitochondrial related oxidative damage. In this regard, exploration of the multitude of mitochondrial mechanisms altered in the pathogenesis of AD constitutes novel promising therapeutic targets for the disease. In this review, we will summarize recent progress that underscores the essential role of mitochondria dysfunction in the pathogenesis of AD and discuss mechanisms underlying mitochondrial dysfunction with a focus on the loss of mitochondrial structural and functional integrity in AD including mitochondrial biogenesis and dynamics, axonal transport, ER-mitochondria interaction, mitophagy and mitochondrial proteostasis. Alzheimer’s disease (AD) is one of the most prevalent neurodegenerative diseases in the world –3. The mMitochondria are conserved organelles that carry out multiple essential functions in different cellular processes : Many neTo date, a large body of research has shown extensive mitochondria abnormalities in the brain of AD patients . ConsistBrain constitutes on average 2% of the total body weight, but utilizes 25% of total body glucose and 20% of body oxygen consumption in resting awake state. As one of the high-energy consuming organs, brain is vulnerable to impaired energy metabolism such that even mild changes in energy metabolism in human brain closely associates with the disturbance in nervous function. In fact, impaired energy metabolism is one of the earliest and most consistent features in AD.Glucose is the predominant substrate for the human adult brain under physiological conditions, and its utilization is widely used as one primary measure to assess energy metabolism in the brain. A large body of evidence demonstrated significantly reduced glucose utilization as an early and consistent feature in AD, which actually occurs decades before the onset of disease –13. UsinMultimodal imaging studies, using both FDG PET and amyloid PET biomarkers, have investigated the relationship between amyloid plaque deposition and glucose metabolism. In autosomal-dominant AD mutations carriers, longitudinal Aβ depositions increase in nearly every cortical region 15–25 years before the estimated age of onset, followed by reduced glucose metabolism in selective cortical region approximately 5–10 years later , suggesting that glucose hypometabolism could be a secondary event after Aβ depositions in AD pathogenesis in these cases , 20, 21.On the other hand, energy hypometabolism may play a primary role in a subset of sporadic AD patients. In a population study, while the majority (60%) incident amyloid-positive subjects followed the amyloid-first biomarker profile pathway, 27% had an abnormal FDG-PET at baseline, suggesting the existence of “hypometabolism-first” biomarker profile pathway to preclinical AD . FDG PETGlucose metabolism is a multi-step process involving glucose transportation and intracellular glucose metabolism. Abnormalities involving almost all of these steps from glucose transportation abnormalities including insulin resistance, abnormal glucose transporter  and/or bNevertheless, glucose hypometabolism in AD brain was generally interpreted as impaired energy metabolism through oxidative phosphorylation, which thus strongly implicates the involvement of mitochondrial dysfunction early in the course of AD. Along this line, glucose hypometabolism in frontal, temporal, and parietal cortices of patients with AD were closely correlated with the reduced levels of blood thiamine diphosphate (TDP), a critical coenzyme of pyruvate dehydrogenase (PDHC) and α-ketoglutarate dehydrogenase (KGDHC) in the Krebs cycle and transketolase in the pentose phosphate pathway . SimilarConsistent with impaired energy metabolism in AD, gene expression studies repeatedly identified defects in mitochondrial related metabolic pathways in AD, which provided direct evidence for impaired bioenergetic machinery in mitochondria of AD. For example, a genome-wide transcriptome study in laser-capture micro-dissected neurons found significantly greater proportion of underexpressed nuclear genes encoding mitochondrial ETC subunits in the posterior cingulate cortex than those in the primary visual cortex, a region that is relatively spared metabolically in AD vs. control . A microProteomic and protein expression studies also confirmed underexpressed proteins in OXPHOS pathway as one of the most affected processes in the cortex of AD patients . QuantitHowever, contradictory results were reported where several groups failed to find difference or even increased expression of OXPHOS genes in AD compared to control, some of which may be attributed to different brain regions and/or the widespread heterogeneity of sporadic AD brain samples used. For example, decreased expression of complexes I, II, IV and V was found in the entorhinal cortex but not in the frontal cortex in AD Braak stages V-VI compared with stages I-II . Cell-tyOverall, these gene and protein expression studies along with biochemical studies clearly demonstrated extensive mitochondria bioenergetics defects in AD brains, which makes it a valuable therapeutic target. It remains to pinpoint specific alterations in oxidative metabolism and to generalize the role of mitochondrial dysfunction in regional vulnerability and pathogenesis of AD, which requires consistent measurement of these parameters across different brain regions. Furthermore, it has been demonstrated that reductions of activity of complex I, III and IV that reach certain thresholds such as greater than 70% in some studies were necessary to produce a significant decrease in ATP production , 55. It Reactive oxygen species (ROS) are unavoidable byproducts during electron transport of aerobic respiration in the mitochondria due to electron leaks at complex I and complex III and it is estimated that mitochondria contribute approximately 90% of the cellular ROS . While RA large body of evidence demonstrated increased oxidative damage to almost all types of macromolecules in the brain of AD patients including proteins, sugar, lipid and nucleic acids . For exaRedox proteomics studies to identify oxidatively modified proteins contributed a great deal to the understanding of the diseased proteome in the various stages of AD and shed light on potential molecular pathways involved . These sMitochondria maintain their own DNA called mtDNA, which is a multicopy (1–10 copies per mitochondrion), extrachromosomal genome that codes for 13 mitochondrial core proteins of the ETC complexes and 2 rRNA and 22 tRNAs necessary for mitochondrial protein synthesis , 66. WhiMany patients with primary mtDNA mutations demonstrated pronounced cognitive deficits quite similar to those commonly seen in AD , which sInvolvement of somatic mtDNA mutations were also extensively studied in AD with a focus on the common 5-kb deletion (mtDNA Δ4977) occurring between positions 8470–8482 and 13,447–13,459, which presumably affects the expression of ETC complex I, III and V in AD. Earlier quantitative PCR studies found age-related accumulation of this deletion in frontal cortex  and a stHowever, conflicting results were reported since some groups found no changes in the aggregate burden of brain mtDNA point mutations between AD and control –85, whicIt was believed that increased mtDNA mutations are due to increased oxidative damage found in AD brain . Indeed,Overall, these studies suggest a likely critical role of mtDNA variabilities, mutations and modification in the pathogenesis of AD. Indeed, it has been proposed by the mitochondrial cascade hypothesis that inherited mtDNA variants determine one’s vulnerability and the accumulation of brain somatic mtDNA modifications and mutations reflecting the influence of the environment along aging determines the manifestation of the phenotype . HoweverThe existence of hypometabolism-first biomarker profile pathway to preclinical AD along with the extensive evidence that mitochondrial abnormalities could lead to AD-related deficits in model organisms suggest that mitochondrial dysfunction could play a primary role at least in a subset of sporadic AD patients Fig. . As to tMitochondria are highly dynamic organelles undergoing continuous fusion and fission in the cytoplasm, a process that is essential for maintaining a healthy pool of mitochondria with proper distribution . The molEarly studies demonstrated ultrastructural damage to the susceptible pyramidal neurons in the biopsied brain tissues of AD . More deIndeed, biochemical evaluation of AD brains demonstrated significantly reduced protein expression levels of all the large dynamin-related GTPases involved in fission and fusion including DLP1, OPA1, Mfn1, and Mfn2 along with significantly increased levels of fission factor, Fis1, in AD brain , 105. GiImpaired mitochondrial fusion and fission balance having an essential role in mitochondrial dysfunction and pathogenesis of AD was corroborated by studies in both in vitro and in vivo experimental models of AD , 109. OvAβ induced abnormal mitochondrial dynamics is an early event during neurodegeneration in vivo in Drosophila models , 116. A These studies pave the road to pursue abnormal mitochondrial fission as a potential therapeutic target for AD, and so far specific attention focused on the extensively studied mitochondrial dynamic protein DLP1 which adds more evidence of a critical role of mitochondrial dynamics in the course of AD. Chemical inhibition of mitochondrial fission by DLP1 specific inhibitor Mdivi-1 and genetic reduction of DLP1 proteins were explored in AD models in vivo and in vitro. By inhibition of ERK-DLP1 signaling, Gan et al. reported a protective effect of DLP1 inhibitor mdivi-1 on maintenance of normal mitochondrial structure and function in AD cybrid cell . ConsistIn addition to mitochondrial morphological abnormality, mitochondrial distribution was also disturbed in AD brain: mitochondria become less abundant in the neuronal processes in the susceptible pyramidal neurons in AD . This unMutations in presenilin 1 impair kinesin-based axonal transport through GSK3β activation, which phosphorylates kinesin light chain and releases kinesin from the cargo at sites of membrane insertion . PrimaryHow may Aβ affect the axonal transport of mitochondria? The finding that Cyclophilin D deficiency rescues Aβ-induced axonal mitochondrial transport deficit  implicatOverexpression and/or phosphorylation of tau is another negative regulator for mitochondrial movement in neurons. Earlier studies demonstrated that tau controls the balance of axonal transport through locally differential modulation of dynein and kinesin motor proteins , predictDespite the consensual view that Aβ and tau alterations impaired mitochondrial transport, it is unclear whether they specifically impaired mitochondrial transport or also affected other organelles. There is also debate on whether Aβ preferentially impacted anterograde axonal transport of mitochondria or retrograde axonal transport of mitochondria or both , 154. ThThere are more than 1000 proteins in neuronal mitochondria, 13 of which are encoded by mitochondrial genome and are hydrophobic proteins that form the core parts of the oxidative phosphorylation complexes of the inner membrane of mitochondria, while the remainder are encoded by nuclear genome . TherefoAs discussed earlier, multiple studies demonstrated reduced levels of critical components of the electron transport chain in the brain tissues of AD, which not only underlies the well-documented energy hypometabolism in AD, but may also suggest impaired mitochondrial biogenesis or enhanced mitochondrial clearance. However, mitochondrial clearance through mitophagy is actually impaired in AD (discussed in more detail later). Therefore, an impaired mitochondrial biogenesis is implicated. PGC-1α is abundantly expressed in tissues with high energy demand including the brain. Qin et al. first demonstrated the reduced expression of PGC-1α in AD patients and transgenic mouse model of AD . mtDNA cConsidering the essential role of PGC-1α impairment on mitochondrial dysfunction and Aβ production in AD, it was attractive to investigate how restoration of PGC-1α expression or its activity would affect mitochondrial and neuronal functions in models of AD. Katsouri et al. studied the potential therapeutic effect of PGC-1α by generating a lentiviral vector to express human PGC-1α in hippocampus and cortex of APP23 transgenic mice  and founAnother strategy to restore mitochondrial biogenesis was to enhance PGC-1α activity by chemical stimulation . The firAn alternative strategy is to focus on mitochondrial biogenesis effectors downstream of PGC-1α. Oka et al. examined the effects of human mitochondrial transcriptional factor A (hTFAM) on the pathology of a mouse model of AD (3xTg-AD). They found that expression of hTFAM significantly improved cognitive function, reduced oxidative stress and intracellular Aβ in 3xTg-AD mice and increased expression of transthyretin, known to inhibit Aβ aggregation .Given that 99% of mitochondrial proteins are encoded by nuclear genome and must be imported into mitochondria, mitochondrial biogenesis is heavily dependent on proper mitochondrial protein import , 175, whSeveral groups had pursued the potential role of APP or Aβ on mitochondrial import machinery –184. AnaBoth endoplasmic reticulum (ER) and mitochondria are continuous tubular networks of membranes in the cytoplasm. Approximately 5–20% of the mitochondrial surface is closely apposed at 10–30 nm distance to ER membrane and form a specialized structure called ER-mitochondria contact sites which provide a stable platform to synergize the function of these two organelles , 187. AnC. elegans gene encoding a PSEN homolog, sel-12 resulted in elevated endoplasmic reticulum (ER)-mitochondrial Ca2+ signaling and an increase in mitochondrial superoxide production [The potential involvement of MAM dysfunction in AD was first implicated by Eric Schon’s finding of the MAM localization of presenilin 1 and 2 as well as the gamma-secretase activity , 192. Laoduction . Moleculoduction .Consistent with the in vivo findings, multiple groups demonstrated aberrantly increased ER-mitochondria contacts and/or enhanced MAM function in various cell models of AD which enables more detailed mechanistic studies: for example, overexpression of APP mutants or exposure to nanomolar concentrations of Aβ increases ER-mitochondria contact points and mitochondrial calcium concentrations , 195. EnHowever, there is controversy on the effects of presenilins on MAM structure and function since in tissues from AD patients carrying PS1 E280A mutation, ER-mitochondria tethering was impaired, a result further confirmed by in vitro studies . Pizzo’sAs metabolic active organelles where more than 90% of reactive oxygen species (ROS) are produced , mitochoMitochondria are key targets of autophagic degradation in the brain of AD patients , 211 andMechanistically, PS1 promotes PINK1 promoter transactivation, mRNA and protein expression through AICD, the cleavage product of APP by gamma-secretase yet PS1 mutations disrupted lysosomal acidification and proteolysis due to the failure of v-ATPase targeting to lysosomes during autophagy . CorsettC. elegans models overexpressing Aβ1–42 or tau in a PINK1-dependent manner [+-boosting compounds such as nicotinamide riboside (NR) potently induce mitophagy [While detailed mechanisms underlying impaired mitophagy in AD remains to be worked out, enhancing mitophagy by either genetic manipulation or pharmaceutical methods appears beneficial across different AD models. Overexpression of PARK2 reversed mitophagy failure and led to the recovery of mitochondrial membrane potential in sAD fibroblasts . Overexpt manner . Importat manner . In thisitophagy , 224. NRitophagy .At protein level of mitochondrial quality control mechanisms, a process called mitochondria proteostasis monitors mitochondrial protein damages through an interconnected network consisting of chaperones and proteases in each compartment of mitochondria: Mitochondrial chaperones are involved in protein translocation and folding reactions while ATP-dependent proteases are responsible for directly removing damaged or misfolded proteins from mitochondria –229. DefAt the suborganelle level, abundant evidence demonstrated accumulation of damaged mitochondrial contents including mtDNA and proteins in AD , which sCurrent studies focused on two mitochondrial proteases related to the processing and metabolism of APP or Aβ. One is the mitochondrial peptidasome, PreP, located in mitochondria matrix which is involved in the cleavage/maturation of presequence of mitochondrial matrix proteins after its import . Falkeva1–42 peptide but independent of the PDZ domain in vitro [The other mitochondrial protease of more interest in AD field is HtrA2/Omi, a serine protease in the mitochondrial intermembrane space (IMS). A weak association between HtrA2 A141S and AD was found in Swedish case control studies and specific protease activity of HtrA2 was found to be significantly increased in AD patients . Using yin vitro . Denselyin vitro , suggestin vitro . It is pin vitro . Interesin vitro .Overall, despite ample evidence demonstrating accumulation of damage mitochondrial proteins, there were sparse studies on alterations in mitochondrial proteostasis in AD. Whether and how specific mitochondrial chaperones and proteases in different mitochondrial compartments are involved is not clear. Importantly, recent studies demonstrated “mitochondria as guardian in cytosol” where mitochondria proteases degrade aggregation-prone cytosolic proteins after their importation , 253, suMitochondrial dysfunction plays a critical role in AD either as a primary or secondary event, which could represent promising therapeutic targets Fig. . One theAlternatively, given that the loss of mitochondrial structural and functional integrity is likely causally associated with impaired energy metabolism and increased oxidative stress during the course of AD, it probably holds greater promise to consider restoration of the integrity of mitochondria for therapeutic development, which requires better understanding of the underlying mechanisms. Indeed, recent advances in the field demonstrated the involvement of impaired mitochondrial dynamics and transport, biogenesis and mitophagy, proteostasis and quality control as well as its interactions with other organelles such as ER, which offers multiple novel intervention sites to target the integrity of mitochondria. Most of the mechanistic studies rely on genetic models of AD in vitro and in vivo, which demonstrated how APP or Aβ causes impaired mitochondrial integrity and dysfunction by impacting these mitochondrial regulatory mechanisms separately. However, the relationship between these deficits and their relative contribution to APP- or Aβ-induced mitochondrial dysfunction and neuronal dysfunction was not elucidated. It is possible that they act together in a downward spiral manner to impair mitochondrial integrity and cause mitochondrial dysfunction that mediates neurotoxic effect of APP/Aβ. This implies that restoration of one deficit may have an overall beneficial effect on mitochondrial integrity and function as repeatedly demonstrated in these studies.More studies are needed to address how mitochondria become dysfunctional in the case when mitochondrial dysfunction plays the primary role in the pathogenesis. Because aging is the only known risk factor for this subset of AD, given the multifaceted nature of biological aging, the lack of specific models is the biggest hurdle. Nevertheless, as stipulated by the “mitochondrial cascade hypothesis”, the accumulation of somatic mtDNA mutations with advancing aging influences brain function. Many of these mitochondrial regulation mechanisms become loose during aging. For example, mitochondrial biogenesis and mitophagy decline while mitochondria fission enhances along aging. It is possible that mild deficit in one or several of these mitochondrial regulation mechanisms determined by one’s genetic background could set the motion of a downward spiral and all of these mitochondrial deficits could come into play at some points that impairs mitochondrial integrity, which causes damage, affects repair and/or replication of mtDNA and thus accelerates the accumulation of mtDNA changes, leading to mitochondrial dysfunction and eventually, the disease Fig. . Therefo