Dormancy Associated MADS-box, Flowering Locus C-like, Flowering Locus T-like and Terminal Flower 1-like genes was found to be modulated under differential chilling. The co-expression network analysis indentified two high chill specific modules that were found to be enriched for “post-embryonic development” GO terms. The network analysis also identified hub genes including Early flowering 7, RAF10, ZEP4 and F-box, which may be involved in regulating chilling-mediated dormancy release and fruit set. The results of transcriptome and co-expression network analysis indicate that chilling availability majorly regulates phytohormone-related pathways and post-embryonic development during bud break.The availability of sufficient chilling during bud dormancy plays an important role in the subsequent yield and quality of apple fruit, whereas, insufficient chilling availability negatively impacts the apple production. The transcriptome profiling during bud dormancy release and initial fruit set under low and high chill conditions was performed using RNA-seq. The comparative high number of differentially expressed genes during bud break and fruit set under high chill condition indicates that chilling availability was associated with transcriptional reorganization. The comparative analysis reveals the differential expression of genes involved in phytohormone metabolism, particularly for Abscisic acid, gibberellic acid, ethylene, auxin and cytokinin. The expression of  FLOWERING LOCUS T (FT) is negatively regulated by FLOWERING LOCUS C (FLC)FLC through epigenetic regulation. Bud break in poplar has been found to be associated with expression of PtFT1, an ortholog of Arabidopsis flowering promoter FTFLC-like genes has been reported in few perennial species, their expression level does not coincide with the dormancy release4evergrowing peach mutant, which was unable to enter dormancy led to identification of DORMANCY ASSOCIATED MADS-BOX (DAM) geneDAM as potential flower repressor in perennial plants78DAM genes was not found to be coinciding with dormancy release in apple, however a possible role of FLC-like genes was proposed in dormancy regulationTemperature plays a crucial role in determining the phenological events of perennial plants. In the changing climate scenario, the productivity and distribution of perennial plants is affected by the phenological traits of the vegetative and reproductive buds. The involvement of molecular mechanism analogous to vernalization process in Arabidopsis, has been proposed for chilling-dependent bud dormancy release in perennial speciesethylene response factors (ERF), which resulted in temporary induction of endogenous ethylene synthesisBetula pendula) was also reportedIn addition, role of various plant growth regulators during bud dormancy establishment and its release has been evaluated in several studiesMalus domestica Borkh) is one of the most widely cultivated fruit trees in the temperate zones of the world. As winter approaches, already developed paradormant floral buds gradually switch to endodormant state and remain dormant until they accumulate sufficient chilling units  during winter. Even after the accumulation of sufficient CUs, persistent unfavorable environmental conditions gradually shift floral buds from endodormant to ecodormant state. Rise in temperature makes the ecodormant buds committed to re-initiate their growth. Change in climatic conditions of North-Western Himalayas led to decreasing trend of low temperature during November to February months10Apple (viz. dormant bud (DB), silver tip (ST), green tip (GT) and initial fruit set (FS) of apple under differential chilling conditions was performed. The sample abbreviations, accumulated chilling units (CUs) and schematic representation of comparative conditions are given in de novo assembly of 90,880,310 high quality reads was performed that yielded 68,455 contigs, with N50 of 766 bp and an average length of 603 bp. Out of 68,455 contigs, 57,603 (84.1%) were successfully annotated against non-redundant, NCBI protein database using BLASTX program.The RNA-seq analysis of four developmental stages In the present analysis, DEGs were identified in ten comparative conditions . The sumIn the present investigation, irrespective of chilling conditions, the GO terms such as response to stimulus and response to stress were highly enriched in STL-vs-DBL and STH-vs-DBH comparisons . In GTL-The DEGs of different comparative conditions were also subjected to KEGG enrichment analysis using KOBAS. In STL-vs-DBL, the enrichment of fatty acid metabolism, plant hormone signal transduction and circadian rhythm pathways was observed . While, NCED3 (Contig_57348), a key gene involved in ABA biosynthesis, was found to be increased along the bud break and fruit set under high chill conditions (NCED1 (Contig_49254 and Contig_62052) were found to be down-regulated in STL-vs-DBL and STH-vs-DBH before bud break, while upregulation of these transcripts was observed during fruit set in FSL-vs-GTL and FSH-vs-GTH. Almost similar expression pattern was observed for NCED5 (Contig_43868), however, up-regulation of this transcript was observed in GTH-vs-STH. In comparison of similar phenological stages under differential chilling conditions, the expression of NCED3 was observed to be downregulated during bud break under low chill conditions. In addition, the high expression of ABA 8′-hydroxylase, involved in ABA degradation, was observed under low chill conditions in comparison to high chill conditions , a key gene in gibberellic acid (GA) biosynthesis, were found to be up-regulated in STH-vs-DBH and GTH-vs-STH, while only one transcript (Contig_33744) was found to be up-regulated in STL-vs-DBL and GTL-vs-STL  of gibberellin 2-oxidase (GA 2-oxd) were found to have higher expression in GTH-vs-STH and GTL-vs-STL, where fold change was found to be more in GTH-vs-STH. In addition, these transcripts were found to be up-regulated in STL-vs-STH, while in GTL-vs-GTH and FSL-vs-FSH, their expression was either down-regulated or unaltered under low chill condition. Moreover, majority of the DELLA transcripts were found to have higher expression in STL-vs-STH and FSL-vs-FSH, while their expression was observed to be either unaltered or down-regulated in GTL-vs-GTH (Three transcripts of L-vs-STL . SimilarL-vs-GTH .ACC synthase (ACS) transcripts , associated with ethylene biosynthesis, were found to be gradually up-regulated along the bud break and thereafter decreased during subsequent fruit set. While, expression of these transcripts was found to be higher in GTH-vs-STH and STL-vs-DBL  transcript (Contig_15267), which oxidizes ACC to ethylene, was found to have down-regulated expression in STH-vs-DBH and STL-vs-DBL. However, slight increase in its expression was observed in GTH-vs-STH and FSL-vs-GTL. In similar stages under differential chilling conditions, DBL-vs-DBH and GTL-vs-GTH were observed to have low relative expression of ACO, while its expression was high in STL-vs-STH , was found to be gradually increased along the bud break with higher expression under high chill condition (YUCCA4 transcripts (Contig_7052 and Contig_46911) was observed, however with maximum expression during GT stage. Thereafter, its expression was found to be either low or undetectable in fruit set stage , auxin response factor3  and auxin response factor6-like (Contig_33446 and Contig_38459) was found to be upregulated under low chill conditions (cytokinin synthase (Contig_25970 and Contig_54321), involved in cytokinin biosynthesis, was found to have upregulated expression in STH-vs-DBH and STL-vs-DBL, with more upregulation under low chill conditions  among the other comparative conditions, therefore, the expression of analyzed . The expondition . The simet stage . Among tnditions . These rnditions . These rDORMANCY ASSOCIATED MADS-BOX (DAM) genes. All these transcripts were found to have decreased expression along with bud break under both chilling conditions, however the downregulation was more in STH-vs-DBH as compared to STL-vs-DBL  under high chill condition as compared to low chill condition (0.85 fold in STL-vs-DBL) (FLC-like gene was found to be higher during the bud break under low chill conditions. The expression of FT-like (Contig_36756), a gene belonging to PEBP family, was found to be gradually increased towards the bud break and fruit set, where higher expression of FT-like gene was observed under low chill conditions. The TERMINAL FLOWER 1 (TFL1)-like (Contig_56235), a member of PEBP family associated with repression of precocious flowering, was found to be expressed only in actively growing buds (GT) with downregulated expression in GTL-vs-GTH  showed high similarity with previously identified L-vs-DBL . The ana-vs-DBL) . MoreoveL-vs-GTH .Histone acetyltransferase1 (HAC1)-like transcripts were found to be upregulated towards the initiation of active growth under high chill conditions in GTH-vs- STH (HAC1-like transcripts were found to be higher under low chill conditions. The expression of histone deacetylase (HDA), involved in transcriptional repression, including HDA14 and HDA19 was found to be either upregulated or unaltered during initiation of active growth under high chill condition in GTH-vs-STH. In contrast, the expression of HDA2, HDA5, HDA6 and HDA8 was found to be either downregulated or unaltered during dormancy release towards the fruit set through initiation of active growth, however, either upregulated or unaltered expression was observed in GTH-vs-STH and FSL-vs-GTL  and phytohormone metabolism . The sequence of the contigs selected for qRT–PCR analysis and their respective primers are given in DAMs, FLC-like, TFL-like, NCED1, NCED5, GA 20-oxd, GA 2-oxd, ACO, ACS, YUCCA4 and cytokinin synthase, observed through the FPKM and qRT-PCR based expression values were moderately correlated with each other, whereas few samples show obscure correlation in FPKM and qRT-PCR expression values  modules against corresponding high chilling (control dataset) modules   ensured  modules . The modsummary statistics  had have very poor overlapping of genes with modules from test dataset , Ki ≥ 0.9 were selected and top 5 genes based on Ki were mapped to the network of each module. Interestingly, most of such genes with high connectivity were either found to specifically interact with hub genes or itself forming the hub genes  is also known to play important role during dormancy in plants. Recently, the exogenous application of GA was found to substitute the chilling requirement in Chrysanthemum, while ethylene insensitive transgenic was unable to undergo dormancy even after exogenous ethylene applicationPopulus, the transient increase in ethylene related genes was observed during dormancy induction in response to short day photoperiod5ACS was found to be increased during bud break. While ACO, a gene encoding key enzyme of ethylene biosynthesis, was found to be down-regulated towards bud break. These results suggest that ethylene biosynthesis decreased towards the bud break, where the low chilling was found to be associated with low abundance of ethylene biosynthetic genes. Moreover, it was suggested that ethylene plays a role in ABA accumulation through up-regulation of the ABA biosynthetic gene NCEDNCED3 under low chill conditions, might suggest a cross talk between ethylene and ABA levels under low chill conditions.The involvement of ethylene has been known in several physiological processes. Exogenous application of ethylene was found to induce the dormancy in cytokinin synthase, a key gene involved in cytokinin biosynthesiscytokinin synthase was also found to be higher under low chill conditions beside the higher expression of genes involved in auxin biosynthesis and auxin signaling. These results indicate that the transport of auxin to the stem region might be responsible for high expression of cytokinin synthase. Moreover, the high expression of cytokinin synthase suggests the high accumulation of cytokinin which might result in early bud break under low chill conditions.In apple, auxin has been suggested to play inhibitory role in dormancy release of lateral budsFT and SOC1 was negatively regulated by FLC, which in turn are epigenetically downregulated by extended cold treatmentFLC in woody perennial plants is still obscure due to absence of close orthologs of Arabidopsis FLC gene in woody plants37FLC-like transcript was found to exhibit higher expression in DBH as compared to DBL, which was further downregulated with the dormancy release. Similarly, the upregulation of FLC-like gene during chilling acquisition in apple buds was also reportedEVG locus of evergrowing mutant of peach revealed that the deletion of four out of six tandemly repeated SVP-like DAM genes was responsible for non-dormant phenotypeDAM genes was found to be associated with dormancy release in various perennial plants939DAM genes  with bud break indicates the dormancy-associated expression of apple DAM genes, similar to that of other perennial plants. The comparative expression of DAM genes showed variation under differential chilling conditions, with higher expression of DAM genes under high chill conditions as compared to low chill conditions, which concomitantly decrease with the bud break. Similarly, the initial higher expression of DAM genes in Japanese pear buds was observed during early chilling accumulation and thereafter concomitant decrease in the expression of DAM genes was observed with the chilling accumulationDAM genes. In leafy spurge, the presence of CBF binding sites in the promoter region of DAM genes was found to be responsible for its low temperature inducible expressionDAM genes appear to play important role in chilling mediated bud dormancy regulation in apple.In Arabidopsis, prolonged cold treatment results in flower initiation, a process known as vernalizationFT was found to move from leaf to shoot apex either as mRNA or protein, where it acts as flower inducing signalSVP and FLC in Arabidopsis negatively regulate FT to repress the flowering initiationFT-like genes, MdFT1 and MdFT2 was reported in the apical bud and reproductive tissues of apple, respectively, as compared to leaf tissueMdFT1 showed early flowering phenotype. In the present investigation, expression of FT-like gene was found to be gradually increased towards bud break, where low chill condition was associated with higher expression of FT-like gene as compared to high chill condition. Moreover, expression of FT-like gene was found to be antagonistic to that of DAM genes, which suggests that DAM might negatively regulate the expression of FT-like gene in apple. In a recent study, the antagonistic expression of DAM and FT-like genes in apple has been reported by our groupDAM and FT-like genes was reported in leafy spurge and Japanese pear72641DAM and FT-like genes indicate their role in dormancy release and flowering initiation regulation in apple. A recent study reported detection of quantitative trait loci (QTLs) associated with chilling mediated bud break and flowering time regulation in apple, which was found to be co-localized with DAM and FT genesTFL1 in Arabidopsis, is involved in the suppression of floral induction through negative regulation of floral meristem identity genes LEAFY and APETALA1MdTFL1 gene, exhibit delayed flowering phenotype similar to TFL1 overexpressing Arabidopsis lines, which suggest analogous function of MdTFL1 to Arabidopsis TFL1MdTFL1 in apple exhibit precocious flowering with short juvenile phase, which reduced plant vigour due to cessation of vegetative growth, therefore, it was suggested that MdTFL1 affects the plant architecture as well as flower development in applePpTFL1-1α was found to be higher in buds which received higher chilling durationTFL1-like gene was found to be downregulated under low chill conditions as compared to high chill conditions, which might result into precocious flowering that might affect the plant vigour under low chill conditions.In Arabidopsis, the FLC after extended cold exposure to promote flowering has been well established in Arabidopsis5152DAM6 with concomitant histone methylation and deacetylation in its promoter region, corroborate the analogous function of DAM and FLCFLC-like and DAM transcripts was also found to be strongly upregulated in dormant bud, thereafter downregulated during bud break. Therefore, these results indicate that downregulation of FLC-like and DAM genes in apple might also involve similar epigenetic mechanism. Moreover, the increased expression of Histone acetyltransferase1 (HAC1) transcripts towards the initiation of active growth under high chill condition , HDA08 and HDA14 were found to be upregulated during dormant periodHDA08 and HDA06 were found to be downregulated towards the dormancy release and fruit set, while in contrast, HDA14 and HDA19 were found to be upregulated towards the dormancy releases and initiation of active growth. The expression of histone acetylases and deacetylases during dormancy release and fruit set suggested the dynamic balance in their opposing activities to attain appropriate transcriptional level of target genes. The counterbalance between these activities has also been suggested previouslyThe role of epigenetic control in the downregulation of ondition , might bde novo DNA methyltransferase, DRM2 during apple bud breakDRM2 was higher under low chill condition. Also the basic transcript level of MET1 and CMT3 was found to be higher under low chill conditions as compared to high chill conditions during bud break. The similar increase in expression of peach maintenance DNA methyltransferase, PpMET1, in lateral bud meristem, was suggested to be associated with DNA replication in actively proliferating meristematic cells to maintain the DNA methylation pattern in cell lineagesDME-like and ROS1-like transcripts under high chill conditions which was found to corroborate the reduction in DNA cytosine methylation during bud break under high chill conditionDME-like in hybrid aspen during dormancy inductionIn addition, the DNA methylation level has been reported to be higher in dormant state as compared to active growth phase in chestnutThe co-expression network analysis reveals the differential behavior of two comparable datasets under differential chilling conditions during winter dormancy in apple. From the total identified six non-conserved high chill associated modules, only yellow and purple modules showed significant GO terms enrichment and also clearly differentiated their expression behavior under differential chilling conditions . The cri6263EARLY FLOWERING 7 (ELF7) has been shown to be involved in flowering time regulation by regulating the expression of FLC in ArabidopsisRAF10, a mitogen-activated protein kinase kinase kinase (MAP3K), has been reported to act as positive regulator of ABA response and seed dormancy in Arabidopsis69ZEP4, encodes a zinc finger protein was suggested to negatively regulate ABA signalling during seed germination in ArabidopsisMoreover, the hub genes identified in yellow and purple modules might frequently interact with other genes involved in specific pathway. The hub gene from the yellow module, DAM, FLC-like, FT-like and TF1-like genes suggest that early flowering was induced under low chill condition. The plant has to prepare itself  before entering into the reproductive phase, therefore early flowering might adversely affect the plant vigour in the reproductive phase of the phenological cycle. In addition, the co-expression network analysis revealed the presence of chilling associated gene modules. The enrichment of “post-embryonic development” for two high chill specific modules, indicates their involvement in vigorous re-initiation of meristematic activity in bud tissue during dormancy release, under high chill condition.In the present study, transcriptome analysis of samples collected during bud break and fruit set in apple under differential chilling availability in field conditions has been carried out. The higher number of DEGs during active growth phases under high chill condition, suggests that chilling availability drives the transcriptional changes during dormancy release. It is apparent that, chilling availability affects the expression of genes involved in regulation of phytohormones and epigenetic mechanism, such as DNA methylation and histone modification. The expression profiles of Plant material was collected from apple trees of Royal delicious cultivar, selected from two apple orchards situated at Palchan  and Seobag  of Kullu district, Himachal Pradesh (HP), India. From each selected location, the samples of four developmental stages, namely dormant bud (DB), silver tip (ST), green tip (GT) and initial fruit set (FS) were collected during January 8, 2013 to April 15, 2013 from Seobag and during January 8, 2013 to May 8, 2013 from Palchan . Each sade novo assembled using CLC Genomics Workbench 6.5 (http://www.clcbio.com). Various parameters like K-mer and N50 were optimized to get the best assembly results. The assembled contigs were annotated against non-redundant protein database of NCBI using BLASTX program. The assembled contigs were also subjected to GO and KEGG analysis using AgriGOTotal RNA from each plant sample was extracted as described previouslyThe 1 μg of total RNA was converted to first strand cDNA using RevertAid H minus cDNA synthesis kit (Fermentas), following the manufacturer’s instructions. The primers for qRT-PCR analysis were designed using Primer Express software version 3.0.1 (Invitrogen) with default parameters. The qRT-PCR assays were performed with 1:10 diluted cDNA in 20 μl reaction using SYBR green PCR master mix (Thermo Scientific). The relative fold change in the expression of target genes was calculated by comparative delta-delta Ct method using GAPDH as internal control genes for qRT-PCR data normalizationi and j) is described as:Normalized RNA-seq datasets of apple under four developmental conditions were analyzed through network biology approach to explore their differential behavior towards high- and low-chill conditions. Top hit BLASTX annotation for the complete transcriptomic sequences against Arabidopsis proteome, at the e-value cutoff of 1e–05, was considered for network analysis. Transcripts with identical TAIR identifier were analyzed to obtained unique transcripts with maximum statistical varianceaij is the network adjacency. Finally, hierarchical clustering along with DistTOM measure was used to obtain comparable modules from both datasets with appropriate value of deepsplitsummary score using module preservation function in the WGCNA librarywhere, Functional annotations of the non-conserved modules were performed using AgriGO and KAAS. Cytoscape v3.0 was used to visualize the co-expression networksHow to cite this article: Kumar, G. et al. Chilling Affects Phytohormone and Post-Embryonic Development Pathways during Bud Break and Fruit Set in Apple . Sci. Rep.7, 42593; doi: 10.1038/srep42593 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


DNA-based vaccine strategy is increasingly realized as a viable cancer treatment approach. Strategies to enhance immunogenicity utilizing tumor associated antigens have been investigated in several pre-clinical and clinical studies. The promising outcomes of these studies have suggested that DNA-based vaccines induce potent T-cell effector responses and at the same time cause only minimal side-effects to cancer patients. However, the immune evasive tumor microenvironment is still an important hindrance to a long-term vaccine success. Several options are currently under various stages of study to overcome immune inhibitory effect in tumor microenvironment. Some of these approaches include, but are not limited to, identification of neoantigens, mutanome studies, designing fusion plasmids, vaccine adjuvant modifications, and co-treatment with immune-checkpoint inhibitors. In this review, we follow a Porter’s analysis analogy, otherwise commonly used in business models, to analyze various immune-forces that determine the potential success and sustainable positive outcomes following DNA vaccination using non-viral tumor associated antigens in treatment against cancer. Advances in immune understanding have enhanced optimism towards DNA-based vaccine therapies against cancer. While the traditional treatment approaches such as tumor resection, radiotherapy, and anti-cancer chemotherapy have shown success in early stage localized tumors, they have only limited role against later staged metastatic malignancies. Furthermore, these standard agents have shown to cause extensive damage to normal tissues leading to hair loss, blood cell destruction, and debilitating side effects such as decreased appetite, hair loss, and immune-suppression. All of these lead to more dangerous secondary infections in these patients. Thus, immune-based therapeutic strategies offer viable long-tern success by specifically eliminating cancer cells and inducing relapse-free quality survival in cancer patients .Recently, DNA-based vaccines have been developed as a concrete and viable approach and anti-cancer treatment strategy . AdvanceDNA vaccines have a promising application both in the prevention of cancer and also in the treatment of already existing cancers. The ability of DNA-based vaccines to mount an effector T-cell-based immune responses make them an attractive anti-cancer therapeutic tools. For example, a major breakthrough was achieved in the prevention of cervical cancer with recombinant protein-based human papilloma virus (HPV) vaccination, as HPV (strains 16 and 18) have been strongly correlated with the development of cervical cancer. This approach has demonstrated an upregulation of humoral (B-cell based) immune responses as evidenced by increased titers of anti-capsid antibodies that could neutralize HPV capsid and prevent the development of HPV infection. However, this approach did not mount an efficient cellular (or T-cell-based) immune response. While efficient prevention of cervical cancer with HPV vaccination was effected through induction of potent antibody response, the vaccine did not demonstrate significant protective effect on already infected patients since they had already undergone antigen-expression changes in infected cells . Therefowww.clinicaltrials.gov, accessed on 17 January 2017). The long-term success of DNA vaccines as an efficient anti-cancer tools is dependent upon multiple immunological factors. To comprehensively analyze the impact of multiple immune-factors, in this review, we adopt a Porter’s strategy analysis analogy to discuss specific immunological factors that affect the potential success of this novel anti-cancer therapeutic strategy. The Porter’s model is a standard method to analysis various factors affecting the success of a new business [Currently there are 340 clinical trials on cancer DNA vaccines that are either actively recruiting or have approval for open recruiting  primarily depending upon the solid organ and cell-type from where the cancer originates. The TAAs are either expressed in the tumor tissue, overexpressed by oncogenes or selected as differentiation antigens during cancer development. In 2009, the National Cancer Institute (NCI) has published 75 cancer antigens that are used in developing DNA-based vaccines . Unfortuhi T-cells in Mam-A vaccinated breast cancer patients [In our studies, we have identified mammaglobin-A (Mam-A), a breast cancer specific TAA, as a viable option to induce immune responses following DNA vaccination in breast cancer patients. Previous dendritic cells–based studies with TAAs, human epidermal growth factor-2 (HER2/neu), andmucin-1 (MUC1) have demonstrated expansion of peptide-specific CD8+ cytotoxic T-lymphocytes (CTLs) in breast cancer patients ,26. Howepatients  and alsopatients .Theoretically, the most potent antigen to be used in the development of DNA vaccine would be completely non-native to the patient, so that there is no pre-existing central tolerance. Neo-antigens are the self-antigens that mutate to form novel non-self-antigens . The mutImportant characteristics of a DNA vaccine plasmid backbone would include a strong promoter sequence, an antibiotic selection marker, and a poly-A sequence to stabilize the mRNA transcript . The traDNA vaccine should be administered with an aim to deliver the TAA gene to the dendritic cells so that there will be efficient antigen presentation. This is effectively achieved when DNA vaccine is delivered by intramuscular (i.m.) route. However, the efficient T-cell activation will depend on the dendritic cell’s homing ability to the nearest lymph node. Recent evidence suggests that mucosal cancers are more efficiently treated when vaccine-activated T-cells can home-in to mucosa-associated lymph nodes, thus suggesting alternate routes  as viable options . Other DDNA vaccine design is done such that there is concentrated antigen delivery to dendritic cells . These aFollowing administration of a DNA vaccination, the expressed TAAs, when presented in the context of MHC class I proteins, induce activation of CD8+ T-cells. These newly activated CD8 T-cells, upon contact with tumor cells, exert their effector function by lysing the tumor cells through the release of pore-forming perforin in a calcium dependent manner onto the target cell membrane . The actOur studies with the Mam-A DNA vaccine, utilizing a tetramer-based assay approach, have demonstrated that Mam-A cDNA vaccination to HLA-A2 patients specifically expanded the CD8+ T-cells specific to HLA-A2 immunodominant epitope of Mam-A (LIYDSSLCDL). The effector response of these activated CD8 T-cells has been thought be mediated by the inflammatory cytokines (IFN-γ and TNF-α)  and alsoIn humans, NKG2D has been shown to exert its signaling through association with the adaptor molecule DNAX-activation protein 10 (DAP-10) . Furthern = 8) analyzed in our study. Further, in the same cohort, the ICOS expression on CD4+ T-cells remained constant (approximately 21%) in breast cancer patients prior to and following vaccination similar to that seen in normal subjects. However, interestingly, the ratio of ICOS+ CD4+ T-cells to FoxP3+ CD4+ T-cells significantly increased from 7% pre-vaccination to 23% post-vaccination, which is also accompanied by a three-fold increase in the IFN-γ response in CD4+ T-cells following vaccination to breast cancer patients. This data strongly suggests that following Mam-A cDNA vaccination there is increased activation of anti-tumor CD4+ T-cells [The CD4+ T-helper cells, which are predominantly activated by epitopes in the context of MHC class II molecules, have a major role in production of antibodies by B cells and induction of memory CD8+ T-cell responses. It is also known that a subset (5%–15%) of CD4+ T-cells can act as regulatory cells (Treg), which specifically in the context of cancer unfortunately promotes cancer growth . Therefo T-cells . In nutsTo induce activation of CD4+ T-cells from the non-tolerized CD4+ T-cells naïve repertoire, several research groups have fused the variable region genes of the immunoglobulin called single-chain Fv (scFv) sequence with the TAA gene in the recombinant plasmid . As prevHRAS oncogene, suggesting a direct correlation between cancer cell transformation and downregulation of antigen presentation pathway. A consequential phenomenon of lowered vaccine induced CD8+ T-cell activation was observed against HER2 oncogene expressing breast cancers [As previously discussed MHC class I molecules, expressed on all nucleated cells, play crucial role in the antigen presentation and activation by cytotoxic CD8+ T-cells. Several studies have shown that specifically in the tumor there is decreased cell surface expression of MHC class I proteins . Along w cancers .Recently, a subset of CD8+ T-cell were identified that possess a capability to recognize and eradicate cancers with impaired antigen presentation machinery (APM). Interestingly, these CD8+ T-cell’s immune detection of APM deficient cells is linked to T-cell receptor (TCR) and not natural killer (NK) cell receptors. The TAAs recognized by these TCR were shown to have been immaturely process as so these unique epitopes of are called TEIPP, T-cell epitopes associated with impaired peptide processing . The TEIResearch evidence has established that the tumor microenvironment has a higher concentration of immune-suppressive, pro-tumor, alternatively activated M2 macrophages. These M2-type macrophages participate in TH2 cell responses, dampen inflammation, suppress immunity, and promote tumor progression . A stronThe immature myeloid cells called MDSCs are found in large numbers in the blood and tumors of patients with cancer. These tumor localization of MDSCs is favored by the production of cytokine GM-CSF by tumor cells . These tThe Tregs are a subset of CD4+ T-cells with immune-suppressive cell type present in the tumor microenvironment. Furthermore, Tregs have been shown to exert there immune-suppressive effect both in tumor-specific and tumor-nonspecific manner . SeveralThe cytokines, Type I interferons (IFNs) and interleukin-12 (IL-12) are required for an optimal CD8+ T-cell activation and effector cytotoxic responses. These inflammatory cytokines are produced by both innate and adaptive immune cells. However, the local concentration and kinetics of the cytokine release depend on the nature of immune cell activation and the type of cells activated during immune response . IL-12 hA critical pre-requisite for successful DNA vaccination strategy is the correct choice and dose of vaccine adjuvant. The adjuvants work by induction of costimulatory molecules such as toll like receptor factors (TLR 3 and 9) through binding with its ligands such as poly I:C and CpG islands on cellular DNA . Just liAnti-cancer chemotherapy targets tumors based on cancer cell’s unique genetics which selectively promotes rapid cell growth over normal terminally differentiated cells. Further, several studies have shown that these anti-cancer drugs can promote reactivation of tumor-specific immune responses by enhancing T-cell infiltration to the tumor microenvironment, and also by making tumor cells more amenable to cytotoxic CD8+ T-cell effector response . Drugs sThe signaling mechanisms mediated by co-stimulatory molecules, such as CD28, and OX-40, play an important role in T-cell engagement with antigen presenting cells and its subsequent activation and maturation . SeveralDNA-based non-viral cancer vaccines offer a viable complement to the current anti-cancer therapeutic regimen by inducing robust effector and memory T-cell responses. However, there still remain several challenges requiring more careful optimization of vaccine design to overcome immunosuppressive tumor microenvironment. Therefore, a better understanding of tumor–host immune interactions helps us design more efficient immunotherapeutic strategies. Recent advances in immune checkpoint therapy have revealed an important futuristic combinatorial therapeutic strategy. More research in the identification of specific TAs and neo-antigen could induce strong effector T-cell responses. DNA-based cancer vaccine strategies have promising future applications. Complemented with a better understanding on the TAA selection and immune-checkpoint therapy, they will emerge as viable next-generation therapeutic strategy against cancers.


Plasmodium falciparum circumsporozoite protein (PfCSP).Previous studies have shown that the baculovirus-vectored vaccine based on the “baculovirus dual expression system (BDES)” is an effective vaccine delivery platform for malaria. However, a point of weakness remaining for use of this vaccine platform in vivo concerns viral inactivation by serum complement. In an effort to achieve complement resistance, the gene encoding the human decay-accelerating factor (hDAF) was incorporated into the BDES malaria vaccine expressing the Plasmodium berghei sporozoites expressing PfCSP.The newly-developed BDES vaccine, designated BDES-sPfCSP2-Spider, effectively displayed hDAF and PfCSP on the surface of the viral envelope, resulting in complement resistance both in vitro and in vivo. Importantly, upon intramuscular inoculation into mice, the BDES-sPfCSP2-Spider vaccine had a higher protective efficacy (60%) than that of the control vaccine BDES-sPfCSP2-Spier (30%) against challenge with transgenic DAF-shielded BDES-vaccines offer great potential for development as a new malaria vaccine platform against the sporozoite challenge.The online version of this article (doi:10.1186/s12936-017-2039-x) contains supplementary material, which is available to authorized users. Plasmodium parasites and transmitted by the bite of infected anopheline mosquitoes, has a profound impact on affected human populations. The World Health Organization (WHO) estimates that there were 212 million new cases and 429,000 deaths attributable to malaria in 2016  after the forth immunization with BDES-sPfCSP2-Spier  and BDES-sPfCSP2-Spider  were significantly higher than that of BDES-PfCSP1-gp64   and BDES-sPfCSP2-Spider (60%), but not BDES-PfCSP1-gp64 (10%), compared with the control mice Table , but theP. falciparum) were introduced into a BDES vaccine, and its protective efficacy against challenge infections in mice with a transgenic parasite line expressing P. falciparum CSP via the natural infection route (mosquito bites) was evaluated. The present study clearly showed that the DAF-shielded baculovirus-vectored vaccine enhances protection against malaria sporozoite challenge infections in mice.The present study examined the potential of BDES as a new malaria vaccine platform. In addition to its high vaccine efficacy, this platform has several advantages, especially from a biological safety perspective including (i) low cytotoxicity, (ii) an inability to replicate in mammalian cells, and (iii) an absence of preexisting antibodies. One of the major obstacles in the in vivo use of baculovirus vectors is the virus inactivation by serum complement . Hence, DAF, a GPI anchored protein, is broadly distributed among haematopoietic and non-haematopoietic cells. DAF acts in both pathways either by promoting the decay of C3 convertases in the complement cascade or by catalyzing the permanent inactivation of C3 convertases via factor I-induced proteolytic cleavage , 26. SevIn the present study, codon-optimization as well as the display platform with VSV-G TM could enhance not only the expression levels of the antigen but also humoral immunity against PfCSP. Compared with other recombinant protein-based malaria vaccines , the BDES vaccine platform not only displays the relevant antigens on its envelope, with native confirmation –16, but Recently, outbreaks of emerging and re-emerging infectious diseases have become a major threat to human health and global stability. Quick actions must be taken to develop effective vaccines to combat such infectious agents. To this end, the development of a novel vaccine platform is, undoubtedly, now urgent. The present study suggests that the DAF-shielded BDES vaccine platform, which has safety and large-scale manufacturing/bioreactor technologies at the heart of its development, has tremendous potential as a generic “next generation” vaccine candidate for malaria as well as for other infectious pathogens.Additional file 1: Figure S1. Schematic representation of pFast-Spider. Table S1. Primers used in this study. Supplementary materials and methods.


Available genomic sequences for all members of the UCC provide opportunity to investigate the metabolic routes of carbon transfer between autotroph and heterotrophs. Here, we applied a subsystem-based comparative genomics approach to reconstruct carbohydrate utilization pathways and identify glycohydrolytic enzymes, carbohydrate transporters and pathway-specific transcriptional regulators in 17 heterotrophic members of the UCC. The reconstructed metabolic pathways include 800 genes, near a one-fourth of which encode enzymes, transporters and regulators with newly assigned metabolic functions resulting in discovery of novel functional variants of carbohydrate utilization pathways. The in silico analysis revealed the utilization capabilities for 40 carbohydrates and their derivatives. Two Halomonas species demonstrated the largest number of sugar catabolic pathways. Trehalose, sucrose, maltose, glucose, and beta-glucosides are the most commonly utilized saccharides in this community. Reconstructed regulons for global regulators HexR and CceR include central carbohydrate metabolism genes in the members of Gammaproteobacteria and Alphaproteobacteria, respectively. Genomics analyses were supplemented by experimental characterization of metabolic phenotypes in four isolates derived from the consortia. Measurements of isolate growth on the defined medium supplied with individual carbohydrates confirmed most of the predicted catabolic phenotypes. Not all consortia members use carbohydrates and only a few use complex polysaccharides suggesting a hierarchical carbon flow from cyanobacteria to each heterotroph. In summary, the genomics-based identification of carbohydrate utilization capabilities provides a basis for future experimental studies of carbon flow in UCC.Two nearly identical unicyanobacterial consortia (UCC) were previously isolated from benthic microbial mats that occur in a heliothermal saline lake in northern Washington State. Carbohydrates are a primary source of carbon and energy for most heterotrophic bacteria. Since CO Previous experimental data suggest the presence of numerous metabolic interactions between the heterotrophs and the cyanobacterium, which makes these consortia excellent models for defining the metabolic interactions potential in the community  were derived from a photosynthetic microbial mat from Hot Lake, Washington  and theiommunity .Cyanobacteria excrete numerous organic compounds including fermentation products , osmolytin silico approach was shown by our previous reconstructions of sugar utilization networks in Bacteroides, Shewanella, and Thermotoga genera . Completeness of genomic content for most of the analyzed metagenomic bins, as previously estimated by presence/absence of 100 conserved single-copy genes, was at least 98%, with the exception of one member, bin09, whose estimated coverage is 88% . The annotated genomic sequences were downloaded from the Integrated Microbial Genome (IMG) expert review database . In addienically  (Table 1e is 88% . In addiGlycoside hydrolases (GHs) were predicted by analyzing the deduced proteome sequence from all 19 UCC organisms on the dbCAN server . CellulaThe UCC genomes were previously annotated via the following two pipelines: (i) the DOE-JGI Microbial Genome Annotation Pipeline , and ii. First, Reconstruction of carbohydrate utilization pathways in 17 heterotrophic UCC members was performed using the subsystem-based comparative genomics approach combined with genomic reconstruction of carbohydrate-specific transcription factor (TF) regulons and identification of candidate carbohydrate-specific transporters as previously described . TypicalFor reconstruction of novel TF regulons, we used the bioinformatics technique based on identification and comparative analysis of candidate TF-binding sites in closely related genomes  and implHalomonas sp. HL-48 and HL-93 strains, Roseibaca calidilacus HL-91 and Marinobacter sp. HL-58 were tested for their ability to grow on a panel of various carbon sources as a sole carbon and energy source. Cells were grown in Hot Lake Heterotroph (HLH) medium . The HLH media for starter cultures was supplemented with 5 mM glycerol (for HL-91) or 5 mM sucrose . The mid-log phase grown cells were further washed three times with HLH medium without any carbon source to eliminate carry over of carbon substrates from starter media prior to inoculation and washed cells were used as inoculums in Api50 CH strip. Utilization of carbon sources were indicated by the growth on the well. For each strain, two independent repetitions were performed. The incubation time for Api50 CH strip measurements was 3 days  and 7 days (for HL-91). Growth phenotype of Api50 CH strip results were further validated by growth measurements using selected carbohydrates. An optical density (OD600) was measured to monitor cell growth during 60–100 h using a plate reader instrument Norden Lab Professional-Bioscreen. 250 μL culture volumes in the 100 well Bioscreen plate, and each growth experiments were performed in 10 replicates.An ultimate validation of the genomics-based metabolic reconstructions was attained by experimental testing of growth phenotypes. Four UCC isolates including the ) medium , containTable 1). The majority of the identified GHs have a predicted cellular localization either in the cytoplasm (for 217 GHs) or in the periplasm (for 178 GHs), with an additional 29 GHs found in the inner membrane  that are involved in breakdown of oligosaccharides (and polysaccharides) into monosaccharides. Overall, 441 proteins containing at least one GH domain were found unevenly distributed in the studied UCC genomes  we analyzed potential function of 374 GHs identified in the heterotrophic UCC organisms. As result, we tentatively assigned substrate specificity and metabolic pathway to 338 GHs . Overall, we identified pathways for utilization of six hexoses , two amino sugars , two pentoses (arabinose and xylose), 10 sugar acids and diacids (see below) and six sugar alcohols including inositol, arabinitol, mannitol, sorbitol, erythritol, and glycerol. In addition to monosaccharides, we reconstructed catabolic pathways for several oligosaccharides including α- and β-glucosides, α- and β-galactosides, maltose, sucrose, and trehalose. Finally, we predicted a novel putative pathway for utilization of mannoheptulose (a heptose).We applied subsystem-based comparative genomics approach to reconstruct peripheral carbohydrate utilization pathways in the 17 heterotrophic UCC members. Our analysis revealed highly diverse capabilities of UCC organisms to utilize carbohydrates and their derivatives (Table 1). M. excellens HL-55, Erythrobacter sp. HL-111, Rhodobacteriaceae bin12, and Oceanicaulis bin04 lack any complete carbohydrate utilization pathway although some of them possess some catabolic genes in the absence of predicted carbohydrate-specific transporters. UCC members with a limited number of carbohydrate utilization pathways include A. calidilacus HL-53 (predicted to utilize glucose and β-glucosides) and Rhodobacteriaceae bin07 (only has sorbitol utilization pathway). In contrast, two Halomonas species (HL-48 and HL-93) and Rhodobacteriaceae bin08 have the largest numbers of identified carbohydrate utilization genes and pathways. For instance, HL-93 has the predicted capabilities to utilize 25 carbohydrates and their derivatives, whereas HL-48 and bin08 have 18–19 individual pathways.Most heterotrophic UCC members were predicted to catabolize at least one carbohydrate (Table 1). The complete list of 798 proteins involved in the reconstructed pathways across 16 organisms along with their deduced functional annotations is provided in Supplementary Table Table 1 and Supplementary Table The peripheral carbohydrate utilization pathways include between eight proteins in HL-111 to up to 150 proteins in HL-93 . It encodes proteins that are orthologous to (i) the ABC-type arabinose uptake transporter system AraFHG and (ii) the AraA, AraC, and AraE enzymes from the oxidative arabinose degradation pathway in Azospirillum brasiliense (L-arabonate dehydratase (AraD) and a second isozyme of arabinose-1-dehydrogenase (AraY). A member of aldose 1-epimerase family encoded in the ara gene cluster was previously assigned the functional role L-arabinose mutarotase (AraM), which interconverts alpha and beta anomers of L-arabinose. The ara gene locus in Halomonas encodes two novel TFs from the LysR and GntR families . Reconstruction of their cognate regulons using these and other Halomonas genomes has revealed two different DNA motifs  transporter family and a hypothetical lactonase, which was assigned the missing arabinolactonase function (named AraB). Nearly all known TRAP-family transporters have specificities to organic acids , A. marincola HL-49 and Rhodobacteriaceae bin08 . HL-48 possesses genes encoding the last two steps of this pathway, COG0179 and COG1028. However, the absence of genes for the first two steps of the fucose pathway suggests that HL-48 can only utilize the L-fuconate intermediate. In contrast, HL-93 and bin08 have the complete L-fucose utilization pathway, whereas in HL-49 L-fuconate dehydratase FucD is missing and L-fucose dehydrogenase is substituted with a novel non-orthologous dehydrogenase (named FucOII). The four UCC genomes have different transporters encoded in the fuc loci. HL-49 has a gene encoding an ortholog of fucose permease FucP from X. campestris. HL-93 and bin08 have two non-orthologous ABC systems that we predict to be involved in uptake of L-fucose.The oxidative pathway for utilization of mpestris . We obseL-fucose/L-fuconate utilization gene loci in both Halomonas species contain the lgoD gene encoding L-galactonate-5-dehydrogenase, which is the signature gene of L-galactonate utilization (uxaA and uxaB (or uxaF) genes involved in the downstream steps of L-galactonate catabolism, as well as a novel TRAP-family transporter operon . A similar L-galactonate catabolic gene locus in Chromohalobacter salexigens  encodes the same set of catabolic enzymes and a non-orthologous TRAP transporter, which was previously characterized to have a dual specificity toward L-fuconate and L-galactonate  of genes that are involved in the shared carbohydrate utilization pathways.The lization , the uxaactonate . Based oD-Galacturonate and D-glucuronate are hexuronic acids that are commonly found in pectins, proteoglycans and glucuronans. D-Galactarate and D-glucarate are the ring opened hexose diacids  that serve as a growth substrate to many microorganisms. Both Halomonas species (HL-48 and HL-93) contain a gene cluster encoding enzymes involved in the galactarate/glucarate utilization, as well as a novel predicted transporter from the tripartite tricarboxylate transporter (TTT) family (named TctABC) and a novel GntR-family TF (termed GguR). The TctABC transporter is predicted to be involved in galactarate/glucarate uptake. The reconstructed GguR regulon in both Halomonas genomes includes the galactarate/glucarate utilization operon, whereas HL-93 has an additional GguR-regulated operon, which encodes the glucoronate/galacturonate utilization enzymes Udh, Gli, and Gci, as well as an ortholog of the known hexuronate transporter UxuPQM. We concluded that HL-93 (but not HL-48) has an additional capability to utilize galacturonate and glucoronate using the pathway partially shared with the galactarate/glucarate pathway .R. calidilacus HL-91 and Rhodobacteriaceae bin08 and bin18 genomes encode a different variant of the glucuronate utilization pathway, which starts from the UxaC isomerase and continues through 2-dehydro-3-deoxygluconate (KDG) and its phosphorylated derivative, KDG-6P. The corresponding glucoronate utilization gene loci include a novel ABC-family transporter operon, which was predicted to be co-regulated with the glucuronate utilization genes by a novel GntR-family regulator (UxuR). However, the corresponding transporter and regulator are missing in HL-91, thus the mechanism of glucuronate uptake in yet unknown in this organism. We speculate that it can take up glucuronides that are hydrolyzed in the cytoplasm by the LfaA glucosidase, thus providing glucoronate to feed the catabolic pathway.The Algoriphagus marincola HL-49 has two separate loci with galacturonate and glucuronate utilization genes. Both of these gene loci are controlled by a novel LacI-family TF (UxuR2) and encode a novel or TRAP-family transporter (UxuPQMII) and an uncharacterized glycosyl hydrolase from the GH109 family. UxuPQMII is distantly related to the previously characterized UxuPQM transporters in various Proteobacteria . The galactose gene locus in HL-46 contains a predicted galactose permease from the SSS family, which is orthologous to the GalPII transporter previously identified in the galactose catabolic gene loci in Shewanella spp. . In this pathway, D-galactose is first oxidized to D-galactonate, which is then converted to pyruvate and GAP through the subsequent action of a dehydratase, a kinase and an aldolase  family that mostly known to transporter di- and oligo-saccharides, according to the TCDB database  and a novel TTT-family transporter. Known transporters from the TTT family are specific to tricarboxylate and sugar acids , which can serve as a non-orthologous gene displacement for D-galactono-1,4-lactone lactonase GalA. The reconstructed DgoR regulon in the Halomonas genomes includes additional candidate co-regulated gene encoding a SSS-family transporter with predicted galactose specificity . Although orthologs of known D-galactose dehydrogenase  are missing in Halomonas spp., we tentatively assigned them the galactose utilization capability, which is supported by growth phenotype testing (see below). Further similarity searches revealed one possible candidate for the missing GalD reaction in Halomonas spp. – a D-xylose dehydrogenase (XylD) from the xylose utilization gene cluster . Thus, we tentatively propose that XylD in Halomonas spp. has specificity to multiple substrates including D-galactose and D-xylose.The DeLey-Doudoroff pathway genes for II), galactose , and L-galactonate (UxaF) are described in details above. Other proposed cases of non-orthologous enzymes include a novel N-acetylglucosamine kinase (COG1070) in bin09, the putative sorbitol dehydrogenase SorDII in bin07, and the predicted fructokinase MtlZ involved in utilization of arabinitol, sorbitol, and mannitol in both Halomonas spp.The reconstructed peripheral pathways in heterotrophic UCC members contain 171 novel genes distinguishing them from those previously described in model species. These include 22 genes encoding novel enzymes with assigned function, 107 genes encoding components of novel sugar transporters and 42 novel sugar-specific transcriptional regulators. Most common are numerous cases of non-orthologous gene displacement, when a novel functional role is encoded by a gene that is not orthologous to any of the previously known genes of the same function. Several predicted non-orthologous enzymes involved in utilization of arabinose , fucose , oligosaccharides , as well as glucoronate, sorbitol, erythritol, and mannoheptulose. All five newly predicted TRAP systems and three TTT-family transporters are specific to sugar acids  and hexose diacids . We also identified 12 novel single-component sugar permeases located in the inner membrane , three TonB-dependent outer membrane transporters (with predicted specificities to sucrose and β-glucosides) and two novel outer membrane porins (possibly involved in uptake of glucose and glycerol).Carbohydrate uptake transporters constitute the largest group of newly functionally assigned genes in UCC genomes. Most of these genes encode components of 27 multicomponent transport systems from the ABC, TRAP, and TTT families  and two kinases (COG1940 from the ROK family and COG0529 from the adenylylsulfate kinase family). The substrate-binding component of this ABC transport system has 83% similarity to Avi_5339 from Agrobacterium vitis, which was previously found to bind mannoheptulose . Mannoheptulose is a heptose, which is structurally similar to D-tagatose. Based on these observations and known substrate specificities for other kinases from the COG1940 and COG0529 families, we propose the following hypothetical pathway for mannoheptulose utilization. The COG1940 kinase first phosphorylates the substrate to produce mannoheptulose-7-phosphate, then the COG0529 kinase further produces mannoheptulose-1,7-biphosphate, which is subject to the COG3684 aldolase reaction producing glycerone phosphate and erythrose-4-phosphate.In Halomonas species have a different pathway for xylose utilization including xylose dehydrogenase XylD. In Caulobacter crescentus, xylose is converted to α-ketoglutarate by xylose dehydrogenase, xylonolactonase, and xylonate dehydratase  and a Gfo/Idh/MocA-family oxidoreductase (COG0673), however, their exact biochemical functions require further experimental characterization.Three UCC members are predicted to utilize xylose though the classical isomerase pathway , whereas both ydratase . HoweverRhodobacteriaceae bin08. Its putative rhamnose catabolic locus contains genes encoding the RhaFGHJ transporter and the RhaM mutarotase, however, bin08 lacks other candidate genes required for utilization of rhamnose . Both Halomonas spp. are able to grow on arabinose, xylose, galactose, arabinitol, mannitol, sorbitol and gluconate, whereas HL-91 grow on N-acetylglucosamine, mannose, and cellobiose (α β-glucoside). In addition, HL-93 has growth phenotypes on fucose, mannose, erythritol, inositol, glucoronate, and galacturonate. With a single exception of the fructose and mannose utilization in HL-91 when we were unable to predict specific catabolic pathways, the measured phenotypes are consistent with the reconstructed catabolic pathways.All four tested organisms demonstrated the ability to grow on two hexoses  and three disaccharides , as well as on glycerol . To understand downstream parts of the reconstructed catabolic pathways, we searched the genomes of 17 heterotrophic UCC members for known CCM genes  pathway and the Entner-Doudoroff (ED) pathway . The RpiR-family regulator HexR that responds to 2-keto-3-deoxy-gluconate-6P . The Halomonas strains HL-48 and HL-93 have capabilities to utilize over 20 substrates including pentoses, hexoses, disaccharides, sugar acids, and alcohols. Rhodobacteriaceae bin08 is able to grow on 18 substrates including mannoheptulose, a heptose for which we proposed a novel catabolic pathway/transporter/regulon. Each catabolic pathway includes a specialized, often multicomponent, transport system and a set of intracellular enzymes catalyzing biochemical transformations of a particular sugar into one of the common CCM intermediates . For the majority of reconstructed pathways we also mapped their cognate TFs that are involved in transcriptional regulation (induction) of catabolic genes, and reconstructed the TF regulons, often allowing the identification of missing transporters and enzymes. Further assessment of two Halomonas strains and two other UCC organisms for the growth on a large number of carbon sources allowed us to confirm the majority of the in silico predicted catabolic phenotypes. The results of the Api50 testes and extended growth profiling revealed a remarkable consistency between the predicted and observed phenotypes.By applying the subsystem-based comparative genomics approach, we reconstructed carbohydrate utilization pathways and predicted catabolic potential for heterotrophic members of UCC consortia. Overall, the reconstructed sugar utilization subsystems include almost 800 genes unevenly distributed across 17 analyzed genomes. Functional roles of 171 genes were first proposed in this study. 13 UCC members were predicted to utilize at least some carbohydrates as a source of carbon and energy using the dedicated catabolic pathways . Secreted GHs (such as glucosidases) identified in the two Bacteroidetes members could benefit the other carbohydrate-utilizing heterotrophs by producing transportable mono- and oligosaccharides. Utilization pathways for disaccharides , β-glucosides and glucose are the most abundant among UCC members (present in 8–10 genomes). All other peripheral pathways are present in <30% of the UCC organisms, and among them, 15 pathways are present only in 1–2 genomes. The obtained carbohydrate utilization profiles of UCC heterotrophs are in agreement with the carbohydrate composition of cyanobacterial EPSs  Cyanobacteria that provide both EPS and osmoprotectants, and (ii) heterotrophic bacteria that could use the cyanobacteria-generated substrates to synthesize their own osmoprotectants and in turn share them with the community. Four UCC members do not rely on carbohydrates for growing. These organisms could use other by-products (such as lactate) secreted by Cyanobacteria and other heterotrophs and thus serve as yard cleaners for the community.Exopolysaccharides produced by autotrophic ial EPSs . Cumulatial EPSs . Indeed,Our ability to predict a phenotype of organism from its genome is one of the key goals in microbiology. A systematic application of this omics approach for metabolic reconstruction in a growing number of microbial genomes would allow us to establish the capability of highly accurate automated annotation and assertion of carbohydrate catabolic phenotypes in microbial communities.SL performed the majority of bioinformatics analysis and wrote the paper; YM performed experimental validation of UCC isolate phenotypes; MR annotated the analyzed genomes; DR designed the study, analyzed the obtained data and wrote the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Drosophila metamorphosis, nascent testis myotubes migrate from the prospective seminal vesicle of the genital disc onto pupal testes and then further to cover the testes with multinucleated smooth-like muscles. Here we show that DWnt2 is likely required for determination of testis-relevant myoblasts on the genital disc. Knock down of fibroblast growth factor receptor (FGFR) heartless by RNAi and a dominant-negative version revealed multiple functions of Heartless, namely regulation of the amount of myoblasts on the genital disc, connection of seminal vesicles and testes, and migration of muscles along the testes. Live imaging indicated that the downstream effector Stumps is required for migration of testis myotubes on the testis towards the apical tip. After myoblast fusion, myosin II is needed for migration of nascent testis myotubes, in which Thisbe-dependent fibroblast growth factor (FGF) signaling is activated. Cadherin-N is essential for connecting these single myofibers and for creating a firm testis muscle sheath that shapes and stabilizes the testis tubule. Based on these results, we propose a model for the migration of testis myotubes in which nascent testis myotubes migrate as a collective onto and along the testis, dependent on FGF-regulated expression of myosin II.During  Summary:Drosophila testes and mammalian seminiferous tubules are surrounded by a muscle layer. Drosophila myotubes migrate towards testes in dependence of the FGF receptor Heartless, myosin II and Cadherin-N. Drosophila, various single as well as collective cell migration processes have been described, such as border cell migration in the ovary and embryonal mesoderm migration  in segments A8/A9/A10  signaling. The FGF receptor (FGFR) Breathless (Btl) and its ligand Branchless recruit larval mesodermal cells, which become epithelial and give rise to paragonia and seminal vesicles , becomes activated by its ligands Thisbe (Ths) and Pyramus (Pyr)   and is ius (Pyr) , where ius (Pyr) . Htl is us (Pyr) . Htl-depogenesis . During ogenesis , while nogenesis .Drosophila, cadherins such as Shotgun  and Cadherin-N (Cad-N) can mediate adhesion between neighboring cells. Catenins, such as beta-catenin, mediate the link between cadherins and the cytoskeleton  was expressed in the epithelium of the prospective seminal vesicle but was hardly detectable in myoblasts  with the driver line UAS-Dcr-2;;Mef2-Gal4 specifically in myoblasts. This resulted in a disturbed morphology of the testis. In the wild type, the adult testis is a long, thin tubule of 2.5 coils , Cad-N expression is largely restricted to FC-like myoblasts . mefGAL4-driven knock-down of Cad-N was efficient in FC-like myoblasts, whereas expression was hardly affected in myoblasts lying over the paragonia . At 44 h APF, cad-N knock-down testes displayed nascent myotubes, which were distributed all over the testes . In contrast to wild-type testes , the nascent myotubes in these cad-N knock-down testes are less elongated and their number seemed to be reduced . Nevertheless, adult males with this phenotype were able to produce offspring .Hence, we then knocked down .5 coils F. In theone coil G. The teone coil G′. Genitone coil . In diffDrosophila . Cad-N expression between adjacent myotubes was preserved upon arm knock-down, whereas no Cad-N expression was detected when testis myotubes did not adhere to each other , the beta-catenin homologue in osophila , specifiwo coils H. The tewo coils H′. Thesech other I.We conclude that Cad-N in cooperation with Arm is involved in testis myotube migration and is necessary both to stabilize the testis muscle sheath and to shape the testis.Many migratory processes depend on the actin-myosin network . Using mspaghetti squash (sqh), the regulatory light chain of non-muscle myosin II .When we myoblast-specifically knocked down yosin II . The tesyosin II A, and hayosin II C. The muyosin II C′ than iyosin II B. These zipper (zip), the non-muscle myosin II heavy chain . This was surprising because the sqh and zip knock-down phenotypes resembled each other and are components of myosin II. The observed differences in male fertility might be due to an unpredicted off-target in the sqh RNAi line.Testes morphology and musculature were also disturbed when vy chain , was dowvy chain D, compara sheath D′. In cosqh knock-down testes in more detail. Cad-N was expressed between the few remaining adjacent myotubes , which suggested that the muscle sheath defect was not due to faults in Cad-N-mediated attachment of single myotubes. Instead, it was likely caused by insufficient population of the testis with muscles. We further analyzed the status of the extracellular matrix by monitoring Terribly reduced optic lobes (Trol) , which indicated extracellular matrix integrity. At 44 h APF, pupal testes with myoblast-specific sqh knock-down were populated with fewer nascent myotubes than wild-type testes . However, the testis shape was not affected at this time of development.As an example, we first analyzed adult s (Trol) , which is (Trol) . ExpressTaken together, these data led us to conclude that non-muscle myosin II is required for testis myotube migration and that correct shaping of the testis depends on the presence of an intact tight muscle sheath.DWnt2 alleles in trans . By contrast, we observed very drastic defects when the FGFR Htl was knocked down. Specifically, all flies were pharate lethal, and most testes were not attached to the seminal vesicles and did not elongate . This might be due to myoblast determination defects, since htl knock-down genital discs 24 h APF contained fewer myoblasts than wild-type genital discs 24 h APF. This was especially the case in the posterior part of the genital discs, where myoblasts for the muscle sheaths of the ejaculatory duct and sperm pump are localized . At 30 h APF, prospective seminal vesicles of htl knock-down genital discs contained fewer nascent myotubes than the wild type . Myoblast-specific expression of a dominant-negative (DN) version of Htl lacking the kinase domain . The adult testes were elongated and coiled, but had bulky tips as observed with the DN-version of Htl . The testis muscles covered the organs except for the tips . To gain further insight into which FGF ligand activates Htl in nascent myotubes, we analyzed the testis shape and musculature of hypomorph e02026ths/Df(2R)ths238 mutants. Adult testes had approximately two coils and a very bulky tip . After stumps knock-down – in contrast to the htl knock-down  – we observed many myoblasts lying over the seminal vesicles . Stumps expression on htl knock-down genital discs 24 h APF remained unchanged in testis-relevant myoblasts  in our analysis, which resembles the Htl-DN  phenotype and the thisbe hypomorph mutant and thus had muscles on the testes. As RNAi-mediated knock-down of Stumps  was very efficient , we focused on stumps in our analysis in parallel to the htl-RNAi line BL35024.So far, we gained evidence for Heartless-dependent migration of nascent myotubes along the testes and showed that knock-down of Cad-N G-G′ and We observed Cad-N expression in nascent myotubes migrating along the testis and in the mature musculature D-E. We ahtl-RNAi knock-down situation, however, nascent myotubes were less densely packed and had started to stretch while encircling the testis tube. In this situation, Sqh expression was not obviously disturbed  resulted in adult testes that were not fully covered with muscles, which indicated that non-muscle myosin II is necessary for the migration of nascent testis myotubes. Myosin II is also required for proper detachment and migration of border cells during egg chamber development  After myoblast fusion, when Stumps and Cad-N are distributed along the plasma membrane of nascent testis myotubes that are in close contact to the adjacent epithelium of the prospective seminal vesicles, FGF signaling initiates collective migration of nascent testis myotubes from the prospective seminal vesicle onto the testis B. (ii) TWe propose that the ligand Ths and possibly also Pyr binds Htl in nascent myotubes and thereby activates FGF signaling via Stumps during both phases of migration; the source of these ligands needs to be determined. Downstream, components of MAPK signaling, such as Erk, could be activated, which might cause changes in transcription of different target genes. Indeed, we observed Htl-dependent expression of Sqh. We hypothesize that Thisbe/Heartless-induced MAPK signaling might link to the actin cytoskeleton via non-muscle myosin II, thereby facilitating cytoskeletal changes needed for cell migration B.Our data provide first insights into the Thisbe/Heartless-dependent regulation of myotube migration via myosin II expression from the seminal vesicle to and along the testes, which is dependent on successful connection of these tissues. Future studies aiming at identifying missing components and functions to fully elucidate testis myoblast specification and myotube migration will require the establishment of more specific tools, such as driver lines specifically active in testes myoblasts and myotubes.1118 (BL6326) was used as the wild-type reference. The following transgenic flies were used: Mef2-Gal4 , UAS-arm-RNAi , UAS-sqh-RNAi , UAS-zip-RNAi , UAS-btl-RNAi , UAS-htl-RNAi , and UAS-stumps-RNAi . We conducted all RNAi experiments with at least two different fly lines per gene; the differences were in knock-down efficiencies or effects of second site insertions, as described in Table S1. The ths mutant was generated by crossing e02026ths/CyO.actin-GFP (Df(2R)ths238/CyO.actin-GFP , and v flies were obtained from Vienna Drosophila RNAi Center .Flies were kept and RNAi crossings were carried out on standard medium at 25°C. wef2-Gal4 , Htl-Galctin-GFP  and Df over seven days. After two weeks, the efficiency of the matings was examined. All fertility tests were carried out at 25°C.One adult male of the F® 488 , anti-rabbit DyLight 488 , anti-rabbit DyLight 549 . For visualization of F-actin, we used Phalloidin-Atto 565 ; to visualize nuclei, Hoechst 33342 was used .Pupae selection, genital disc dissection, and immunofluorescence were carried out as described elsewhere  and were35mm,high  containing 2 ml culture medium Shields and Sang M3 Insect Medium  was made according to −1 Ecdysone . Culture medium worked successfully for up to 4 days when stored at 4°C. A Zeiss AxioObserver Z.1 inverse microscope was used for live imaging. A Z-stack was acquired every 15 min for up to 10 h.Male white prepupae were collected at 0 h APF and aged on a moistened filter. Genital discs and pupal testes were dissected in culture medium and transferred into a tissue culture treated µ-DishConventional fluorescent images and optical sections were captured with a Zeiss AxioObserver Z.1 inverse microscope with ApoTome function. Images were taken and processed with AxioVision LE64 , figures were assembled with Adobe Photoshop CS6 , and models were generated in Adobe Illustrator CS6 (Adobe Systems Incorporated). Charts were generated in Microsoft Excel 2016 .


No Abstract available. We describe the incidental postmortem findings in an 83-year-old female brain donor. She had a history of stroke, from which she recovered ad integrum. The brain showed mild Alzheimer’s disease changes with a mild to moderate density of neuritic plaques, capillary amyloid angiopathy, mild τ-positive neurofibrillary pathology , and mild age-related temporo-medial astrogliopathy (ARTAG) with subventricular thorn-shaped astrocytes. At the level of the thoracic spinal cord there was a small, well delineated nodular lesion within the posterior root A in an eSchwannomas, also known as neurinomas or neurilemmoma, are homogeneous and slowly growing benign tumors of the nerve sheath, originating from Schwann cells . They ocThe authors report no conflict of interest.


Cancer is caused by uncontrollable growth of neoplastic cells, leading to invasion of adjacent and distant tissues resulting in death. Cancer cells have specific nutrient(s) auxotrophy and have a much higher nutrient demand compared to normal tissues. Therefore, different metabolic inhibitors or nutrient-depleting enzymes have been tested for their anti-cancer activities. We review recent available laboratory and clinical data on using various specific amino acid metabolic pathways inhibitors in treating cancers. Our focus is on glutamine, asparagine, and arginine starvation. These three amino acids are chosen due to their better scientific evidence compared to other related approaches in cancer treatment. Amino acid-specific depleting enzymes have been adopted in different standard chemotherapy protocols. Glutamine starvation by glutaminase inhibitior, transporter inhibitor, or glutamine depletion has shown to have significant anti-cancer effect in pre-clinical studies. Currently, glutaminase inhibitor is under clinical trial for testing anti-cancer efficacy. Clinical data suggests that asparagine depletion is effective in treating hematologic malignancies even as a single agent. On the other hand, arginine depletion has lower toxicity profile and can effectively reduce the level of pro-cancer biochemicals in patients as shown by ours and others’ data. This supports the clinical use of arginine depletion as anti-cancer therapy but its exact efficacy in various cancers requires further investigation. However, clinical application of these enzymes is usually hindered by common problems including allergy to these foreign proteins, off-target cytotoxicity, short half-life and rapidly emerging chemoresistance. There have been efforts to overcome these problems by modifying the drugs in different ways to circumvent these hindrance such as (1) isolate human native enzymes to reduce allergy, (2) isolate enzyme isoforms with higher specificities and efficiencies, (3) pegylate the enzymes to reduce allergy and prolong the half-lives, and (4) design drug combinations protocols to enhance the efficacy of chemotherapy by drug synergy and minimizing resistance. These improvements can potentially lead to the development of more effective anti-cancer treatment with less adverse effects and higher therapeutic efficacy. Uncontrollable cellular proliferation, invasion and metastasis are the characteristics of cancer cells. Due to the absence of cellular contact inhibition, cancer cells can form a huge mass (as in solid tumors) and also migrate to other parts of the body through either blood or lymphatic circulation . In hemaWhen mammalian cells experience amino acid starvation, they would undergo a homeostatic response to amino acid shortage . BrieflyMYC-mediated apoptosis [Glutamine is a non-essential amino acid, which means it can be synthesized within human cells. Cancer cells are known to be having enhanced glutaminolysis (conversion of glutamine into glutamate), suggesting glutamine is a very important nutrient for cancer cells . Glutamipoptosis . Glutamipoptosis , 36. TheAlthough the importance of glutamine metabolism in cancer has been known for decades, the development of glutamine depletion agents was just started in recent decades. The key concept of glutamine starvation on cancer cells is to stop the cancer cells from obtaining glutamine. The current development of drug targeting on glutamine metabolism of cancer cells focuses on glutamine depletion, glutaminase inhibition, and membrane glutamine transporter inhibition –41.Glutamine depletion was reported effective in suppressing some cancer growth or enhancing cancer-killing activity of immune system . The mosOther than glutamine depletion, inhibition of glutamine transporter was also studied for anti-cancer drug design, up-regulated glutamine transporter targets SLC1A5/38A2 are recently reported in different cancer types , 49. TheAdvanced development of glutamine starvation approach has been focusing on the specific glutaminase inhibitors, 968, BPTES and CB-839, in which the anti-cancer activity have been studied in vitro or in vivo , 53–59. Like any other chemotherapeutics, cancer cells may have resistance to glutaminase inhibitors. There are two glutaminase inhibitor resistance mechanisms reported , 65. OneAsparagine is a non-essential amino acid for normal human cells, which means that human cells can synthesize asparagines by themselves. Inside the cells, asparagine is the precursor of aspartate for conversion into malate as a tricarboxylic acid cycle intermediate for respiration or acts as a neuro-transmitter in neuro-endocrine tissues . AsparagE. coli L-asparaginase which had the same anti-tumor activity as guinea pig serum [The study of asparagine metabolism inhibition for cancer treatment can be traced back to four decades ago. It had been known that leukemia and lymphoma require asparagine for growth in cell culture due to absence of ASNS expression , 72. A sig serum . Such brig serum . Currentig serum , 44. L-aig serum . There hig serum , 77. L-aig serum . L-asparig serum . Even thig serum . In an eig serum . Combinaig serum . Currentig serum , 83.Despite high remission rate, relapse is still common in ALL patients. L-asparaginase resistance is one of the important reasons for relapse. There are different resistance mechanisms reported in both pre-clinical and clinical studies. Normal and leukemic lymphoblasts may degrade L-aspraginase and potentiate antigen processing, leading to immune reactions against L-asparaginase . ProductE. coli L-asparaginase, pegylated E. coli L-asparaginase and native Erwinia chrysanthemi L-asparaginase [E. coli or Erwinia chrysanthemi. Pegylated form of E. coli L-asparaginase involved the addition of polyethylene glycol group into the native enzyme that can reduce immunogenicity and prolonged half-life (1.24 day for native and 5.73 days for pegylated formulation) [E. coli L-asparaginase may cross-react with pegylated E. coli L-asparaginase [Erwinia chrysanthemi L-asparaginase is an alternative to E. coli-origin L-asparaginase. It may induce less complications and toxicities  than L-asparaginase in leukemia patients [E. coli vs 0.65 day for Erwinia) of Erwinia L-asparaginase may be much shorter than that of E. coli L-asparaginase [E. coli L-asparaginase would not cross-react with Erwinia L-asparaginase [Erwinia L-asparaginase is preferred for patients with allergy to E coli L-asparaginase [Erwinia arginase is also developed recently for improving the clinical efficacy of Ewinia L-asparaginase [E. coli L-asparaginase are still the first-line drug while Erwinia L-asparaginase is used when patients show allergic responses to native and pegylated E. coli L-asparaginase.Not only drug resistance to L-asparaginase is an issue, therapy related side effect is another problem. Glutamine depletion is a common side effect of L-asparaginase due to its dual asparaginase and glutaminase activity. Glutamine depletion may cause acute pancreatitis, thrombotic complication, and immune-suppression –46. The raginase . These tulation) . Howeverraginase . This mapatients , 103. Horaginase . It was raginase . Thereforaginase . Pegylatraginase . But, itraginase . After aIn summary, L-asparagine depletion approach by L-asparaginase treatment may suppress cancer growth in different cancer types under pre-clinical settings. Exploring the chemo-resistance mechanism(s) of cancer cells to L-asparaginase may help in designing or improving multi-drug chemotherapy protocols. After weighing the therapeutic benefits and the adverse drug effects, L-asparaginase seems to be clinically beneficial only in treating selected hematologic malignancies. To reduce the side effects and optimize the benefits on patients using L-asparaginase, it is important to develop versions of L-asparaginase with longer half-life, low-immunogenicity, and low glutaminase activity. Allergy test before treatment and monitoring of blood anti-asparaginase antibodies level during treatment may also help to manage the complications caused by the use of L-asparaginase. Resolving the problem of anaphylaxis may allow the use of L-asparaginase in treating a broader spectrum of cancer types. To resolve the problem of immunogenicity and anaphylaxis-related complications, formulation of human-derived L-asparaginase may be a solution in the future.Arginine is a versatile semi-essential amino acid. It can be synthesized from the body using glutamine, glutamate and proline in adult but not childhood . Dietaryhttp://www.proteinatlas.org). Responses of cancer types sensitive to different therapeutic arginine-depleting agents will be discussed in Tables The role of arginine in cancer has been explored for quite a long time. Way back in the 50s, arginine had been shown to have a bipolar effect in either growth stimulation (for large tumors) or inhibition  in different rat sarcoma models . This suADI breaks down arginine intro citrulline, cells without ASS-1 expression may encounter arginine starvation upon arginine deiminase treatment. Arginine deiminase is derived from micro-organisms and human cells do not produce such enzyme . As a foThere have been clinical trials of pegylated arginine deiminase in melanoma, hepatocellular carcinoma, mesothelioma, and some other advanced cancers patients , 139. Inlow sarcoma in vivo model reported, ADI-PEG20 showed significant reduction in tumor growth compared to control, but a slow increase in tumor size was still observed over time and the tumor lysate showed significantly enhanced ASS1 expression [Other than neutralizing antibody production in patients receiving ADI-PEG20 treatment, there are other intrinsic ADI-PEG20 resistance mechanisms found in cancer cells discovered in pre-clinical studies. Autophagy and enhanced ASS1 expression are known to be associated with ADI-resistance , 144. Inpression . In the pression . The autpression . Such fiSome other conditions like hypoxia and enhanced glutaminolysis may induce ASS1 expression in ASS1-ve cancer cells , 146. A In another recent study on mesothelioma, a weak point of ASS1-deficiency was reported . In the Interestingly, there may be conditions that ASS1 does not re-express even upon arginine depletion by ADI-PEG20 . In a BRAnother arginine depleting agent under clinical trial is pegylated arginase I. Pegylated arginase I breaks down arginine to ornithine. Pegylated arginine deiminase targets cancer cells defective in ASS1 expression, while pegylated arginase I targets a broader spectrum of cancer cells, which are defective in OTC and/or ASS1 expression. Pegylated arginase I has advantage over the native human arginase because pegylated arginase I have extended half-life from a few hours to 3–4 days . There aJust like pegylated arginine deiminase, pegylated arginase I may induce growth suppression and probably autophagy and/or apoptosis, depending on cancer types. There are pre-clinical data showing pegylated arginase I may show stronger anti-cancer effect in combination with other chemotherapeutic agents. Pegylated arginase I and 5-flurouracil showed more potent tumor suppression than pegylated arginase I alone in a subcutaneous hepatocellular carcinoma mouse model . PegylatThere is one clinical trial of pegylated arginase I on hepatocellular carcinoma. The form of pegylated arginase I used is the pegylated formulation without modification of prosthetic group. In the 8 week study of advanced hepatocellular carcinoma patients, patients taken pegylated arginase I displayed mainly grade 1 and grade 2 toxicities without severe adverse effects I. These adverse effects disappear after discontinuing pegylated arginase I. Unlike patients treated with pegylated arginine deiminase, no neutralizing antibodies were detected in sera of patients receiving pegylated arginase I. Throughout the 8 weeks of study, arginine depletion was found sustainable in all patients treated with pegylated arginase I, apparently the patients did not develop resistance to pegylated arginase I. However, due to the insufficient patient samples as well as the relatively advanced stage of the disease being treated, there is no definite conclusion on the treatment response of the patients to pegylated arginase I . The lowE. coli [Apart from ADI-PEG20 and pegylated arginase I, there are some other formulations recently developed for arginine depletion therapy. They are the ADI-cell penetrating fusion protein complex, engineered arginase-fc protein and arginine decarboxylase. Unlikely pegylated arginase I, ADI-PEG20 is poorly internalized in cells and it mainly allows extra-cellular arginine depletion, therefore ASS1-expressing cells may be resistant to ADI-PEG20 , 154. ADE. coli –159. ArgE. coli , 159. ThIn summary, arginine depletion may be a feasible approach for cancer treatment because of the anti-cancer effects reported in pre-clinical studies, the low adverse reactions and the treatment-achieved stable disease in clinical trials of advanced stage cancers. However, the choice of arginine depleting agent for such use may require further clinical investigation. A therapeutically useful arginine depleting agent for cancer treatment should have low toxicity, non-immunogenic  and fast-acting (to delay emergence of drug resistance) with long circulation half-life (to achieve sustained arginine depletion). Furthermore, there is no study showing complete remission of tumor by using arginine depletion alone. This suggests the possibility of emergence of resistance to arginine depletion, probably by re-expression of ASS1 or autophagy. For the better benefit of patients, it is important to investigate the resistance mechanisms of arginine depletion and also the potential synergy of arginine depletion with other drugs to maximize therapeutic efficacy and minimize the therapy related toxicity.With different approaches in interrupting amino acid metabolism in cancer cells, enzymatic depletion strategy is the most well-studied and promising. The hydrolytic enzymes can extensively eliminate the target amino acids to ensure specific amino acid depletion in blood circulation. With the previous research outcome mentioned above, we come up with several conclusions. Firstly, among all the glutamine metabolism inhibition treatments, glutaminase inhibition is apparently feasible due to the high specificity, efficacy and well tolerance shown in pre-clinical studies and clinical trials. The side effects of glutamine depletion using hydrolytic enzymes are too severe, and there isn’t glutamine-specific hydrolytic enzyme yet, making it currently not a good approach for treating cancer patients. Glutamine transporter inhibitors currently developed require too high dosage and development of transporter inhibitors with higher potency may be required. Secondly, asparaginase depletion using L-asparaginase has been a very effective approach in treating hematologic malignancies with detailed toxicity profile and well-studied resistance mechanism. L-asparaginase demonstrated anti-cancer function even to solid tumors. However, the usage of L-asparaginase is currently limited to hematologic malignancies as the side effects may out-weight the benefits especially in adult patients. These side effects are mainly due to the anaphylaxis induction and off-target glutamine depletion in clinical use. Thirdly, although pegylated arginine deiminase and pegylated arginase I are both arginine depletors, arginine deiminase is foreign to human body and may not achieve sustainable arginine depletion in blood circulation. Further modification on ADI-PEG20 or proper drug combination may help overcoming the antibody-induced resistance to ADI-PEG20 and allow sustainable plasma arginine depletion. Pegylated human arginase I, though lacking clinical data on efficacy over a broad range of cancer types, it shows good safety profile and sustainable arginine depletion in patients. This suggests pegylated arginase I will be a suitable candidate for anti-cancer therapy, but more research is required to explore the potential use of pegylated arginase I in treating different cancer types.In the future design of amino acid starvation treatment against cancer, there are several important aspects to consider. Firstly, the auxotrophy of cancer cells to particular amino acid(s) should be thoroughly investigated for the choice of amino acid target. The most ideal target will be an amino acid auxotrophic to cancer cells but not normal cells, and at the same time the amino acid is very important for cancer growth. Next, the enzyme is preferably of human origin. This may reduce the anaphylactic response and also antibody reaction. The use of human originated enzymes may help to reduce the side-effects and also drug resistance due to autoantibodies. Furthermore, the enzyme for amino acid depletion should be pegylated to prolong the half-life and reduce the immunogenicity. This may increase the durability of the enzyme in patients’ circulation to allow sustainable amino acid depletion. The side-effects due to anaphylaxis may also be reduced. Last but not least, amino acid depletion should combine with other chemotherapeutic agents to enhance efficacy. Multi-drug protocols should be designed to provide effective and safe treatment regimen based on their complementary mechanisms.