MGMT, RAD17, and USP44) show prior evidence of a tumour suppressive function. Other candidate tumour suppressors, such as MAFTRR, KIAA1551, and IGF2BP2, are novel. Our study demonstrates how rare tumour suppressors can be identified through copy number meta-analysis.Homozygous deletions are rare in cancers and often target tumour suppressor genes. Here, we build a compendium of 2218 primary tumours across 12 human cancer types and systematically screen for homozygous deletions, aiming to identify rare tumour suppressors. Our analysis defines 96 genomic regions recurrently targeted by homozygous deletions. These recurrent homozygous deletions occur either over tumour suppressors or over fragile sites, regions of increased genomic instability. We construct a statistical model that separates fragile sites from regions showing signatures of positive selection for homozygous deletions and identify candidate tumour suppressors within those regions. We find 16 established tumour suppressors and propose 27 candidate tumour suppressors. Several of these genes (including  MAFTRR, KIAA1551, and IGF2BP2.Homozygous deletions are rare in cancers and often target tumour suppressor genes. Here, the authors conduct pan-cancer analyses and apply statistical modelling to identify 27 candidate tumour suppressors, including  Many of these somatic changes are neutral passenger events, yet some confer a clonal selective advantage on cancer cells and drive oncogenesis1. The genes involved can be oncogenes, which are frequently hit by activating point mutations or amplifications and behave in a dominant fashion, or tumour suppressor genes, which are recurrently targeted by inactivating mutations such as truncating point mutations or deletions and display a recessive pattern4.The genomes of cancer cells are shaped by somatic mutations, including base substitutions, small insertions and deletions, genomic rearrangements, and copy number changes5.The most frequently used approach to identify cancer genes is assessing recurrence of non-synonymous somatic point mutations above background level. This is an extensive field that has been highly successful in the past. The power to detect cancer genes varies based on sample size and background mutation frequency: for most tumour types, current sample sizes are inadequate to reliably detect rare cancer genes mutated at ≤ 5% above backgroundHomozygous deletions require two independent hits and, in addition, any homozygous deletion covering a gene that carries out an essential function or confers a survival advantage is swiftly eliminated by negative selection. Therefore, homozygous deletions are rare and often focal in cancers. We reasoned that a systematic screen for homozygous deletions in a large series of cancer samples would be a powerful orthogonal way to specifically identify tumour suppressors, hypothesising that some tumour suppressors may be more prone to inactivation by homozygous deletions than by truncating point mutations. As traditional recurrence analysis has identified most or all frequently mutated tumour suppressors, our novel candidates are likely rare cancer genes, inactivated only in specific contexts.6. However, this model system has important limitations: cancer cell lines are biased towards late stage cancers and metastases, they have accumulated mutations to adapt to growth in culture, their numbers are limited, and some cancer types do not lend themselves to growth in culture7.Tumour samples contain both tumour cells and admixed normal cells in unknown proportions, complicating the distinction between homozygous and hemizygous deletions and hampering the discovery of tumour suppressors. Cancer cell lines represent a simplified model system that does not show this normal cell admixture and a comprehensive catalogue of homozygous deletions in cancer cell lines has been constructed8. This prompted the development of several computational methods and mathematical models, leading to mature approaches that can infer tumour purity and ploidy and separate copy number profiles from tumour cells and admixed normal cells12, opening novel avenues for studying tumorigenesis and tumour evolution13. These methods are able to reliably separate homozygous from hemizygous deletions, and have been able to pinpoint driver homozygous deletions in known and newly identified tumour suppressors4.The advent of SNP array technologies brought the realisation that it is theoretically possible to infer the fraction of admixed normal cells in primary tumours from array dataHere, we perform a systematic screen for homozygous deletions over a compendium of 2218 SNP arrays across 12 cancer types, aiming to identify rare tumour suppressors. We find 96 genomic regions recurrently targeted by homozygous deletions, overlapping 16 established tumour suppressors. However, homozygous deletions also occur frequently over fragile sites, chromosomal regions of increased genomic instability. We therefore construct a novel statistical model to separate fragile sites from regions containing tumour suppressors. This analysis extends the landscape of cancer genes by identifying 27 candidate tumour suppressors, adding to the emerging evidence for several tumour suppressor genes recently proposed in the literature and highlighting several novel candidates.10 to infer tumour purity and ploidy and derive copy number profiles. ASCAT failed on 81 samples (3.7%) due to excessive noise, and these samples were excluded from further analyses. Of the 2137 cases that passed ASCAT analysis, 273 (12.8%) showed only few or no copy number aberrations, resulting in high variance tumour purity estimates. These samples were therefore excluded from the analysis of tumour purity and ploidy. As expected, the proportion of non-aberrant samples varied considerably between tumour types, with leukaemia showing the highest proportion of non-aberrant cases (55.6%), followed by lymphoma (22.5%) and sarcoma (20.1%)  and the highest in brain cancers (70%). These findings are broadly consistent with previous ploidy measurements from chromosome counts14, as well as with a previous SNP array study on an overlapping series of samples12.Most cancer types displayed extensive variation in purity and ploidy Fig. . All tumWe performed a systematic screen for homozygous deletions across our 2137 primary tumours that passed ASCAT analysis. We identified 1865 homozygous deletions in total (median length: 315 kb), involving 826 tumours, each having on average 2.0 Mb (median 524 kb) homozygously deleted , while the size distribution of the deletions is the same for both . CDKN2A was the dominant homozygously deleted tumour suppressor, with 108 homozygous deletions across nine cancer types. Virtually all of these homozygous deletions inactivate the adjacent “backup” tumour suppressor CDKN2B as well, rationalising the frequent loss of the combined locus17. Homozygous deletions of CYLD and BIRC3 (cIAP2) are found exclusively in multiple myeloma and are both linked to aberrant NF-κB signalling18. All BIRC3 deletions include its adjacent homologue BIRC2, consistent with previous observations that only loss of both was shown to induce alternative NF-κB activation in B-cells19. Similarly, we observed six homozygous deletions over VHL, five of which take out the nearby tumour suppressor FANCD2 as well. We detected 16 homozygous deletions over PTEN in multiple cancer types and 5 homozygous deletions over NF1. Homozygous deletions of RB1 were most frequently found in tetraploid sarcomas. We further observed four homozygous deletions each over TP53 (three sarcomas), CDKN2C, and FAT1, and six homozygous deletions each over MAP2K4 (three breast carcinomas) and CDH1 (five lung cancers). Homozygous deletions of SMARCB1 are found only in brain cancers, while homozygous deletions of TET1 were almost exclusively observed in lung cancer (seven out of eight cases). Interestingly, the four homozygous deletions over BRCA2 included two sarcomas, a cancer type not previously linked to BRCA2 mutations.We observed 15 peaks of homozygous deletions over 16 established tumour suppressors  fragile sites Fig. .6. Large hemizygous deletions, in contrast, are due to several other mechanisms, such as whole-chromosome or whole-arm loss6. As a result, fragile sites are characterised by frequent small deletion events, while regions containing tumour suppressors more frequently show large deletion events6. This difference was exploited to construct three metrics that discriminate fragile sites from regions containing tumour suppressors  the ratio of homozygous deletions to small hemizygous deletions over the peak ; and (R2) the ratio of large hemizygous deletions to small hemizygous deletions . Indeed, both ratios are significantly larger for the identified known tumour suppressors when compared to the known fragile sites . The densities of R1 and R2 for fragile sites (or tumour suppressors) can be estimated via a resampling and simulation approach using the known sites, which allows calculation of p-values for any peak under a fragile site null model (Methods). In addition, we leveraged tumour-type specificity as a third element in our statistical model. Tumour-type specificity of homozygous deletions can be explained either by selection or by increased genomic instability in the originating tissue or cell type. However, higher genomic instability in a certain tissue or cell type would result in enrichment of both homozygous and small hemizygous deletions in a certain tumour type. Therefore, we can use differences in tumour-type specificity between homozygous and small hemizygous deletions as an indication that tissue-specificity of homozygous deletions is driven by selection , do we reject the null model and consider the presence of a tumour suppressor.Homozygous deletions over tumour suppressors are enriched due to positive selection, whereas homozygous deletions over fragile sites are enriched due to a local increase in genomic instability. Consequently, the structural signature of deletions is distinct in fragile sites, compared to regions harbouring tumour suppressors. Small hemizygous deletions reflect local fragility, as they require two DNA breakage events in close proximityors Fig. . The firs); and R the ratiVHL/FANCD2 and CDKN2C being the exceptions. Notably, VHL shows frequent biallelic inactivation by a hemizygous deletion combined with a point mutation, while homozygous deletions are rare. A possible reason is that for some tumour suppressor genes, homozygous deletions, which often also affect neighbouring genes or regulatory regions, are selected against, whereas point mutations may be better tolerated. Alternatively, a protein may have multiple functions, and while biallelic deletions abolish all functions, point mutations may abolish one while leaving others intact. Two of the established tumour suppressors, CDH1 and MAP2K4, fall within known fragile sites FRA16B and FRA17A, respectively. In both cases, however, our model is able to pick up the signature of positive selection due to a strong signal of tumour-type specificity for CDH1 and a high ratio of large to small hemizygous deletions for MAP2K4.Our model is able to reject local fragility for 13 of the 15 peaks containing known tumour suppressors Table , VHL/FAN6, as well as 9 (sub)telomeres showing enrichment of both homozygous and hemizygous deletions telomeric losses show more complex rearrangements compared to cases that do not show such losses  scores24; Fig. Hemizygous and homozygous deletions in these (sub)telomeric regions represent the signature of a prior telomere crisis triggering genomic instability. Ongoing cell division in the absence of telomerase expression leads to telomere attrition and subsequent removal of subtelomeric sequences. The exposed chromosome ends are often resolved by end-to-end chromosome fusions, with further loss of subtelomeric sequences25 and with scientific literature, aiming to identify candidate tumour suppressors. Two regions were intergenic, and for four additional regions the targets remain unknown , as well as novel candidates .In addition to the 15 regions containing established tumour suppressors, our model identified 32 regions that are unlikely to be explained by local fragility, showing a signature of positive selection , a suggested tumour suppressor gene that encodes a protein component of the BRCA1–A complex that leads the BRCA1-BARD1 heterodimer to sites of DNA double-strand breaks, targeting these for homologous recombination44. We therefore hypothesise that homozygous deletions of this region on chromosome 4 coordinately inactivate two tumour suppressor genes, involved in different DNA damage repair pathways.Our screen identified six genes involved in DNA damage response and repair. CASP3 and CASP9, or regions close by, in various cancer types. Both genes encode pro-apoptotic and anti-apoptotic splice isoforms of the respective cysteine/aspartic acid proteases46. CASP8, another member of the caspase family, is a tumour suppressor in breast cancer4. The pro-apoptotic isoforms of caspase-8 and caspase-9 induce apoptosis through cleavage of caspase-3. It is therefore likely that perturbation of any of these three caspase genes may abrogate apoptosis. We detected a peak of homozygous deletions in lung cancer close to SETD1B, a member of a family of histone methyltransferases that also includes SETD2, known to be involved in renal carcinoma. SETD1B specifically methylates H3K4, thereby playing a role in epigenetic control of transcription. We identified several large and smaller homozygous deletions targeting ARHGEF10, a Rho guanine nucleotide exchange factor regulating mitotic spindle formation that has been proposed as a tumour suppressor48. Interestingly, we also observed homozygous deletions affecting its closest paralog ARHGEF10L, suggesting that both genes may be tumour suppressors.We also find candidate tumour suppressors that are related to known cancer genes. We detected homozygous deletions affecting PDE4D, encoding phosphodiesterase 4D. PDE4D has previously been reported to be targeted by internal microdeletions that are hypothesised to function as tumour-promoting factors49. Therefore, these homozygous deletions may represent gain-of-function rather than loss-of-function mutations.Some homozygous deletions may drive oncogenesis through other mechanisms than inactivation of a tumour suppressor gene. We observed intragenic deletions of LEPROTL1, KIAA1551, MIDEAS, MAFTRR, and IGF2BP2. Together with its homologue Leptin Receptor Overlapping Transcript (LEPROT), LEPROTL1 negatively regulates leptin receptor surface expression and thus the response to leptin, a pleiotropic hormone51. Lung tissues both produce and respond to leptin, and leptin is required for proliferation of various non-small cell lung cancer cell lines, at least in part due to activation of downstream Notch and JAK/STAT signalling52. Four homozygous deletions in lung cancer suggest a role for LEPROTL1 in keeping this feedback loop in check in normal lung cells. MIDEAS is part of a recently discovered class I histone deacetylase complex, dubbed MiDAC (Mitotic Deacetylase Complex)53. The complex associates with cyclin A and is upregulated in cells blocked in mitosis54. While the co-repressor MIDEAS couples inositol phosphate signalling to activation of histone deacetylation, its precise role in cell cycle regulation is still unknown55. Two homozygous deletions each in myeloma and sarcoma targeted MAFTRR , a long intergenic noncoding RNA gene. MAFTRR was recently shown to recruit chromatin modifiers LSD1 and EZH2 to the upstream MAF oncogene in a long-distance chromatin interaction, downregulating its expression56. MAF overexpression is a frequent oncogenic event in multiple myeloma, stimulating cell cycle progression and altering bone marrow stromal interactions57. Note that one homozygous deletion in sarcoma abolishes both MAF and MAFTRR, suggesting MAFTRR may still have other functions. Aside from MAFTRR, we identify three additional lncRNA genes as potential tumour suppressors: LINC01060, LINC00375, and LINC00662. Unfortunately, their precise functions are yet to be elucidated. IGF2BP2  encodes a post-transcriptional modulator implicated in mRNA localisation, stability, and translational control. It is part of the frequently aberrated IGF/PI3K-AKT/mTOR pathway and controls the translation of mitochondrial mRNAs59. Depletion of IGF2BP2 decreases oxygen consumption while increasing mitochondrial mRNA translation and possibly mitochondrial biogenesis58. Our observation of a peak of homozygous deletions suggests it could act as a rare tumour suppressor, controlling a switch in cancer nutrient and energy metabolism.Several peaks of homozygous deletions point to genes not previously implicated as tumour suppressors, e.g., In this study, we aimed to identify rare tumour suppressors through a systematic pan-cancer analysis of homozygous deletions in primary tumours. Our screen detected 16 established tumour suppressors, 3 immune regions, 15 known (named) fragile sites, 24 additional intrachromosomal fragile sites, 9 regions of telomeric instability, and 32 regions showing signatures of positive selection for homozygous deletions . Particularly in kidney cancer, homozygous deletions are very rare: we only detect 25 in 73 cases, 3 of which overlap PTPRD.We developed a statistical model to test if enrichment of homozygous deletions in a region can be explained by local fragility alone, or whether there may be positive selection Fig. . As illuDMD (encoding dystrophin), we observe seven in sarcomas. Although DMD is the largest gene in the human genome and is part of known fragile site FRAXC, this suggests homozygous deletions of DMD may play a role in sarcoma. Indeed, DMD was recently validated as a tumour suppressor and anti-metastatic factor in myogenic sarcomas60 and dystrophin/dysferlin double mutant mice show a high incidence of rhabdomyosarcoma61. We also observe a peak of homozygous deletions targeting MXRA5, a poorly studied gene that was recently suggested to be frequently mutated in non-small cell lung carcinoma62. While we are unable to reject local fragility as the underlying cause of both peaks, this may be due to lack of power, and further studies are needed to conclusively determine if these genes play a tumour suppressive role in some cancers.Our model is considerably more conservative on the X chromosome (see Methods) and we do not infer any tumour suppressors on X. Out of eight homozygous deletions affecting 63 to identify recurrent focal gains and losses64, some of which could be attributed to oncogenes and tumour suppressors. Due to our strict focus on homozygous deletions rather than focal losses in general, we aim to identify tumour suppressors recurrently targeted by two independent copy number changes. This approach is not well suited to identify haploinsufficient tumour suppressors or tumour suppressors showing mainly recurrent point mutations combined with copy number losses of the other allele, as discussed above for VHL. However, both our study (on 2218 cancers) and the Zack et al. study on a larger number of cases (4934) identify the same 13 known tumour suppressors , suggesting our focused approach is competitive at identifying tumour suppressors.A previous pan-cancer study performed GISTIC analysisTP53, found mutated in a very high proportion of cases across cancer types, but homozygously deleted in less than 0.2% of cancers in this study. Other known tumour suppressors, such as RB1 and PTEN, can readily be inactivated by both homozygous deletions and inactivating point mutations . Adjacent tumour suppressor pairs  may be especially rewarding targets for homozygous deletion in some cases, as two mutation events can abolish all tumour suppressor activity. We conjecture that this study identifies a class of predominantly rare tumour suppressors, such as CPEB3 and MGMT, that are more prone to be inactivated by homozygous deletions than point mutations, a proportion of which therefore may not be readily identifiable through mutation analysis given current sample sizes.Our results provide a view on the landscape of tumour suppressors that is complementary to sequencing screens for recurrent single-nucleotide substitutions and small insertions and deletions. Many genes frequently targeted by inactivating mutations in cancers do not seem to be frequently targeted by homozygous deletions. This is exemplified by 65, the MetaCGH database66, and from literature searches, with the aim of collecting all studies of primary tumours performed on Affymetrix 250K StyI arrays with publically available raw data. All pre-cancerous lesions and metastases were excluded. Pairwise comparison of all samples with Pearson correlation was used to identify duplicate samples (Pearson correlation above 0.8), in which case duplicates were removed. Samples were stratified into 12 broadly defined tumour types: breast cancer, ovarian cancer, colorectal cancer, hepatocellular carcinoma, renal cancer, lung cancer, cancers of the brain and nervous system, oesophageal cancer, sarcoma, multiple myeloma, leukaemia and lymphoma, each containing >50 samples. Other tumour types with fewer samples available were excluded. Our final data set included 2218 unique primary tumour samples from 27 different studies (Supplementary Table SNP array studies were selected from the CaSNP database67. GRCh37/hg19 probe annotation (version 32) was obtained from the Affymetrix website (http://www.affymetrix.com). Only probes that mapped to unique locations of the genome were retained. GC wave correction was performed as previously described68 and as implemented in ASCAT 2.210. Copy number analysis was performed using ASCAT10 version 2.2, with default parameters for tumour samples without matched normal data. The default value γ = 0.55 was used in ASCAT and has previously been shown to fit the Affymetrix data well. In order to remove any adverse influence of germline copy number variants (CNVs), all SNPs within known germline CNVs were removed prior to ASCAT analysis. Positions of known germline CNVs were obtained from the Database of Genomic Variants69, version hg19.v10, from which all CNVs identified on Affymetrix 500K and SNP6 platforms were selected. This reduced the number of analysed probes from 228,586 to 208,786.Total copy number (LogR) and B allele frequency (BAF) values were obtained from CEL files using PennCNV-AffyStep 1: Identification of hotspots of homozygous deletions: After ASCAT analysis, homozygous deletions were straightforwardly identified as regions having zero copies of both alleles in the tumour cells, each region extending from the position of the first probe being homozygously deleted to the last probe being homozygously deleted. Therefore, the reported positions of a homozygous deletion can be considered the minimal region that is homozygously deleted, as supported by the SNP array data of that sample. The genome was then segmented into bins of constant number of observed homozygous deletions by considering all start and end points of every homozygous deletion to be a breakpoint. Each bin thus defined was then associated with the total number of homozygous deletions over that region observed across all samples .To determine the significance of the number of homozygous deletions in each bin, a permutation test was performed. We initially reasoned that each observed homozygous deletion is the result of two distinct hemizygous deletion events, and we therefore treated both events separately in the simulations. We identified within each parental chromosome and within each sample all hemizygously and homozygously deleted regions. Each such region represents the result of a hemizygous deletion event, and a homozygous deletion occurs when two such regions overlap within a sample. Deletions were classified into two categories, “small” and “large”, and the positions of the small deletions were randomly assigned to a new position in the genome , while the large deletions were kept fixed in their original positions. For parental chromosomal deletions being part of a homozygous deletion, the small and large deletions are readily identified by comparing their sizes. For parental chromosomal deletions not being part of a homozygous deletions , a classifier was applied to determine whether the deletion was small or large. This classifier was a 2-component mixture model trained on the small and large deletions being a part of a homozygous deletion, and resulted in a probability of being a small deletion which was then used to decide whether the position of the deletion should be kept fixed or randomly drawn in the permutation. Finally, a Even with the larger deletions in fixed positions, this permutation strategy indicated that the incidence of simulated homozygous deletions was much higher than the observed rate of homozygous deletions across the whole genome Fig. . In addip-value was calculated as n/M, where n was the number of permutations resulting in at least as many homozygous deletions in the target bin as were observed, and M was the total number of permutations . P-values were adjusted for multiple comparisons by applying the Benjamini–Hochberg procedure. Bins were considered significant if their false discovery rate-adjusted p-value  ≤0.05. Neighbouring bins with a significant q-value were merged when they lay within 1 Mb of one another and when they had more than half of their underlying homozygous deletions in common . When they were separate peaks  they were kept as such. In line with this rule, non-adjacent bins were merged in the following regions: chr4:39.08–39.15, chr4:182.34–182.70, chr9:133.39–133.62, chr11:101.95–102.05 , in a total of 30,382 protein-coding/lincRNA/miRNA genes (ensemble GRCh37 release 85), we computed the probability of observing at least as many known tumour suppressors as we do (≥16) among the total number of genes falling within a 1 Mb window around the centre of an enrichment peak (1197).Step 2: Identifying peaks containing tumour suppressors: A statistical model was constructed quantifying the probability that the observed signature of deletions in a given peak  can be ascribed to local fragility alone . Each metric captures a largely orthogonal structural or biological feature of the peak, and the three p-values are combined into one final meta-analysis p-value  to small hemizygous deletions over the peak  are estimated via a resampling and simulation approach in which we sample 107 times with replacement from the known sites and simulate a number of homozygous, small and large hemizygous deletions according to a multinomial distribution n the cohort size and P-values for any given peak can then be calculated as P(R1 ≥ r1) and P(R2 ≥ r2) using the estimated fragile site density as a null model . As a third metric, we leveraged tumour-type specificity of homozygous deletions vs. small hemizygous deletions  was then used to combine these partially correlated p-values into a single p-value for each homozygous deletion peak70 and the Benjamini–Hochberg method was used for multiple testing correction. Only when the signature of deletions in a peak is unlikely to be explained by local fragility (q ≤ 0.05) do we consider the presence of a tumour suppressor in the region.The metrics above generated three R1 and R2) were not applied to the X chromosome and tumour-type specificity was assayed only on the cancers in females. Therefore, power to separate fragile sites from tumour suppressors is considerably reduced on X. FRA16B and FRA17A were excluded for density estimation of the fragile site null model as they contain tumour suppressors CDH1 and MAP2K4, respectively. To avoid infinite values of R1 and R2, a pseudocount of 0.5 was added to each of the observed counts.To avoid biases, the two structural models  noise in the signal may result in “misclassification” of a homozygously deleted array probe as being part of the adjacent segment. We computed this probability as follows. For each homozygously deleted segment and its adjacent segments on the same chromosome, we obtained robust estimates of the mean and standard deviation of the LogR signal (LogR was preferred as it is more informative than BAF in homozygously deleted regions). The probability of an array probe on the homozygously deleted segment to be wrongly assigned to the adjacent segment was then calculated as the corresponding overlap between the normal distributions of the signal of both segments. Finally, the probability of the homozygously deleted segment extending 1, 2, 3, …, array probe positions into the adjacent segments was computed using a negative binomial distribution. Results show that homozygous deletions are unlikely to extend more than two array probe positions beyond the identified minimal segments (p = 0.015).https://github.com/jdemeul/HomDels.The R code that was used to run simulations, compute statistics, and generate figures is publically available at The Affymetrix 250K StyI array data that support the findings of this study are described in the studies listed in Supplementary Table Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3


Receiving an R01 grant from the National Institutes of Health (NIH) is regarded as a major accomplishment for the physician researcher and can be used as a means of scholarly activity for core faculty in emergency medicine (EM). However, the Accreditation Council for Graduate Medical Education requires that a grant must be obtained for it to count towards a core faculty member’s scholarly activity, while the American Osteopathic Association states that an application for a grant would qualify for scholarly activity whether it is received or not. The aim of the study was to determine if a medical degree disparity exists between those who successfully receive an EM R01 grant and those who do not, and to determine the publication characteristics of those recipients.We queried the NIH RePORTER search engine for those physicians who received an R01 grant in EM. Degree designation was then determined for each grant recipient based on a web-based search involving the recipient’s name and the location where the grant was awarded. The grant recipient was then queried through PubMed central for the total number of publications published in the decade prior to receiving the grant.We noted a total of 264 R01 grant recipients during the study period; of those who received the award, 78.03% were allopathic physicians. No osteopathic physician had received an R01 grant in EM over the past 10 years. Of those allopathic physicians who received the grant, 44.17% held a dual degree. Allopathic physicians had an average of 48.05 publications over the 10 years prior to grant receipt and those with a dual degree had 51.62 publications.Allopathic physicians comprise the majority of those who have received an R01 grant in EM over the last decade. These physicians typically have numerous prior publications and an advanced degree. For many physician researchers, receiving the National Institutes of Health (NIH) R01 grant is a major accomplishment and serves as an early career milestone from which further granting opportunities arise. However, receiving one of these prestigious grants is a rarity in academic emergency medicine (EM). A 2011 study found that 18 investigators in 2010 had one of these grants despite the relatively large population of practicing emergency physicians.1In that study, however, there was no distinction made with regard to what type of degree the physician researcher held. Recently, a disparity has been noted among osteopathic and allopathic physicians who serve on editorial boards and publish manuscripts in EM.http://projectreporter.nih.gov) for “emergency medicine” as a key word for 2006 through 2016 for R01 grants. Each recipient was then categorized as either having a degree in osteopathic or allopathic medicine. We did this by completing a web-based search for the author and the author’s home institution. Secondarily, we recorded any advanced degrees for each grant recipient.After obtaining institution review board approval, we queried the NIH RePORTER search engine (http://www.pubmed.com) search using the author’s name. The author was then classified as either the first, second or last author for each manuscript.Once the grant recipient was determined to be accurate, we quantified the author’s publication activity by determining the number of peer-reviewed manuscripts published by the recipient in the10 years prior to being awarded the grant. This was accomplished by completing a PubMed  of all grant recipients, and no osteopathic physicians were awarded an EM R01 grant during the study period. Those who were categorized in the “other degree” category comprised the remainder of the grant recipients. Of the allopathic physicians awarded an EM R01 grant, 44.17% (91/206) held a dual advanced degree.In the 10 years preceding the receipt of their award, all recipients had prior research publications that were identified in PubMed. Allopathic physicians accounted for 79.32%  of the primary authors, 73.31%  of secondary authors and 73.80%  of all senior authors. On average, each allopathic physician who successfully obtained an R01 grant had 48.05 publications over the 10-year period prior to receiving the grant. Upon subgroup analysis of allopathic physicians, we found that those with a dual degree comprised 52.90%  of primary authors, 41.69% (488/1173) of secondary authors and 45.82%  of all senior authors. Allopathic physicians who held a dual degree had on average 51.62 publications in the 10 years prior to receiving their R01 grants.This is the first article to describe a medical degree disparity between those physicians who receive EM R01 grants and those who do not. Over the last 10 years, the majority of recipients of an EM R01 grant have been allopathic physicians, and no osteopathic physician has received an EM R01 grant during that same period. It is unclear why this disparity exists, but it appears that prior research publications and advanced research training play a crucial role in determining who receives EM R01 grants.Recent literature has shown that there is a disparity in medical degree designation among those who have published manuscripts in high-ranking EM journals over the last two decades. According to Lammers et al., very few osteopathic EM physicians are either the first or senior author on original research manuscripts in these journals.The current study also shows that almost half of all allopathic physicians who have received an EM R01 grant hold an advanced degree. A dual-degree program offers the physician researcher the opportunity to hone his/her research skills and work directly with a mentor who may have already successfully navigated the granting process. A total of 26 osteopathic medical students were enrolled in a dual-degree program in 2004 as reported by the American Association of Colleges of Osteopathic Medicine.Previous reported data has also shown that the median age to receive an R01 grant in EM was 43 years.Annals of Emergency Medicine.3It has been previously noted that receiving an R01 grant from the NIH is a gateway to increased involvement in peer review at the national level and is regarded as a seminal event for the physician researcher.A core faculty member’s scholarly activity is a key component in determining if a residency program receives a citation from either the AOA or ACGME upon its site review. The current results show that no osteopathic EP has obtained a NIH R01 grant over the last decade, and based upon the new single accreditation standards would not have received any credit for scholarly activity. Previously, however, applying for a grant would have awarded an osteopathic core faculty member scholarly activity. As progression occurs through the merger, osteopathic EM residencies must be aware of these changes and attempt to rectify the situation through increased faculty development in order to develop faculty members who are better candidates for national grants.There are several key limitations to this study. First, the authors only reviewed one specialty’s receipt of R01 grants over the last decade. Other specialties may have had more of an osteopathic presence among those who received R01 grants, which may alter the conclusions we have drawn. Also, the authors used a web-based search to determine the grant recipients’ degree designations. Although the author’s home institution was examined, the data on the website may not have been accurate and would therefore alter the results. We also note that PubMed is unable to distinguish the difference between those physicians who have the same name. This may have led to an increased number of publications being reported. Similarly, PubMed does not include all journals that are currently being published and the authors may have published manuscripts in journals that are not in the PubMed repository. Because we were unable to comment on the years that were not reviewed, it is possible that inclusion of a greater time span might have shown osteopathic physician-researcher involvement.Authors who have received an R01 grant from the NIH in emergency medicine are primarily allopathic physicians who hold a dual degree and have a track record of prior publications. The osteopathic community must continue to further educate physician researchers in order to close the medical degree disparity gap among those who receive grant funding from the NIH.


Investigated input features for each grant holder include gender, degree, targeted category, international mobility, international grants, number of publications, total number of citations, H-index, best publications, impact factors in the last 2 years, and assessment scores provided by the experts. Evaluated performance indicators include cumulative impact factor, number of D1 publications, and number of first/last author D1 publications during the grant running time. Grant holders’ publication output increased by 23 and 52% for life sciences and material sciences researchers. Scientific performance was independent from gender, degree, international grants, category applied for, and citations received for the best pre-grant publication. Those with international mobility had significantly lower scientific output . Scores received from grant review experts were independent from later publication activity. The strongest correlations were observed between scientific output and total number of citations, H-index, and impact factor in the last 2 years pre-grant. In summary, group leaders with a dynamic publication track record were able to attain the most additional momentum. Our results can help accelerate and improve future grant review processes.The Momentum program launched in 2009 provides funding of up to 1 million Euro to establish new, independent research groups at Hungarian academic institutions. Here, our aim was to determine factors associated with the scientific output of these research groups. Publication data were downloaded from the Hungarian Scientific Work Archive ( A cautious question is asked about the overall usefulness of techniques applied in such a selection. Our research hypothesis is similar to the Antitrust Paradox in a certain way  Grant Scheme (MP). This research is partly justified by the need for a performance assessment of MP including more effective tools of MP was launched in 2009, with an annual award budget of up to EUR 1 million. This budget has been financed from resources provided by the government to the Hungarian Academy of Sciences (HAS), a non-governmental public organization, administering MP along with a string of other R&D funding schemes. MP’s innovative character is based mainly on the fact that its priority target is to provide an effective incentive to Hungarian researchers working abroad to return to their home country. Therefore, Momentum is meant to offer internationally competitive financial conditions to its grantees. Grants are awarded for 5 years, with new applications considered once each year.MP’s grant eligibility criteria include a PhD degree from an internationally renowned university, and documented research or teaching experience obtained in internationally prominent research centers of the field. Furthermore, continuous publication activity in leading scientific journals of the field based on original research papers, and a good record of invited speakership at scientific conferences are also requested along with suitable grant-attaining performance. These criteria are in line with international standards of grant eligibility.Momentum does not offer only lucrative financial conditions to its grantees. It also sets truly international standards of scientific performance. This study is the first comprehensive analysis of Momentum-related research performance. Our intent was to base this analysis on a multi-dimensional set of indicators of research output in line with the most recent scientometric standards.total number of publications,total number of citations,total number of publications with at least a number of citations,total number of citations for the most cited papers,average number of citations per paper (ACPP),H-index Hirsch and are used to measure the influence and citation impact of journals or the scientific performance of researchers.Simple and basic bibliometric indicators include:Several bibliometric indicators have been developed to measure the productivity of a researcher and of his or her research  in the United Kingdom have been analyzed recently. It was noted that the use of bibliometrics is used primarily in the initial individual assessment of candidates . Delimiting the number of projects to which one researcher can apply at one time and promoting collaboration between researchers through project subdivisions has been suggested   by means of the three most frequently applied criteria, reliability, fairness, and validity Reinhart . BibliomA timely analysis of participation in the 8th European Framework Programme for Research and Innovation (EU FP) Horizon 2020 by Norway was investigated by Enger and Castellacci . Their dThe peer-review process often is used for assessing scientific output  intend to announce research results when publishing, but applications for research funding are only tentative projects Fang . The queMagyar Tudományos Művek Tára—www.mtmt.hu) to assess all grant holders . The data were used relative to the grant running time . Most grant holders update their database at the end of their grant’s yearly reporting period, which generally is in July. Bibliometric data were downloaded for all papers published in scientific journals for each grant holder. Conference proceedings, correspondence to the editors, and other non-peer-reviewed publications were omitted. Data were collected in November 2017.We took advantage of the Hungarian Scientific Work Archive (www.scimagojr.com). SCImago is a research group dedicated to information analysis from the Consejo Superior de Investigaciones Científicas (CSIC), University of Granada, Extremadura, Carlos III (Madrid) and Alcalá de Henares (Spain). The SCImago page ranking was developed using the Google PageRank™ algorithm and includes 34,100 titles from more than 5000 publishers. A journal was assigned to be in D1 (first decile) if it ranked within the first 10% of journals within its subject area. For example, if one category has 50 journals, the first five of these were allocated into the D1 category. Notably, the D1 category was not established for the present paper but has widespread use by grant agencies in Hungary.IF data were obtained for each journal from Thomson Reuters (jcr.incites.thomsonreuters.com), and journal ranks were extracted from the publicly available Scimago Journal Rank database , number of D1 publications, and number of first/last author D1 publications during grant running time and up to 3 years before the grant submission cutoff. Impact factor was used, as it is the most common measure also listed on the homepage of most journals. D1 was used because it enables the comparison of different scientific disciplines.The above publication output features after receiving the grant were compared to the grant holder’s characteristics at the time of application. For this, a series of questions regarding the scientific qualities of the applicants were collected from their grant applications. These include degree, international grants, number of publications, total number of citations, H-index, citation data of the best publication, and impact factor in the last 2 years. Gender was determined by examining the name of the applicants. Since 2011, the Momentum grant scheme has had two distinct categories:”Momentum I” for early-stage researchers between 30 and 38 years old and “Momentum II” for experienced researchers between 39 and 45 years old. Data for these categories were collected from the online application system. A Momentum grant can only be executed in Hungary, and grant holders who moved to Hungary to execute the grant program were designated as fellows with “international mobility”.Each Momentum grant proposal is evaluated by 3–4 experts. These experts provide a written description as well as a percentage estimation of the applicant’s performance. The percentage estimation can be in the range between 0 and 100. The reviewers’ scores for each grant holder were provided by the Momentum grant administration office.p < 0.05 in each analysis.Grant holders are assigned to three disciplines at the time of application – life sciences, material sciences, and humanities. Grant holders in humanities were excluded from our analysis due to markedly different publication characteristics. In humanities, many publications, such as books, do not have an impact factor and/or cannot be assigned into categories like D1. In particular, the mean number of publications was 33.3 in life sciences, 28.9 in material sciences and 11.4 in humanities, and 87.6, 90.7 and 30.1% of these publications  had an impact factor. An exception was psychology : grant holders in this area were added to the life sciences discipline. Statistical analysis was performed for the two major disciplines separately. Scientific characteristics of the grant holders at the time of application and publication track record after obtaining the grant were compared by Mann–Whitney and Kruskall–Wallis tests and Spearman rank correlation. The threshold for statistical significance was set at A total of 117 research groups were evaluated. Descriptive characteristics of awarded Momentum grants are summarized in Fig. n = 48) was 24.8 ± 5.9, and of female grantees (n = 6) 22.3 ± 14.3. The number of D1 publications were 10.9 ± 2.7 and 8.2 ± 3.9, and the number of first/last author D1 publications were 4.5 ± 1.2 and 3 ± 2.6 in male and female grantees, respectively. Female researchers had numerically higher yearly impact factors in material sciences . Here, the number of D1 publications were 7.9 ± 2.6 and 1.5 ± 1.5, and the number of first/last author D1 publications were 4.0 ± 1.1 and 0.75 ± 1.5 in male and female grantees, respectively.Parameters without a significant correlation to publication efficiency are summarized first. Scientific performance was independent (no significant correlation) of gender in both life sciences and material sciences. In life sciences, the mean yearly impact factor of male grantees . In both cases, those having Momentum II had higher output, but the differences were statistically not significant  or the Academy of Sciences (DSc). By and large, a scientific performance of ten times that of a PhD degree is necessary to receive a DSc title, which is roughly equivalent to tenure in respected U.S. universities but does not offer employment. For example, if a given scientific discipline requires an IF of 6 for a PhD degree, then the requirement for a DSc degree is an IF of 60. In addition, only those who already mentored at least one PhD fellow are eligible for DSc. There was no significant difference when comparing the output of fellows with PhD and DSc degrees  gained a yearly impact factor of 26.9 ± 7.5 and those without an ERC (n = 19) reached a yearly IF of 26.6 ± 13. In material sciences, only one candidate had an ERC with a yearly mean IF of 27.1, and those without an ERC (n = 20) published a yearly IF of 34 ± 27. Again, one must note that this information was only available for a fraction of the applications.A primary focus of the Momentum program is to provide national support to researchers working in Hungary comparable to grants awarded by the European Research Council. In line with this, the applicants are also asked whether they have or had an ERC grant. In life sciences, the ERC grantees (R2 = 0.001). At the same time, a not-far-from-significant moderate positive correlation trend was observed in material sciences (R2 = 0.65).One of the criteria for a Momentum grant is to have highly cited papers in the CV of the applicant. Interestingly, when assessing the correlation between number of citations received for the best publication of the applicant and subsequent papers, there was no correlation in life sciences . Similar results were derived in material sciences . Of note, reviewers who provided maximum scores for an applicant were excluded to avoid potential biases; the actual correlations were even smaller when including these.Quite surprisingly, mean scores received from grant review experts show almost zero correlation to later publication activity see Fig. c. In lifp = n.s.; corr.coeff. = −0.09, p = 0.12.; and corr.coeff. = −0.09, p = 0.13, respectively. See Fig. Finally, when comparing individual scores received from each reviewer for each grantee, a trend towards a negative correlation was observed for score versus yearly impact factor, number of D1 publications, and number of first/last author D1 publications , to number of D1 publication  and to number for first/last author D1 publications .Global metrics of the entire previous publication track record include total citation and H-index see Fig. a, b. In n = 13) displayed significantly lower publication performance when compared to those already in Hungary (n = 39). These results were observed when considering yearly impact factors , number of D1 publications (5.6 ± 3 vs. 12.5 ± 3), and number of first/last author D1 publications  and number of D1 publications , and marginally significant for the number of first/last author D1 publications , and H-index (R2 = 0.44) show a strong positive correlation. As in life sciences, the strongest correlation was observed between impact factor in the last 2 years and yearly impact factor during the Momentum grant , or national funding agencies, a combination of bibliometric/scientometric and peer-review evaluation is needed when deciding upon funding research projects. We aimed to add new elements to diagnoses of peer-review systems used in the allocation of grants for research. Our main findings provide some potentially valuable input to policymakers interested in the design of efficient and effective research funding systems based on real scientific merit to the highest possible extent.There is a natural consensus that research could not take place without funding. There remains, however, disagreement about the role of peer-review systems of funding agencies as influencers of the novelty and impact of research, and the extent of any interaction of funding provisions between different sources .Despite of the surprisingly good performance of Two remarks are in order at this point. First, peer reviews are not zero-cost at all. This means they consume time, human resources, and such money for which, at least in part, a much better use could be found in the funding scheme in question. Second, we do not suggest giving up the practice of pre-grant peer reviewing. We simply recommend a comparative cost–benefit analysis of pre-grant peer reviews, encompassing a wider array of research grant systems both in Hungary and on the international level.Our final finding is related to the original, “strategic” purpose of the Momentum program. It had been launched based on the assumption that Hungarian researchers well established abroad but returning home as Momentum grant holders will affect research performance positively in Hungary. This expectation has materialized without any doubt, but a closer scrutiny of the performance of such “repatriated” scientists is not exempt from surprise. It has been shown that international mobility tends to reduce publication output in this special case. Thus, one might ask whether it is advisable at all to allocate funds to stimulate the return of such researchers instead of prioritizing the support for local researchers.This conclusion may be relativized due to a relatively small sub-sample size for international mobility and the fact that the number of returning Momentum grant holders has decreased from the starting year of Momentum to this day. It is also true that the category of “returning” scientists includes a variety of research careers from tenured professors at a high-ranking university to associate professors at a less quoted teaching institution.Nevertheless, this apparent drift also requires further, more in-depth research. Such research should identify, in the first place, the main factors underlying this surprising trend. The causes of the latter potentially might include the impact of changing research topic as well as work conditions for the returning scholar, the length of time needed for his/her re-positioning in international networks of research or, again, the not as-high-as-expected efficiency of pre-grant peer reviews.In brief, large-scale grant schemes such as the MP are indeed able to enrich the scientific landscape of a country. The MP winners were already the best, and performance further increased by 23–52% during the grant period. In particular, group leaders with a dynamic pre-grant publication track record were able to attain the most additional momentum. It is, however, still an open question as to what extent the MP or a similar grant scheme also involving international mobility is able to appeal to the best researchers determined to establish their new independent research groups in a different country. Our study may be considered an initial effort to shed some light on the ways that grant selection processes can be made more efficient in the future.


Nanoparticles can be used as drug carriers, contrast agents and radiosensitisers for the treatment of cancer. Nanoparticles can either passively accumulate within tumour sites, or be conjugated with targeting ligands to actively enable tumour deposition. With respect to passive accumulation, particles < 150 nm accumulate with higher efficiency within the tumour microenvironment, a consequence of the enhanced permeability and retention effect. Despite these favourable properties, clinical translation of nano-therapeutics is inhibited due to poor in vivo stability, biodistribution and target cell internalisation. Nano-therapeutics can be modified to exploit features of the tumour microenvironment such as elevated hypoxia, increased pH and a compromised extracellular matrix. This is in contrast to cytotoxic chemotherapies which generally do not exploit the characteristic pathological features of the tumour microenvironment, and as such are prone to debilitating systemic toxicities. This review examines strategies for tumour microenvironment targeting to improve nanoparticle delivery, with particular focus on the delivery of nucleic acids and gold nanoparticles. Evidence for key research areas and future technologies are presented and critically evaluated. Among the most promising technologies are the development of next-generation cell penetrating peptides and the incorporation of micro-environment responsive stealth molecules. In the last 20 years, nanoparticle delivery systems have been at the forefront of research for improving cancer therapeutic delivery and efficacy. A number of nano-therapies have been FDA approved for the treatment of cancer such as Doxil® and Vyxeros®, with approved therapies most often being liposomal in nature, delivering pre-approved chemotherapeutics. However, despite the wealth of promising pre-clinical data for the use of nanoparticle therapeutics, successful clinical translation has proved challenging. One example of this is Aroplatin™ a platinum analogue  encapsulated within a liposome. In vivo, Aroplatin was shown to successfully increase survival of mice bearing L1210 leukemia following both intravenous and intraperitoneal administration, compared to cisplatin delivered as a free drug. Aroplatin also increased the survival time of mice bearing reticulosarcoma liver metastasis by 37% over free drug , poor methodology of pre-clinical studies (e.g. randomisation), upscaling and a lack of standardisation with regards pre-clinical validation  and a low intracellular pH (pH between 6.0 and 7.0), both of which impede the delivery and efficacy of current therapeutics  resulted in a 40% increase in 5-FU uptake compared to rats receiving no pre-treatment. Despite this the effect was transient, remaining for a maximum of 1 h post PGE1 administration. This suggests that repeated dosing of PGE1 would be necessary to maintain enhanced 5-FU uptake and efficacy, highlighting practical limitations to this approach  compared to that of free drug alone (13.43 ± 0.8 μg/ml). The authors attribute this increased efficacy to enhanced target cell internalisation, however, uptake studies show less endocytosis of the gelatin-coated nanoparticle compared to the lipid only particle. It is, therefore, likely that the increased toxicity is due to the inclusion of imantinib. Imantinib reduced tumoural IFP by > 40% (17.2 mmHg) in a nude 4T1 xenograft mouse model, with control animals exhibiting an IFP of 29.18 mmHg. Importantly, the impact of both in vitro enhanced toxicity and in vivo reduced IFP translated to a significant anti-tumour effect with a 2.8 fold increase in tumour growth delay and a 72.5% reduction in the formation of pulmonary metastases for GPN treated animals  , Hypoxia-inducible factor-1-alpha (HIF-1-α), PIM-1 oncogene (PIM-1) and endothelin-1 (EDN 1)  is overexpressed under hypoxic conditions, driving a corresponding increase in the expression of downstream targets such as vascular endothelial growth factor (VEGF), involved in angiogenesis, glucose metabolism and invasion  spheroids, with up to a sevenfold increase in intratumoural platinum compared to free cisplatin or cisplatin delivered using a non-pH sensitive system. Furthermore, use of this vehicle translated to significant tumour growth delay in a Bx-PC3 xenografts by 82%, compared to 24% for mice treated with free cisplatin at a 2 mg/kg dose  and tenfold lower than mice treated with saline alone(~ 1000 mm3)  is composed of the basement membrane and interstitial matrix, providing tissue structure, regulating cell proliferation and migration  (PLGA-b-PEG-COOH) nanoparticle. Conjugation via covalent carbodiimide chemistry produced 220 nm nanoparticles determined by dynamic light scattering (DLS) and TEM. At a nanoparticle/LOXAb concentration of 50 μg/ml, a fourfold inhibition of 4T1 cell proliferation and migration was demonstrated. Additionally, the proliferative rate was unaffected when using an equal concentration of free soluble antibody. Importantly, the nanoparticle conjugated antibody resulted in a 25-fold reduction in dose to suppress an orthotropic mammary 4TI mouse tumour model compared to free antibody. Conjugation also resulted in an increased therapeutic index, with a decrease in adverse effects  is an enzyme responsible for the cross-linking of collagen to elastin in the ECM, inducing ECM rigidity. LOX has been shown to be elevated in numerous tumour subtypes including breast and head and neck cancer  to enhance the uptake and therapeutic efficacy of cytotoxic chemotherapeutics. For example, Rijt et al.  conjugatTissue extracted from cisplatin-treated k-ras mutated transgenic mice showed increased cell death of up to 20-fold, with a corresponding increase in apoptosis . RES, a component of the innate immune system, recognises and clears foreign pathogens and particles through opsonisation and phagocytosis. Additionally, DNA is both hydrophilic and anionic in nature, and therefore, is repelled from the phospholipid bilayer of cell membranes, impeding the movement of the DNA into the cytosol : 1,2Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) liposomal nanoparticle, conjugated to an anti-transferrin antibody, enabling tumour specific targeting. Patients with a variety of solid tumours including colorectal and pancreatic received treatment with the nanoparticle conjugates on a dose escalating regime from 0.6 to 3.6 mg of DNA per infusion. Post treatment, PCR analysis highlighted tumour-specific increased expression of p53, with negligible increases observed in normal skin biopsies, suggesting that transferrin confers tumour specificity. 7 out of 11 patients within the study displayed stable disease at a 6-week post-treatment, with an increased median survival of 340 days. However, only one patient displayed evidence of tumour necrosis, demonstrating the limited effect of SGT-53 as a monotherapy . Preliminary results indicated that 2 patients experienced a partial response, with 67% of patients displaying stable disease  are short peptide sequences that facilitate cell internalisation through a variety of endocytic pathways, without the requirement of specific targeting receptors  and Penetratin (sequence: RQIKIYFQNRRMKWKK) are composed of basic amino acids enabling an alpha-helical conformation and translocation of cargo across the cell membrane. Extensive research has been carried out into the key components of a CPP structure. Mitchell et al.  highlighOne such peptide is RALA. RALA was derived from two alternative cell penetrating peptides termed GALA and KALA. GALA (sequence: WEAALAEALAEALAEHLAEALEALAA) is a fusogenic peptide with 7 identical glutamic acid–alanine–leucine–alanine repeats which provide the peptide with pH-responsive properties, enabling embedment within the phospholipid biolayer. However, the use of GALA as a delivery vehicle is limited by the fact that it cannot condense or bind to negatively charged moieties , such as alendronate, which have bone-targeting specificity. This opens up the options of treating metastatic disease which form bone lesions. However, the clinical application of BP’s is impeded by systemic toxicity. Massey et al.  present However, the biodistribution profile of RALA nanoparticles have highlighted high levels of accumulation in normal organs such as the lungs and liver, rather than the intended tumour  can take advantage of the leaky tumour vasculature, accumulating in tissue due to the EPR effect. This has led to a variety of possible applications including radiosensitisers, contrast agents and drug delivery vehicles   are hydrophilic in nature, coating hydrophobic nanoparticles and increasing particle stability through altered polarity  describe a class of extracellularly-expressed, zinc-dependent proteases that have a key role in physiological processes requiring ECM modification (Vartak and Gemeinhart An alternative approach to the cleavage of PEG within the TME is to exploit the acidic nature of the TME as previously discussed “As discussed throughout, exploiting characteristics of the TME for nanoparticle design and therapy has shown significant potential for enhancing drug delivery and improving cancer therapeutics. Tumours can no longer be considered in isolation as a mass of rapidly proliferating neoplastic cells. Instead, tumours are a dynamic network of immune and endothelial cells connected by a chaotic and aberrant vasculature system. High IFP, hypoxia and low pH have now all be shown as indicative of the TME. It is known that these characteristics impede the delivery and efficacy of numerous chemotherapeutics. However, evidence presented within this review suggest that nanoparticle therapies can exploit these aberrant characteristics, actively or passively accumulating within tumour tissue. Consequently, nanoparticle therapies are widely considered to represent the next-generation of cancer therapeutics.Currently, translation of nanoparticle therapies to clinic remains limited due to poor stability, circulation time and internalisation. Conjugation of stealth molecules often improves stability, however, as discussed stealth molecules can themselves impede target cell uptake. This review outlines a number of possible modifications that could circumvent these difficulties, including the use of cell-penetrating peptides and the development of TME responsive nanoparticles. To date, these approaches remain in their infancy with in vitro validation remaining challenging. Yet, as our knowledge and understanding of these relatively new technologies continue to emerge, it is likely that therapeutic strategies which exploit features of the tumour microenvironment will eventually hold prominence routine in clinical practice.


Varicella-zoster virus (VZV) reactivation is common but difficult to predict in HIV-infected persons.Since qualitative VZV antibodies can determine past VZV disease or vaccination, we evaluated whether quantitative VZV antibody levels over time can predict future zoster.US Military HIV Natural History (NHS) participants with a zoster diagnosis at least 5 years after HIV diagnosis (n = 100) were included. Zoster-negative controls (n = 200) were matched by age, race, gender, and CD4 count at HIV diagnosis. Repository plasma specimens collected at baseline and prior to zoster diagnosis were evaluated using a quantitative anti-VZV ELISA assay. Differences in quantitative VZV levels were analyzed by Wilcoxon Mann–Whitney and Fisher’s exact tests.Median CD4 count at HIV diagnosis was similar for cases and controls , but lower for cases at zoster diagnosis . Antiretroviral therapy (ART) use prior to zoster diagnosis was lower for cases (52.0%) compared to controls . Cases had similar mean VZV antibody levels prior to zoster diagnosis compared to controls  with no difference in the change in antibody levels over time .Quantitative VZV antibody levels are stable in HIV-infected persons and do not predict zoster reactivation. Low CD4 count and lack of ART use appear to be better predictors of future zoster diagnosis. Infection with varicella zoster virus (VZV) can cause relapse of disease in those with impaired cell mediated immunity (CMI) known as herpes zoster (HZ) and is seen more frequently in those with HIV infection compared to HIV-uninfected individuals . OverallCell mediated immunity appears to play a role in preventing HZ, as waning CD4 count is associated with development of HZ , 4. ConvWe retrospectively evaluated VZV antibody titers in HIV-infected persons with and without HZ in order to determine if VZV antibody levels can be used to predict HZ.The US Military HIV Natural History Study (NHS) is a prospective observational study of HIV-infected military members and beneficiaries. Enrolled participants are ≥ 18 years of age with documented HIV infection and complete informed consent in this IRB approved study. NHS participants have clinical visits approximately every 6–12 months at select military treatment facilities.The NHS database was queried for participants with (cases) or without (controls) a HZ diagnosis after HIV diagnosis. In order to assess the change in serum VZV antibody titers over time, only participants with a HZ diagnosis greater than or equal to 5 years after HIV diagnosis were included. Participants were required to have a serum sample 30–180 days prior to HZ diagnosis in addition to a sample at least 3 years prior to documented HZ infection. A total of 100 cases and 200 controls meeting inclusion criteria and serum sample availability were selected for analysis. Control participants who did not develop HZ were matched by age, race, gender, and CD4 count at time of HIV diagnosis. Participants with negative VZV antibody or receipt of varicella or zoster vaccines were excluded.Two repository plasma specimens were evaluated using a quantitative anti-VZV ELISA assay . VZV antibody levels are represented for each sample by a calibration cut-off value which is expressed as index value/OD ratio. Specimen time-points for controls were matched to corresponding cases. Differences in quantitative VZV levels were analyzed by the Wilcoxon Mann–Whitney test and Fisher’s Exact test, controlling for time intervals between specimens.10 copies/mL, respectively.Participants were predominantly male (94%) with a median age of 29 [IQR 24–34] years at HIV diagnosis Table . The medCases had similar mean VZV antibody levels prior to HZ diagnosis compared to controls  and there was no difference in the change in VZV antibody levels over time . The range of values for cases was similar to controls [IQR 1.7–2.9 index value/OD vs. 1.7–3 index value/OD]. However, the mean antibody level was higher for those on ART  compared to those who were not on ART  with SD of 0.89 and p < 0.05 (Table Although VZV antibody can identify exposure to Varicella infection or past immunization, we found that neither the magnitude of VZV antibody level nor the change in concentration over time predicted HZ reactivation in HIV-infected persons. CMI most likely plays a larger role in immune protection from HZ and may be a better predictor of HZ than VZV antibodies, however the latter have been shown to be important as described below. Markers of intact CMI are associated with reduced incidence of HZ and reduced PHN , 13. TheIn addition to a T-cell mediated immune response, humoral response also appears to be important as VZV antibodies have been used to demonstrate vaccine effectiveness , 17–19. In the population with HIV infection, the risk for HZ exists even in those less than the age of 40  and it w


Here, we report on a new somatostatin receptor subtype 2 (sst2) antagonist, SS-01 (p-Cl-Phe-cyclo(D-Cys-Tyr-D-Trp-Lys-Thr-Cys)D-Tyr-NH2), with the aim of developing 99mTc-labelled ligands for SPECT/CT imaging. SS-01 was prepared using Fmoc solid-phase synthesis and subsequently coupled to the chelators 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 6-carboxy-1,4,8,11-tetraazaundecane (N4), and 6-hydrazinonicotinic acid (HYNIC) to form the corresponding peptide-chelator conjugates SS-03, SS-04, and SS-05, respectively. SS-04 and SS-05 were radiolabelled with 99mTc and SS-03 with 177Lu. Binding affinity and antagonistic properties were determined using autoradiography and immunofluorescence microscopy. Biodistribution and small animal SPECT/CT studies were performed on mice bearing HEK293-rsst2 xenografts.Somatostatin receptor targeting radiopeptides are successfully being used to image, stage, and monitor patients with neuroendocrine tumours. They are exclusively agonists that internalise upon binding to the relevant receptor. According to recent reports, antagonists may be preferable to agonists. To date, 177Lu-DOTA-SS-01 (177Lu-SS-03) and 99mTc-N4-SS-01 (99mTc-SS-04) demonstrated high cell binding and low internalisation, whereas 99mTc-HYNIC/edda-SS-01 (99mTc-SS-05) showed practically no cellular uptake in vitro. The 99mTc-SS-04 demonstrated impressive tumour uptake at early time points, with 47% injected activity per gram tumour (%IA/g) at 1 h post-injection. The tumour uptake persisted after 4 h and was 32.5 %IA/g at 24 h. The uptake in all other organs decreased much more rapidly leading to high tumour-to-normal organ ratios, which was reflected in high-contrast SPECT/CT images.The conjugates showed low nanomolar sst2 affinity and antagonistic properties. 99mTc-labelled sst2-targeting antagonist. The results demonstrate high sensitivity of the 99mTc-labelling strategy, which was shown to strongly influence the receptor affinity, contrary to corresponding agonists. 99mTc-SS-04 exhibits excellent pharmacokinetics and imaging properties and appears to be a suitable candidate for SPECT/CT clinical translation.These data indicate a very promising The online version of this article (10.1186/s13550-018-0428-y) contains supplementary material, which is available to authorized users. Somatostatin receptors are important biomarkers for imaging and targeted radionuclide therapy of human cancers. They belong to the large family of G-protein coupled receptors, which currently account for 30–40% of marketed drugs  and are 68Ga and 64Cu and other 3+ (radio)metals octreotide) labelled with 64Cu using CB-TE2A, a cross-bridged cyclam-14 derivative, with the antagonist d-Tyr-NH2), labelled using the same strategy octreotide (TOC) (the sst2 agonist) at 37 °C for 30 min. After fixation and permeabilisation, cells were stained with the sst2-specific primary antibody R2-88  as described previously octreotide. The results were expressed as a percentage of the applied radioactivity.The activity in each fraction was measured in a γ-counter (Cobra II). Nonspecific uptake was determined in the presence of 250 pmol/well -D-Tyr-NH2), both SS-03 and SS-04 retained high affinity to sst2.Table 3]octreotide (TOC) triggers massive receptor internalisation, whereas SS-03 or SS-04 at the much higher concentration of 1000 nM does not stimulate receptor internalisation. However, at a concentration of 1 μM together with 10 nM of TOC, the conjugates were able to prevent the agonist-induced receptor internalisation.Immunofluorescence-based internalisation was performed using HEK-sst2 cells to demonstrate the antagonistic property of the conjugates. Figure 177Lu-SS-03 and 99mTc-SS-04 showed high uptake and blocking studies demonstrated that the uptake was receptor-mediated. Within 2 h of incubation, the specifically bound fraction levelled off at 48%, demonstrating rapid binding of 177Lu-SS-03 and 99mTc-SS-04 to the receptors. The internalised fraction was about 8–10% and increased very slowly. Surprisingly, 99mTc-SS-05 showed very low cellular uptake of < 1% even after 4 h of incubation.Figure 177Lu-SS-03 and 99mTc-SS-04 were studied by a temperature shift experiment and showed a plateau at 2 h, indicating that a steady state had been achieved  and kidneys (49.82 ± 4.31 %IA/g) and higher blood pool activity (1.85 ± 0.41 %IA/g) than 177Lu-SS-03 . 99mTc-SS-04 exhibits twofold higher tumour uptake compared to 177Lu-SS-03. Uptake in other sst2 receptor-positive organs such as the stomach and pancreas is lower for 99mTc-SS-04  than 177Lu-SS-03 . At 4 h, the tumour uptake of 177Lu-SS-03 increased to 31.68 ± 4.00 %IA/g, whereas the tumour uptake of 99mTc-SS-04 remained constant. Tumour uptake of 177Lu-SS-03 and 99mTc-SS-04 was significantly reduced by pre-injection of 20 nmol of unlabelled peptide. At 24 h, both conjugates showed clearance from the blood pool (< 0.1 %IA/g) and significant wash-out from the liver (177Lu-SS-03: 0.47 ± 0.07 %IA/g and 99mTc-SS-04: 1.87 ± 0.33 %IA/g) and kidneys (177Lu-SS-03: 7.3 ± 1.6 %IA/g and 99mTc-SS-04: 6.3 ± 1.8 %IA/g). 99mTc-SS-04 showed higher tumour uptake at all time points including 24 h p.i. (32.5 ± 0.78 %IA/g) compared to 177Lu-SS-03 (26.32 ± 4.42 %IA/g). The tumour-to-normal tissue ratios of 177Lu-SS-03 and 99mTc-SS-04 at each time point are illustrated in Table 177Lu-SS-03 shows higher tumour-to-normal tissue ratios compared to 99mTc-SS-04. At 4 h, both radiopeptides showed similar ratios except 177Lu-SS-03 had a twofold higher tumour-to-liver ratio. However, at 24 h, 99mTc-SS-04 exhibited excellent biodistribution with a 4.5-fold higher tumour-to-blood ratio, approximately threefold higher tumour-to-kidney ratio, and approximately twofold higher tumour-to-liver ratio compared to 177Lu-SS-03. All relevant tumour-to-normal organ ratios increased with time and were > 4 at 24 h.Table 99mTc-SS-04. The highest uptake was visible in the tumour and the kidneys. To a lesser extent, uptake was also visible in the abdomen due to tracer accumulation in the sst2-expressing organs.The SPECT/CT images Fig.  were acqRadiolabelled peptides targeting G-protein-coupled receptors have been an important focus in the radiopharmaceutical and nuclear oncology fields over the last 20 years –29. The 111In, 64Cu, 68Ga, and 177Lu) have been described, but only one 99mTc-labelled antagonist has been reported to date octreotide/edda kit (HYNIC-TOC) is registered in some European countries. The [O=Tc=O]+ core is formed with linear and macrocyclic tetraamines such as 1,4,8,11-tetraazaundecane and cyclam-14. This core exhibits high kinetic stability, can be labelled at room temperature, is highly hydrophilic, and with different functions in 6-position, such as carboxylic acid can be easily coupled to the N-termini of biomolecules octreotide but do not trigger internalisation on their own. Somewhat surprisingly, but as previously reported -SS-01 shows practically no cell binding or internalisation in our HEK-rsst2 cell assay. This is contrary to HYNIC conjugated to somatostatin-based agonistic octapeptides such as [Tyr3]octreotide (TOC) and octreotide (TATE). These conjugates showed impressive preclinical [A very important outcome of this work is that [ch as Tyroctreotidclinical –35. The clinical , 7.99mTc-labelled radiopeptide showed superior properties, with very high cell uptake in vitro and about the highest tumour uptake at 1 and 4 h of any somatostatin-based radiopeptide studied to date in this xenograft model [99mTc-SS-04, compared with 177Lu-SS-03, may be attributed to the somewhat slower blood clearance of 99mTc-SS-04, even though at 4 h p.i, the blood values are at the same level for both radiopeptides. In addition, it shows very good tumour retention, no change between 1 and 4 h, and only about 30% release between 1 and 24 h p.i. The target-to-relevant organ ratios are > 5 at 24 h, reflected in excellent SPECT/CT images. For both radiotracers, the kidney uptake is somewhat high but the tumour-to-kidney ratios are still within a reasonable range when compared to potent radioagonists. We are currently studying somewhat different peptide motifs coupled to tetraamine chelators for 99mTc-labelling to improve the overall pharmacokinetics. The hypothesised improvement is based on the properties of 177Lu-SS-03, which exhibits pharmacokinetics somewhat inferior to the previously published 111In-DOTA and 177Lu-DOTA conjugated antagonists [99mTc tricarbonyl strategy and an NSN-type chelator [99mTc-based sst2 antagonist 99mTc-SS-04 is hydrophilic, as indicated by its log D value. Consequently, it has limited accumulation in the abdomen and rather high renal excretion. The biodistribution profile of 99mTc-SS-04, together with its very high tumour uptake, favours this tracer for clinical translation.In contrast, the N4-conjugated and ft model , 7. The agonists . As prevchelator . The Re-chelator . The pro99mTc using two common chelators. Surprisingly, the widely used 99mTc ligand HYNIC along with the co-ligand edda resulted in almost complete loss of sst2 binding affinity, whereas 99mTc-SS-04 demonstrated impressive tumour uptake and high tumour-to-normal organ ratios. Therefore, 99mTc-SS-04 appears to be an excellent candidate for SPECT imaging of sst2-positive tumours and is a promising option for further modification of the peptide motif. The pharmacokinetics of 99mTc-SS-04 demonstrate once again that the 99mTcO2(N4) core offers favourable pharmacokinetic features for small peptides and may potentially be the 99mTc-labelling strategy of choice.Sst2 antagonists were labelled with Additional file 1:Materials and methods. (PDF 484 kb)


Syringomatous tumour (SyT) is a rare type of benign locally infiltrative tumour with debated origin. Because of the growth pattern, SyT can be mistaken for a malignant tumour, and it is therefore important to keep this diagnosis in mind. This case presents a woman with two supernumerary nipples on each side of the abdomen. One of the nipples was inverted with a small palpable firm mass in close relation to the nipple, leading to referral to the breast surgery department. SyT occurring in a supernumerary nipple and presenting with the symptoms described in this case has to our knowledge never been described previously. Syringomatous tumour of the nipple (SyT) is a rare locally infiltrative tumour of the nipple, which histologically resembles skin adnexal tumours , 2. The This case presents a woman with two supernumerary nipples on each side of the abdomen. One of the nipples was inverted with a small palpable firm mass in close relation to the nipple, leading to referral to the breast surgery department.A 61-year-old woman presented with two supernumerary nipples located along the milk line on each side of the upper abdomen. During a few months before referral, the patient had noticed a firm palpable mass in close relations to the supernumerary nipple on the right side . She hadUpon clinical examination, the supernumerary nipple on the right side presented with inversion and a palpable firm mass underneath this nipple. Ultrasound-guided needle core biopsy (16G) was performed . The patFollowing a Multidisciplinary Team Conference, it was decided to recommend resection with a 5 mm rim of normal tissue, which was subsequently performed .Macroscopically the tumour measured 7 x 5 x 5 mm and was described as a firm and grey-white tumour, in close relations to the supernumerary nipple. Microscopically the tumour measured 13 mm in diameter and was localized in the deep part of dermis and underlying subcutaneous tissue with no relation to the epidermis. The tumour was composed of solid trabecular and glandular imitating formations with focal cysts. The epithelium consisted of cells with slightly irregular nuclei and indistinct nucleoli, surrounded by an eosinophilic cytoplasm. The glandular structures were lined by cuboidal cells. The tumour was in close association with the lactiferous ducts and smooth muscle of the nipple . ForeignImmunohistochemical analysis showed positive reaction for CK5, CK14, and P63  in assocA re-excision was performed due to insufficient rim of normal tissue in the cranial direction in order to minimize the risk of recurrence. PIK3CA mutations in LGASC as compared to SyT [Due to the rarity of SyT, this type of tumour may constitute a diagnostic challenge. Primary malignant breast carcinoma as low-grade adenosquamous carcinoma (LGASC) and tubular carcinoma are mentioned as important differential diagnoses , 2. Howed to SyT .With respect to malignant skin adnexal tumours the most closely related tumours are syringoid carcinoma and microcystic adnexal carcinoma . These tSupernumerary nipples are not an uncommon abnormality, with a prevalence ranging from 1.7 to 3.75 % reported in the literature . As descIn conclusion, SyT is a rare type of “benign” locally infiltrative tumour with debated origin. Morphologically it can be difficult to differentiate it from malignant tumours. Only two previous cases in the literature describe this type of tumour occurring within supernumerary breast tissue or nipple. SyT occurring in a supernumerary nipple presenting with the symptoms described in this case has to our knowledge never been described previously.


Sci., 2018, 9, 4142–4151.Correction for ‘The enhancement and suppression of immersion mode heterogeneous ice-nucleation by solutes’ by Thomas F. Whale  The authors regret that in the original article they didn't acknowledge the main grant which supported this research. The missing grant was from the European Research Council .The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers.


Salmonella spp. in animals and humans make it as the most studied pathogen with regards to anti-tumour therapy. The present study aims to construct a genetically modified S. Agona auxotroph as an anti-tumour agent. LeuB and ArgD metabolic genes in ΔSopBΔSopD double knockout S. Agona were successfully knocked out using a Targetron gene knockout system. The knockout was confirmed by colony PCR and the strains were characterized in vitro and in vivo. The knockout of metabolic genes causes significant growth defect in M9 minimal media. Quadruple knockout ΔSopBΔSopDΔLeuBΔArgD (BDLA) exhibited lowest virulence among all of the strains in all parameters including bacterial load, immunity profile and histopathology studies. In vivo anti-tumour study on colorectal tumour bearing-BALB/c mice revealed that all strains of S. Agona were able to suppress the growth of the large solid tumour as compared with negative control and ΔLeuBΔArgD (LA) and BDLA auxotroph showed better efficacy. Interestingly, higher level of tumour growth suppression was noticed in large tumour. However, multiple administration of bacteria dosage did not increase the tumour suppression efficacy. In this study, the virulence of BDLA knockout strain was slightly reduced and tumour growth suppression efficacy was successfully enhanced, which provide a valuable starting point for the development of S. Agona as anti-tumour agent.The fight against cancer has been a never-ending battle. Limitations of conventional therapies include lack of selectivity, poor penetration and highly toxic to the host. Using genetically modified bacteria as a tumour therapy agent has gained the interest of scientist from the past few decades. Low virulence and highly tolerability of  Cancer is one of the leading causes of morbidity and mortality worldwide. Conventional anti-cancer therapies often encounter significant side effects and fail to achieve complete tumour remission. Heterogeneous tumour microenvironment, including gradients in chemical concentration and tissue hypoxia make it particularly resistant to systemic treatment . These sSalmonella, Bifidobacterium, Clostridium, and Listeria possess unique features to overcome many of the limitations of chemotherapy  and a genetically modified Ag 1 were used in this study. The food source strain S. Agona was recovered from indigenous vegetables in Selangor state, Malaysia  at 37 °C and stock stored in 15% glycerol at −80 °C.A food source strain Malaysia . The genknockout . This paMus musculus, mouse colon carcinoma cell line, CT-26 was obtained from American Type Culture Collection (ATCC), USA, and cultured in Roswell Park Memorial Institute 1640 (RPMI 1640)  complete medium supplemented with 10% fetal bovine serum , 1% v/v penicillin/streptomycin  and 2% of L-glutamine  in a humidified incubator with 5% CO2. Cell line was detached from the culture flask using a 0.25% trypsin-EDTA solution and re-suspended as monolayer cell suspension in RPMI 1640 culture medium.Salmonella Agona leuB and argD metabolic genes were amplified using polymerase chain reaction (PCR) in the present study followed by gene disruption using Targetron Gene knockout System . A computer algorithm at Sigma-Aldrich Targetron design website  was used to identify the potential L1.LtrB insertion sites. Three primers were designed to modify the RNA portion of intron template which base pair optimally to corresponding insertion sites, referred to as IBS, EBS1d, EBS2. The designed primers are listed in HindIII (1U) and BaeGI (0.5U)  followed by ligation into the pACD4-C intron donor chloramphenicol-resistant linear vector (2 ng) . The ligation reaction was performed using Quick-link T4 Ligation Kit  according to the manufacturer’s instruction. The ligated product was first transformed into E.coli QIAGEN EZ competent cells due to low transformation competency of S. Agona strain. The pooled plasmid from E. coli was then co-transformed with plasmid pAR1219 which expressed T7 RNA polymerase into S. Agona via electroporation. The intron insertion in the transformant was identified by colony PCR using gene specific primers which amplifies the entire intron inserted target gene. To adapt the system for multiple gene knockouts, the first constructed leuB knockout was applied to sequentially mutate the second argD metabolic gene for the construction of ΔleuBΔargD double knockouts. The ΔsopBΔsopD double knockout was subjected to the same experiment as mentioned above to construct the quadruple knockout ΔsopBΔsopDΔleuBΔargD.S. Agona (W), double knockout ΔleuB ΔargD (LA), double knockout ΔsopB ΔsopD (BD) and quadruple knockout ΔsopB ΔsopD ΔleuB ΔargD (BDLA) was carried out in M9 minimal media. Each overnight culture in Nutrient broth was washed twice with phosphate-buffered saline (PBS) prior to inoculation in M9 minimal media. M9 minimal media was prepared according to recipe describe by Sambrook . After infection, cells were washed three times with PBS and then treated with 100 µg/mL of gentamicin  to kill the external bacteria, for 1 h at 37 °C. After the gentamicin incubation, the cells were washed for three times again and then incubated with 0.1% Triton X-100  for 10 min at 37 °C. The number of internalized bacteria was determined by plating ten-fold serial dilutions of the cell lysates on xylose lysine deoxycholate (XLD) agar plates.The CT-26 cells at number of 1 × 101 to 107 cfu in 100 µl PBS. Animal deaths were recorded for 30 days.Mice used in this study were purchased from a local supplier  and adapted for one week prior to the experiment (see ‘Ethics Statement’ below). All mice were maintained in an enriched environment and in accordance with institutional animal care and guidelines. Consent for experiments involving animal was obtained from the appropriate University Putra Malaysia (UPM) Ethics Committee. Six-to-eight week old male, BALB/c mice were randomly divided into five groups and treated with W, BD, LA, BDLA and PBS as negative control. The inoculums were administered intraperitoneally with different concentrations of bacteria ranging from 10All animal experiments conformed to the Institutional Animal Care and Use Committee of Universiti Putra Malaysia using the protocol UPM/IACUC/AUP-R056/2013.4 cfu in 100 µl of PBS. PBS was used as negative control. Mice were sacrificed at day-1, day-3, day-10, day-21 and day-36. The potential side effects and toxicities induced by the treatments were evaluated in the aspect of bacterial distribution, histopathology and full blood count analysis.Six-to-8 week-old male, BALB/c mice were divided into five groups and treated with W, LA, BD, and BDLA at concentration of 10The isolation and titration of bacteria from mice were performed as described with slight modification . BrieflyPortion of liver, spleen, lung, kidney and intestine were fixed immediately with 10% paraformaldehyde/0.1 M phosphate buffer (PB) with fixative volume of 15–20 times greater than tissue volume. The subsequent embedding and staining processes were proceeding according to standard histological procedures. Inflammation and infectious foci of the tissues were observed under a light microscope to evaluate the severity of inflammation induced by bacteria.Blood samples were collected from mice at day-1, 3, 10, 21, 36 days post infection. Blood samples were collected through cardiac puncture and were stored immediately in EDTA blood collection tubes. The level of neutrophils, lymphocytes and white blood cells were determined subsequently by Haematology and Clinical Biochemistry Laboratory, Faculty of Veterinary, Universiti Putra Malaysia.6 cells in 100 µl of PBS. Tumour was allowed to grow for 2 weeks before treatment. Mice were examined daily until the tumour size was approximately 250 mm3 and 450 mm3 for small and large tumour respectively. Different Salmonella strains, W, BD, LA and BDLA were administered intraperitoneally at concentration of 104 cfu in 100 µl of PBS. Anti-tumour activity of the treatments was evaluated by measuring tumour size every 2 days. Tumours were measured individually with a digital caliper. Tumour volumes were determined by the formula: tumour volume = length × width2 × 0.52 .Statistical significance of experimental results was determined by Student’s S. Agona. The potential targets sites for leuB (9 sites) and argD (5 sites) were predicted by Sigma-Aldrich Targetron. Targetron integration was successful for the 6th design for leuB and 2nd design for argD  and quadruple knockout ΔsopB ΔsopD ΔleuB ΔargD (BDLA) were unable to grow in M9 minimal media throughout 12 h. Both of the metabolic gene knockout strains were able to grow partially in M9 minimal media supplemented with leucine only reaching final cell densities OD600 ∼ 0.4 compared with OD600 ∼ 2.0 for the parental strain after incubation period of 12 h. However, both genetically modified LA and BDLA strains were still unable to grow in M9 minimal media supplemented with arginine only. Growth inhibition of LA and BDLA mutant strains were able to be fully reversed with the supplementation of both leucine and arginine in M9 minimal media. The corresponding cultures reached final cell densities (OD600 ∼ 1.9) that were similar to the parental strain S. Agona. Growth ability of double knockout ΔsopB ΔsopD (BD) is not affected as it reaches similar cell density as the parental strain throughout 12 h of incubation period. Salmonella is an intracellular bacteria and invasion is essential to cause the infection. We next examined the invasiveness of these modified S. Agona strains and found no significant difference in the recovered bacterial colony forming unit  and white pulp (WP) (S. Agona (S. Agona (S. Agona showed higher number of infection foci per field (S. Agona (Histology was performed to examine the inflammatory response of spleen and liver to ulp (WP) . As for S. Agona , the whiS. Agona  as compaer field  comparedS. Agona . This phS. Agona into BALB/c mice led to significant increment in total white blood cell concentration at day-10 post-infection as shown in Salmonella infection also induced a significant increase in neutrophil level in mice on day 1 post-infection and subsequently declined on day-3 post-infection followed by normal level on day-21 post-infection  per gram of bacteria in tumour was found to be 2 ×106-fold higher than liver, indicating that genetically modified S. Agona auxotrophic strain prefer to colonize in tumour tissue . However, in small tumour model there was no significant tumour growth suppression observed for all the treatments. We also investigated if multiple administration of bacteria dosage could enhance the effectiveness of tumour suppression but found no improvement in the therapy  is a food borne pathogen for human and animal and yet very little information about this bacterium has been reported. To date, there is no genetically modified S. Agona reported except triple knockout strain ΔsopBΔsopDΔPipD from our previous work  to survive and cause lethal infection in mice  is responsible for gastroenteritis by increasing the fluid secretion in the intestine of infected host. sopB mutant of S. Typhimurium and S. Dublin blocked their ability to cause gastroenteritis but they were still able to cause diarrhea and death in calves at the higher dosage  contribute to certain level of Salmonella virulence in systemic infection of the mouse model. Furthermore, present work demonstrated the use of auxotrophic S. Agona in CT-26 colon tumour treatment for the first time. Double and quadruple knockouts LA and BDLA were able to show lower T/C% value when compared to parental strain. Auxotroph S. Agona was attracted to nutrient-rich tumour resulting in tumour suppression. sopB and sopD virulence genes do not contribute to tumour fitness since double knockout BD showed almost similar T/C% with parental strain. Notably, the current study has shown that multiple administration of treatment was unable to enhance the anti-tumour efficacy significantly. Overall, the findings in this study help to gain an understanding of virulence and the role of metabolic gene deletions in fine-tuning Salmonella pathogenecity for successful tumour colonization and suppression.In conclusion, this study demonstrated the use of the group II intron in constructing quadruple knockout pathogens for the first time. The intron insertion in 10.7717/peerj.5989/supp-1Figure S1Click here for additional data file.10.7717/peerj.5989/supp-2Figure S2sopB gene (1170); Lane 3: sop B mutated gene (2170 bp) in double knockout ΔsopBΔsopD; Lane 4: sopB mutated gene (2170 bp) in quadruple knockout ΔsopB ΔsopDΔleuBΔargD; Lane 5 and 6: Wild type control template of sopD gene (310 bp); Lane 7: sopD mutated gene  in double knockout ΔsopBΔsopD; Lane 8: sopD mutated gene  in quadruple knockout ΔsopB ΔsopD ΔleuB ΔargD; Lane 9 and 10: Wild-type control template of leuB gene ; Lane 11: leuB mutated gene  in double knockout ΔleuBΔargD; Lane 12: leuB mutated gene  in quadruple knockout ΔsopBΔsopDΔleuBΔargD; Lane 13 and 14: Wild-type control template of argD gene (887 bp); Lane 15: argD mutated gene  in double knockout ΔleuB ΔargD; Lane 16: argD mutated gene  in quadruple knockout ΔsopBΔsopDΔleuBΔargD.(A) Lane 1 and 2: Wild-type control template of Click here for additional data file.10.7717/peerj.5989/supp-3Figure S3P < 0.01 and (▾) P < 0.05 when compared with no tumour group. (B): Blood neutrophil percentage in tumour-free mice and CT-26 tumour-bearing mice. Mice were sacrificed and blood was collected post treatment at indicated time point. Each bar represents the mean ±SEM of four mice per group. (*) P < 0.01 and (▾) P < 0.05 when compared with no tumour group.(A) Bacterial load in the spleen of tumour-free mice and CT-26 tumour-bearing mice. Mice were sacrificed and spleen was collected post treatment at indicated time point. Each bar represents the mean ±SEM of four mice per group. (*) Click here for additional data file.10.7717/peerj.5989/supp-4Figure S4S. Agona. CT-26 mice were administrated intraperitoneally with different strains of Salmonella and the body weight of mice was taken every two days. No significant difference observed in the body weight throughout the study. (B) Tumour growth curve of CT-26 tumour-bearing mice post treatment with wild-type and engineered strains of S. Agona. CT-26 mice were administrated intraperitoneally with different strains of Salmonella and the tumour size was taken every two days.(A) Body weight of CT-26 tumour-bearing mice post treatment with wild-type and engineered strains of Click here for additional data file.10.7717/peerj.5989/supp-5Figure S5Click here for additional data file.10.7717/peerj.5989/supp-6Figure S6Click here for additional data file.10.7717/peerj.5989/supp-7Data S1Click here for additional data file.10.7717/peerj.5989/supp-8Data S2Click here for additional data file.10.7717/peerj.5989/supp-9Data S3Click here for additional data file.10.7717/peerj.5989/supp-10Data S4Click here for additional data file.