Understanding how a pathogen colonizes and adapts to a new host environment is a primary aim in studying emerging infectious diseases. Adaptive mutations arise among the thousands of variants generated during RNA virus infection, and identifying these variants will shed light onto how changes in tropism and species jumps can occur. Here, we adapted Coxsackie virus B3 to a highly permissive and less permissive environment. Using deep sequencing and bioinformatics, we identified a multi-step adaptive process to adaptation involving residues in the receptor footprints that correlated with receptor availability and with increase in virus fitness in an environment-specific manner. We show that adaptation occurs by selection of a dominant mutation followed by group selection of minority variants that together, confer the fitness increase observed in the population, rather than selection of a single dominant genotype. When RNA viruses replicate, they do so with a high rate of error; hence, their populations are not composed of a single genotype, but of a swarm of different, yet related, genomes. This mutant spectrum has been described as the viral quasispecies, and its composition has important consequences for evolution, adaptation and emergence. In this study, we analysed adaptation in fine detail thanks to the use of the deep sequencing, and we determined the adaptative pathway of a model RNA virus, Coxsackievirus B3, to a new environment, A549 cells. Our results demonstrate that adaptation occurred in response to a differential expression of the virus receptors in the new cellular environment, compared to the former. Our experiments and mathematical analyses established that the corresponding increase in fitness resulted from the selection and contribution of a group of genotypes, including low frequency variants, and not to the effect of a single, dominant genome. Our work underscores the importance of considering group effects when studying RNA virus biology and evolution. The extreme mutation rates of RNA viruses and the highly diverse populations they generate in few replication cycles are considered the basis for their rapid adaptation to new environments ,2. Such One of the goals in the field of emerging infectious diseases is to determine whether adaptation to novel hosts  can be identified for a recently introduced pathogen that is confronted with a less than optimal host environment –11. ViruTo monitor the evolution of CVB3 towards novel and less permissive host environments, we selected human lung A549 cells, which gave similar final virus yields as the highly permissive HeLa cells, but after two days rather than one day of infection. CVB3 was thus serially passaged 40 times in six biological replicate series in both cell types. Virus yields were constant throughout the passage series suggesting that no significant genetic drift or accumulation of detrimental mutations through population bottlenecking had occurred Fig  and 1B. in vitro transcribed RNA derived from an infectious clone. The data also confirmed that severe bottlenecking did not occur during the passage series where expansion of diversity should have been stalled or lost.Whole-genome deep sequencing of these populations revealed significant increases in overall genetic variation, throughout the genome, between the 1st, 20th and 40th passages in both HeLa  and A549Although the HeLa and A549 cell passage series presented similar mean genetic diversity at passage 40 Fig . In bothConsidering these CAR/DAF-specific differences, we hypothesized that expression levels of these molecules must vary between these cells. There were no significant differences in CAR expression in either cell by both flow cytometry  or WesteIt has been shown that adaptation is often a multi-step process, especially in asexual populations like viruses. Usually, until the mutation with the largest beneficial effect becomes fixed, secondary beneficial mutations with less effect are unable to compete, thereby rendering the adaptation process sequential –19. GiveDespite the clear fitness advantage conferred by the E76G mutation alone (approximately 10-fold increase with respect to wildtype), it could not account for the more significant fitness increases observed in the passaged populations, particularly at late passage stages (nearly 100-fold increases). Following the fixation of this first mutation, the frequency of VP3-E76G plateaued until a second increase after passage 27, in each of the six replicates, coinciding with the emergence of different combinations of mutations, all mapping to the DAF footprint . Closer To distinguish between the above cases, phylogenetic trees describing the mix of haplotypes at each passage were inferred from the longitudinal data using maximum likelihood estimation in a Bayesian model. The best-fit, predicted haplotypes were then generated by reverse genetics and their relative fitness values were measured. As expected, this analysis illustrated the quick rise of the E76G genotype that continues to coexist with, although generally dominating over, the original WT genotype Fig –5F, withFinally, shortly after the appearance of residue 138 and 234 variants, a third mutation appeared during the passage series at residue 63 Fig  and 5E. in vitro transcribed RNAs corresponding to some of these variants and assayed virus production at 8 hours (before a new round of infection can occur). The data revealed that the fitness advantages  and disadvantages  observed during infection of cells , which has been regarded as a caveat to properly describe RNA virus quasispecies or mutant spectra. This problem was recently resolved by elegant molecular biology techniques to remove background error ,25. By ain vivo), that the emergence of such minority variants would be delayed or impeded.In this work, adaptation to a novel host environment was a multistep process involving the emergence of an initial mutation (E76G), followed by selection of a number of minority variants on the E76G background. Initially, we expected mutations to accumulate in a step-wise manner in a single genotype; however, fitness assays revealed that genotypes harboring double and triple combinations of these mutations presented fitness decreases relative to the wildtype and/or E76G backgrounds. Instead, computational inference of haplotypes suggested that such variants existed as a heterogeneous population of distinct genotypes. An intriguing observation was that no single variant (nor combination of two variants) conferred the high fitness values observed in the passage 40 populations. Strikingly, only the combination of the four most predominant genotypes, as an artificial quasispecies with the same frequencies observed in the passage 40 populations, was able to confer the same fitness values as the p40 replicate samples. This phenomenon is reminiscent of previous work that suggested that minority variants within an RNA virus quasispecies may contribute significantly to phenotype . In thatin vitro transcribed using T7 RNA polymerase. It should be noted that unlike other Nancy strains, this infectious clone already has VP2-138D and VP3-234Q residues, known to facilitate binding to DAF. 4 μg of transcript were electroporated into 4 x 106 Vero cells that were washed twice in PBS and resuspended in PBS at 107 cells/ml. For A549 cells, 15 μg of transcript were electroporated into 10 x 106 cells. Electroporation conditions were as follows: 0.4mm cuvette, 950 μF, 250V, maximum resistance, exponential decay in a Biorad GenePulser XCell electroporator. Cells were recovered in DMEM-10% NCS. For each passage , virus was titrated by TCID50 and 400 μl of medium containing 2 x 105 TCID50 was used to infect 2 x 106 Hela or A549 cells in 6 well plates using a multiplicity of infection (MOI) of 0.1. Cells were incubated with virus for 45 minutes with frequent rocking, the supernatant was removed and monolayers were washed twice with 2ml PBS, then replenished with 2ml of complete medium. For each passage, virus was harvested at total cytopathic effect (CPE) by one freeze-thaw cycle, representing 2–3 viral generations. Six biologically independent stocks and passage series were generated.HeLa and A549 cells (American Type Culture Collection) were maintained in DMEM medium with 10% new-born calf serum. Coxsackie virus B3 (Nancy strain) was recovered from a pCB3-Nancy infectious cDNA plasmid  that was4 Vero cells plated in 100 μl of DMEM-10% NCS. After 5 days living cell monolayers were colored by crystal violet. TCID50 values were determined by the Reed and Muensch method. No significant differences were observed between TCID50 values when using Vero, HeLa or A549 cells as the cellular substrate.Ten-fold serial dilutions of virus were prepared in 96-well flat-bottom plates in DMEM. Dilutions were performed in octuplate and 100 μl of dilution were transferred to 108 virion from passaged samples were RNA extracted and RT-PCR amplified by RT (Superscript III) and PCR (Phusion) using primers sets that covered the whole genome, in 3–4 kb fragments. For consensus sequencing, the resulting PCR products were purified, sequenced and analyzed using Lasergene software (DNAStar Inc). For deep sequencing, PCR fragments were purified via the Nucleospin Gel and PCR Clean-up kit (Macherey-Nagel) and total DNA was quantified by Nano-drop. PCR products were then fragmented (Fragmentase), linked to Illumina multiplex adapters, clusterized and sequenced with Illumina cBot and GAIIX technology. Sequences were demultiplexed by CASAVA with no mismatches permitted. Clipping was performed using the fastq-mcf tool, removing common adapter contaminants and trimming low quality bases (Phred<30). Clipped reads were aligned to the Coxsackie virus B3 Nancy sequence as reference with a maximum 2 mismatches per read, and no gaps, using BWA v0.5.9. Alignments were processed using SAMTools to obtain a pileup of the called bases at each position. An in-house pipeline, termed ViVAN  [i:5x10Nalysis)  was usedHela and A549 cells were plated onto coverslips, fixed with 2% paraformaldehyde for 20 minutes at room temperature, and then washed with PBS. Before staining, non-specific staining was blocked by incubating the cells with 5% FCS and 0.05% saponin in PBS during 10 minutes. Staining antibodies were diluted also in this buffer, and it was used for washes between antibodies. Cells were incubated with either CAR (Santacruz) or DAF (Abcam) primary antibody, washed, stained with a secondary antibody coupled to the appropriate fluorophore and washed. Cells were analyzed using a Zeiss LSM-700 confocal microscope. 3D reconstruction of the images was performed using the Imaris software.One million cells of HeLa and A549 cells were lysed using a buffer containing 1% Triton-X and 1% sodium deoxycholate with protease inhibitors (Sigma). A fraction of the lysate was run for 1 hour on a 4–15% gradient gel (Biorad) on denaturalizing conditions. After the run, we performed the transfer to a nitrocellulose membrane. We washed the membrane with PBS-T (PBS 1X and 0.1% Tween-20) and blocked for 1 hour in PBS-T plus 5% milk. After the blocking we washed again with PBS-T and left overnight with each one of the antibodies, anti-CAR and anti-DAF (Santacruz Biotechnology). We washed the membrane and we added the secondary fluorescent antibodies  for 1 hour. We washed the membrane one last time and measure the fluorescence in the Odyssey system (Li-Cor).In order to prepare cells for cell cytometry analysis, cells were washed twice with PBS 1x and trysinized, washed again twice in PBS. Cells were stained for 30 minutes with either CAR-PE or DAF-FITC antibodies (Millipore and Abcam) on ice. Unbound antibody was discarded and cells were washed again with PBS 1x. Cells were resuspended in 1% Parafolmaldehyde  and kept in the dark for 15 minutes. After the incubation PFA was discarded and cells were resuspended in 200 μl of PBS 1x. Cells were kept at 4C until analysed. For each cell type used (HeLa and A549) specific instrument settings were set according to the size and complexity of the cell type, as well as antibodies fluorescence. Samples were analysed using the MACSquant flow cytometer (Miltenyi Biotec) using 96 well plates and obtaining 10,000 events per sample. Mock samples were also used in each plate to setup the baseline. Results were analysed using Flowjo software v10.Relative fitness values were obtained by competing each virus population with a marked reference virus that contains four adjacent silent mutations in the polymerase region introduced by direct mutagenesis. Co-infections were performed in triplicate at an MOI of 0.01 using a 1:1 mixture of each variant with the reference virus for 24 hours. The proportion of each virus was determined by real time RT-PCR on extracted RNA from the infection supernanant, using a mixture of Taqman probes labelled with two different fluorescent reporter dyes. MGB_CVB3_WT detects WT virus (including the fidelity variants) with the sequence CGCATCGTACCCATGG and labelled at the 5’ end with a 6FAM dye (6-carboxyfluorescein) and MGB_CVB3_Ref containing the four silent mutations: CGCTAGCTACCCATGG was labelled with a 5’ VIC dye. Each 25 μl-reaction contained 5ul of RNA, 900nM of each primer  and 150 nM of each probe. The relative fitness was determined by the method described by Carrasco et al. [CVB3 parental strain, and CVB3-E76G were propagated in HeLa cells and purified as described previously . The memX be a phylogenetic tree, taken to include relative population sizes of the branches, and let Xt = (stpt)T be the state of the tree at time t = 1, 2,…,T. Here st denotes the structure of the tree, i.e. the set of branches existing at time t and pt is a vector with the relative population sizes of all existing branches. Furthermore, let Y be the set of measurements, where Yt ∊ ℤ+xN64 is the number of reads supporting each of the 64 codons at each of the N different positions in the genome. By Bayes' theorem,Y | X) follows a multinomial distribution for each position. The dynamics of the phylogenetic tree are naturally assumed to be Markovian, i.e.pt | st, pt-1), the population change from t−1 to t is taken to be lognormally distributed with standard deviation σh where h is the length of the time step. The structural part, P, depends on the mutation frequency of the virus and the population size of the haplotype in which the mutation occurs. The posterior probability is thus, assuming that s1 and p1 are known,Let X from the posterior tractable, some approximations are made. The attention is limited to a small set of variants, greatly reducing the number of possible tree structures. The most prevalent variants in the data should be included as they dominate the dynamic behaviour, but minority variants of particular interest can also be added.To make a maximum likelihood estimation (MLE) of pt | st, Yt) is then computed for each tree and time point. The rationale is that there is very little freedom for the population sizes to deviate from a point which can explain the output data. By construction of the tree structure, the number of non-reference haplotypes equals the number of variants at each time point in every tree. Hence, there is at most one convex combination of the haplotypes that match the event probabilities of the multinomial distribution for the output data. Due to the high number of reads in the deep sequencing data, moving away from the optimal point will cause a quick drop in the posterior probability. Dependencies between variants that are close enough to be covered by a single read are included in the model (amino acid residues 63 and 76).Every possible tree structure matching the selected set of variants is generated. The time of appearance for each variant is set to where it is first seen in the measurements, i.e. the first time the frequency is above a small threshold. An MLE of P cancels out. Hence, P is simply proportional to the population size of the haplotypes in which the mutations occur.The MLE of S1 FigLocalization of DAF, in green, in HeLa (A) or A549 (B) cells by confocal microscopy. Nuclear staining was done with DAPI (blue). (B) DAF could only be detected when the exposure was increased 5 times compared to the HeLa settings.(PDF)Click here for additional data file.S1 MovieHela cells were plated onto coverslips and processed for confocal microscopy analysis, as described in Materials and Methods. Z stack images acquired for Fig (AVI)Click here for additional data file.S2 MovieA549 cells were plated onto coverslips and processed for confocal microscopy analysis, as described in Materials and Methods. Z stack images acquired for Fig (AVI)Click here for additional data file.S1 DatasetThe following tables reveal all of the minority variants identified by bioinformatic analysis that are present at frequencies above 0.001 . The limit of detection where variants could not be distinguished from background error was always above 0.0001 frequency, but for clarity, variants at 0.0001 to 0.001 frequencies are not shown. The total number of variants in each population are quite similar, ranging from 1779 to 2005. The majority of these are very low frequency variants that do not change over the passage series. As would be expected by the generally neutral nature of synonymous mutations, nearly half of low frequency variants are synonymous. In some cases however, a number of synonymous mutations are found at increasing frequency from passage 1 to 40 (eg. nt. position 920 in replicate 4), or at significantly higher, yet relatively constant, frequency over the passage series (eg. nt. position 1283 in replicate 4). Their presence may indicate variants altering RNA structure, codon usage or other factors influencing fitness, but may also result from stochastic fixation of neutral mutations since they generally appear in only one of six replicates. Some variants with amino acid changes are present at considerable frequencies, without significant increase or decrease over the passage series (eg. VP1 position 104–106). Their contribution to the fitness of the population has not been studied here, but lack of increase in frequency suggests that they do not play a role in adaptation to A549 cells. Data from these tables was used to generate panels in (XLSX)Click here for additional data file.


Artemia to arsenic  and found that infection consistently reduced mortality across a range of arsenic concentrations. An increase from 25°C to 29°C, simulating the change in mean temperature expected under climate change, increased arsenic toxicity, but the benefits of infection persisted. Infected individuals showed higher levels of catalase and glutathione reductase activity, antioxidant enzymes with a very important role in the protection against oxidative stress. Levels of TBARS were unaffected by parasites, suggesting that infection is not associated with oxidative damage. Moreover, infected Artemia had a higher number of carotenoid-rich lipid droplets which may also protect the host through the “survival of the fattest” principle and the antioxidant potential of carotenoids. This study illustrates the need to consider the multi-stress context (contaminants and temperature increase) in which host-parasite interactions occur.Parasites and pollutants can both affect any living organism, and their interactions can be very important. To date, repeated studies have found that parasites and heavy metals or metalloids both have important negative effects on the health of animals, often in a synergistic manner. Here, we show for the first time that parasites can increase host resistance to metalloid arsenic, focusing on a clonal population of brine shrimp from the contaminated Odiel and Tinto estuary in SW Spain. We studied the effect of cestodes on the response of  Artemia. We found that tapeworms make Artemiamore resistant to As, a major pollutant in aquatic environments, even under increased temperature conditions. These parasites increase the capacity of antioxidant enzymatic defenses, allowing infected individuals to cope better with As. Moreover, tapeworms increase fat reserves in their hosts, which may be advantageous due to the ability of lipids to sequester pollutants . Although our results may be unusual, we find a clear explanation for them. This makes them of broad significance and general interest.Virtually all free-living organisms are infected by parasites. Moreover, both parasites and hosts may be exposed to increasing levels of pollution and might be affected by climate change. However, few studies have considered the environmental context in which parasites and hosts interact, and the relationships between these factors remains poorly understood. It is assumed that infection with parasites increases mortality under a cause of stress such as pollution. We studied the combined effect of arsenic (As) pollution, temperature increase and infection by tapeworms on the health of the economically and ecologically important brine shrimp  Many aspects of host-parasite interactions have been studied in detail, from molecular mechanisms to adaptive strategies and their ecological and evolutionary consequences  of protecting against the parasite. Conversely, the epidemiology of the parasite may be altered negatively if the pollutant impacts the life history of the host , thus compromising parasite transmission. Moreover, parasites and pollution can interact to affect the health of the host, the central topic of the emerging field of “Environmental Parasitology” . Most stHowever, parasites can influence multiple facets of host phenotype , includiFurthermore, all these effects are likely to depend on physiological variation which is influenced by temperature and hence by climate change. The projected temperature increase this century  is likelArtemia spp.  are economically and ecologically important. They are used as model organisms in aquatic toxicology [Artemia are the dominant macroinvertebrate in hypersaline ecosystems around the world, and many waterbirds depend on them as food [Artemia [Brine shrimps xicology , 18 and xicology . Artemia as food ,21,22. T as food  and are  as food . These p[Artemia ,26,27 whArtemia parthenogenetica population from the Odiel and Tinto estuary, SW Spain, one of the most polluted estuarine systems in Western Europe [A. parthenogenetica from Odiel have high levels of cestode infection [Flamingolepis liguloides which uses flamingos as final hosts, and Confluaria podicipina which infects grebes [-1 and 23 t yr-1 of As is transported by the Tinto and Odiel rivers (respectively) into the Atlantic Ocean [We studied the n Europe . A. partnfection , especias grebes . Arsenics grebes . Arsenics grebes ,33,34. Aic Ocean . Previouic Ocean , exceediic Ocean ), and alic Ocean ).Artemia to acute As exposure. We conducted toxicity tests to compare As median lethal concentrations at 24 h (24-h LC50) for infected and uninfected individuals on two separate dates one month apart, in order to test the effect of different parasite assemblages on arsenic sensitivity. Since cestode parasites in A. parthenogenetica have a strong seasonal pattern [Artemia, in order to measure the capacity of infected animals for detoxification of reactive oxygen species caused by factors such as pollution or climate change. We included the activity of four important enzymes: superoxide dismutase (SOD) responsible for the detoxification of the highly reactive oxygen species superoxide anions; catalase (CAT) which catalyses the decomposition of hydrogen peroxide in 02 and H20; glutathione peroxidase (GPX) which acts as a scavenger for high concentrations of hydrogen peroxide; and glutathione reductase (GR) implicated in the reduction of glutathione disulphide to the sulphydryl form glutathione, which is a critical molecule in combating oxidative stress. In order to evaluate the efficacy of the above enzymatic mechanisms for control of reactive oxygen species, we also measured the levels of thiobarbituric acid reactive substances (TBARS), which indicates the extent of lipid peroxidation as a consequence of oxidative damage. Finally, we quantified the carotenoid-rich lipid droplets of infected and uninfected individuals. These droplets serve as intracellular lipid storage but also play a protective role in protecting cells against oxidative stress.This study was designed to test the individual and combined effects of infection by cestode parasites and a 4°C temperature change on the sensitivity of  pattern , and difArtemia to As, two experiments were conducted in different months (April and May 2014). Infected and uninfected individuals were separated on the basis of their colour ,,Artemia infected group varied significantly between the two experiments  for six of nine cestode species recorded in the eriments .Flamingolepis liguloides (68%) and Confluaria podicipina (65%), with 36% of individuals being infected by both these parasites  with no C. podicipina. Other minority cestodes  present in one or both of the experiments were Flamingolepis flamingo and Gynandrotaenia stammeri , Eurycestus avoceti, Anomotaenia microphallos and A. tringae (shorebirds), Fimbriarioides tadornae (shelducks) and Wardium stellorae (gulls). Total parasite loads were significantly higher in April, as was the number of cestode species present per individual Artemia  in infected and uninfected individuals at a single temperature (25°C in April) or at two temperature conditions (25 and 29°C in May). In April, when Artemia starts the first generation of the year, there were not enough individuals to conduct the experiment at 29°C. However it was a good moment to conduct the experiments because at the beginning of the season a high proportion of Artemia are infected by only one parasite species, making results easier to interpret ..50 was s50 of the uninfected individuals at 25°C did not change between the two dates, that for infected individuals was significantly higher in May  compared with the experiment conducted in April   . An incr(Artemia .Artemia that died) was significantly influenced by temperature (p = 0.015), date (p = 0.007), parasitic status (p < 0.0001) and As concentration (p < 0.0001) , mortality rate  . The intF. liguloides, and its prevalence did not differ from the sampling of May 2014 . Prevalence and abundance of minority cestode species varied among dates.It was impossible to analyse oxidative stress in individuals used in acute toxicity tests because of the need to crush the specimens in order to check for parasites, so we conducted a separate sampling in May 2015. Parasite prevalence has a seasonal pattern , so in oArtemia revealed the association of parasites with strong changes in the antioxidant capacity of the host. Infected individuals showed significantly higher levels of CAT  is related to the increased levels of hemoglobins in F. liguloides-infected Artemia compared with uninfected individuals . Decrease in antioxidant status enhances short-term survival prospects [This enhancement of the anti-oxidative defense mechanisms is probably connected with the trophic transmission mode of cestodes that infect rospects , so poterospects ).F. liguloides increases total lipid levels [C. podicipina increases triglyceride levels in Artemia [survival of the fattest [Pisidium amnicum [Artemia, additional lipids accumulate in the host, not in the parasite as in the case of Pisidium.We also found that there were more lipid droplets in infected individuals, which is consistent with previous work indicating that d levels  and that Artemia  Fig 2B)F. ligulo Artemia . Neutral Artemia ,55—a pri fattest . Althoug amnicum . Clams i amnicum . In paraArtemia are associated with carotenoids  by increasing visibility [Artemia. Given that oxidative stress is a common marker of toxicity, not only for As in plants, invertebrates and vertebrates [Artemia against a broader range of pollutants.Lipid droplets in infected oids see  and thisoids see . Both F. Artemia . In cont Artemia  and with Artemia . Caroten Artemia  and are  Artemia . The accsibility  and enhasibility . Carotensibility ,63,64 sotebrates ,66,67 butebrates ), cestodUnlike carotenoids, a positive effect of parasites on host lipid content is common in nature, e.g. in acanthocephalans infecting gammarids  or tremaArtemia collected on different dates have several possible explanations, including a negative effect of the generally higher infection levels, or a higher pathogenicity of C. podicipina which was absent in May. Alternatively, it could be related to seasonal changes in the ages of the parasites or their hosts, or changes in the lipid or carotenoid levels in the host. Increased heavy metal accumulation with age in cysticercoids has been shown in other cestodes [The differences in sensitivity to As observed in infected cestodes ,72. Prev50 for both infected and uninfected Artemia. The decrease in dissolved oxygen in hypersaline waters with increasing temperature coincides with higher respiratory demands in Artemia. Water permeability and drinking in Artemia increase markedly with temperature [Artemia increases with temperature owing to increased activity and diffusion rate [The toxicity of As is highly dependent on its bioavailability, which in turn is dependent on its chemical form and the capacity to be released from environmental matrices . In mariperature , hence uion rate . Differeion rate .Parasites and pathogens are conventionally considered as detrimental for a host, but they can also have positive impacts with consequences for non-host species and even the whole ecosystem . We provOur results provide an important advance in our understanding of host-parasite interactions and underline the importance of considering interactions between parasites, pollutants and temperature in combination, particularly given expected climate change and the likelihood that toxicity will increase with temperature.A. parthenogenetica were collected with a plankton net (0.5 mm) within the Odiel saltpan complex  at which the relative abundance of different cestode parasites changed considerably. On the 14th of April, Artemia were collected from three ponds with salinities of 140, 150 and 190 g/l (measured with a refractometer). On the 15th of May, samples were collected from another pond with a salinity of 200 g/l. These four ponds were all within the same stage of the solar evaporation process, were hydrologically interconnected, and had similar sediment type, depth, and invertebrate and bird communities [Artemia. Artemia were transported to the laboratory in 25 litre containers. Infected and uninfected Artemia individuals of a similar size were then selected on the basis of their colour . Among individuals allocated to the uninfected group, 98% were truly uninfected (n = 988).Sampling was carried out on two dates . Two experiments were carried out. The first (with Artemia collected on 14th of April) was conducted at 25°C  was conducted at both 25 and 29°C. During summer months, Artemia are often exposed to temperatures ≥ 29°C [Toxicity experiments were conducted after 24 h of acclimation of the alt pans ). The ses ≥ 29°C , 22 and 50) was used to quantify As toxicity in infected and uninfected adult Artemia. Arsenic, as reagent-grade sodium arsenate, NaAsO (CAS No. 10048-95-0) was used to prepare a concentrated stock solution. The study design concentrations were prepared by mixing different proportions of the stock solution and saltwater (Instant Ocean prepared with MilliQ). The saltwater used for the dilutions was prepared within 24 h of the start of the experiment and As added one hour before conducting the experiment (to prevent oxygen depletion). Ten concentrations of As between 5 and 140 mg/l were used for the experiments at 25°C , and ten between 4 and 67 mg/l were used for the experiment at 29°C  in order to estimate the LC50. Experimental concentrations were adjusted to the observed mortality . These concentrations were selected after preliminary tests. Three replicates were used per concentration, with each replicate made up of a group of 10 individuals. Infected (red) and uninfected (transparent) individuals were transferred to 25 ml beakers  and placed in climatic chambers at the chosen temperature, with a 12:12 photoperiod and without food. After a 24 h exposure period, dead individuals (considered to be those with no movement of the appendages observed within 10 seconds) were counted and all  individuals were mounted on slides to confirm parasitic status under the microscope. After observations of the cysticercoids  in situ, each infected specimen was gently pressed under the coverslip. If the identification of the cysticercoids recorded was not possible at this stage, whole infected brine shrimps or isolated cysticercoids were prepared as permanent mounts in Berlese’s medium in order to facilitate observations on the morphology of rostellar hooks. Cysticercoids were identified following Georgiev et al. [et al. [infected group” on both dates.Median lethal concentration  before the experiment. A subsample was used to characterize the exact parasite composition  and quantify the number of lipid droplets (n = 20 infected and 20 uninfected Artemia). The numbers of cysticercoids, prevalence, mean abundance and mean intensity . Infected and uninfected A. parthenogenetica of the same size range were allocated to 1L glass vials  with 600 ml of experimental solution (either control (no As) or 4.69 mg/L As) during 24h at 25°C (12:12 photoperiod) without food.The rest of the specimens were acclimated during 24h to the experimental salinity with continuous aeration and fed Artemia availability. The different biomarkers were determined in the whole soft tissues after homogenization and centrifugation. Tissues were homogenized with an electrical homogenator (Miccra D-1 Art Moderne Labor Technik) in cool homogenization buffer  using 200 μl of buffer per sample . The sample was centrifuged at 14,000 rpm at 4°C for 30 minutes and supernatant stored at −80°C until enzymatic determination.After 24h exposure, individuals were gently washed in distilled water and, after removing excess water, stored at ‒80°C until biochemical analysis. All operations were performed at 4°C to prevent enzyme or tissue degradation. We performed the biochemical analysis on pools of 20 individuals per treatment. Number of replicates varied from 1 to 12 depending on Artemia: activity of four enzymes  and lipid peroxidation levels (thiobarbituric acid reactive substances TBARS). Total protein content in the supernatant fluid was determined following a standard Bradford’s procedure [We quantified five parameters as a proxy for oxidative status of rocedure . Enzyme et al. (1970) [2O2 by converting it to H2O and O2 as follows:.Catalase activity was analyzed according to the method of Cohen . (1970)  by monitH2O2 → 2H2O + O2Potassium permanganate (KMnO4) is a powerful oxidizing agent that decomposes hydrogen peroxide through the following reaction:2 KMnO4 + 5 H2O2 + 3H2SO4 → 2 MnSO4 + 8 H2O + 5O2 + 3K2SO44 is added to the samples. Catalase activity is quantified by extrapolating in a standard curve of commercial catalase (SIGMA-60634), and is expressed as U/mg of total proteins.This produces a red end product. Absorbance at a wavelength of 480 nm is read five minutes after KMnOSuperoxide dismutase catalyses the dismutation of superoxide radicals to oxygen and hydrogen. SOD was estimated using the xanthine-oxidase cytochrome c method as described by McCord and Fridovich (1969) . The inhSelenium-dependent glutathione peroxidase (Se-GPx) exerts its protective role by reducing organic peroxides and hydrogen peroxide to alcohols and water, respectively, using glutathione (GSH) as a hydrogen donor. When GSH is oxidized to GSSG it loses its protective function. The GPx activity was determined by estimating nicotinamide adenine dinucleotide phosphate (NADPH) oxidation following the method of Carmagnol et al. (1983) , which iGlutathione reductase (GR) plays an essential role in protection against oxidative damage by catalysing the reduction of glutathione disulphide (GSSG) in a reaction dependent on NADPH. The activity of GR was determined, as for GPx, by measuring the decrease in absorbance at 340 nm due to the oxidation of NADPH, which acts in the reduction of GSSG to GSH, following the method of Cribb et al. 1989 . GR actiLipid peroxidation was estimated by the formation of thiobarbituric acid reactive substances (TBARS) following Buege and Aust (1978) . Lipid pAll biochemical analyses were performed at the Ecophysiology Laboratory of the EBD-CSIC in a Victor 3 multiplate reader .50) and 95% confidence limits were estimated and compared between infected and uninfected individuals, different temperatures and dates using Trimmed Spearman-Karber (TSK) analysis for lethal tests [non-overlapping 95% confidence limits was used to determine a significant difference (p < 0.05) between LC50 values [The median acute lethal concentration (LCal tests . The cri0 values .To test the effect of date, temperature, parasitic status and As concentration on mortality we performed GLZ with a Poisson error distribution, log link function and correction for overdispersion. Date, temperature and parasitic status were included as categorical factors, and As concentration as a continuous variable. Stepwise backwards removal was used to obtain a final model containing only significant factors.Differences in prevalence of different cestode species between the two sampling dates were evaluated with Z-tests. Comparisons of cestode abundance were performed with Mann-Whitney U tests. We also used Mann-Whitney tests to compare enzymatic activity and lipid peroxidation, as well as lipid droplets between infected and uninfected individuals. Statistica 12 software for Windows was used for all statistical analyses .


The relationship between obesity and cardiovascular disease (CVD) remains unclear. This study aims to describe the relationship between arterial stiffness and obesity in order to investigate the effects of obesity on CVD.We collected data from 5,158 individuals over 40 years of age from a cross-sectional study in Nanjing, China. Anthropometric, demographic, hemodynamic measurements and arterial stiffness measured through brachial-ankle pulse wave velocity (baPWV) were obtained. Subjects were grouped by body mass index (BMI), waist circumference (WC) and visceral adiposity index (VAI), a sex-specific index based on BMI, WC, triglyceride (TG) and high-density lipoprotein cholesterol (HDL-C).The multivariate regression analysis revealed a negative but weak effect of BMI  on baPWV, but failed to demonstrate any significant effect of WC on baPWV while VAI was a positive independent indicator of baPWV . The unadjusted baPWV significantly increased across groups with higher obesity categories (P<0.01). Although the positive association was lost after adjustments for confounding factors in the BMI or WC categories (P>0.05), it was still obtained between baPWV and VAI quartile (P<0.01). No differences were observed among the metabolically healthy groups or the metabolically abnormal groups in the BMI and WC categories (P>0.05). However, baPWV significantly increased across groups with higher VAI categories even in the same metabolic category (P<0.01).This study supports the concept of heterogeneity of metabolic status among individuals within the same obesity range. Obese individuals are at an increased risk of arterial stiffness regardless of their metabolic conditions. VAI may be a surrogate marker for the assessment of obesity and the effects of obesity on arterial stiffness. The prevalence of obesity is increasing at a dramatic rate in developed and developing countries, with data indicating that this will affect more than one billion people by 2030 Recent studies have shown that visceral adipose tissue participates in the pathogenesis of CVD A total of 5,158 community-dwelling individuals over 40 years of age living in the Gulou district, Nanjing, Jiangsu Province, China, from June 2011 to December 2011, were recruited for the study. The study was approved by the institutional review board of the First Affiliated Hospital of Nanjing Medical University and all participants provided written informed consent before initiation of the study protocol.2), or obese (BMI≥28 kg/m2). According to WC, individuals were divided into three groups with the cut-off points of 85 cm/80 cm and 90 cm/85 cm.A questionnaire was used to obtain information about demographic factors, medical history, medication use, and personal health habits, such as smoking habits. Anthropometric parameters were obtained by trained technicians using standard methods. Standing height was measured to the nearest millimeter, without shoes, using a wall-mounted vertical ruler. Weight was assessed to the nearest 0.1 kg without outdoor clothing or shoes. BMI was calculated as the weight in kilograms divided by the square of the height in meters. WC was measured in the standing position as the midpoint between the lower rib margin and the iliac crest according to the WHO's recommendation −×I−)]∧0.5 Venous blood samples were taken after an overnight fast for measurement of fasting plasma glucose (FPG), HbA1c, TG, cholesterol (CHOL), HDL-C and low-density lipoprotein cholesterol (LDL-C). A 75 g oral glucose tolerance test was performed and blood samples were taken at 0, 30, and 120 min for the measurement of plasma glucose and insulin concentrations. All measurements were analyzed by an autoanalyzer . Serum insulin concentrations were determined with the Phadebas Insulin Test  using a radioimmunosorbent technique. Insulin resistance was estimated using homeostasis model assessment-insulin resistance (HOMA-IR) calculated as fasting plasma insulin (µU/ml) × fasting plasma glucose (mmol/L)/22.5 Males: VAI = (WC/(39.68+(1.88×BMI)))×(TG/1.03)×(1.31/HDL)Females: VAI = (WC/(36.58+(1.89×BMI)))×(TG/0.81)×(1.52/HDL)Blood pressure (BP) was measured in a seated position using a standard mercury sphygmomanometer after a 5–10-min rest period under quiet environmental conditions according to a standardized protocol. BP measurement was repeated three times at 5 min intervals by a trained physician. Average BP was then calculated from the three consecutive measurements. Hypertension is defined as an average systolic BP (SBP) ≥140 mmHg or diastolic BP (DBP) ≥90 mmHg. BaPWV was measured in the supine position after at least 5 min of bed rest using a volume-plethysmographic apparatus with an automatic waveform analyzer , which simultaneously records PWV, the electrocardiogram and heart sounds, and was calculated as distance/time (cm/s). The distance between sampling points of the baPWV was calculated automatically according to the height of the subjects. The time interval between the brachium and the ankle was defined as the time interval between the wave front of the brachial waveform and that of the ankle waveform. Mean values of right and left baPWV were used for analysis.Continuous data were expressed as mean ± s.d., while categorical data were described as frequencies or percentages. The analyses of continuous variables to assess the differences among obesity categories were determined using ANOVA, followed by Bonferroni's test for multiple comparisons. Step-wise multiple linear regression analysis was performed to determine the correlation and independent variables for baPWV. Variables selected for multivariate regression were based on their clinical and biological plausibility and after a significant correlation had been obtained. PG120 was found to be a stronger independent predictor of arterial stiffness than FPG 2 and >23.9 kg/m2, respectively. The prevalence of individuals with a WC >85 cm/80 cm and >90 cm/85 cm was 23.4% and 39.4%, respectively.The clinical and laboratory characteristics of 5,158 subjects are presented in The step-wise multivariate regression analysis was performed to assess the correlation of baPWV with other clinical and laboratory variables in all subjects. The relationship between obesity category  and baPWV is showed in In order to further assess the effect of obesity on arterial stiffness, body size phenotypes were defined based on the combined consideration of the absence or presence of the abnormal metabolic components and the obesity categories. Compared with the metabolically abnormal category, metabolically healthy individuals had a lower value of baPWV regardless of their obesity category ; P<0.01.The associations between HOMA-IR, Matsuda ISI and VAI category are depicted in Our study presents new data investigating the relationship between obesity and arterial stiffness. BMI and WC are not independent predictors of arterial stiffness. VAI, an indirect indicator of fat distribution and function, is independently associated with increased arterial stiffness, even in metabolically healthy individuals, and may be suitable as an evaluation index of the effects of obesity on arterial stiffness.A population-based study has demonstrated that BMI and WC are not related to aortic stiffness in the fully adjusted model Both BMI and WC are clinical parameters reflecting the total visceral and subcutaneous fat mass Although obesity has been widely recognized as an important risk factor for the development of CVD, to what extent the impact of obesity on arterial stiffness remains unknown. Arnlöv et al. A major confusion may have arisen from the difference in the follow-up duration. In the study by Arnlöv et al. We noted several strengths and limitations of our study. The advantages of this study include the larger population sample and it addresses not only findings related to total fat, but also to visceral fat and fat distribution indirectly reflected by VAI, a newly extrapolated index that may be used as a surrogate marker of adipose tissue dysfunction. However, our study is limited. First, the cross-sectional design of this study cannot directly establish any causal associations between obesity categories and arterial stiffness. Second, some  portions of baPWV may be determined by ‘peripheral’ (or muscular) arterial stiffness, and baPWV is not the gold standard for the assessment of arterial stiffness. Nevertheless, previous studies have shown that there is a significant positive correlation between baPWV and CVD In conclusion, our study supports the concept of heterogeneity of metabolic status among individuals within the same obesity range. Obese individuals are at an increased risk of arterial stiffness regardless of their metabolic condition. Further studies are required in order to obtain a more suitable indicator to evaluate the impact of obesity on arterial stiffness. However, to a certain extent and considering the simplicity of the measurement and detection of the related indicators, VAI may be a surrogate marker for the assessment of obesity and the effects of obesity on arterial stiffness in daily clinical practice and population studies.


The aim of this study was to assess whether chronic exposure to volcanogenic air pollution by hydrothermal soil diffuse degassing is associated with respiratory defects in humans. This study was carried in the archipelago of the Azores, an area with active volcanism located in the Atlantic Ocean where Eurasian, African, and American lithospheric plates meet. A cross-sectional study was performed on a study group of 146 individuals inhabiting an area where volcanic activity is marked by active fumarolic fields and soil degassing  and a reference group of 359 individuals inhabiting an area without these secondary manifestations of volcanism . Odds ratio (OR) and 95% confidence intervals (CIs) were adjusted for age, gender, fatigue, asthma, and smoking. The OR for restrictive defects and for exacerbation of obstructive defects (COPD) in the hydrothermal area was 4.4 (95% CI 1.78–10.69) and 3.2 (95% CI 1.82–5.58), respectively. Increased prevalence of restrictions and all COPD severity ranks  was observed in the population from the hydrothermal area. These findings may assist health officials in advising and keeping up with these populations to prevent and minimize the risk of respiratory diseases. About 10% of the worldwide population inhabits or lives in the vicinity of some active or historically active volcano –4 and lo2-rich springs, and soil diffuse degassing areas [2) and radon (222Rn), this last one a radioactive gas. Carbon dioxide is one of the most abundant volcanic gases and is amongst the most important diffused gases released by soil degassing in Furnas Volcano  [2 is released permanently to the atmosphere from soils in volcanic areas not only during eruptive periods, but also during quiescent periods of activity. Considering that CO2 released by soils may enter the buildings through pipes, cracks in the floor, and/or the contact between floor and walls, it is considered important to assess the CO2 flux in buildings. Carbon dioxide level is usually greater inside a building than outside, and it can act as an indicator of ventilation efficiency, showing whether the supply of outside air is sufficient to dilute indoor air contaminants [The Azores archipelago  comprises nine volcanic inhabited islands, located between 36°45′–39°45′N and 24°45′–31°17′W , where tng areas , 13. Gas968 t/d) ; this ga968 t/d) . Previouaminants . Accordiaminants  that est2 from volcanic origin [Furnas and Ribeira Quente are two villages located, respectively, inside the caldera and in the south flank of Furnas active volcano, where the ground gas emissions that characterize the diffuse degassing areas occur permanently and, thus, inhabitants of such areas are often exposed to elevated concentrations of COc origin . Previouc origin , 20 and c origin . Moreovec origin  showed tTo perform this study, two areas were selected: an area with no secondary manifestations of volcanism, therefore a nonhydrothermal area (Ponta Delgada), and an area where volcanic activity is marked by active fumarolic fields and degassing soils  (Ribeira Quente). A diagnosis campaign was established for evaluation of pulmonary function by spirometry tests for the inhabitants of both areas. Spirometry tests were carried out in either the participants' workplaces or their homes. A standard questionnaire was applied to each individual that volunteered to participate. Medical history data for respiratory symptoms were taken using a standard questionnaire modified from a standardized respiratory symptom questionnaire from American Thoracic Society (ATS)  and Brit2 degassing with volcanic-hydrothermal origin [2 is anomalously high and can reach lethal values during extreme weather events [2 concentrations independently of the anthropogenic contribution.The study group consisted of 146 participants (94 women and 52 men), residents in Ribeira Quente village . Ribeiral origin . In addil origin . Thus, Rr events . PermaneThe reference group comprised 359 individuals (204 women and 155 men) working in downtown of Ponta Delgada city . Ponta DThe Ethics Board of Divino Espirito Santo Hospital  approved the study. All individuals signed a written informed consent, in compliance with the Helsinki Declaration and Oviedo Convention, to participate in this study. 1) and the forced vital capacity (FVC) values were obtained by spirometry in all participants. Spirometry tests were conducted with the participants in an up position wearing a nose clip and a disposable mouth piece using the EasyOne automated portable spirometer , which meets ATS/ERS spirometry standards [The forced expiratory volume in one second , according to the third United States National Health and Nutrition Examination Survey (NHANES III) for adult Caucasians. According to the GOLD guidelines, COPD was further classified in the following ranks given by the spirometer output: mild (FEV1 ≥ 80%), moderate (FEV1 50–79%), and severe (FEV1 30–49%).Spirometry data were classified categorically as being consistent with either a normal pulmonary function, a restrictive defect, or an obstructive defect. Spirometric detection of restriction was considered in all subjects with normal FEVCarbon dioxide degassing maps may be useful for volcanic/seismic monitoring purposes as they represent a reference for future variations on the state of activity of the volcano .2 released by soil degassing in Ribeira Quente village were carried out recently by Viveiros et al. [2 degassing zone (DDS), with soil CO2 fluxes that can reach values higher than 25000 g/m2/d [2 released by soils may enter the buildings , CO2 degassing maps produced for Ribeira Quente village [2 flux value to each building. Even if no indoor measurements were performed, the CO2 released from soils is positively correlated with the indoor CO2 concentrations [2 concentrations that can be found indoors [2 flux measurements were performed at Ponta Delgada in the areas surrounding the buildings occupied by individuals from the reference group. The 68 measurements performed showed that CO2 flux values were lower than 25 g/m2/d and thus representative of biogenic origin and without hydrothermal contribution.Measurements of soil COs et al. , 26. Thes et al. . Almost 0 g/m2/d . Conside village , 26 weretrations , so meas indoors . SporadiPearson Chi-Square test was used to compare restrictions and COPD prevalence between individuals inhabiting the environment with volcanic degassing and individuals from the reference group. To estimate the association between chronic exposure to an environment with volcanic air pollution and restrictive and obstructive defects (no versus yes), odds ratio (OR) and 95% confidence intervals (95% CIs) were calculated using a binary logistic regression model, adjusting for age, gender , fatigue (yes versus no), and smoking status (yes versus no).To estimate the association between chronic exposure to an environment with volcanic air pollution and the increase in the severity COPD, odds ratio (OR) and 95% confidence intervals (95% CIs) were calculated using an ordinal logistic regression model, adjusting for age, fatigue (yes versus no), asthma (yes versus no), and smoking status (yes versus no). The occurrence of obstructions was graded on scales, according to their occurrence and severity: 1: without obstruction, and 2 to 4: with obstruction, from the least severe (2) to the most severe (4).U test was used to compare soil CO2 fluxes released by diffuse degassing between the reference and the study group.Mann-Whitney P ≤ 0.05.All statistical analysis was performed using IBM SPSS Statistics 20.0 for Windows , and the2 versus 25.7 kg/m2, resp.). On the other hand, the reference group has a higher percentage of smokers than the study group (37.6% versus 19.1%).The general characteristics of the study populations are presented in 2 flux was significantly different (P < 0.001) between the studied areas (2 flux < 25 g/m2/d), while in Ribeira Quente 1.9% of the buildings were located in moderate susceptibility areas (soil CO2 flux between 25 g/m2/d and 50 g/m2/d) and the remaining 98.1% were in a high susceptibility area (soil CO2 flux ≥ 50 g/m2/d) .Soil COed areas . AccordiP = 0.001). Similarly, the prevalence of COPD was significantly higher in the study group than in the reference one   . The preP = 0.001) (P < 0.001)  . Also, a= 0.001) . Exacerb< 0.001) .The analyzed confounding factors did not show any significant association with respiratory restrictions , but res2-rich springs as well as degassing soils, release into the environment metals, hazardous aerosols, and gases that daily affect the quality of the environment and the health of human populations [The association between exposure to air pollution and adverse respiratory effects has been widely demonstrated , 32. Suculations .2 flux revealed that the study group  is chronically exposed to elevated volcanogenic air pollution by soil diffuse degassing compared to the reference group  versus 15.36 g/m2/d (≈1.08 ppm/s), resp.). Carbon dioxide levels are described to be usually greater inside a building than outside [2 than the presented values. Also, independently of the biological CO2 contribution, the hydrothermal CO2 emission is permanent and thus the level of CO2 in the Ribeira Quente buildings must be always higher than the “normal” values and sporadically reach anomalous high values as recognized in previous works, only due to meteorological changes [2 is 5000 ppm (WHO), and possibly the average CO2 concentrations encountered in the buildings are below this limit and are not expected to cause health symptoms such as headaches, fatigue, and eye and throat irritation. However, the consequences of the permanent presence of indoor CO2 in concentrations higher than the outdoor environment are still far from being understood and may eventually lead to some stress effect in the organisms that needs to be better studied. In addition, the CO2 emission may be also associated with other volcanic emissions, as it is the case of the radioactive gas radon. The measurements performed showed that the study group is chronically exposed to elevated volcanogenic air pollution, namely, high CO2 soil diffuse degassing; high concentrations of CO2 can cause headaches due to hypercapnia and the decrease of O2 in the bloodstream [The measurements of CO outside , 35; the changes . The thrResults also showed significant associations between the increase in the severity of COPD and the exposure to soil diffuse degassing in the study group when compared to the reference one, given as 3.2 times higher in Ribeira Quente inhabitants (study group). Age, asthma, fatigue, and smoking status were also significantly associated with the increase in COPD severity.1 and FVC decrease with age, and thus the observed increase in COPD associated with age was expected . Also recently results from Eurostat [Age and smoking habits are well associated with COPD prevalence , 38. AccEurostat  demonstrRegarding asthma, there might be a significant overlap with COPD, since both are characterized by an underlying airway inflammation, although the structural and pathophysiologic findings in both diseases can be usually differentiated . AccordiFatigue is considered the second most common symptom of COPD, after breathlessness . The incIt is well established that tobacco is the most important causative factor for COPD, with individual susceptibility continuously interacting synergistically with other risk factors , such as10, NO2, SO2, and O3 [It is well known that air pollution can perpetuate a chronic inflammatory process that potentially leads to lung diseases . However, and O3 , 51, whi, and O3 , 52. Lon, and O3  reported, and O3  showed t, and O3  that obs2 emissions in hydrothermal areas on the development of respiratory defects. The results of this study show that long-term exposure to air pollution by soil diffuse degassing increases the risk of developing restrictive defects as well as the exacerbation of COPD in the inhabitants of hydrothermal volcanic areas. Therefore, mitigation measures should be implemented in populations inhabiting elevated diffuse degassing areas, such as the construction of natural/forced ventilation systems, as well as follow-up health programs in order to provide medical counseling when necessary. The results of the study also support the need to particularly follow up the asthmatic and the older individuals, since these factors are associated with exacerbation in COPD severity in individuals chronically exposed to volcanogenic soil diffuse degassing.To our knowledge, this is the first study assessing long-term effects of CO2 in the atmosphere, areas as Ribeira Quente village can be used as natural analogous to studying the possible effects of these increases on the population.In addition, considering the positive trend of the CO2 diffuse degassing map from Ribeira Quente village . The dots represent the 146 studied individuals and their respiratory test outcomes .Supplementary Material 1 represents the volcanogenic soil CO


PMM2-CDG (formerly known as CDG Ia) a deficiency in phosphomannomutase, is the most frequent congenital disorder of glycosylation. The phenotype encompasses a wide range of neurological and non-neurological manifestations comprising cerebellar atrophy and intellectual deficiency. The phenotype of the disorder is well characterized in children but the long term course of the disease is unknown and the phenotype of late onset forms has not been comprehensively described. We thus retrospectively collected the clinical, biological and radiological data of 29 French PMM2-CDG patients aged 15 years or more with a proven molecular diagnosis . In addition, thirteen of these patients were reexamined at the time of the study to obtain detailed information. 27 of the 29 patients had a typical PMM2-CDG phenotype, with infantile hypotonia, strabismus, developmental delay followed by intellectual deficiency, epilepsy, retinitis pigmentosa and/or visceral manifestations. The main health problems for these patients as teenagers and in adulthood were primary ovarian insufficiency, growth retardation, coagulation anomalies and thrombotic events, skeletal deformities and osteopenia/osteoporosis, retinitis pigmentosa, as well as peripheral neuropathy. Three patients had never walked and three lost their ability to walk. The two remaining patients had a late-onset phenotype unreported to date. All patients (n = 29) had stable cerebellar atrophy. Our findings are in line with those of previous adult PMM2-CDG cohorts and points to the need for a multidisciplinary approach to the follow up of PMM2-CDG patients to prevent late complications. Additionally, our findings add weight to the view that PMM2-CDG may be diagnosed in teenage/adult patients with cerebellar atrophy, even in the absence of intellectual deficiency or non-neurological involvement. PMM2 gene [PMM2 sequencing.Congenital Disorders of Glycosylation (CDGs) are rare metabolic diseases with various clinical manifestations and a wide range of severity ,2. PhospMM2 gene ) which cMost articles reporting series of PMM2-CDG patients focused on the pediatric population because the diagnosis is usually established during childhood . Though PMM2 mutations. Clinical, biological, radiological and electrophysiological data were obtained from the medical files of all patients and 13 of them were reexamined during the previous two years, in accordance to the law and ethical requirements. Motor disability was evaluated with the Spinocerebellar Degeneration Functional Score  [We retrospectively collected the clinical observations of French PMM2-CDG patients aged 15 years or more with biallelic dridden) ,18.All blood samples were collected after obtaining informed consent. For all the patients at the time of diagnosis, whatever their age, screening of PMM2-CDG was performed in the Laboratory of Biochemistry of the Hospital Bichat-Claude Bernard in Paris (N. Seta) and evidenced abnormal glycosylation of serum glycoproteins and reduced activity of leukocytes (N > 6.0 U/g protein) and/or fibroblasts (N > 4.0 U/g proteins) phosphomannomutase (PMM) . The mol29 patients from 25 families aged from 15 to 49 years were included (mean 27.4 years). One patient (#13) died at 25 years of age. There were 16 males and 13 females (sex ratio of 1.2) and three sib pairs. Most patients were from France, 3 patients were from Spain, Portugal and West Indies.Normal pregnancies and deliveries were reported in 20/29 patients, in three cases where pregnancies included hydramnios, eclampsia and/or prematurity. Six patients had low birth weight, three with normal pregnancy and normal fetal growth parameters.First signs of PMM2-CDG were identified during the first months of life in 10/20 patients, and before the end of the first year in all but one. Visceral signs  were present in 8/20 patients and were always associated with truncal hypotonia and strabismus during the first year. When visceral signs were absent (n = 12/20), hypotonia, strabismus and psychomotor delay were the main revealing signs and remained in adulthood.Stroke-like episodes with full recovery occurred in two patients. A single stroke-like episode occurred at 3 years of age in one patient during a venous thrombosis. The second patient presented with three episodes between 3 and 13 years of age. No episode was reported for any patient after 15 years.Patients #11 and #14 had no reported signs suggesting PMM2-CDG during childhood, except patient #11 who had an isolated esotropia starting at two months of age.Cerebellar ataxia was the leading sign for diagnosis and was present in all patients. Gait ataxia was present during childhood in all walking patients and did not appear to worsen with time. 15/27 patients acquired independent walking (without aid), including two before the age of 18 months, and 13 between 18 months and 14 years of age . Eleven patients acquired walking with aid and three patients never walked. In the former group, three patients lost their ability to walk during adulthood because of severe kyphoscoliosis causing respiratory insufficiency, depression and possible lack of stimulation and exercise. During the course of the disease and at last examination, slowness and dysmetria were more often noticed than intentional tremor. Usual rating scales of ataxia, such as the Scale for the Assessment and Rating of Ataxia, could not be used with the 13 patients reexamined for this study because of their marked slowness and difficulties with comprehension. At last examination, patients who acquired independent walking were still walking without aid , seven patients needed aid (SDFS = 4 or 5), five patients were wheelchair bound (SDFS =6) and two were bedridden (SDFS =7).Brain MRI or TDM showed cerebellar atrophy in all patients and was sometimes associated with mild pons atrophy  showed diffuse or focal slow background activity in three patients and anterior fast rhythms (without benzodiazepine therapy) in another. Seizures were well-controlled with antiepileptic therapy, mostly with a single drug and with two drugs or more in two-thirds of the cases. Only occasional seizures occurred in adult patients.22/24 patients had delayed development and intellectual disability (ID) of variable severity. 10/15 patients with informative data achieved the ability to read at least a few words and six patients could read correctly and write by hand or with the use of a keyboard. None of these patients had a typical education: four patients followed an adapted program in “normal” schools but the others were enrolled in specialized schools for mentally and physically disabled children. Among 17 patients of working age, one worked as a farm-worker and another obtained an internship in business. Three patients were still working in specialized employment for people with disabilities and four others were waiting for such employment. The eight other patients were dependent on their family at home or attending occupational centers.Signs of peripheral neuropathy were reported for 22 patients and found in 13 after the age 15, mainly abolished osteotendinous reflexes, distal amyotrophy and sensitive alteration. Nerve conduction velocity (NCV) had been measured in nine patients. Two patients had a normal exam, axonal neuropathy was diagnosed in one and demyelinating neuropathy in the six others (exam performed between 1 and 49 years of age). NCV recordings were repeated in three cases, and demonstrated a slow worsening of demyelination over a period of 9 years in one patient and over a period of 3 years in adulthood for the other, with additional involvement of the upper limbs.Psychological aspects. Although PMM2-CDG patients were often described as affable and cheerful, eight patients in our cohort developed psychiatric symptoms, including aggressive behaviour (n = 5), apathy (n = 2) and depression (n = 1). These difficulties could contribute to the loss of autonomy, as exemplified by the depressed patient #22 who lost the ability to walk.Coagulation parameters were altered in 15/17 patients (88%) including significant decrease of factors IX, XI and XII, and less frequently of antithrombin III, protein C and protein S. Five patients presented blood count abnormalities, i.e. neutropenia (n = 2) or thrombocytopenia (n = 4).Five patients had venous thrombotic events in lower or upper limbs. The earliest venous thrombosis occurred in patient #7 at 3 years of age while the four others occurred in adolescent or adult patients at 17, 22, 41 and 44 years of age. Two patients had a single event and three had recurring events. Clotting factors IX and XI, as well as antithrombin III, were decreased during these events in all five patients, but the measures were not significantly lower than those collected before or after these events during routine biological assessments. Chronic low levels of protein C and protein S and thrombocytopenia were found in 2/5 with thrombotic events.One patient was treated with long-term anticoagulant therapy, another with antiplatelet therapy after a period of anticoagulant therapy and the third had a temporary anticoagulant treatment. There was no evidence of recurrence of thrombosis with antithrombotic therapy.25/28 patients had strabismus at some point during their lifetime and which persisted to adulthood for 24 patients. At least four patients underwent one or two strabismus surgeries, with poor outcome. No worsening was observed but one patient developed amblyopia. Severe associated refraction problems were observed in three patients: two with severe myopia (patients #4 and #8) and one with severe hypermetropia.Retinitis pigmentosa was diagnosed in 10/19 patients in whom fundus oculi and/or an electroretinogram (ERG) was available. It was progressive in one patient with a normal ERG at age 45 and a diagnosis of retinitis pigmentosa at age 49.Kyphoscoliosis was noticed in 17/24 patients and appeared before adolescence in all cases. Kyphoscoliosis worsened with time, causing severe thoracic deformities during adolescence in three patients aged 30 years or more. A fourth patient had an earlier thoracic deformation with severe respiratory failure at age 15.Bone densitometry was carried out in five patients aged 15 to 25 years and showed osteopenia in all of them and osteoporosis in two. Osteoporosis manifested as hip or tibial fracture in two patients.Head circumference was reported to be normal in all 14/29 patients where data was available. Thirteen patients had growth retardation . Growth parameters of others were around or beyond the mean. Correlation between height and weight remained harmonious, without obesity or underweight. No patients had thyroid dysfunction (n = 0/13); 2/6 patients with cortisol assessment had a low cortisol rate at 8 am.All female patients (n = 16) had primary ovarian insufficiency. At least six patients received hormone replacement therapy (commencement between 12 to 15 years of age). Hypocholesterolemia was found in 6/15 patients, hypoalbuminemia in 3/7 and elevated liver enzymes in 2/16.Inverted nipples and abnormal fat repartition, which are typical morphological signs present in the first months of life, were reported in 3/11 and in 7/19 adult patients of the cohort respectively  while all but two patients had signs before the end of the first year of life. In other adult patients in the available literature, the diagnosis was made at a mean age of 14 years ,12,16 anPregnancy, delivery, and the first years of life of our patient cohort were similar to those in the available literature ,22. FirsThe functional motor outcome of PMM2-CDG patients tended to differ according to the extent of the involvement of differing systems at onset: 7/8 patients with the neurological-multivisceral form required aid for walking or were unable to walk at last evaluation (SDFS ≥5), whereas 9/12 patients with the neurological phenotype walked independently (SDFS ≤3). This suggests that neonatal/infantile visceral PMM2-CDG is generally followed by a more severe neurological outcome. However, visceral manifestations do not always imply severe CNS involvement, particularly when onset is later in life .It has been reported repeatedly that cerebellar ataxia is not progressive in PMM2-CDG ,23, whicThough 35% of the patients of our cohort were epileptic, no patient had a first seizure in adolescence/adulthood and epilepsy was well-controlled with one or more anti-epileptic drugs in all patients, as has been shown in other adult samples ,15. StroOnly one third of adult patients reported here had short stature, which is less than in other cohorts  . All femDepression or behavioral issues reported in patients of our cohort point to the deleterious effect of the lack of stimulation in this vulnerable population of patients.Five patients of our cohort had venous thrombotic events, including three during adulthood. It is well-known that the prevalence of thrombosis in PMM2-CDG patients, evaluated at 14% for all ages ,27, is pOne third of the patients reported here had retinitis pigmentosa, which is less than in other reports  and in ophthalmological paediatric samples ,29. ThisScoliosis and thoracic deformities were recorded in 70% of our patients and in 80-100% of other adult patients in the literature ,15. OsteAll of these data underline the need for attentive, multidisciplinary approach to adult PMM2-CDG patients with teams that include neurologists, orthopedic surgeons and experts in rehabilitation medicine .The diagnosis of PMM2-CDG in patients #11 and #14 of our sample shows that the absence of specific signs in childhood, or abnormal or subnormal intellectual functioning, does not rule out the presence of the condition. A single pediatric patient with cerebellar hypoplasia and borderline cognitive dysfunction has been reported . Other PWe confirm that CDG screening should be a part of the routine workup of adolescent/adult patients with sporadic or presumably autosomal recessive cerebellar ataxia, even in the absence of other suggestive signs. We would also recommend that PMM2-CDG patients have: an annual biological workup , an annual ophthalmological and neurological clinical assessment, and an evaluation in physical and rehabilitation medicine. Bone densitometry should be proposed at least once in adulthood and we recommend calcium supplementation. Thrombosis prevention should be discussed in any predisposing situations.Written informed consent was obtained for the publication of this report and any accompanying images.OMIM: http://www.omim.org/entry/601785.


Bacterial penetration of dentinal tubules via exposed dentine can lead to root caries and promote infections of the pulp and root canal system. The aim of this work was to develop a new experimental model for studying bacterial invasion of dentinal tubules within the human oral cavity. Sections of human root dentine were mounted into lower oral appliances that were worn by four human subjects for 15 d. Roots were then fixed, sectioned, stained and examined microscopically for evidence of bacterial invasion. Levels of invasion were expressed as Tubule Invasion Factor (TIF). DNA was extracted from root samples, subjected to polymerase chain reaction amplification of 16S rRNA genes, and invading bacteria were identified by comparison of sequences with GenBank database.Granulicatella, Streptococcus, Klebsiella, Enterobacter, Acinetobacter, and Pseudomonas as dentinal tubule residents. All root dentine samples with patent tubules showed evidence of bacterial cell invasion  to depths of 200 mm or more. A spectrum of Gram-positive and Gram-negative cell morphotypes were visualized, and molecular typing identified species of in vivo model is described, which provides for human root dentine to be efficiently infected by oral microorganisms. A range of bacteria were able to initially invade dentinal tubules within exposed dentine. The model will be useful for testing the effectiveness of antiseptics, irrigants, and potential tubule occluding agents in preventing bacterial invasion of dentine. A novel  Root caries, pulpitis, and dentine hypersensitivity are becoming increasingly more problematic as the dentate human population agesEnterococcus and StreptococcusEnterococci in particular readily penetrate dentinal tubules,,in vitro dentine infection models employ conditions that are quite different from the natural in vivo infection environment.Bacterial persistence within the dentinal tubules may exacerbate development of root caries, and the formation of complex microbial communities within deeper dentine or root canal spacein vitro have also included testing various compounds for ability to occlude tubules as desensitizing agentsin vivo conditions. For example, West, et al.in vivo. This will provide a suitable platform by which to investigate bacterial invasion of dentine within a clinical environment, and to test for effectiveness of tubule-occluding or antimicrobial agents to prevent bacterial invasion of dentine.Dentine studies 2O at 4°C until required. Roots were sectioned using a water cooled steel bladed cutting machine . In brief, the crown and root tip were removed, the remaining root was cut into 0.5 cm lengths, and the cervical segments were longitudinally sectioned in such a way that the root canal was exposed. The root sections were then autoclaved  in distilled H2O, which did not visually affect tubule structuresNon-carious, unrestored human canine or pre-molar teeth with single root canals were obtained from orthodontic extractions. Teeth were obtained with informed consent and the study was approved by Central and South Bristol Ethics Committee (REC ref. 04/Q2006/50). Following extraction, teeth were soaked in 2% sodium hypochlorite (NaOCl) for 48 h and any soft tissue remaining was removed. Prior to sectioning, roots were washed in copious amounts of water, and rinsing was repeated following sectioning to ensure no traces of NaOCl remained. Teeth were stored in sterile distilled H® self curing acrylic . Adams cribs were constructed to fit the mandibular first molars to aid retention and wire loops were constructed in an anterior and posterior trench region to hold the dentine samples in place . Inclusion criteria were healthy volunteers aged 18 or over that could accommodate a lower buccal appliance. Exclusion criteria were: pregnancy, lactation, gross caries, unstable periodontal disease, antimicrobial medication within 7 d previously, orthodontic appliances that would interfere with the study evaluations, and tongue or lip piercing. Four subjects, age range 20-26 years, with informed consent, were fitted with the appliances which were then worn over the course of the next 15 d between 09:00 and 21:00 h. They were removed for 1 h twice a day for mealtimes and also for the period over which any drink other than water was consumed. At 21:00 h, subjects removed the appliances, brushed them with tap water, rinsed them in running water for 20 s, and then stored them overnight in an airtight container. Subjects brushed their teeth morning and evening with fluoridated dentifrice. On completion of the trial on day 15, root samples were removed from the appliances at 15:00 h. There were no adverse events.Six pieces of dentine from the appliances were fixed in 10% neutral buffered formalin for 7 d before being demineralized in 10% formic acid containing 2% formalin for 7 d. Samples were then dehydrated  before being blocked in wax. Fifteen transverse sections, 6 mm thick and 60 mm apart from the next section, were cut from each dentine sample, mounted on poly-lysine slides, and heat fixed prior to staining using the Brown and Brenn methodper field) invaded scored 1; 21-50 tubules invaded scored 2; and >50 tubules invaded scored 3. These scores were then converted to Tubule Invasion Factor (TIF) that took depth of invasion of tubules into accountper field showed invasion depth >50 mm; and x3, where ≥5 tubules per field were invaded to depth of ≥100 mm, as previously described Penetration of bacteria into dentine was visualized by light microscopy at x400 magnification. The central point of the root section was identified and five fields of view radiating out from this point were examined for each of the 15 root sections. The extent of invasion was initially expressed as the tubule invasion index (TI)2O and transferred to tubes containing Gene Releaser  for DNA extraction according to the manufacturer’s instructions.Two root pieces from each appliance were rinsed in sterile distilled water and stored at -80°C. One of the specimens in each case was used to optimize the methods for DNA extraction and amplification. Once this had been achieved, the second sample from each subject was transferred into a microfuge tube containing 0.1 mL sterile 10% EDTA (pH 6.5), vigorously vortex-mixed, and incubated at room temperature for 30 min to partially decalcify. Samples were then transferred into 0.1 mL 2 M citric acid (pH 1.6) and incubated for 30 min. Samples were extensively rinsed in sterile distilled HThe DNA extracts were used as templates in Polymerase Chain Reaction (PCR) amplification with universal 16S rRNA gene primers: DGGE F3(5’CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGGCCTACGGGAGGCAGCAG) with the GC clamp for Denaturing Gradient Gel Electrophoresis (DGGE)Escherichia coli XL1-Blue by standard procedures. Plasmids were extracted from transformant colonies using QIAprep Spin MiniPrep Kit (Qiagen), checked by agarose gel electrophoresis, and the 160-bp inserts were dideoxy-sequenced . The partial 16S rRNA gene sequences were then compared with 16S rRNA gene sequences in GenBank using the standard nucleotide NCBI/BLAST program.Each of the eluted gel bands was then subjected to further PCR amplification using primers F3 (5’CCTACGGGAGGCAGCAG) (as above minus GC clamp), and R2 (above). Presence of amplified fragments was confirmed by agarose gel electrophoresis . The frain vivo, the root pieces were removed and processed as described in Material and Methods for microscopic analyses (A-F). All samples containing patent dentinal tubules showed high levels of bacterial invasion, with TIF values in the range of 5 to 9  varied enormously across the dentine samples. We examined all dentine samples to confirm bacterial invasion, but in the following descriptions we have included only representative micrographs exhibiting distinct features of the invasion processes.For subject 1, root sample D, there was invasion of purple-stained Gram-positive cocci  provided sequences 100% identical to Acinetobacter and Streptococcus database entries, and 99% to Enterobacter spp. . Only a limited number of specimens were employed here because we were interested in first establishing a model system. The results suggest that the model can be applied to future studies of dentine hypersensitivity agents, determining their clinical efficacy and their ability to occlude tubules and block bacterial invasionE. faecalis was not found in our analyses. We identified Granulicatella, S. oralis, S. mitis, and S. gordonii which, with the exception of Granulicatella, have been previously implicated in tubule invasionGranulicatella are organisms that have been linked with infective endocarditis. Therefore, there could potentially be an association between ability to invade dentine and ability to cause endocardial or intravascular infectionsOne of the samples in the study described here was invaded by several species of Gram-positive cocci, which corroborates the notion that these organisms are often some of the first to invade dentinein vivoWe have thus identified organisms that were present within dentinal tubules that have been exposed to many hundreds of different bacteria in vivo model for studying the infection of dentine by oral microorganisms. Dentine specimens exposed to the human oral environment become infected with microorganisms to similar extent and depth to dentine infected in vitro under laboratory conditions. In addition to streptococci, bacteria from the genera Enterobacter, Klebsiella and Pseudomonas were identified as primary invading organisms. This in vivo model should provide the means to confirm in vitro experimental results on the effects of antiseptics, irrigants, or tubule occluding agents on dentine invasion by oral bacteria.In summary, this study has established a novel


Squamous cell carcinoma of the head and neck can present as a cervical metastasis from an unknown primary site. Recently, transoral robotic surgery (TORS) and transoral laser microsurgery (TLM) have been incorporated in the workup of unknown primary tumors.We searched MEDLINE, EMBASE, Cochrane, and CINAHL from inception to June 2015 for all English-language studies that utilized TORS, TLM, or lingual tonsillectomy in the approach to an unknown primary.Of 217 identified studies, eight were reviewed. TORS/TLM identified the primary tumor in 111/139 (80 %) patients overall, and 36/54 (67 %) patients with no remarkable findings following physical exam, radiologic imaging, and panendoscopy with directed biopsies. Lingual tonsillectomy identified the primary tumor in 18/25 (72 %) patients with no findings. Hemorrhage (5 %) was the most common perioperative complication.Lingual tonsillectomy using new approaches such as TORS/TLM may improve the identification of occult primary tumors. Cervical metastases from an unknown primary tumor site account for 2 to 5 % of all squamous cell carcinoma of the head and neck , 2. IdenThe standard workup of an unknown primary consists of a history, physical examination with flexible endoscopy, and diagnostic imaging such as computed tomography (CT) and/or magnetic resonance imaging (MRI). Positron-emission tomography (PET), alone or fused with CT images (PET-CT), may improve the diagnostic sensitivity when traditional imaging modalities fail to localize a primary tumor , 4, 8, 9In the absence of a visible or palpable lesion, a palatine tonsillectomy may improve the diagnostic yield of an occult primary tumor compared to deep tonsil biopsies –14, as mRecently, Transoral Laser Microsurgery (TLM) and Transoral Robotic Surgery (TORS) have emerged as effective modalities to aid in the identification and treatment of an unknown primary tumor. These techniques provide enhanced visualization and maneuverability, allowing for a complete resection of the entire tongue base mucosa and lingual tonsils, a procedure which is challenging to perform using traditional instrumentation and visualization , 16. RecThe present study aims to conduct a systematic review of the literature to determine the incremental benefit of lingual tonsillectomy using TORS/TLM in localizing the primary tumor site of regionally metastatic head and neck squamous cell carcinoma of unknown origin.A systematic review of published reports on TORS or TLM for the workup of CUP was performed. MEDLINE, EMBASE, Cochrane Central Register, and CINAHL were searched from inception to June 2015 for all relevant English-language studies. Medical Subject Headings and keywords specifying histopathology (e.g. squamous cell carcinoma), location , unknown primary, and diagnostic approach  were used to identify studies. Bibliographies of all included studies were also searched for relevant articles.Two reviewers (T.F. & A.F.) independently screened all identified studies by title and abstract for further full text review, and then independently reviewed these studies for eligibility Fig. . StudiesData were extracted in duplicate by two reviewers (T.F. & A.F.). The primary outcome was the identification rate of an unknown primary site using TORS, TLM, or lingual tonsillectomy performed using TORS/TLM. Information on study design, patient and tumor characteristics, diagnostic workup, margin status, and perioperative complications was also extracted. Subgroup analysis of identification rates were performed based on the presence or absence of positive findings on preoperative investigations including : physicaκ = 0.92).The literature search identified a total of 217 articles  underwent lingual tonsillectomy by TORS or TLM. Of the 90 patients with available information, 36 (40 %) had ipsilateral lingual tonsillectomy and 54 (60 %) had bilateral lingual tonsillectomy. Three studies , 19, 26 A total of 70 of 103 (68 %) patients underwent palatine tonsillectomy by TORS or TLM. Palatine tonsillectomy was either not performed or not reported in the remainder of the patients (36 of 139) for the following reasons: (i) 20 patients in one series  did not Overall, TORS/TLM successfully localized the primary tumor in 111 of 139 (80 %) patients, as shown in Table Identification rates for subgroups of patients with positive or negative findings during preoperative investigations are shown in Fig. Similarly, identification rates were recorded for a subgroup of patients who underwent lingual tonsillectomy performed using TORS or TLM Fig. . A primaTable The most common complication was hemorrhage in 5 of 97 (5 %) patients, of which three (3 %) required return to the operating room for hemostasis. None (0 %) of the 22 patients with available outcomes required tracheostomy, and only 1 of 36 (3 %) patients required a gastrostomy tube. In this single patient, the requirement for a permanent gastrostomy tube was due to adjuvant chemoradiation and heavy tobacco use in the post-operative period . FurtherLocalization of the primary tumor in patients with cervical metastasis of unknown origin remains a challenging yet important goal. When available diagnostic modalities fail to detect a primary tumor, treatment typically consists of large volume radiation to the neck as well as potential primary mucosal sites with or without chemotherapy, or neck dissection with or without adjuvant chemoradiation –7. Head The goal of this systematic review was to determine the effectiveness of TORS and TLM in localizing an occult primary tumor and to elucidate the role of these techniques within the traditional diagnostic paradigm. Our findings demonstrated that TORS/TLM can increase the detection of occult primary tumors at all stages of the diagnostic workup. We also aimed to determine the incremental benefit of using these techniques by analyzing the identification rate of unknown primaries in a subgroup of patients undergoing lingual tonsillectomies. Many of the patients who are managed with TORS and TLM undergo a palatine tonsillectomy in addition to lingual tonsillectomy. While a palatine tonsillectomy can be performed using more cost-effective traditional approaches, a lingual tonsillectomy, on the other hand, may require the superior visualization and exposure afforded by these techniques. In the present study, the identification rate of a primary tumor using lingual tonsillectomy was 60/108 (56 %).Currently, there is no standard diagnostic algorithm for an unknown primary tumor. The typical workup includes physical examination and diagnostic imaging consisting of CT and/or MRI. The addition of PET and PET/CT have resulted in improved detection rates ranging from 15 to 28 % , 11, 31 Surgical evaluation of an unknown primary involves the use of examination under anesthesia with biopsies of clinically and radiologically suspicious sites. Studies also show that palatine tonsillectomy improves the detection rate compared to tonsil biopsy in patients with demonstrable tonsillar tissue , 14. OveIn comparison, our present study demonstrates significantly higher identification rates using TORS/TLM compared to traditional diagnostic techniques, suggesting that evaluation of the lingual tonsil with the aid of TORS/TLM has clinical benefit in the work-up of unknown primary tumors. In contrast to the detection rates reported above, our review of the literature revealed an identification rate of 79 % in the presence of remarkable findings on physical examination and imaging, and a 92 % detection rate in the presence of remarkable findings on panendoscopy. Most importantly, the detection rate remained high at 71 % in the absence of findings on physical examination and imaging (including PET/CT), and 67 % even after failed EUA with directed biopsies. This highlights a potential role for TORS/TLM in the diagnostic algorithm of these patients as a “final step” after failed panendoscopy.Similar findings were noted in the subgroup of patients undergoing lingual tonsillectomy using TORS or TLM. The detection rate was 61 % among patients with remarkable findings on physical examination and radiological imaging, and remained at 59 % among patients with unremarkable findings. Furthermore, lingual tonsillectomy was successful in identifying the primary tumor in 18 of 25 (72 %) patients even after failed EUA with biopsies. These data also support the use of TORS and TLM to perform a lingual tonsillectomy as a “last resort” when all other diagnostic modalities have failed to localize a primary tumor site.Some authors advocate for upfront lingual tonsillectomy in the initial management of occult primary tumors rather than awaiting the results of directed biopsies of the pharynx , 17. ThiOur findings corroborate previous studies that suggest that subsites of the oropharynx such as the palatine tonsil and tongue base are the most common sites of occult primary tumors , 12, 15.The issue of “bilaterality” or contralateral tumor resection is one that warrants discussion. In our study, we report a contralateral primary tumor in the tongue base in 6 % of the contralateral tongue base and 15 % in the contralateral tonsil. This proportion is comparable to previous reports which have found bilateral or contralateral palatine tonsil disease in 10 to 23 % of identified primary tumors , 37, 38.This study is limited by the small sample size of included studies, particularly for the subgroup of patients receiving lingual tonsillectomy, as well as the heterogeneity between diagnostic workup performed at different institutions. This is not surprising given the relatively recent advent of this expanded surgical paradigm in the investigation of head and neck carcinoma of unknown primary. Our study highlights the need for a standardized diagnostic and treatment approach to unknown primary tumors that also considers emerging transoral surgical procedures such as TORS and TLM. Inter-institutional and inter-surgeon variation in the technique used to perform lingual tonsillectomy could have also affected our findings, particularly given that only three studies , 19, 26 This systematic review supports the use of TORS and TLM to aid in the identification of a primary head and neck squamous cell carcinoma of unknown origin, with superior detection rates compared to the traditional diagnostic workup. We also demonstrate that the addition of formal lingual tonsillectomy using TORS/TLM is a safe and effective option that can increase the yield of localizing an occult primary tumor. Identification of the primary tumor using minimally-invasive transoral techniques reduces treatment-induced morbidity and permits directed management, thereby potentially improving survival and functional outcomes.