Heroin may have been accidentally contaminated by an animal-derived source along a major drug trafficking route. Bacillus anthracis strain from a heroin user (Ba4599), developed Ba4599-specific single-nucleotide polymorphism assays, and genotyped all available material from other heroin users with anthrax. Of 34 case-patients with B. anthracis–positive PCR results, all shared the Ba4599 single-nucleotide polymorphism genotype. Phylogeographic analysis demonstrated that Ba4599 was closely related to strains from Turkey and not to previously identified isolates from Scotland or Afghanistan, the presumed origin of the heroin. Our results suggest accidental contamination along the drug trafficking route through a cutting agent or animal hides used to smuggle heroin into Europe.In December 2009, two unusual cases of anthrax were diagnosed in heroin users in Scotland. A subsequent anthrax outbreak in heroin users emerged throughout Scotland and expanded into England and Germany, sparking concern of nefarious introduction of anthrax spores into the heroin supply. To better understand the outbreak origin, we used established genetic signatures that provided insights about strain origin. Next, we sequenced the whole genome of a representative  Bacillus anthracis is a gram-positive endospore-forming bacterium that causes the disease anthrax in livestock, wildlife, and humans. Because of its hardy spores, B. anthracis can survive for extended periods in the environment, a trait that likely contributed to the successful global spread of this organism  from Norway on postmortem examination  genotyping against heroin anthrax samples and an extensive collection of diverse worldwide samples   . Most saB. anthracis isolate obtained from a Scottish heroin user in December 2009 (Ba4599) was cultured for 18 hours on 5% sheep blood agar, and genomic DNA was obtained by using phenol/chloroform extraction. All other B. anthracis isolates obtained during the outbreak were extracted by using a QIAamp DNA Mini Kit . We extracted 337 geographically diverse B. anthracis isolates from the Trans-Eurasian (TEA) group of the B. anthracis phylogeny  with previously published genomes , SNP1967560 , and SNP1118831 . Underlined nucleotides indicate the SNP position, and lowercase nucleotides indicate deliberately incorporated penultimate mismatches for enhanced allele specificity  to place Ba4599 into a broader phylogenetic context and to accurately establish the evolutionary relationships among these strains. We also sought to identify heroin strain–specific SNPs that could be used to rapidly determine whether xl instrument (Applied Biosystems). Read mapping was performed by using BWA and BWA-SW 0.5.9 , SNP3287006 AGGTTGCCTTCCCCATCTATT (A), and SNP3836105 AATCGTAAAGTGGCTGTATTT (C). Primers for the first SNP (1530761) assay are listed in order of derived, ancestral, and common primers as follows (5′ → 3′): SNP1530761 . This assay was the most robust of the 3 tested assays.We did not have whole genome sequencing data for isolate A0264 from Turkey. We therefore used canSNP genotyping assays to determine the approximate phylogenetic location of this strain relative to Ba4599 and A0149. CanSNPs along the branch leading to A0149 and Ba4599 were identified from whole genome sequencing data by searching for SNPs with allelic states shared only between these 2 genomes. We designed assays for 3 of these SNPs . The posATAGTAA, FAM-CTGCATAAATACCAGGTAGTAA) and SNP1173928 . Ninety-three TEA isolates unrelated to the heroin outbreak and 2 non-TEA isolates belonging to the Ames (A0462) and Western North America (A0303) groups were screened to confirm specificity of these 2 SNP assays for the Ba4599 heroin genotype.We then used whole genome comparisons to identify SNPs along the branch leading to the Ba4599 genome. These SNPs were tested against A0264 and all 36 clinical samples from the heroin outbreak. We targeted 2 of the 13 identified SNPs  for TaqMB. anthracis have demonstrated that particular genotypes correspond with geographic regions, a trait made possible by the relatively recent emergence of this highly clonal pathogen . Ba4599 has derived alleles for the first 2 SNPs but an ancestral allele for 1118831; we were therefore interested in identifying other strains with the same canSNP profile as Ba4599. We tested these 3 SNP signatures across all 120 A.Br.008/011 strains within our B. anthracis collection, comprising isolates from Albania (n = 5), Argentina (1), Austria (1), China (47), Ethiopia (1), Hungary (3), Iran (1), Italy (21), Norway (1), Pakistan (2), Poland (1), Republic of Georgia (5), Russia (2), Scotland (12), Slovakia (2), Turkey (6), Ukraine (1), or unknown origin (8). Notably, Ba4599 was indistinguishable from only 2 isolates, A0149 and A0264, both of which originated from Turkey. Twelve isolates linked to a fatal case of anthrax in a drum maker in Scotland in 2006, which originated from contaminated animal skins from Ghana, did not match the Ba4599 genotype. Similarly, the bioweaponized B. anthracis used on Gruinard Island, Scotland, during World War II  before being smuggled into Europe. Heroin produced in Afghanistan is thought to be trafficked through 2 major routes: the Silk Route and, more commonly, the Balkan Route. Both of these routes pass through numerous countries where anthrax is endemic and where isolates belonging to the TEA group have been found. Although we were unable to exhaustively test B. anthracis isolates along these routes to find a precise match to Ba4599, Turkey is definitely a possible origin, given that this country is central to the heroin smuggling trade from Afghanistan into Europe along the Balkan Route  or destination (Scotland). How it was contaminated is highly speculative, but it may have involved the addition of an animal-derived cutting agent, e.g., bone meal, or, alternately, wrapping in animal hide contaminated with B. anthracis–positive (by culture or PCR) samples from the heroin-associated outbreak, including an isolate obtained from a heroin user with anthrax in Germany . It is therefore possible that future cases of anthrax may arise, should the contaminated batch of heroin recirculate in the user population.Two Ba4599-specific SNP assays were used to rapidly screen 35 other In this study, we used a high-resolution molecular epidemiologic approach to identify the geographic source of anthrax spores responsible for the largest outbreak of injectional anthrax observed to date. Using all available clinical material, we found that the 2009–2010 anthrax outbreak that affected heroin users across Scotland, Germany, and England was caused by a single anthrax strain. Further, genetic and genomic analyses demonstrated that the anthrax spores, while not an exact match, were most closely related to isolates from Turkey. Turkey is a central country along the Balkan Route, a common route for trafficking heroin from its primary source in Afghanistan into European countries. Given the commonality of the TEA group throughout the world, and the close relationship of Ba4599 to isolates from Turkey, we do not possess any evidence to suggest that the heroin was contaminated with nefarious intent. Our results suggest accidental contamination from an animal-derived source, such as bone meal (a cutting agent used to dilute heroin) or animal hides. Although no new cases of injectional anthrax have been reported since a case was diagnosed in November 2010 in Maidstone, England, this outbreak may indicate an emerging infectious disease in drug addicts in Europe and that illicit drugs such as heroin are a novel point of entry for this pathogen into non–anthrax-endemic regions. Educating users of the potential risks of acquiring anthrax from their drug use, coupled with maintained vigilance of public health investigators toward identifying anthrax cases, would help mitigate the public health effects of future outbreaks in this susceptible population.


Antibiotic consumption is a major driver of bacterial resistance. To address the increasing burden of multi-drug resistant bacterial infections, antibiotic stewardship programmes are promoted worldwide to rationalize antibiotic prescribing and conserve remaining antibiotics. Few studies have been reported from developing countries and none from Africa that report on an intervention based approach with outcomes that include morbidity and mortality.An antibiotic prescription chart and weekly antibiotic stewardship ward round was introduced into two medical wards of an academic teaching hospital in South Africa between January-December 2012. Electronic pharmacy records were used to collect the volume and cost of antibiotics used, the patient database was analysed to determine inpatient mortality and 30-day re-admission rates, and laboratory records to determine use of infection-related tests. Outcomes were compared to a control period, January-December 2011.During the intervention period, 475.8 defined daily doses were prescribed per 1000 inpatient days compared to 592.0 defined daily doses/1000 inpatient days during the control period. This represents a 19.6% decrease in volume with a cost reduction of 35% of the pharmacy’s antibiotic budget. There was a concomitant increase in laboratory tests driven by requests for procalcitonin. There was no difference in inpatient mortality or 30-day readmission rate during the control and intervention periods.Introduction of antibiotic stewardship ward rounds and a dedicated prescription chart in a developing country setting can achieve reduction in antibiotic consumption without harm to patients. Increased laboratory costs should be anticipated when introducing an antibiotic stewardship program. Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) is also rising internationally and in South Africa  18 years), compared to 2517 patients, mean age of 50 years (SD 18 years) during the intervention period. A total of 311 (12.8%) inpatients died and 226  were re-admitted to the hospital within 30 days in the control period compared to 315 (12.5%) inpatient deaths and 213  patients re-admitted during the intervention period.The key finding of this study is that the introduction of AS ward rounds and a dedicated antibiotic chart resulted in 19.6% reduction in the total volume of antibiotic use and a cost reduction of 35% of the pharmacy budget in 2 general medical wards in a developing country setting. Importantly, the intervention was not associated with ‘collateral damage’ i.e. unpredicted adverse effects, in terms of inpatient mortality or 30-day readmission rate to the hospital. However, a concomitant increase in laboratory costs was incurred, mainly due to an increase in the use of procalcitonin tests. This study adds to the pertinent body of knowledge by demonstrating the utility of an AS intervention in a developing country such as South Africa. In the latest Cochrane Systematic Review of interventions to improve antibiotic prescribing practices for hospital inpatients, none of the 89 studies included in the review were from Africa and only 5 were from developing countries i.e. Brazil, Colombia and Thailand .The effect of the AS programme on the profile of Gram-negative bacterial antibiotic resistance in our hospital is being studied longitudinally. However, in the face of increasing countrywide antibiotic resistance and transfer of patients into our hospital already colonized or infected with MDR infections, demonstrating a reduction due to the intervention will be challenging.We witnessed a steady decline in the number of patients still on antibiotics when seen on the weekly ward rounds due to early discontinuation of the antibiotics initially started in the EU or ward. The commonest reasons for this were inadequate indication for antibiotics or an inappropriate duration of antibiotics charted by the admitting physician. For example, prior to the intervention, it was commonplace for ceftriaxone to be prescribed for 2 weeks for all patients admitted with community-acquired pneumonia or sepsis of unknown cause. Following the introduction of AS ward rounds, this duration decreased to a standard of 5 days, except for infections such as meningitis and others requiring prolonged duration of antibiotics. We also demonstrated an increase in initial use of oral over parenteral antibiotics, and an increase in earlier parenteral to oral switch. This was particularly evident for ciprofloxacin, clarithromycin and co-amoxiclav.Ideally, one would use a stepwise introduction of a single intervention to judge benefit. On this occasion, we opted for a dual intervention strategy as the antibiotic prescription chart also acted as an audit tool. Although we cannot disregard the possibility that the chart itself may have contributed to the reduced antibiotic use, we hypothesize that the major factor effecting change was the AS ward round. Ward rounds were held once weekly, yet the early stopping of antibiotics commonly occurred prior to the weekly ward round, indicating change in prescribing practice. Furthermore, the AS ward round was used to train and transfer skills, particularly in complex cases where there is often a question of equipoise. A number of studies from different healthcare settings in the United States support our hypothesis, having shown reduction in antibiotic prescribing and cost following input by trained specialists in the decision making process -19.Antibiotic restriction reduces antibiotic use and cost . HoweverA number of studies have documented benefit from introduction of dedicated antibiotic prescription charts. Closest to our own chart design, Durbin et al reported reduction in inappropriate prophylactic antibiotic use and mean duration of prophylactic antibiotics, as well as an increase in appropriate antibiotic prescribing in urology patients from 38% to 89%, following an introduction of a new chart . SimilarLaboratory costs associated with the introduction of the AS programme at GSH increased. A gatekeeping strategy was in place to limit the use of CRP. However, no such strategy was in place for PCT at the start of our intervention. The majority of the excess costs were attributed to inappropriate PCT use, often with repeated or serial measurements. Although serial measurement of PCT has been shown to be of benefit in guiding when to stop antibiotics, particularly in the intensive care unit setting ,24, in aThere are several limitations to our study. As highlighted, the use of a 2-part intervention makes it difficult to dissect out the contribution made by the chart or the ward rounds. Secondly, the AS team described in this intervention is not reproducible outside of a tertiary, or rarely a secondary level hospital in South Africa or many developing countries. Infectious diseases specialists and microbiologists are usually domiciled at tertiary centres, and although they perform outreach and support to primary level facilities, the same composition of AS team is unlikely to be replicated. However, the principle of stewardship and a team constructed around key role players such as pharmacists and physicians with appropriate training could be introduced at any health care facility. One of the objectives of the South African Antibiotic Stewardship Programme  is to deWe did not record baseline demographic patient information or severity of illness indicators, which leaves us unable to accurately assess differences in the inpatient populations during the control and intervention years. However, we did not witness any gross differences in patient profile during the 2 years, both of which had mild influenza seasons. Our results to date do not allow us to determine what effect the intervention has had on antibiotic resistance rates in our hospital. Although antibiotic consumption is a major driver of resistance, introduction of resistant organisms from other hospitals will confound the issue. Our intervention on 2 medical wards, does not allow us to generalize in terms of its applicability in different clinical settings. However, preliminary results from a similar ongoing intervention in the general surgical wards at our hospital, is also showing a reduction in total volume of antibiotic use and pharmacy cost (data not shown). Future research questions will be aimed at addressing the issues of reproducibility of the intervention in different healthcare settings, change in resistance patterns, and optimizing individual patient outcomes through AS. Reducing hang time (the time from writing up the prescription to entry of antibiotic into the patient), optimizing antibiotic dose and rational therapeutic drug monitoring are important research questions to address. We face an uncertain future of MDR Gram-negative bacterial infections. The rise in CRE and its characterization has highlighted the broad resistance genes these bacteria carry and the ability of most antibiotics to select out these resistant organisms. Hence, interventions to reduce antibiotic consumption are urgently needed to limit the emergence of resistant organisms and must be introduced alongside strengthening of basic infection control practices, such as hand washing. Antibiotic prescription charts and more importantly the rollout of AS ward round activity by AS teams in every healthcare institution will be critical in reducing the volume of antibiotic use and slowing the march towards a post-antibiotic era.


G76V variant to the N-terminus of MelanA results in rapid proteasomal degradation via the endoplasmic reticulum-associated degradation (ERAD) pathway and, consequently, leads to an increased MHC-I antigen presentation. While lysine residues within Ub are dispensable for these effects, the presence of one single lysine residue, irrespectively of its location along the fusion protein, is sufficient to induce degradation of MelanA. These results show that the ubiquitination, ER to cytosol relocation and proteasomal degradation of a transmembrane protein can be increased by N-terminal fusion of Ub at the presence of at least one, position independent lysine residue. These findings are in contrast to the conventional wisdom concerning the UFD and indicate a new concept to target a protein into the ubiquitin-proteasome system (UPS) and thus for enhanced MHC-I antigen presentation, and might open up new possibilities in the development of tumor vaccines.N-terminal stable in frame fusion of ubiquitin (Ub) has been shown to target the fusion protein for proteasomal degradation. This pathway, called the Ub fusion degradation (UFD), might also elevate MHC class I (MHC-I) antigen presentation of specific antigens. The UFD, mainly studied on cytosolic proteins, has been described to be mediated by polyubiquitination of specific lysine residues within the fused Ub moiety. Using the well characterized melanoma-specific antigen MelanA as a model protein, we analyzed the requirements of the UFD for ubiquitination and proteasomal degradation of a transmembrane protein. Here we show that fusion of the non-cleavable Ub Ubiquitin (Ub) is attached to Lys, or, in rare cases, other residues Stable in frame fusion of Ub to the N-terminus of proteins has been shown to augment their proteasomal degradation and thus enhances their MHC-I antigen presentation The accumulation of misfolded proteins in the endoplasmic reticulum (ER) is prevented by a process termed ER-associated degradation ERAD, . MisfoldTo analyze the mechanism of the UFD for a transmembrane protein, we used MelanA  as a model protein. MelanA has been discovered as a melanoma-associated antigen targeted by tumor-reactive cytolytic T lymphocytes G76V to the N-terminus of the transmembrane protein MelanA converted the otherwise stable protein into a substrate for both the UFD and the ERAD pathway, resulting in the rapid proteasomal degradation of the fusion protein. The increased turnover correlated with an enhanced entry into the MHC-I pathway. These effects were, in contrast to the current working model for the UFD, independent of Lys residues within the fused Ub moiety. Both the primary sequence of Ub as well as a position independent single Lys residue in the entire sequence of the fusion protein are sufficient to target MelanA for rapid destruction by the 26S proteasome and thus to enhance its MHC-I antigen presentation. This finding reveals a yet unknown mechanism for the UFD and could be useful in the optimisation of tumor vaccination and augmentation of the immunogenicity of transmembrane antigens.In this study, we demonstrate that stable fusion of UbG76V was fused to its N-terminus. To analyze if this stable Ub fusion correlates with an enhanced MHC-I antigen presentation of a MelanA-derived epitope, the model epitope SIINFEKL (SL) was introduced as an indicator for antigen processing into the N-terminal region of MelanA and UbG76VMelanA  were inhibited. When steady-state protein levels in whole cell lysates were analyzed by Western blot using an anti-MelanA Ab, Ubdetected . Followiumulated . This fielanA-SL , lane 4.bG76VMelanA-SL. Flow cytometry analysis using the mAb 25D1.16, which specifically recognizes H2-Kb-bound SL, revealed that cells expressing UbG76VMelanA-SL displayed higher numbers of H2-Kb-SL complexes at the cell surface compared to cells expressing the wt protein . The cumulative results of 36 independent experiments reveal a highly significant two-fold increase in SL-presentation following fusion of UbG76V to MelanA-SL  G76VMelanA-SL is still inserted within intracellular membranes and not degraded because of a mislocalization G76VMelanA-SL and ubiquitinated species thereof became clearly detectable in the pellet fraction, showing that this protein is indeed inserted within cellular membranes. Another fraction of UbG76VMelanA-SL however accumulates in the soluble, cytosolic fraction  or 29 (K29) within the fused Ub moiety G76VMelanA-SL had no influence on its metabolic stability or MHC-I antigen presentation (data not shown). Moreover, none of the individual Lys residues within the fused UbG76V  was essential for the rapid proteasomal degradation or enhanced MHC-I antigen presentation of UbG76VMelanA-SL (data not shown).The stable N-terminal fusion of UbG76V were exchanged for Arg, this mutant, called UbG76V(K0)MelanA-SL, was still efficiently degraded by the 26S proteasome, preventing its accumulation to detectable levels MelanA-SL as well as putative ubiquitinated species thereof could be detected after addition of PIs MelanA-SL in HeLa cells. MelanA-SL was immunoprecipitated from denatured cell lysates using an anti-MelanA Ab, and ubiquitinated species thereof were specifically detected by anti-HA in Western blot analysis. Following treatment with PIs, both UbG76VMelanA-SL and UbG76V(K0)MelanA-SL displayed a ladder of bands with reduced mobility, showing that HA-Ub molecules were covalently attached to those variants. Additionally a high-molecular weight smear could be detected, which most probably represents polyubiquitin-chains which consist of several ubiquitin molecules that are linked with each other via K48 and are covalently attached to one of the remaining lysine residues within the MelanA part of the fusion protein (G76VMelanA-SL nor UbG76V(K0)MelanA-SL could be detected in the Western blot. In contrast, MelanA-SL shows ubiquitinated species in the absence and presence of PIs, most probably representing polyubiquitin-chains, which consist of several ubiquitin molecules that are linked with each other via K63 and are also covalently attached to a lysine residue within MelanA-SL. This modification is crucial for the transport of the protein throughout the trans-Golgi network, as shown previously by others To confirm that the MelanA species between ∼30 and 43 kDa represent ubiquitinated species of MelanA-SL, N-terminally HA-tagged Ub (HA-Ub) was coexpressed with MelanA-SL, Ub protein . In the G76V(K0)MelanA-SL or UbG76VMelanA-SL displayed a two-fold increase in the amount of H2-Kb-SL complexes at the cell surface when compared to cells expressing the original MelanA-SL MelanA-SL  to Arg had no influence on the stability or MHC-I antigen presentation (data not shown). This indicates that there is no unique, specific ubiquitination site within MelanA-SL.As the Lys residues within the fused Ub are dispensable for an efficient proteasomal degradation of Ubtopology . The Lystopology . IndividG76V(K0)MelanA-SL should be polyubiquitinated. To test this assumption, all Lys residues within UbG76V(K0)MelanA-SL were successively mutated to Arg from the C- to the N-terminus. The Lys residue at position 19 was not exchanged, as it is essential for the recognition of H2-Kb-SL complexes by the mAb 25D1.16. Mutation of Lys residues 116-70 had no effect on the stability of UbG76V(K0)MelanA-SL (G76V(K0)MelanA-SL.The ER-resident E3 Ub ligases identified so far have their catalytic RING domain at cytosolic face of the ER membrane elanA-SL . AdditioelanA-SL . This inG76V(K0)MelanA-SL from the N- to the C-terminus had very similar effects on protein levels as shown by Western blot analysis (G76V(K0)MelanA-SL with only one lysine residue (K19) is still highly significantly enhanced when compared to the wt MelanA-SL fusion protein were exchanged to alanine, except one. The mutants were called UbG76V(K0)MelanA-SLK24/25only, UbG76V(K0)MelanA-SLK70only, UbG76V(K0)MelanA-SLK95only, UbG76V(K0)MelanA-SLK101only and UbG76V(K0)MelanA-SLK116only.We have shown above that the Lys residue at position 19 of MelanA is sufficient to induce its proteasomal degradation . We now Western blot analysis of the whole cell lysates show that the stability of these mutants is only slightly increased. Nevertheless, after treatment with PIs, all mutants are obviously stabilized, indicating that they are still rapidly degraded by the 26S proteasome .G76V(K0)MelanA-SL into the UPS.Taken together, this cumulative data clearly illustrate that one position independent lysine residue is sufficient to target UbG76V(K0)MelanA-SL is targeted for proteasomal degradation by such atypical polyubiquitination or independently of polyubiquitinylation G76V(K0)MelanA-SL to Arg (UbG76V(K0)MelanA(K0)-SL). Western blot analysis of whole cell lysates demonstrates that the steady-state protein levels of UbG76V(K0)MelanA(K0)-SL and MelanA-SL are similar, revealing that the enhanced proteasomal degradation is completely abolished when all Lys residues are mutated. In addition, no ubiquitinated adducts were detectable and the mutant did not further respond to the treatment with PIs MelanA-SL for degradation by the 26S proteasome.This result clearly shows that the presence of at least one Lys residue is required to target UbG76VMelanA-SL accumulates in cytosol MelanA-SL are efficiently degraded MelanA-SL are due to unspecific inhibitory effects on the UFD pathway or the UPS.Besides the ERAD pathway, VCP/p97 is also an important component of the UFD pathway n of PIs , lane 5.G76VMelanA-SL is degraded by the 26S proteasome. Treatment with ExoA blocked the degradation of UbG76VMelanA-SL and caused an accumulation of the protein similar to the phenomenon obtained after addition of PIs , as described previously n of PIs . To veriG76VMelanA-SL, we measured the H2-Kb-SL presentation following treatment with ExoA. To remove the H2-Kb-SL complexes from the cell surface, which had already been loaded before treatment with ExoA, an acid wash of cells was performed and 4 hours later the H2-Kb-SL presentation was measured. While the acid wash removed most of the H2-Kb-SL-complexes from the cell surface .It was shown previously that epitopes generated for MHC-I presentation from an ER-targeted protein are presented by MHC-I molecules without being processed by the ERAD pathway t levels . The totG76VMelanA-SL is degraded via the ERAD pathway which is crucial for an efficient MHC-I antigen presentation of SL epitopes derived from this ER resident protein.These results show that UbG76V to its luminal N-terminus converts the transmembrane model protein MelanA-SL into a substrate for both the UFD and the ERAD pathway. In contrast to the current working model of the UFD pathway, the degradation and MHC-I antigen presentation of UbG76VMelanA-SL were independent of Lys residues within the fused Ub moiety, but dependent on the presence of the primary sequence of Ub and at least one position independent Lys residue within the entire fusion protein.We show here that a stable fusion of UbG76V to MelanA has enhancing effects on its proteasomal degradation and MHC-I antigen presentation further underlines results obtained for a number of cytosolic G76VMelanA-SL were essential to induce rapid proteasomal degradation of the fusion protein. Although, to our knowledge, this requirement for Lys residues has not been studied for transmembrane substrates of the UFD pathway so far, this result is in contrast to findings reported for cytosolic UFD substrates Our finding that N-terminal in frame fusion of UbG76V fusion, did not interfere with the insertion of MelanA into the ER membrane, as suggested by in silico prediction. Following inhibition of proteasomal activity, UbG76VMelanA-SL accumulated in the ER, but also in the cytoplasm. This indicates that at least a fraction of the protein is degraded via the ERAD pathway. Indeed, degradation of the transmembrane UFD model substrate UbG76VMelanA-SL, but not the soluble UFD substrate UbG76VGFP G76VMelanA-SL more effectively than treatment with Eey1 alone. Though, alternative p97/VCP- and ubiquitination-independent routes to exit the ER have been reported G76VMelanA-SL that has not been inserted into the ER membrane yet, or has already been extracted from the ER membrane prior to addition of Eey1. In addition, it cannot be completely ruled out that the concentration of Eey1 used in our experiments might be insufficient for complete inhibition of the retranslocation from the ER to the cytosol. To further substantiate the results obtained with Eey1 we used as another inhibitor of the ERAD pathway ExoA as described before G76VMelanA-SL in the same way as Eey1 providing another proof that UbG76VMelanA-SL is indeed degraded by the ERAD pathway  presented by MHC-I molecules completely occurs in the cytoplasm before the protein is inserted into the ER and thus without any involvement of the ERAD pathway G76VMelanA-SL the ERAD pathway is crucial, as the blockade of this pathway by the addition of ExoA clearly reduces its MHC-I antigen presentation almost to wt levels  and BstEII-SL-rw (5′- GTA ACC TAG CTT TTC GAA GTT GAT GAT CGA-3′). For the construct pUbG76VMelanA-SL, overlapping DNA fragments were fused by PCR using the oligonucleotides EcoRI-UbV fw (5′-TGC TGG AAT TCG CCG CCA CCA TGC AGA TCT TCG-3′), UbV rev (5′-GCA TCT TCT CTT GGC ATC ACC CCC CTC AAG CG-3′), MelanA-UbV fw (5′-TTG AGG GGG GTG ATG CCA AGA GAA GAT GCT CAC-3′), MelanA-XbaI rev (5′-GAT GCA TGC TCG AGG TAA TTA AGA ATG TGC AGC-3′) and cloned into pcDNA3.1 using EcoRI and XbaI. Point mutations were introduced by site directed mutagenesis using the Quikchange kit (Stragene) and following oligonucleotides: UbV K6R fw (5′-GAT CTT CGT GAG GAC CCT GAC CGG C-3′), UbV K6R rev (5-′GCC GGT CAG GGT CCT CAC GAA GAT C-3′), UbV K11R fw (5-′CCC TGA CCG GCA GGA CCA TCA CCC TGG-3′), UbV K11R rev (5-′CCA GGG TGA TGG TCC TGC CGG TCA GGG-3′), UbV K27R fw (5-′CCA TCG AAA ATG TGA GGG CCA AGA TCC AGG-3′), UbV K27R rev (5-′CGA AAA TGT GAA GGC CAG GAT CCA GGA TAA AGA AGG-3′), UbV K29R rev (5-′CCT TCT TTA TCC TGG ATC CTG GCC TTC ACA TTT TCG-3′), UbV K33R fw (5-′GCC AAG ATC CAG GAT AGA GAA GGC ATC CCC CC-3′), UbV K33R rev (5-′GGG GGG ATG CCT TCT CTA TCC TGG ATC TTG GC-3′), UbV K48R fw (5-′GCT CAT CTT TGC AGG CAG ACA GTT GGA AGA TGG C-3′), UbV K48R rev (5-′GCC ATC TTC CAA CTG TCT GCC TGC AAA GAT GAG C-3′), UbV K63R fw (5-′CAA CAT CCA GAG AGA GTC GAC CCT GCA TCT GG-3′), UbV K63R rev (5-′CCA GAT GCA GGG TCG ACT CTC TCT GGA TGT TG-3′), MelanA K19R fw (5-′CGA TCA TCA ACT TCG AAA GAC TAG GTT ACC CC-3′), MelanA K19R rev (5-′GGG GTA ACC TAG TCT TTC GAA GTT GAT GAT CG-3′), MelanA K24/25R fw (5-′GGT TAC CCC AGA AGA GGG CAC GGC C-3′), MelanA K24/25R rev (5-′GGC CGT GCC CTC TTC TGG GGT AAC C-3′), MelanA K70R fw (5-′CCT TGA TGG ATA GAA GTC TTC ATG TTG GC-3′), MelanA K70R rev (5-′GCC AAC ATG AAG ACT TCT ATC CAT CAA GG-3′), MelanA K95R fw (5-′CGG GAC AGC AGA GTG TCT CTT CAA G-3′), MelanA K95R rev (5-′CTT GAA GAG ACA CTC TGC TGT CCC G-3′), MelanA K101R fw (5-′CTC TTC AAG AGA GAA ACT GTG AAC CTG TGG-3′), MelanA K101R rev (5-′CCA CAG GTT CAC AGT TTC TCT CTT GAA GAG-3′), MelanA K116R fw (5-′CCT GCT TAT GAG AGA CTC TCT GCA GAA CAG TCA CC-3′), MelanA K116R rev (5-′GGT GAC TGT TCT GCA GAG AGT CTC TCA TAA GCA GG-3′).The sequence coding for the SL epitope, corresponding to amino acids 257–264 of ovalbumin, was introduced into the MelanA-coding sequence using a unique Bst EII site and oligonucleotides BstEII-SL-fw , MG-132  or Eey1  were added for 4 h. ExoA  was added for 18 h.HeLaSS6 cells were cultured in DMEM supplemented with 10% (v/v) inactivated FCS, 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin. For HeLa-KCells were lysed in RIPA ). Proteins were separated by SDS-PAGE, blotted onto PVDF membranes  and probed with following Abs prior to ECL detection: monoclonal anti-MelanA (Novocastra), monoclonal anti-GFP (Roche), monoclonal anti-ß-actin (Sigma), polyclonal anti-ExoA (Sigma) and monoclonal anti-Hsp90 (Santa Cruz).G76VMelanA-SL respectively. 24 h post transfection the total RNA was extracted using the RNeasy Mini Kit (Quiagen) and its quality and quantity was assessed using the BioPhotometer plus (Eppendorf). The qRT-PCR analysis was conducted using the SuperScript®III Platinum® SYBR® Green One-Step qRT-PCR Kit (Invitrogen) according to the manufacturer's instruction. The primer sequences for MelanA-SL were as follows: forward 5′- GGA TCG GCA TCC TGA CAG TGA TC -3′ and reverse 5′- GCA TTG GGA ACC ACA GGT TCA CAG -3′. Each sample was amplified in duplicate and normalized for the housekeeping gene GAPDH. The quantitative RT-PCR was conducted using the Abi 7500 PCR machine (Applied Biosystems).HeLa cells were transiently transfected with expression plasmids that code for MelanA-SL or UbFollowing coexpression of MelanA-SL and variants thereof with HA- tagged Ub, MelanA-SL contained in ∼800 µg whole cellular proteins was precipitated from the whole cell lysates using anti-MelanA Ab prebound to protein G-Sepharose. Cells were lysed in RIPA . The Western blot analyses were conducted as described before.b-bound SL-epitope, cells were stained with the allophycocyanin (APC)-conjugated 25D1.16 mAb (eBioscience) diluted 1∶100 in FACS buffer . For detection of total H2-Kb molecules, cells were incubated with hybridoma cell culture supernatant containing the mAb B8-24-3 For detection of H2-Kb cells were transiently transfected with expression plasmids that code for MelanA-SL or UbG76VMelanA-SL respectively. 24 h later ExoA was added for 18 h. Subsequently newly H2-Kb-SL-complex formation at the cell surface was followed by flow cytometry after an acid wash procedure as described previously HeLa-KCells were fixed in 3% (w/v) paraformaldehyde and permeabilized using 0.1% (v/v) TritonX-100 in PBS. MelanA and the TGN were detected using a mouse anti-MelanA Ab (Novocastra) and a rabbit anti-TGN46 Ab (Abcam), respectively, followed by staining with secondary Alexa488-conjugated anti-mouse and Alexa647-conjugated anti-rabbit Abs (Invitrogen), respectively. The nucleus was visualized by staining with DAPI. Images were obtained using a Leica TCS SP5 confocal microscope with the 63× objective and analyzed by Adobe Photoshop software.The plasma membrane was permeabilized using 20 µM digitonin as described previously For the isolation of ER resident proteins an ER isolation kit (Sigma) was used according to the manufacturer's instruction. The ER fraction was analyzed by Western blot as indicated.Statistical analysis was performed using the Graph Pad Prism software. P-values were calculated by a paired two-tailed student's t-test after confirming that the measured values followed the Gaussian distribution (Kolmogorov-Smirnov)


Immunoglobulin G4 (IgG4)-related disease is a systemic syndrome, characterized by sclerosing lesions and usually associated with a raised serum IgG4 level; the pancreas, salivary glands, and lacrimal glands are typically affected. Recently, it has been suggested that IgG4-related sclerosing disease represents a subset of cases previously diagnosed as idiopathic hypertrophic pachymeningitis. This rare inflammatory disorder causes localized or diffused thickening of intracranial dura mater. Headache, cranial nerve palsy, and ataxia are the most common clinical manifestations.Herein, we report the clinical and histopathological features of a rare case of IgG4-related intracranial hypertrophic pachymeningitis involving cranial hyperostosis.A 52-year-old man presented with refractory generalized tonic-clonic seizure. Magnetic resonance imaging revealed thickening of the meninges with enhancement near the superior sagittal sinus; skull bone defect was also noted. Extensive excision of affected skull bone and dura was performed, providing the diagnosis of IgG4-related pachymeningitis. After the surgery, the patient’s seizure stopped and he was smoothly tapered off antiepileptic medication.To our knowledge, this is the first reported case of IgG4-related pachymeningitis with concomitant skull hyperostosis. Immunoglobulin G4 (IgG4)-related disease is a systemic syndrome, characterized by sclerosing lesions and usually associated with a raised serum IgG4 level ; the panHerein, we report the clinical and histopathological features of a rare case of IgG4-related intracranial hypertrophic pachymeningitis involving cranial hyperostosis.A 52-year-old man with a history of treated hypertension and diabetes mellitus presented to our hospital following a single seizure attack; he regained consciousness shortly before arrival. Two weeks before this, he had a head injury that resulted in some minor scalp abrasion and bleeding. No obvious neurological deficit was noted at that time, and he was discharged following a period of observation without imaging. However, on the day after the discharge, he experienced another seizure attack that lasted for 5–6 minutes with subsequent loss of consciousness. He was then admitted to our hospital.Initially, the patient experienced generalized tonic-clonic seizures twice daily. Persistent right calf paresthesia was also noted; this presented as a focal seizure when aggravated and often progressed to a generalized tonic-clonic seizure. Under antiepileptic medications, focal seizures remained persistent but progression to secondary generalized seizures became rare.On physical examination there were no clinical signs to suggest other organ involvement, in particular there was no glandular enlargement or orbital disease. Abdominal ultrasound showed a normal liver, gallbladder, pancreas and kidneys.Magnetic resonance imaging showed thickening of the meninges with enhancement near the superior sagittal sinus; skull bone defect was also noted Figure A-D. We iLeft parietal skull and dural nodule excision were then performed for both pathological diagnosis and symptomatic relief Figure A. DuroplMicroscopically, sections of the dura and galea showed mixed inflammatory cell infiltration, composed mainly of lymphoplasma cells, in a stroiform fibrous background with occasional eosionphils. Lymphoid follicles with germinal center formations were also observed in the galeal tissue sections Figure A-C. SkulImmunohistochemical analysis using the avidin-biotin-complex (ABC) stain revealed an increased IgG4 (+) plasma cell count, which accounted for more than 50% of the IgG (+) cells with a mean more than 30 in 3 high power fields (HPFs); this was assessed by counting 20 HPFs  (Figure After total excision of the affected skull bone and meninges, the patient’s seizures stopped; he was smoothly tapered off antiepileptic medication. In addition, his serum IgG4 levels returned to normal, and no obvious sequela was noted after the operation. Follow-up imaging study and physical examinations in 3 months later revealed that the infiltration of the arachnoid membrane had vanished and there was no involvement of other organs.Hypertrophic pachymeningitis is a rare disorder that causes localized or diffused thickening of the dura mater . NumerouIgG4-related sclerosing disease was initially recognized as a form of autoimmune pancreatitis. However, it is now known that this disease can affect the bile duct, gallbladder, salivary glands, retroperitoneum, kidneys, lungs, and even, prostate. Pathologically, this disease is characterized by extensive infiltration of IgG4-positive cells in various organs . ClinicaMeningeal biopsies are frequently performed for a differential diagnosis of pachymeningitis, especially for those with infectious etiology ,7,9. In Although it has been reported that pachymeningitis responds well to steroid treatment and other immunosuppressants, we did not prescribe the above medications owing to concerns about the adverse effects of their long-term use ,9,11,12.The patient’s symptoms disappeared following surgical treatment. Thus, surgical excision to decrease the affected region may benefit patients with IgG4-related pachymeningitis, especially for patients with a solitary mass and without other organ involvement. Beyond our expectation, the infiltrative pattern of the involved arachnoid membrane disappeared following surgery. The self-limiting course may be related to the decrease in systemic IgG4 levels. It will be important to follow-up the patient for signs of recurrence and further organ involvement in the future.Despite its extremely rarity, IgG4-related sclerosing disease may present as a solitary intracranial condition. Surgical biopsy may be the only means of obtaining a differential diagnosis. Extensive excision of the intracranial lesion may be performed for symptomatic patients.Written informed consent was obtained from the patient for publication of this case report and any accompanying images.A copy of the written consent is available for review by the Editor of this journal.IgG4: Immunoglobulin G4; CSF: Cerebrospinal fluid; ABC: Avidin-biotin-complex.The authors declare that they have no competing interests.C.K.L: Collections of the clinical data of the case and writing of the manuscript. D.M.L: Instructions and responsibility for the completion of this manuscript. Both authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2482/13/37/prepub


The HIV/AIDS epidemic has evolved with an increasing burden in older adults. We assessed for knowledge about aging and HIV/AIDS, among clinicians in Kampala district, Uganda.A cross-sectional survey of 301 clinicians complemented by 9 key-informant interviews between May and October 2011. Data was analyzed by multivariable logistic regression for potential determinants of clinician knowledge about HIV/AIDS in older adults, estimating their adjusted Odds Ratios (aOR) and 95% confidence intervals (95% CI) using Stata 11.2 software.Two-hundred and sixty-two questionnaires (87.7%) were returned. Respondents had a median age of 30 years (IQR 27–34) and 57.8% were general medical doctors. The mean knowledge score was 49% (range 8.8%–79.4%). Questions related to co-morbidities in HIV/AIDS (non-AIDS related cancers and systemic diseases) and chronic antiretroviral treatment toxicities (metabolic disorders) accounted for significantly lower scores  compared to HIV/AIDS epidemiology and prevention . Determinants of clinician knowledge in the multivariable analysis included : clinician age , number of persons with HIV/AIDS seen in the past year  and clinical profession . Having diploma level education had a marginal association with lower knowledge about HIV and aging (p = 0.09).Our study identified gaps and determinants of knowledge about HIV/AIDS in older adults among clinicians in Kampala district, Uganda. Clinicians in low and middle income countries could benefit from targeted training in chronic care for older adults with HIV/AIDS and long-term complications of antiretroviral treatment. HIV/AIDS is a major public health problem globally, which disproportionately affects sub-Saharan Africa The world population is aging Although evidence on aging and HIV/AIDS is largely from America and Europe, Low– and Middle Income Countries (LMICs) are beginning to observe this phenomenon. Studies from Uganda showed that age at HIV sero-conversion was associated with more rapid progression to AIDS Health care worker knowledge on HIV/AIDS has been shown to correlate with attitudes, practices and outcomes This was a mixed methods study conducted between May and October 2011 Since the expected level of aging and HIV/AIDS related knowledge among clinicians in Uganda was unknown, a maximum variability of 0.5 was assumed adjusting for a small population, N = 500, and 0.1 proportion of incomplete or non-response; 0.05 precision and 1.96 standard normal deviate. The total required sample size was estimated at 239 clinicians using Cochran’s formula The second method constituted 9 key informants who were purposively sampled and interviewed. Snowball sampling was used to further recruit more respondents until no new information was obtained. These included: senior clinicians working in HIV/AIDS clinics (n = 3); HIV/AIDS activists (n = 3); and policy makers in the relevant government institutions (n = 3).The study questionnaire adopted aspects of instruments previously used for similar evaluations These included: clinician age, gender, education level, professional cadre, work setting, interest in geriatrics, history of taking an HIV test, years of working experience and additional training in HIV/AIDS.Clinician knowledge about aging and HIV/AIDS was the main outcome measure. This examined HIV/AIDS epidemiology and prevention, immunology, antiretroviral treatment outcomes and laboratory results, non-communicable co-morbidities and long term complications of antiretroviral treatment scored as “True, False or Don’t Know”. Responses to the 34-questions on knowledge about aging and HIV/AIDS were scored (one point for correct and zero for incorrect), then aggregated into those who scored 50% or below, and those above 50%. The key informant guide captured respondents’ knowledge, experiences and recommendations about prevention, care and treatment of HIV/AIDS among older adults in Uganda.P<0.05. Potential predictors with P<0.1 at univariate level were further explored in a multivariable logistic regression model to estimate the adjusted Odds Ratios and their 95% confidence intervals. Multiple imputations by chained equations was employed for sensitivity analysis of the missing data using the Stata™ command “ice” and “mim”, assuming a Missing At Random mechanism. In the adjusted analysis, all missing baseline variables and the outcome, HIV/AIDS knowledge, were included to create twenty imputed data sets. Qualitative data from key informant interviews were transcribed and analyzed for thematic content on clinician knowledge and training, then triangulated with the survey findings.Data was coded, entered in Epi Info™ version 3.5.3  and analyzed using Stata™ version 11.2 . This data was summarized using frequencies, percentages and appropriate measures of central tendency. The association between exposure variables and knowledge score was explored using Student’s t-test, one-way analysis of variance and univariate logistic regression. Tests were two-sided and significant if This study was approved by the Joint Clinical Research Centre Institutional Review Board and Uganda National Council for Science and Technology (HS 990). Clinicians were given a written informed consent guide and asked to tick yes or no before completing the anonymised survey.Three hundred and one questionnaires were distributed of which 262 (87.7%) were returned. Clinicians who completed the survey had a median age of 30 years (IQR: 27–34). The majority of respondents (59.7%) were male and 57.8% were general medical doctors. Most respondents worked in a hospital setting (69.7%) and the median time of clinical experience was 4 years (IQR: 2–7). Seventy two percent qualified with a bachelor’s degree and 88.8% had received additional short-term training in HIV/AIDS covering the following topics: prevention of mother-to-child transmission (45.8%), TB/HIV co-infection (44.3%), antiretroviral-therapy (40.8%) whilst only 23.6% completed modules in HIV/AIDS co-morbidities which predominantly cover non-communicable diseases and complications of chronic antiretroviral-therapy. At least 90% of the respondents had been tested for HIV before and 87% said they had interest in geriatrics whilst about a half (52.4%) had attended to over 100 PHLWAs in the preceding year .The mean knowledge score was 49% (range 8.8%–79.4%). In the univariate analysis  cliniciaCompared to medical officers, clinical nurses of various levels of qualification were less knowledgeable . More clinicians who had attended to less than 100 and less than 50 PHLWAs respectively in the preceding year scored 50% and below on the knowledge scale compared to those who saw over 100, .However, in the multivariable logistic regression model only clinician age , PHLWAs seen in the past year , and professional cadre level  were independently associated with knowledge scores about HIV/AIDS in older adults . The assThe following baseline variables were missing arbitrarily : years of clinical experience (24), work setting (21), educational qualification (15), professional cadre (13), PLWHAs attended to in the past year (10), gender (9), age (8) and additional HIV/AIDS training (2) totaling to 65 (22.9%) incomplete cases in the adjusted regression model. We found small to moderate correlations between missingness of a given baseline variable and specific questions with the largest absolute values ranging from 0.16 to 0.22, consistent with our assumption of Missing At Random. The imputed model was not adversely different from the complete cases analysis .Three themes emerged from content analysis namely, a need for clinician training for chronic care, and the associated enablers and barriers.Respondents had varying views about training clinicians to care for older adults. These were either specific to older adults or general issues of patient care. On the one hand, a respondent acknowledged that curricula offered in the training schools did not pay sufficient attention to the care for older persons:…You go to the training institutions in health, (and you find) their knowledge (and awareness) on (care for older persons) geriatrics is very low. There is no special attention (given to) geriatrics (care for older persons) and as a result the older people suffer more…” [Policy maker and researcher]“On the other hand, a different respondent recalled her experiences as a medical student and observed that while clinician training had generally evolved, clinician–patient interaction could be improved irrespective of the patients’ age category:…No! Clinician training needs to (change); it has changed a bit…they are doing a bit of psychology now in the medical school which we didn’t do; we are now trying to get them to do a bit of communication skills, communication! 50% of our patients need just talking therapy which is counseling. The clinicians never do it…” [Clinician and policy maker]“Respondents identified three prospects for training clinicians to care for older adults. Foremost, one respondent cited the current HIV/AIDS epidemic which has matured and resulted in more chronic users of antiretroviral drugs and in older adults too. Another referred to an existing short course in advanced antiretroviral therapy management provided by one centre of excellence in HIV/AIDS care whilst the third alluded to training efforts by three government agencies in Uganda, as illustrated below:…Then of course, a priority is training of health workers from the emergency mode to a sustainable mode. I think the emergency mode has been mainly to get people (with HIV/AIDS) to treatment; you tell them about adherence, what the drugs are for, and we off-load them to the treatment unit. The sustainable (HIV/AIDS care plan) is now considering the possible complications of the long-term therapy. That by giving consideration to the management of the elderly or aged people on the treatment; how can we manage?” “…I think it’s important that the people (clinicians) get the training now so that the mind set is already there; once these (aging in HIV/AIDS) reach a certain critical point. The (HIV/AIDS care centre of excellence) offers an advanced ART course within which we talk about some co-morbidities (including non-communicable diseases); we talk about toxicities, Hepatitis B … what might be lacking is consolidating this into a module that looks at aging in particular…” [Clinician and researcher]“…we are now developing a manual and in this manual we are integrating geriatrics with gerontology. We have got support from World Health Organization…but we have worked hard with the (relevant policy body) to ensure that issues of geriatrics and gerontology are included in the training programs. In (a particular university), has a course unit in social gerontology under the department of social work and social administration, and also under the department of community based rehabilitation of people with disabilities?” [Policy maker and researcher]“Respondents identified various potential barriers to training clinicians to care for older individuals with HIV/AIDS. Some appeared to arise from low awareness and poor attitudes towards care for older persons, while others seemed to be more fundamental beyond a clinicians influence. Responses varied from having little interest in caring for the elderly, to clinicians declining to take courses in geriatrics even when the opportunities were at their disposal. One respondent noted:…I carried out a study there (clinician training institution) about the aging issues; one respondent said…we are more interested in the under – fives; why bother with those, the expired (older adults)… and then another one on HIV and AIDS… they think that older persons are not sexually active and therefore they are not targeted…” [Policy maker and researcher]“And on interest of healthcare workers in being trained:…In fact one time we wanted to sponsor a nurse to go for a course in Malta in geriatrics but they  seemed not to be interested. I went to the NGO  that’s where I got a nurse from … and she went (for the training)…” [Policy maker and researcher]“A policy maker observed that while absenteeism from work was an important obstacle to training of health workers, those who were trained did not optimize their skills. In addition, she lamented over staff attrition in search for greener pastures outside the health sector and abroad.…Those who are trained are not there ; we have no retention strategy, we have no motivation strategy. We have continuously trained apparently but (there is) no recruitment…” [HIV/AIDS policy maker]“since most of the internationally accepted HIV/AIDS indicators are based on the population aged 15–49…,” Aging in HIV/AIDS is an emerging area of interest that has received more attention in High- and Middle-Income Countries than in sub-Saharan Africa, including Uganda In this study we report areas of sub-optimal clinician knowledge about older adults with HIV/AIDS. We also identified that extremes of age; fewer number of PLWHAs seen; being a clinical nurse or having diploma education level were independently associated with lower knowledge scores about aging and HIV/AIDS.Although most clinicians accurately stated Uganda’s national HIV sero-prevalence rate of 6.4%, very few pointed out that the burden is higher in 50–59 year older adults than the youth aged 15–24 years Despite a high self-reported interest in care of older individuals, most clinicians scored 50% or below. Questions on HIV/AIDS co-morbidities and long term antiretroviral-therapy toxicities attracted lower scores compared to epidemiology and prevention. The data also suggest that more clinicians with additional HIV/AIDS training had a knowledge score above 50%. However, this was not sustained in the multivariable model indicating that a critical mass is yet to be reached in order to make a difference in terms of transfer of knowledge on care for aging PLWHAs. Only 23% had completed short courses in HIV/AIDS co-morbidities and toxicities following chronic antiretroviral-therapy use, demonstrating that this topic should be given priority in future comprehensive HIV care trainings.Clinician age independently predicted knowledge scores, consistent with two previous studies among physicians in New York, USA Clinical nurses had significantly less knowledge scores compared to general medical doctors. In addition clinicians with a diploma qualification had lower knowledge, with borderline significance in the adjusted analysis. This group typically consisting of clinical nurses and clinical officers, are increasingly taking over duties from medical doctors as HIV clinics become more congested, cohorts become clinically stable, grow older, HIV/AIDS funding becomes less available and to some extent due to brain-drain The estimated number of PLWHAs seen in the preceding 12 months independently predicted knowledge scores. Those who estimated that they had seen fewer than 50 PHLWAs had less knowledge scores irrespective of work setting. In this line, clinicians who practiced mainly in HIV/AIDS specialist centres did not have higher knowledge scores in the regression model. On the one hand, this finding may imply that quality HIV/AIDS care and knowledge has diffused into the primary health care system in Uganda so that it does not matter where clinicians practice. On the other hand, selection of target groups for trainings should not assume clinicians in HIV/AIDS clinics are more knowledgeable and they should be equally prioritized.Findings from the qualitative interviews suggest that medical and nursing schools may have lagged behind in pre-service training of clinicians in the care for older adults. These institutions should optimize the mature HIV/AIDS cohorts and budding training opportunities to kick-start short – courses in HIV/AIDS care for older adults in Uganda. In addition, communication skills’ training is vital. Good communication facilitates managing concerns of older adults whilst providing HIV/AIDS prevention and care. For example, during HIV counseling and testing younger clinicians eliciting sexual history from older adults in an African cultural context presents a challenge.This study was not without limitations. The urban study sample of clinicians may not represent those in rural HIV care centres. However, it is likely that clinicians isolated in rural settings are less knowledgeable, since access to updated medical evidence and opportunities for consultation or training are limited making our study findings even more relevant. Secondly, a human resources for health survey showed that 71% of medical doctors are in the central region where Kampala falls, making our study representative Our study demonstrated gaps and differences in clinician knowledge that need to be addressed. Clinician HIV/AIDS training and practice has focused on management of acute opportunistic infections, antiretroviral therapy initiation and acute toxicities, and prevention of mother-to-child transmission. This situation is likely to be similar in other low and middle income countries affected by HIV/AIDS. With increased access to antiretroviral drugs and improved patient outcomes, the government health and education departments in sub-Saharan Africa should consider training clinicians to meet the challenges of comprehensive chronic care for older adults with HIV/AIDS.Figure S1Survey Questionnaire for the Aging & HIV/AIDS in Older Adults Study.(DOC)Click here for additional data file.


We aimed to compare the incidence of fatal and non-fatal AIDS and non-AIDS events occurring at CD4 counts >350 cells/mm3 and viral load was measured in the 6 months prior. Poisson regression investigated the relationship between viremia and clinical events, after adjustment for confounding variables.Patients contributed person years at risk if the most recent CD4 count was >350 cells/mm10998 patients were included contributing 43524 person-years of follow-up (PYFU). The majority of follow-up (80%) was with a viral load ≤500, 12% between 501-10000 and 8% >10000. 95%, 72% and 64% of the follow-up in each strata respectively was in patients who had started cART. 379 AIDS events (14 deaths) occurred. There was a lower incidence of AIDS events in patients with a viral load ≤500  compared to a viral load >10000 . 532 non-AIDS events (131 deaths) occurred. Patients with a viral load ≤500 had an incidence of non-AIDS events of 1.50 per 100 PYFU (95%CI 1.36-1.64), and 1.43 per 100 PYFU (95%CI 0.96-1.89) when viral load >10000. As shown in Figure 3 an increased incidence of fatal and non-fatal AIDS and non-AIDS events was found in patients with uncontrolled viral replication, even after adjustment for current CD4 count and use of cART. The association between viral replication and AIDS events was clear and consistent with a biological effect, but with non-AIDS events was less clear without a difference between intermediate and high viral replication.In patients with a CD4 count >350 cells/mm


Optimal antibiotic prescriptions rely on evidence-based clinical guidelines, but creating such guidelines requires a time-consuming systematic review of the literature. We aim at facilitating this process by proposing an innovative tool to extract antibiotic treatments from the literature.what antibiotic is used to treat cystitis caused by E. coli?) to which the system answers by retrieving a set of MEDLINE records from which the most frequently associated antibiotics are extracted and returned in a relevance-ranked list. The users can then access the annotated abstracts of the publications supporting the antibiotic as being a potential treatment, thus allowing them to use their expert judgment to accept or reject the assumption.We develop a web application, embedding a question-answering (QA) module based on EAGLi (Engine for Question-Answering in Genomics Literature), which has been specifically tuned for antibiotherapy. The users ask questions . In addition, infectious disease specialists from the University Hospitals of Geneva evaluated KART with several clinical scenarios. Despite an overall appreciation of the system and the recognition of its usefulness, improvements are required to use it when generating or updating clinical practice guidelines.The tool is accessible at KART seeks to facilitate medical knowledge building by providing an advanced retrieval engine. It provides a novel approach to cope with high volumes of literature generated over systematic reviews by facilitating access to pertinent information on antibiotic-related treatments.None declared.


Sensitivity to obesity is highly variable in humans, and rats fed a high fat diet (HFD) are used as a model of this inhomogeneity. Energy expenditure components  and variations in substrate partitioning are possible factors underlying the variability. Unfortunately, in rats as in humans, results have often been inconclusive and measurements usually made after obesity onset, obscuring if metabolism was a cause or consequence. Additionally, the role of high carbohydrate diet (HCD) has seldom been studied.Rats (n=24) were fed for 3 weeks on HCD and then 3 weeks on HFD. Body composition was tracked by MRI and compared to energy expenditure components measured prior to obesity. Results: 1) under HFD, as expected, by adiposity rats were variable enough to be separable into relatively fat resistant (FR) and sensitive (FS) groups, 2) under HCD, and again by adiposity, rats were also variable enough to be separable into carbohydrate resistant (CR) and sensitive (CS) groups, the normal body weight of CS rats hiding viscerally-biased fat accumulation, 3) HCD adiposity sensitivity was not related to that under HFD, and both HCD and HFD adiposity sensitivities were not related to energy expenditure components , and 4) only carbohydrate to fat partitioning in response to an HCD test meal was related to HCD-induced adiposity.The rat model of human obesity is based on substantial variance in adiposity gains under HFD (FR/FS model). Here, since we also found this phenomenon under HCD, where it was also linked to an identifiable metabolic difference, we should consider the existence of another model: the carbohydrate resistant (CR) or sensitive (CS) rat. This new model is potentially complementary to the FR/FS model due to relatively greater visceral fat accumulation on a low fat high carbohydrate diet. Rats, and humans, exhibit variability in their individual body fat gains in response to diets of varying macronutrient composition ,2. TheseDifferences in the sensitivity of body fat gain to macronutrient composition have been tentatively linked to various defects in the components of energy expenditure (EE), including basal metabolic rate (BMR), thermic effect of feeding (TEF), respiratory quotient (RQ), metabolic flexibility of muscles and the level of uncoupling in muscular contraction ; 5,6]. ,6. 5,6].The goal of the present study was to investigate potential early predisposing factors to dietary obesity. We measured, during low-fat feeding, before any significant adiposity could develop and using a fasting-refeeding indirect calorimetry procedure, several components of total EE that are suspected of being linked to the predisposition to dietary obesity, including BMR, TEF and substrate partitioning (as derived from RQ). These would then be compared to longitudinal body composition measured by magnetic resonance imaging (MRI) during 3 weeks of HCD then 3 weeks of HFD.The results validated and extended the previously described CR/CS model as adiposity gain varied substantially under HCD. Detailed analysis of the components of total EE under low-fat feeding and in response to ingestion of a single high fat test-meal did not reveal any link with adiposity gain under HFD as well as under HCD. However, we observed that RQ responded differently between CR and CS rats to ingestion of a HC meal, with adiposity gain under HCD correlated with the amplitude of the post-meal increase in RQ.48 male Wistar rats (Harlan), arrival weight ~225g (range 193-252g) and age 7 weeks (according to breeder’s data), were delivered as 6 groups of 8, with groups arriving sequentially over a 9 month period. Rats recovered in the laboratory for 1 week on a synthetic high carbohydrate diet HCD;  prior toThe design of the experiment is shown in After the first MRI measurement, the rats were fed for 3 weeks on the synthetic HCD and re-scanned by MRI at the end of this period. They were then fed for 3 weeks on a high fat diet HFD;  prior to-3, and fat-free mass (FFM) determined by subtracting this from the weight of the rat on the day of the scan.Body composition was measured by MRI at the onset of the study and the end of the HCD period , plus whP=0.53). A total of 48 calorimetry sessions were performed . The goal of these measurements was to get from each animal values for BMR, TEF, Rest-RQ, Act-RQ and Act-cost (see next paragraph for definitions) in the fed and fasted state. For this purpose, the rats were initially housed at 18: 00 in the metabolic cages without food but with free access to water. Cage volume is 16L but the apparent dilution volume estimated from injection of a N2/CO2 mixture in the cage gives a practical dilution volume of 10.5L. The cage was ventilated at 1.5L/min. Temperature was set at 26±1°C to minimize thermoregulatory effects [During weeks 2 and 3, while fed the HCD, on a randomly chosen day each rat experienced a meal-response indirect calorimetry session, one in week 2 and the other in week 3. They randomly received either an HC or an HF meal for the first session, and the previously unchosen meal for the second session. The mean body weight of the rats was not different between HC and HF test meals , and via the Weir formula, energy expenditure (EE) [≈ basal metabolic rate (BMR), while post-meal increase in REE above BEE represents the thermic effect of feeding (TEF). RQ can also be separated into resting (Rest-RQ) and activity-specific (Act-RQ) components. All of these parameters were correlated to the propensity of the rats to gain adiposity during HCD and HFD. The data was binned into 15min time periods before statistical analysis. VCOure (EE) ,11. The ure (EE) ,12,13. TThe correction described in this section was only ever applied to BMR and RMR (including glucose and lipid oxidation), never to any of the other metabolic parameters where it is not needed . In the case of TEF, this is because TEF is related to the size and composition of the meal, not to the size and composition of the subject , so TEF 2=1.000, P=0.000.As shown in Since we had small sample sizes, rather than using FM and FFM directly as independent variables in the statistical models of the metabolic parameters, we decided to use normalization to FFM + 0.2 × FM ,14. AlthHere, post-processed data means body composition values extracted from MR images, biologically meaningful parameters derived from raw calorimetry data and so on. This was all analyzed using R (version 2.15.3) , with grWhere used, the linear mixed effects command modeling a metabolic parameter as a function of other data took this general form:model = lme where lme is the linear mixed effects function called in R, mp is the metabolic parameter , adchg is the percentage change in adiposity under HCD or HFD, and mealtime is pre- or post-meal. Thus four fixed effects are estimated: intercept , adchg , mealtime  and interaction . The nested random effects are ratID (the unique identification of each rat), sessionID  and mealtimeID . The type of meal (HC or HF) and the type of adiposity change (under HCD or HFD) were not included in the model; the data for each of the HC and HF meals and for each of the HCD and HFD adiposity gains were analyzed separately. We also tested models where adchg was replaced with the absolute adiposity, in particular the starting adiposity measured at MRI1, as this might have an effect on metabolic parameters. However, these did not yield any interesting relationships and so are not reported.P≤0.01 for significance, and the interval 0.01<P≤0.05 as marginal significance worthy of discussion.Where and as appropriate, results are stated as mean±SEM or (only in the case of the adiposity ratios) median±0.5IQR. A large number of statistical tests were carried out for this study. Although this can sometimes justify multiple comparison correction, many of the comparisons here were of parameters that are not fully independent of each other. This makes it very difficult to carry out a fair correction so we have left the statistics uncorrected and chosen a relatively conservative threshold of P=0.000) than HCD , although the correlation under HCD did remain significant . Still, the overall indication is that BW gain can be taken as a proxy of adiposity gain under a high fat diet but not under a carbohydrate-rich one; this necessitates the use of techniques able to measure adiposity in vivo.We first studied if body weight (BW) and adiposity or body fat gain correlated under high carbohydrate (HCD) and/or high fat (HFD) diets see , adiposiP=0.017, see also individual classification in P=0.000) or after HFD . This does of course have to be considered in the context of absolute fat pad weights  confirming that “palatability” can be maintained only when the organoleptic properties of the diet are frequently modified as is done for example to maintain hyperphagia under cafeteria feeding.We determined how each of Rest-RQ, Act-RQ, activity, Act-cost and RMR measured pre- and post-meal with either HC or HF meals were correlated to adiposity gain under either HCD or HFD  and HFD (FR/FS difference). In the vast majority of cases, no relationships were found: if a particular comparison is not explicitly mentioned here, it can be safely assumed that no significant relationship was observed. However, for readers who may be interested, ≈0.80 before meal-delivery and also did not correlate with propensity to adiposity gain.BMR and TEF values did not correlate to adiposity gain during either HCD or HFD. Otherwise stated, rats with a low BMR and/or a low TEF did not exhibit any greater propensity to become obese than rats with a high BMR or TEF. We also observed that the cost of activity increased rapidly in response to ingestion of either the HC or HF meals see  and S5 bP=0.003) but not with adiposity changes under HFD . In general, the Rest-RQ response increased above HCD food quotient  only in CS rats (P<0.01 for both around the 105-240 min period). No differences were observed in the 3 other experimental conditions  and high fat diet (HFD) was longitudinally measured by MRI in male Wistar rats, and assuming thermoregulation was negligible because the rats were housed at 26° C, compared to all the sub-components of TEE, namely REE, and Act-cost and TEF as well as Rest-RQ and Act-RQ measured by indirect calorimetry and extracted by a previously described process of modeling and filtering of raw data ,12,13. TFirstly, it needs to be stressed that the HCD used in this study was not designed to induce overfeeding and/or insulin resistance through introduction of a high proportion of sucrose as for example in . It was Using sequential measurements of body composition by MRI, the present study confirmed a previous one  in whichInterestingly, an obesity prone  model was recently described in chow fed C57BL/6 mice . At the in vivo adiposity measurement techniques to study the model.Crucially, differences in sensitivity to adiposity gain under HCD were not very predictive of those under HFD, and vice-versa. Thus individuals needed to be classified not only by their HFD-sensitivity but also by their HCD-sensitivity. An important characteristic of the CR/CS model is that increased adiposity during HCD was difficult to spot from weight measurements as the larger fat deposition in CS rats was not paralleled by an increase in lean mass gain as under HFD, such that overall BW gain was only marginally greater. This is indeed a methodological drawback that imposes the use of Finally, it must be noted that body fat gain under HCD was characterized in both CR and CS rats by a relatively larger accumulation of fat in the visceral vs. subcutaneous depots; under HFD, while more fat accumulated overall than under HCD, it tended to be more subcutaneous. This suggests that even if overall fat accumulation is lower under HCD than HFD, the adiposity that develops under HCD could be as or more deleterious in the long-term than that which develops in response to HFD ,24.In general this study found very little link between the various sub-components of TEE and adiposity gain under either HCD or HFD. Among the most important and often suspected parameters involved with resistance/sensitivity to obesity are BMR and TEF. Low BMR values have been suspected to increase the sensitivity to HFD-induced obesity in various animal models as well as in humans ,25,26, tin- or ex vivo measurement of resting muscles [2-VCO2 and the intensity of activity by the Kalman method [Differences in activity have been reported between FR and FS rats or between lean and obese rats ,39,40. F muscles –46. In tn method . This ca2-VCO2 allowed us to study Act-RQ, meaning to follow along the time the fuel mix used by working muscles. The currently available data estimating lipid oxidation by muscles are often contradictory. For example, Dourmaskin and col. reported that before becoming obese, FS rats suffer metabolic disturbances favoring fat storage and in particular a decline in lipid transport and capacity for lipid oxidation in muscles [Together with the cost of activity, the processing of the short-term changes in activity and VO muscles , while C muscles . Here, w2 and VCO2 values, increased more in response to HC feeding in CS than in CR rats. Converted into rates of glucose and lipid oxidation, this difference reflected the fact that the post-meal increase in Rest-Gox and decrease in Rest-Lox were of larger amplitude in CS rats. Accordingly, we previously reported that under free feeding there is a positive correlation between adiposity gain and 24h RQ in rats fed HCD [In contrast, Rest-RQ, meaning RQ computed from resting VO fed HCD . Taken t fed HCD , RQ neveIt has been suggested that nutrient partitioning or metabolic flexibility (meaning the capacity of the organism to adapt fuel oxidation to food composition) may be responsible for establishing different adiposity levels in the long-term –51. ThisThis study firmly establishes recent observations that significant differences in body fat accumulation develop under HCD in rats. The sensitivity to HCD was correlated to that under HFD, but not strongly, indicating that HCD- and HFD-sensitivities do not necessarily share common underlying metabolic defects. This study also indicates that FS rats do not exhibit any defect in any components of TEE and RQ before being submitted to HFD; to our knowledge this leaves for now circulating triglycerides after a high-fat preload as the only good predictor in rats of later sensitivity to HFD . In contFigure S1Adiposity measured during the initial MRI session showing the greater initial adiposities of the rats selected for inclusion in the study.(TIF)Click here for additional data file.Figure S22: VO2. Middle: data of the top panel processed by Kalman filtering. VO2 and VCO2 are transformed into metabolic rate  according to Weir formula =((VO2×16.3)+(VCO2×4.57))/60. Modeling and processing of the changes in VO2 and VCO2 in relation to Act gives computation of RMR. RQ, Resting RQ (Rest-RQ) and Activity RQ (Act-RQ) are computed from resting and activity VO2 and VCO2 respectively. Bottom: metabolic cost of activity computed by the Kalman filter as the amplitude of changes in VO2 and VCO2 relative to intensity of activity. Vertical grey bar at 10: 00 shows the time interval during which data acquisition was frozen to introduce the test-meal.The time frame is reduced to from 0: 00 to 17: 00 for clarity. Top: original raw data as recorded by the gas analyzers and activity monitors. RQ is the ratio of VCO(PDF)Click here for additional data file.Figure S3The data are sorted into rat adiposity sensitivity or resistance groups as defined in (TIF)Click here for additional data file.Figure S4(TIF)Click here for additional data file.Figure S5The six panel groups show meal response metabolic cage data collected during the HCD period . X axis labeled in minutes vs. meal onset. Rest-RQ, Act-RQ, Activity, Act-cost and RMR (BCC = body composition corrected) are all defined as described in the Materials and Methods. The data are sorted into rat adiposity sensitivity or resistance groups as defined in (TIF)Click here for additional data file.Table S1Results are in the simple form of mean±SEM and t-tests for CR vs. CS and FR vs. FS rats after ingestion of the HC or HF test meal and as appropriate during the pre- or post-meal period. BW = body weight, FFM = fat-free mass, FM = fat mass, BMR = basal metabolic rate, TEF = thermic effect of feeding, AUC = area under the curve , W/AUA = Watts per Arbitrary Unit of Activity, NS = not significant (p>0.01). BMR and other basal metabolic data were acquired during the 120 minute pre-meal period. The post-meal period covered the 300 minutes following meal provision.(DOCX)Click here for additional data file.


Head and neck cancer is the sixth most common type of cancer worldwide. Despite advances in aggressive multidisciplinary treatments, the 5-year survival rate for this dreadful disease is only 50%, mostly due to high rate of recurrence and early involvement of regional lymph nodes and subsequent metastasis. Understanding the molecular mechanisms responsible for invasion and metastasis is one of the most pressing goals in the field of head and neck cancer. Met, also known as hepatocyte growth factor receptor (HGFR), is a member of the receptor protein tyrosine kinase (RPTK) family. There is compelling evidence that Met axis is dysregulated and plays important roles in tumorigenesis, progression, metastasis, angiogenesis, and drug resistance in head and neck cancer. We describe in this review current understanding of Met axis in head and neck cancer biology and development of therapeutic inhibitors targeting Met axis. Cancer from the head and neck region is the sixth most common cancer and accounts for some 350,000 cancer deaths worldwide each year ,2. More Traditional treatment for HNSCC has been extensive surgery, which often yields a poor functional outcome. In recent years, improved therapy has been developed involving intensity-modulated radiation and concurrent use of chemotherapy . In addiin vivo [Hepatocyte growth factor (HGF), also known as scatter factor, is active in numerous tissues . HGF is in vivo ,15. HGF in vivo ,17.C-terminal domain of the β subunit, with concomitant activation of downstream effector molecules [C-terminus, tyrosine residues 1234 and 1235, located within the catalytic domain, are required for tyrosine kinase activity [Met gene, located on chromosome 7q31, encodes a receptor protein tyrosine kinase (RPTK). Native Met protein is proteolytically processed to a heterodimer composed of a 50 kDa α subunit and a 145 kDa β subunit, linked together by disulfide bonds . In addiolecules ,21,22. Tolecules ,24. Signolecules ,25. Amonactivity ,27. Tyroactivity ,29,30. Factivity .CD44 is a cell surface adhesion molecule that is involved in cell-cell and cell-matrix interactions. A number of CD44 isoforms, which differ in their extracellular domain, can be generated via alternative splicing . The extThe extracellular domain of Met shares structural homology with plexins, which are transmembrane receptors for semaphorins. Semaphorins function to control axonal guidance in the nervous system. Plexins are also expressed in other tissues where Met is present . The higAlthough α6β4 integrin does not possess an intrinsic catalytic activity, it has been shown to promote invasive growth. α6β4 integrin physically interacts with Met and Met activation by HGF leads to tyrosine phosphorylation of the β4-subunit cytoplasmic domain. The resultant phosphorylated tyrosines provide supplementary docking sites for the recruitment of PI3 kinase, Shc, and Shp2 ,45. For Several studies have demonstrated that Met can be trans-activated by other receptor protein tyrosine kinases (RTKs) . For exaC-terminal tyrosines, which serve as docking sites for down-stream signaling molecules. Therefore, tyrosine phosphorylation is synonymous with Met activation. Net tyrosine phosphorylate of Met is determined by dynamic equilibrium of intrinsic tyrosine kinase activity of Met and counteracting protein tyrosine phosphatases (PTPs). Several PTPs have been shown to regulate Met tyrosine phosphorylation. Palka et al. has demonstrated that DEP-1  preferentially dephosphorylates tyrosines 1349 and 1365. However, DEP-1 does not appear to affect Met-mediated MAP kinase activation [Met plays important roles in both physiological and pathological conditions. The function of Met is determined by phosphorylation of tivation . Antisentivation . PTP1B ativation . RPTP-β regulation of Met tyrosine phosphorylation and biological functions has been extensively studied. Purified RPTP-β directly dephosphorylates purified Met and substrate-trapping mutant of RPTP-β specifically interacts with Met in intact cells. In addition, expression of RPTP-β reduces both basal and HGF-induced Met phosphorylation at tyrosine 1356 and inhibits downstream MEK1/2 and Erk activation, while shRNA-mediated knockdown of RPTP-β increases basal and HGF-stimulated Met phosphorylation at tyrosine 1356 . FurtherNormal epithelial cells express Met, but not HGF . HGF is Met internalization is a major negative regulatory mechanism for desensitizing Met signaling pathway. HGF-induced endocytosis of Met, terminates signaling by sequestering Met in lysosomes, where it is degradated and prevented from recycling to the plasma membrane. Tyrosine 1003, located in the juxtamembrane domain of ligand-activated Met, recruits E3 ubiquitin ligase Cbl, resulting in mono-ubiquitination of Met at multiple sites . UbiquitN-terminal extracellular domain is first cleaved by a disintegrin and metalloprotease (ADAM), resulting in a membrane-anchored cytoplasmic tail. The membrane-associated cytoplasmic region is further processed by γ-secretase mediated proteolysis into labile intracellular fragments for ultimate degradation by proteosomes [Under steady-state conditions, Met is also subjected to low level of proteolytic cleavage independent of ligand or ubiquitination. The teosomes .In addition, LRIG1, a transmembrane leucine-rich repeat and immunoglobulin (Ig)-like domain-containing protein, interacts with Met and negatively affect Met protein stability in HGF and Cbl independent manner, thereby, negatively regulating Met function .miRNAs are small noncoding endogenous RNAs that can negatively regulate protein expression by targeting transcripts for degradation and by blocking translation . A numbein vivo and in vitro studies point toward the importance of Met in human malignancy. Stimulation of Met by HGF enhances cell proliferation, survival, dissociation, migration, morphogenesis, formation of blood vessels, and degradation of extracellular matrix, all characteristics that are associated with invasive cell phenotype [In addition to its key participation in many physiological processes, numerous henotype . Met pathenotype . Many tyhenotype ,70,71.C-terminal sequence in chromosome 7 [Aberrant Met signaling can be achieved by Met or HGF gene over-expression, Met point mutations, amplification, or gene rearrangement, leading to constitutive tyrosine kinase activity. Met was originally isolated as TPR-Met oncogene, which possesses ligand-independent tyrosine kinase activity, due to chromosomal rearrangement of translocated promoter region (TPR) in chromosome 1 fused to Met mosome 7 ,72. Thismosome 7 .Met point mutations have been identified in hereditary and sporadic papillary renal carcinoma, hepatocellular and gastric carcinomas and head and neck squamous cell carcinomas . Large bi.e., with Met gene amplification) can escape EGFR inhibition. These cells become resistant to anti-EGFR therapy and can undergo rapid clonal expansion [Met can also be responsible for resistance of tumors against therapies. For example, in the presence of EGFR inhibitors, such as gefitinib, a subset of tumor cells with high Met expression  production ,102. TheIn vitro experiment showed that HGF treatment of HNSCC cells increased IL-8 and VEGF production [in vitro and in vivo. This capability is further enhanced by the presence of CD44 which facilitates Met activation. In addition, these Met positive cells have enhanced metastatic ability and are resistant to cisplatin treatment [Higher serum HGF levels correlate with higher levels of angiogenic factors such as interleukin-8 (IL-8) and vascular endothelial growth factor (VEGF) in patients with HNSCC . In vitroduction . Furtherreatment . EGFR is over-expressed in over 80% of HNSCC tumors  and antiIn view of its high expression in various human malignancies and its involvement in mediating invasive growth of many types of cancers, Met has been identified as an attractive target for cancer treatment. Many types of inhibitors have been developed to inhibit this signaling pathway, including receptor antagonists, small-molecule tyrosine kinase inhibitors (TKIs), and monoclonal antibodies (mAbs). Among them, mAbs such as rilotumumab and onartuzumab, and TKIs such as tivantinib, crizotinib, foretinib and cabozantinib have recently been tested in phase I and II clinical trials . N-terminal 4 kringle domains of the α-chain of HGF. NK4 competes with HGF binding to Met, thereby inhibits HGF-dependent Met tyrosine phosphorylation, down-stream signal pathways, cell proliferation, and invasion [NK4 is a receptor antagonist consisting of invasion ,117,118.Rilotumumab , is a humanized monoclonal antibody that selectively binds and neutralizes HGF, thereby preventing HGF binding to Met and interfering with Met activation ,120. An Onartuzumab is a humanized mono-valent anti-Met antibody (OA5D5/MetMAb). It binds Met with high affinity, thereby preventing HGF binding to Met and consequent Met phosphorylation, and activation of downstream signaling events. In preclinical studies, onartuzumab has been shown to inhibit tumor growth in multiple tumor models ,125. OnaTivantinib (ARQ197) is a non-competitive, ATP-site selective, small molecule inhibitor of Met tyrosine kinase . Phase ICabozantinib (formerly known as XL184) is another multi-kinase inhibitor against Met, VEGFR2, and Ret. Phase I and II studies have shown promising results for improvement on patient survival . A randoMet targeted therapy is a relatively new area of clinical investigation in HNSCC treatment. Met pathway inhibitors might be used in combination with chemotherapy, radiotherapy, or immunotherapy reagents to overcome potential tumor resistance and to improve overall clinical outcome. Several clinical trials are ongoing to investigate the effects of monoclonal antibodies and small molecular TKI against Met axis in patients with recurrent HNSCC.Crizotinib (PF-02341066) is a potent and selective dual specific TKI for both Met and ALK. It exhibits inhibitory effects on Met-dependent proliferation, migration, and invasion of human tumor cells and is also effective against tumors with an activating ALK gene rearrangement . In a prForetinib (formerly known as XL880) is a multi-targeted TKI whose primary targets are Met and VEGF . It is tSince the discovery of Met more than 25 years ago, much has been learned about its roles in a broad spectrum of cellular and biological functions, including mitogenesis, morphogenesis, angiogenesis, migration, and invasiveness. While these biological processes are tightly regulated during embryogenesis, tissue homeostasis in adulthood, wound healing, and liver regeneration, aberrant activation of Met by gene amplification, gene re-arrangement, point mutations, protein over-expression, or loss of negative regulatory mechanisms can contribute to tumor initiation, progression, metastasis and resistance to therapy. Therefore, Met axis is an attractive target for cancer treatment and a number of Met pathway inhibitors have been developed and are currently in clinical trials. Anti-Met clinical studies for HNSCC are ongoing and results from these studies will shape the landscape for future use of Met inhibitors in HNSCC. Further clinical trials on the efficacy of Met inhibition in HNSCC and other types of solid tumors are warranted, given the important role of Met axis in HNSCC invasive growth.


We conducted a survey of 928 New York City area residents to assess knowledge and worry about AIDS and SARS. Specific sociodemographic groups of persons were more likely to be less informed and more worried about contracting the diseases. Informed behavior change may be needed to control disease transmission. Negative public reactions, such as stigmatizing persons at risk for the disease, may greatly hamper prevention and treatment efforts . AIDS may be considered the prototype of an emerging infectious disease. While AIDS has received considerable public attention since the early 1980s, the strong emotions associated with it create the possibility of nonrational information processing. The stigmatization of persons with or at risk for AIDS has persisted despite public information about the disease  who lived in metropolitan New York City (NYC). The cohort was recruited through a random digit dial telephone survey conducted from March 25 to June 25, 2002. Additional details on the sampling are provided elsewhere . A strong relationship existed between being very worried about both diseases. Of the respondents who reported being very worried about AIDS, 16% reported also being very worried about SARS; 5% of the respondents who were not very worried about AIDS were very worried about SARS (p = 0.016).Finally, we examined the relationships between being informed and worried about contracting AIDS/SARS. These analyses were confined to respondents who reported having some information about AIDS/SARS; respondents who reported that they had not heard about the diseases were not asked the follow-up questions. In these respondents, no relationship between having heard and being worried about getting the diseases was shown.Given the widespread disparities in health among racial/ethnic and socioeconomic groups in the United States (The data from this study were collected in a major city of an industrialized country and should not be generalized to developing and transitional countries. Nevertheless, if obtaining and evaluating information is adversely affected by factors such as low education level, low income, and ethnic minority status, then properly informing the public may be particularly difficult in developing and transitional countries. The epidemiology of AIDS and SARS has been very different in NYC (>58,097 AIDS cases [The limitations of this study included using single items to measure knowledge and worry about AIDS and SARS and the standard limitations of telephone surveys, e.g., inability to contact households without telephones, moderate refusal rates. However, this study strongly suggests that adequate public knowledge and emotional assessment may be critical to control these diseases.Our data suggest that socioeconomic class and race/ethnicity factors may help shape public understanding of emerging infectious diseases. Targeted communication to different population subgroups may be required to achieve public understanding of an emerging infectious disease.