Tandem C2H2-type zinc finger proteins (ZFPs) constitute the largest transcription factor family in animals. Tandem-ZFPs bind DNA in a sequence-specific manner through arrays of multiple zinc finger domains that allow high flexibility and specificity in target recognition. In tetrapods, a large proportion of tandem-ZFPs contain Krüppel-associated-box (KRAB) repression domains, which are able to induce epigenetic silencing through the KAP1 corepressor. The KRAB-ZFP family continuously amplified in tetrapods through segmental gene duplications, often accompanied by deletions, duplications, and mutations of the zinc finger domains. As a result, tetrapod genomes contain unique sets of KRAB-ZFP genes, consisting of ancient and recently evolved family members. Although several hundred human and mouse KRAB-ZFPs have been identified or predicted, the biological functions of most KRAB-ZFP family members have gone unexplored. Furthermore, the evolutionary forces driving the extraordinary KRAB-ZFP expansion and diversification have remained mysterious for decades. In this review, we highlight recent studies that associate KRAB-ZFPs with the repression of parasitic DNA elements in the mammalian germ line and discuss the hypothesis that the KRAB-ZFP family primarily evolved as an adaptive genomic surveillance system against foreign DNA. Finally, we comment on the computational, genetic, and biochemical challenges of studying KRAB-ZFPs and attempt to predict how these challenges may be soon overcome.The online version of this article (doi:10.1186/s13100-015-0050-8) contains supplementary material, which is available to authorized users. Tandem C2H2-type zinc finger proteins (ZFPs) make up the single largest transcription factor family in mice and humans with approximately 600 and 700 genes, respectively . The larKRAB-ZFPs contain a potent KRAB repression domain and tandem arrays of zinc fingers (ZNFs) that mediate DNA binding. What makes KRAB-ZFPs exceptional among other DNA binding transcription factors is their ability to bind to long stretches of DNA by combinatorial use of up to several dozen ZNFs that serve as modular DNA binding units. These exceptional modular DNA binding properties were co-opted for use in gene-editing applications, forming the basis of the first generation of engineered sequence-specific DNA modifying enzymes called zinc finger nucleases , 4. HoweKRAB-ZFPs are also unique among transcription factor families in that a large fraction of their members have DNA binding domains that are rapidly evolving. This rapid evolution may contribute to morphological and behavioral evolution by controlling expression of developmental genes , 6. HoweKRAB-ZFPs are believed to have evolved from the Meisetz (PRDM9) gene , which hKRAB-ZFPs with a SCAN domain have been found in mammals and lizards but are absent in frog and chicken, indicating that this domain was acquired in KRAB-ZFPs around the root of the amniote branch but subsequently got lost in some species . InteresTo date, the vast majority of KRAB-ZFP research has focused on human and mouse KRAB-ZFPs. Therefore, it is important to point out that the findings from these studies may not always be applicable to tetrapod KRAB-ZFPs in general. Nevertheless, all KRAB-ZFPs contain tandem arrays of up to 36 C2H2-type ZNFs, usually encoded by a single exon at the 3’ end of the gene . Each ZNSeveral DNA binding prediction models for tandem-ZFPs have been developed by using bacterial one-hybrid systems, empirical calculations of pairwise amino acid–nucleotide interaction energies, and knowledge from the X-ray crystal structure of a three-fingered, C2H2-type ZFP (Zlf268 or Egr1) , 25–27. About 30–40 % of mammalian tandem-ZFPs have a KRAB domain  that, inTandem-ZFP genes are predominantly organized in genomic clusters , 6. For Although the KRAB domains and amino acids required for ZNF structure are generally well conserved amongst mammalian KRAB-ZFPs, positive selection at the residues that confer DNA binding specificity is common, especially between recently duplicated gene pairs , 49–51. Although the progenitor of the KRAB domain apparently dates to the last common deuterostome ancestor of chordates and echinoderms , KRAB-ZFSome KRAB-ZFPs have been associated with metabolism, differentiation, apoptosis, and human diseases , 57, 58,personal communication), a fraction of KRAB-ZFPs are differentially expressed in adult tissues . Mo. Mo64]. Some KRAB-ZFPs may even restrict retroviral activity without the help of transcriptional corepressors. The KRAB-ZFP associated SCAN domain is not only believed to be derived from a retrotransposon but is also structurally similar to the HIV C-terminal capsid . FurtherThe hypothesis that KRAB-ZFPs evolved to defend host genomes from parasitic DNA implies that many of these transcription factors bind to ERVs and other EREs. Using computational motif prediction tools, it was predicted that many human KRAB-ZFPs bind EREs that entered the human genome around the time these KRAB-ZFPs appeared . RecentlEvidence for an ongoing evolutionary arms race between host KRAB-ZFPs and transposable elements also came from the few studies that identified individual ERE-repressing KRAB-ZFPs. Both Gm6871 and KAP1 bind predominantly to L1 elements that entered the mouse genome 4–7 million years ago . SimilarZNF91 and ZNF93 emerged in the last common ancestor of apes and Old-World monkeys and are members of a KRAB-ZFP cluster that has amplified and diversified throughout the evolution of apes and humans . Both ZNAltogether these findings imply that evolutionarily young KRAB-ZFPs repress retroviruses and EREs, whereas older and well-conserved KRAB-ZFPs fulfill other functions. To gain insight into the evolutionary history of ERE repressing KRAB-ZFPs, we generated a phylogenetic tree of murine and human KRAB-ZFPs. Indeed, ZFP809 and Gm6871 are closely related to other murine KRAB-ZFPs but do not phylogenetically cluster with any human KRAB-ZFPs. On the other hand, the L1 and SVA repressors ZNF91/93 form a cluster with many other human KRAB-ZFPs but are not closely related to any mouse KRAB-ZFP  [unpublished data). Although target motifs similar to the PBSpro can be found at these ZFP809 binding sites . However, the weak binding affinity of ZFP809 to these elements also shows that imperfect binding sites for an ERV-repressing KRAB-ZFP can appear by chance in unrelated ERVs, possibly because of the general GC richness in these elements. ERV-targeting KRAB-ZFPs might have a general potential to bind weakly to other ERVs. If such a KRAB-ZFP is duplicated or becomes available after its original target ERV was inactivated over time, mutations of the ZNFs might allow stronger binding and therefore functional silencing of newly emerged ERVs.ZFP809 binds to VL30 and MmERV elements (both ERVs of the ERV1 family) that contain a PBS(PBSgly)  Fig. 4)pro, but EREs and especially ERVs have a profound impact on patterns of mammalian gene expression. Retroviral LTRs contain strong promoter elements to ensure efficient expression of their proviral genome. When integrated near a cellular gene, transcription from these LTRs can drive the expression of that gene , 152. FuAs discussed above, KRAB-ZFPs that repress newly emerged EREs may result from a duplication of an existing ERE repressor, followed by mutations in the DNA binding domain that leads to recognition of the new targets. Alternatively, gene-targeting KRAB-ZFPs might duplicate and change their binding specificity towards new EREs Fig. . Furtherunpublished data) may enhance the expression of this key protein during brain development [It has been also reported that an IAP LTR that drives gene expression is silenced by a gene located in a KRAB-ZFP cluster . Althougelopment , possiblelopment .One of the best-characterized KRAB-ZFPs, ZFP57, is required to maintain a subset of genomic imprints in mice , and mutRsl) locus encodes two KRAB-ZFP genes, Rsl1 and Rsl2, which are regulated directly in the kidney by androgen or indirectly in the liver by growth hormones [Sexually dimorphic gene expression in liver is a complex phenomenon in mice and humans. Sex-specific expression of growth hormones can induce gene expression, and genes can be repressed in a sex-specific manner. The mouse regulator of sex-limitation  that contained an ancient ERV insertion near the promoter region [One ynthesis . Cyp2d9 ut liver , confirmut liver . BecauseRsl, which encodes sex-limited protein (Slp) was reported to be controlled by an ancient ERV LTR located 2 kb upstream of this gene [Rsl1 is located within a cluster of recently duplicated KRAB-ZFP genes found only in the Mus lineage [More importantly, another target of his gene , 169. Inhis gene . Intrigu lineage .Mus species [In most mammals, homologous recombination during meiosis tends to occur at specific segments of the genome. Interestingly, the placement and activity of these so-called hotspots varies greatly between closely related  species , between species , 174, an species . These h species . The KRA species –179 thro species , 14. Int species , and acc species –183. PRD species , 181. In species .Could this indicate that PRDM9 originally evolved as an ERV repressor? Although PRDM9 predates the emergence of human PRDM9-bound THE1 LTR elements, MaLRs colonized the genomes of eutherian mammals at least 80–100 million years ago . An anciDespite recent progress, KRAB-ZFPs are not only one of the largest but also one of the least understood transcription factor families in mammals. In fact, many functional KRAB-ZFP genes might not even be annotated yet, whereas some predicted KRAB-ZFPs will turn out to be pseudogenes. The highly repetitive nature of KRAB-ZFP genes makes conventional annotations difficult, and estimates of their copy numbers vary as they strongly depend on the inclusion criteria used , 6, 15. Moreover, the assumption that all KRAB-ZFPs are DNA binding transcription factors might be premature and it is possible that some tandem-ZFPs function outside the nucleus. Binding of C2H2-type ZNFs to RNA and proteins has been reported , 188, supro [pro, the fraction of overlapping nucleotides was rather small . Nevertheless, binding to imperfect target sites is likely to occur. We speculate that such binding is kept to a minimum by selectively expressing KRAB-ZFPs at relatively low levels, ensuring that only the preferred binding sites are occupied. However, it is possible that, by regulating KRAB-ZFP expression levels, different cell types can determine the number of functionally relevant KRAB-ZFP binding sites. A powerful strategy to circumvent KRAB-ZFP overexpression in genome-wide binding assays will be tagging of endogenous KRAB-ZFP genes with epitopes using the CRISPR/Cas9 system.Ultimately, loss-of-function studies will be required to determine the functional roles of KRAB-ZFPs. However, the repetitive and clustered characteristics of KRAB-ZFP genes—especially of evolutionarily young ones that have recently duplicated—remain a major obstacle. Conventional gene targeting by homologous recombination as well as RNAi- and CRISPR/Cas9-based approaches depend on unique sequences to ensure that only the right target is affected by knockdown or knockout. Alternatively, gene-trap collections of ESC clones might be used to generate KRAB-ZFPs knockout mice. However, mapping of gene-trap insertions at repetitive genes is problematic as well.Thus new strategies will be needed to test the biological requirements of individual KRAB-ZFPs. We recommend targeting the rather unique regions between KRAB domains and ZNFs with CRISPR/Cas9 guide RNAs to mutate KRAB-ZFP genes. However, in the case of recently duplicated KRAB-ZFP genes, even these regions might be too similar for specific targeting by CRISPR guide-RNAS or siRNAs. Since recently duplicated KRAB-ZFP genes are usually located in close proximity, one might consider genetic depletion of several KRAB-ZFP genes within a cluster at once by inserting loxP sites in the unique flanking regions using CRISPR/Cas9 or conventional gene targeting, followed by Cre-mediated recombination. Stepwise rescue experiments and ChIP-seq with epitope-tagged proteins may be used to assign the observed phenotypes in such KRAB-ZFP cluster knockout cells or animals to a single gene.Once a larger number of KRAB-ZFPs have been characterized in detail, several important questions about the function and evolution of KRAB-ZFPs can be addressed. For instance, little is known about the time it takes for KRAB-ZFPs to evolve to bind newly emerged sequences such as ERVs. It is also somewhat puzzling how KRAB-ZFPs can keep up with active and therefore mutating ERVs. One possibility is that KRAB-ZFPs (such as ZFP809) that bind to retroviral sequences that are at least partially conserved in various ERV groups are preferably selected as repressors of parasitic elements. Alternatively, KRAB-ZFPs might primarily repress ERVs and other retrotransposons that have already lost the ability to replicate, whereas other repression mechanisms act on newly emerged active ERE families, as previously suggested . NeverthThe current KRAB-ZFP sets in mammals are likely a mixture of KRAB-ZFPs that are under purifying or positive selection and KRAB-ZFPs that arose by recent gene duplications and subsequent mutations but remain nonessential for the host. These KRAB-ZFPs will become pseudogenes and eventually disappear by genetic drift. Additionally, one might expect that duplicating and mutating KRAB-ZFPs sometimes reduce host fitness and are therefore rapidly removed by negative selection. How many useless or harmful KRAB-ZFPs are necessary before a beneficial one evolves? This question could be addressed by analyzing the genomes of very closely related species . One might even expect that there are differences in the number of KRAB-ZFPs between individuals of the same species. More importantly, germ-line mutations and segmental duplications of KRAB-ZFPs might influence human development or cause disorders and disease. Indeed, the KRAB-ZFP ZNF568 exist as three different alleles in humans, and a correlation between these alleles and the brain size of newborns was reported .According to a recent study, natural occurring nonsynonymous single nucleotide polymorphisms (SNPs) at “specificity residues” of human tandem-ZFPs are rare, indicating that ZNF mutations that change the binding specificity of tandem-ZFPs are rapidly removed from the population by negative selection . FurtherLittle is known about how a loss-of-function mutation of a single ERE-targeting KRAB-ZFP would affect the host organism. While reactivation of a replication-competent ERV or a high-copy retrotransposon could have immediate deleterious consequences for the host, de-repression of non-autonomous ERVs or low copy transposons might only have subtle effects. In support of this idea, the drastic upregulation of a small subset of non-autonomous VL30 elements in ZFP809 knockout mice did not seem to impair their health or fitness . NeverthInterestingly, it seems that SVA and SVA-related LAVA elements have expanded in some primates that lack ZNF91 , 194–196It is increasingly evident that transposable elements have a profound impact on mammals. Therefore, understanding the factors that keep these elements under control is of high importance for both basic and applied medical research. The recent evidence summarized in this review strongly supports the hypothesis that a large fraction of KRAB-ZFPs evolved to bind and possibly repress mobile parasitic DNA in mammals. However, since only a very small number of KRAB-ZFPs have been thoroughly investigated, the true spectrum of KRAB-ZFP functions cannot be anticipated yet. Without doubt, future research will yield exciting and unexpected insights into this enigmatic protein family.


The fear circuitry orchestrates defense mechanisms in response to environmental threats. This circuitry is evolutionarily crucial for survival, but its dysregulation is thought to play a major role in the pathophysiology of psychiatric conditions in humans. The amygdala is a key player in the processing of fear. This brain area is prominently modulated by the neurotransmitter serotonin . The 5-HT input to the amygdala has drawn particular interest because genetic and pharmacological alterations of the 5-HT transporter (5-HTT) affect amygdala activation in response to emotional stimuli. Nonetheless, the impact of 5-HT on fear processing remains poorly understood.The aim of this review is to elucidate the physiological role of 5-HT in fear learning via its action on the neuronal circuits of the amygdala. Since 5-HT release increases in the basolateral amygdala (BLA) during both fear memory acquisition and expression, we examine whether and how 5-HT neurons encode aversive stimuli and aversive cues. Next, we describe pharmacological and genetic alterations of 5-HT neurotransmission that, in both rodents and humans, lead to altered fear learning. To explore the mechanisms through which 5-HT could modulate conditioned fear, we focus on the rodent BLA. We propose that a circuit-based approach taking into account the localization of specific 5-HT receptors on neurochemically-defined neurons in the BLA may be essential to decipher the role of 5-HT in emotional behavior. In keeping with a 5-HT control of fear learning, we review electrophysiological data suggesting that 5-HT regulates synaptic plasticity, spike synchrony and theta oscillations in the BLA via actions on different subcellular compartments of principal neurons and distinct GABAergic interneuron populations. Finally, we discuss how recently developed optogenetic tools combined with electrophysiological recordings and behavior could progress the knowledge of the mechanisms underlying 5-HT modulation of fear learning via action on amygdala circuits. Such advancement could pave the way for a deeper understanding of 5-HT in emotional behavior in both health and disease. The amygdala, an almond-shaped structure in the medial temporal lobe, is thought to be critical for emotional processing , which arise from the midbrain raphe nuclei  session to elicit fear responses. Although 5-HT neurons have been hypothesized to encode aversive cues and to generate aversive prediction error signals  with an unconditioned stimulus . The Nonetheless, some DRN 5-HT neurons display phasic increases in firing rates at the onset of footshocks in anesthetized rats  gives rise to long (L) and short (S) allele variants. The S allele is associated with reduced transcriptional efficacy, and hence lower transporter expression, leading to reduced 5-HT re-uptake and high extracellular 5-HT levels  mice display higher extracellular 5-HT levels  5-HT neurotransmission in adulthood; or (2) altered neuronal circuit development; or both of these factors.Supporting the developmental account, several lines of evidence suggest that life-long changes in 5-HTT expression result in compensatory changes in 5-HT receptor expression and function in rodents. Namely, altered 5-HT1A  of cued conditioned fear. It remains to be confirmed whether the optogenetic activation of DRN 5-HT axons in the BLA could alter fear memory acquisition and expression, thereby demonstrating not only the necessity but also the sufficiency of BLA 5-HT. Undoubtedly, the link between 5-HT-dependent stress and fear learning also deserves further attention.To understand how BLA 5-HT neurotransmission influences fear learning, it is important to use circuit-based approaches and determine how 5-HT modulates BLA microcircuits. In the last decades, research in rodents has provided unprecedented knowledge about the neurobiological mechanisms underlying fear conditioning.A simple circuit model of fear learning  INs, which promote disinhibition of pyramidal cells dendrites  INs largely co-express calretinin (CR) in the BLA. In contrast, “large” CCK INs express CB but not VIP. In the HPC, CCK INs are a central target for 5-HT input and they express ionotropic 5-HT3 receptors  from thalamic afferents in LA PNs via suppression of glutamate release mediated by 5-HT2 receptors . Furthermore, the impact of this plasticity for learning still needs to be understood.A parallel action of 5-HT in the BA is depolarization of GABAergic INs via 5-HT2 and 5-HT3 receptors Rainnie, , resultiIt is not known whether 5-HT excites or inhibits SOM INs. The answer to this question is crucial, because SOM INs control the integration of excitatory inputs in PN dendrites and their inhibition promotes fear learning  occur in the BLA A positive modulation of fear-driven BLA theta oscillations by 5-HT is suggested by data from LFP recordings in 5-HTT mutant mice during fear conditioning. Namely, 5-HTTOE mice, which exhibit low extracellular 5-HT levels, show attenuated fear-evoked theta oscillations in the BLA during fear memory acquisition and expression Figure . ConversIn mPFC and HPC, PV INs can play an essential role in controlling theta oscillations  influence the acquisition (and likely the expression) of cued fear memories. Selective manipulation of BLA 5-HT neurotransmission with lesions and optogenetics demonstrate that 5-HT modulation of fear learning could be caused, at least in part, by actions on BLA neurons. Specifically, 5-HT could orchestrate the BLA microcircuitry in several ways. First, 5-HT could shape LTP induction at LA PN synapses and control the formation of PN ensembles encoding the CS. This could occur via alterations in glutamatergic transmission from sensory thalamus and cortex to LA PNs and via PN depolarization. Second, 5-HT facilitates LTP induction at BA PNs excitatory synapses from axons in the external capsule. Third, 5-HT could increase theta oscillations and BA PNs spike synchrony via recruitment of PV INs. These scenarios need to be validated with further empirical evidence. We propose that examining the effects of endogenous 5-HT release on discrete GABAergic IN classes and subcellular compartments of PNs is key to comprehend the complex effects of 5-HT on BLA circuits and fear learning. Notably, accumulating evidence suggests that the amygdala does not only encode aversion, but more generally affective significance  should be confirmed with reversible, spatially and temporally precise manipulations. This is because in these animal models 5-HT neurotransmission is altered during gestation, brain development and early-life experiences. Given the role of 5-HT in neuronal circuit formation  and a Wellcome Trust Senior Fellowship award (Grant No. 087736). TS is a recipient of collaborative research grants from Lundbeck. DMB is a member of Lilly UK’s Centre of Cognitive Neuroscience.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To investigate the efficacy of polymyxin-B direct hemoperfusion (PMX-DHP) in terms of improving clinical conditions and life prognosis in severe sepsis cases.The subjects were 88 cases admitted to our hospital ICU between April 2010 and July 2014 with the diagnosis of severe septic shock requiring continuous norepinephrine injection (Cathecholamine Index≧10). We investigated PMX-DHP efficacy by retrospectively comparing 63 cases that underwent PMX-DHP with 25 cases that did not.The two groups did not differ significantly in age, APACHE II score, SOFA score, infection focus, and bacterial species. The 28-day mortality was 32% in the PMX-DHP group and 40% in the non-PMX-DHP group, but this difference was not significant. The ⊿CAI (the change in Cathecholamine Index 24 hours after PMX-DHP administration) and the infusion volume 24 hours after PMX-DHP administration were significantly different between the two groups. Multivariate analysis identified ⊿CAI and SOFA score as independent prognostic factors. Sub-analysis showed no significant difference in 28-day mortality in sepsis cases involving the lower-gastrointestinal tracts, but a trend for improvement in 28-day mortality was observed in sepsis cases where the infection focus could not be removed, such as pneumonia, and in severe sepsis cases whose APACHE II score exceeded 25.PMX-DHP may improve life prognosis of septic shock cases under certain conditions. We have to clarify the indication criteria for PMX-DHP and accumulate data by additional investigations of severe septic cases to pursue the possibilities for further improvements in life prognosis.


Lynx rufus) population sizes, which motivated the development of a new "hybrid" mark-resight model as an alternative to traditional methods. We deployed a sampling grid of 30 cameras throughout the urban southern California study area. Additionally, we physically captured and marked a subset of the bobcat population with GPS telemetry collars. Since we could identify individual bobcats with photos of unique pelage patterns and a subset of the population was physically marked, we were able to use traditional mark-recapture and mark-resight methods, as well as the new “hybrid” mark-resight model we developed to estimate bobcat abundance. We recorded 109 bobcat photos during 4,669 camera nights and physically marked 27 bobcats with GPS telemetry collars. Abundance estimates produced by the traditional mark-recapture, traditional mark-resight, and “hybrid” mark-resight methods were similar, however precision differed depending on the models used. Traditional mark-recapture and mark-resight estimates were relatively imprecise with percent confidence interval lengths exceeding 100% of point estimates. Hybrid mark-resight models produced better precision with percent confidence intervals not exceeding 57%. The increased precision of the hybrid mark-resight method stems from utilizing the complete encounter histories of physically marked individuals (including those never detected by a camera trap) and the encounter histories of naturally marked individuals detected at camera traps. This new estimator may be particularly useful for estimating abundance of uniquely identifiable species that are difficult to sample using camera traps alone.Abundance estimation of carnivore populations is difficult and has prompted the use of non-invasive detection methods, such as remotely-triggered cameras, to collect data. To analyze photo data, studies focusing on carnivores with unique pelage patterns have utilized a mark-recapture framework and studies of carnivores without unique pelage patterns have used a mark-resight framework. We compared mark-resight and mark-recapture estimation methods to estimate bobcat ( Reliably estimating abundance of carnivores can be difficult because many populations exist in low densities and are wide-ranging, nocturnal, and secretive –3. ConsePanthera tigris) )s paper) . While mS1 AppendixThis file also contains Figure A and Figure B. Models include individual sighting heterogeneity  or no individual sighting heterogeneity .(DOCX)Click here for additional data file.S2 AppendixTable A, Examination of closure assumption for mark-recapture analysis using left-sided data. Table B, Examination of closure assumption for mark-recapture analysis using right-sided data.(DOCX)Click here for additional data file.S3 AppendixTable A, Individual model results for the mark-recapture analysis. Table B, Individual model results for the mark-resight analysis. Table C, Individual model results for the hybrid mark-resight analysis.(DOCX)Click here for additional data file.


It is particularly useful for detection of resistance mechanisms based on mutations in ribosomal RNA. Approaches based on MALDI-TOF-MS, alone or in combination with molecular techniques, like PCR/electrospray ionization MS or minisequencing provide the fastest resistance results from pure colonies or even primary samples with a growing number of protocols. This review details the various approaches of rapid resistance testing, their pros and cons, and their potential use for the diagnostic laboratory.Atypical and multidrug resistance, especially ESBL and carbapenemase expressing Enterobacteriaceae, is globally spreading. Therefore, it becomes increasingly difficult to achieve therapeutic success by calculated antibiotic therapy. Consequently, rapid antibiotic resistance testing is essential. Various molecular and mass spectrometry-based approaches have been introduced in diagnostic microbiology to speed up the providing of reliable resistance data. PCR- and sequencing-based approaches are the most expensive but the most frequently applied modes of testing, suitable for the detection of resistance genes even from primary material. Next generation sequencing, based either on assessment of allelic single nucleotide polymorphisms or on the detection of nonubiquitous resistance mechanisms might allow for sequence-based bacterial resistance testing comparable to viral resistance testing on the long term. Fluorescence  Pseudomonas aeruginosa and Acinetobacter baumannii) [ Staphylococcus aureus (laMRSA) [Generation of antimicrobial susceptibility patterns remains one of the most important tasks of clinical microbiology laboratories. The effective calculated antimicrobial therapy of infectious disease patients is consistently challenged by the rapidly rising prevalence of resistant and multidrug- or even pandrug-resistant pathogens worldwide. In recent years, this trend was accompanied by a shift from Gram-positive to Gram-negative bacteria like multidrug-resistant Enterobacteriaceae strains  as well as multidrug-resistant nonfermenters (umannii) –4. In paumannii) –8. Colonumannii) . ESBL coumannii) . Long laumannii) , 12. A mumannii) . An Englumannii) . Furtherumannii) . Howeverumannii) , while aumannii) . Farm an(laMRSA) . Mycobacterium tuberculosis has posed a serious health threat as a result of multidrug resistance. In its 2013 global report on tuberculosis, WHO estimates that 3.6%  of new cases and 20.2%  of previously treated cases had multidrug-resistant (MDR) tuberculosis (defined as tuberculosis caused by M. tuberculosis isolates that are resistant to rifampicin and isoniazid) and 1.3 million TB deaths [The multiresistances of the Gram-negative bacteria represent a major challenge for the traditional culture-based microbiology. Furthermore, the limited treatment options for a calculated therapy and therewith the risk of an inappropriate therapy are an intensifying factor of this problem . As a coB deaths , 22.Information on antimicrobial susceptibility aids a clinician in prescribing an appropriate antimicrobial drug for a particular infection. Due to the rapid rise in antimicrobial resistance worldwide , it is bIn response to the limitations of phenotypic methods and the desires to improve patient management and curb the spread of antimicrobial resistance, rapid antimicrobial susceptibility testing methods are continuously developed. These methods have been found to identify a pathogen and its antimicrobial susceptibility profile within a short period of time. There are basically five different ways to accelerate susceptibility testing in clinical diagnostics: (I) bypassing conventional culture by direct detection of the pathogen or resistance mechanism in the primary sample; (II) bypassing plate or broth culture dependent susceptibility testing (secondary culture); (III) avoiding time consuming work steps/methods; (IV) increasing the sensitivity to the detection of the infectious agent; that means detecting the infectious agent in earlier disease stages at lower viral or microbial loads; and (V) earlier detection of an evolving drug resistance during treatment in spreading less susceptible quasispecies.For example, real-time quantitative PCR (qPCR) has made it possible to detect multidrug-resistant tuberculosis (MDR TB) in a sample within an hour, hence immediately initiating appropriate treatment and control measures . Also, M in situ hybridization (FISH); and mass spectrometry-based methods, for example, phyloproteomics, assays using stable isotope labeling of amino acids, mass spectrometric beta-lactamase assays, PCR/electrospray ionization-mass spectrometry (PCR/ESI MS), minisequencing, and mass spectrometry-based comparative sequence analysis (MSCSA). In addition, we discuss the impact that these techniques are likely to bring for the patient management and the reduction of antimicrobial resistance.In this review, we detail the rapid antimicrobial susceptibility testing methods that have been developed recently. They include classical agglutination assays; molecular testing methods, for example, qPCR, DNA microarrays, Luminex xMAP assays, and next generation sequencing; fluorescenceAgglutination assays are based on a suspension of microparticles coated with specific antibodies, leading to agglutination in contact with their specific antigens. Such procedures are useful for a preliminary resistance screening from pure bacterial colonies if the resistance mechanism of interest is associated with a single antigen only, which is expressed on the surface of the pathogen. Accordingly, agglutination assays are unfeasible for the screening for complex resistance patterns, which may be associated with multiple structurally different families of enzymes as in the case of extended-spectrum beta-lactamases (ESBL) or carbapenemases in Gram-negative rod-shaped bacteria. Staphylococcus aureus (MRSA). Different agglutination kits show specificities of 91.3% to 100% if applied to MRSA colony material [Agglutination assays for the rapid identification of bacterial resistance patterns are widely restricted to the identification of the penicillin binding protein 2a (PBP-2a), the major resistance determinant of Methicillin resistantmaterial –32. The material –32. If smaterial .SCC-mecA) types with type IV scoring particularly poor [ mecA gene. If mecC, a divergent mecA homologue, is the cause of the resistance, agglutination usually fails as observed for 10 out of 10 mecC-positive, live-stock associated MRSA strains [The lack of sensitivity seems to be associated with certain staphylococcal cassette chromosome  nucleic acid amplification methods, particularly real-time quantitative PCR (qPCR); (ii) DNA hybridization based methods, particularly DNA microarrays; (iii) Luminex xMAP technology; and (iv) next generation sequencing methods. Below is a brief description on the application of each of these molecular methods for the rapid detection of antimicrobial resistance. mecA and mecC, which confer methicillin resistance in S. aureus; the vanA and vanB genes, which confer glycopeptide resistance; and genes that encode extended-spectrum β-lactamases  and New Delhi metallo-β-lactamase (blaNDM) in Gram-negative rod-shaped bacteria [OXA-48, blaVIM, and blaIMP carbapenemase genes in Enterobacteriaceae have been established [Recently, one of the PCR techniques that has received a wide application in clinical microbiology is the quantitative real-time PCR (qPCR) technique . This hain, 2012 ). One oue to INH . This asbacteria –43 have ablished –46.CTX-M beta-lactamases and only partially ESBL-associated blaTEM and blaSHV as well as OXA1-type carbapenemases (the latter combined in a consensus run) were introduced  [PCR-based MRSA testing has found wide applications in microbiological routine laboratories. Next to in-house assays, commercially available molecular MRSA testing platforms comprise, for example, BD GeneOhm MRSA , GT MRSA Direct/GQ MRSA , Hyplex StaphyloResist , LightCycler  kits like LC MRSA Advanced, Cepheid Xpert/Gene Expert , and TIB Molbiol LightMix MRSA . All test systems showed reliable results in a recent external laboratory control evaluation in Germany . SimilarGermany) . SimilarGermany) , 50. TheGermany)  allows f M. tuberculosis and rifampicin (USAN: rifampin) resistance. To evaluate its usefulness in POCT, a large multicentre study involving 6069 cases from six unrelated sites was performed. In this study, Xpert MTB/RIF detected rifampicin resistance cases in 1 hour as compared to line-probe assay and phenotypic drug susceptibility testing that detected the same cases in 20 days and 106 days, respectively [Nevertheless, in addition to rapid and simultaneous providing of reliable results, qPCR has been found to be affordable, sensitive, specific, user friendly, not space demanding, and deliverable –39, 51. ectively . As ment S. aureus directly from clinical specimen within 6 hours [ mecA (methicillin resistance), aacA-aphD (aminoglycoside resistance), tetK, tetM (tetracycline resistance), ermA, ermC (macrolide-lincosamide-streptogramin B resistance), vatA, vatB, and vatC (streptogramin A resistance) [Multiplex PCR assays have also been developed to rapidly and simultaneously identify multiple pathogens in clinical specimens as well as the presence of antimicrobial resistance genes in the identified pathogens. Strommenger and coworkers developed a multiplex PCR, which simultaneously detects 9 resistance genes in 6 hours . These 9istance) . Pneumocystis jirovecii and 22 resistance markers from respiratory specimens (http://www.curetis.com/). In one of the studies showing its suitability for POC testing, the UPA assay detected multiple antibiotic resistances within 1 hour (as compared to phenotypic methods that took 96 hours) in a group of 56 hospitalized patients with respiratory tract infections who were under treatment. This finding influenced the modification of treatment in fifteen patients with severe pneumonia leading to their recovery [Like qPCR, multiplex PCR assays were used as POC tests to facilitate patient management. One example is the multiplex PCR-based Unyvero Pneumonia Application (UPA) assay  that has been developed to rapidly and simultaneously detect 18 bacterial species,recovery . The UPAThe continuous development of PCR-based assays with the capability to rapidly and simultaneously detect pathogens and presence of resistance genes in specimens coupled with their application in POCT may further improve the management of patients as long as appropriate quality control is ensured. Mycobacterium species and detect mutations that confer isoniazid (INH) and rifampicin (RMP) resistance in specimens collected from spinal tuberculosis patients as compared to conventional culture and drug susceptibility testing which took a mean time of 56.8 days [ Mycobacterium species based on 16S rRNA sequences and mutations of rpoB, inhA, and katG that confer INH and RMP resistance, were covalently linked to the surface of aldehyde-activated slides. DNA was extracted from specimens. PCR was used to amplify the resistance genes and amplicons hybridized on the slides. The emitted fluorescent signals were analyzed. Guo and coworkers evaluated the ability of a biochip, which is based on the same principle to rapidly and simultaneously identify multidrug-resistant M. tuberculosis (MRTB) and mutations of rpoB, inhA, and katG that confer INH and RMP resistance in clinical sputum specimens [ M. tuberculosis and detect mutations that confer INH and RMP resistance.The biggest challenge associated with the unprecedented rise of antimicrobial drug resistance worldwide is the scarce availability of assays that are able to rapidly and simultaneously identify a causative pathogen and generate its antimicrobial resistance profile. Recent oligonucleotide-based DNA microarrays match this challenge. In a recent study, Zhang and coworkers described that CapitalBio DNA microarray  could in a mean time of 5.8 hours simultaneously identify6.8 days . Brieflyβ-lactamases (ESBLs) and Klebsiella pneumoniae carbapenemases (KPC carbapenemases) [TEM, blaSHV, and blaCTX-M) and blaKPC genes  and extended-spectrum β-lactamase- (ESBL-) related gene families  has been developed. The evaluation of the rapidness of the Check-MDR CT 102 DNA microarray to detect these genes has shown that it yields results 5 hours faster than Check-Point's ESBL/KPC DNA microarray [Recent reports have also reported the availability of Check-Point's ESBL/KPC DNA microarray for the identification and detection of extended-spectrum nemases) , 58. Thiails see ). In a seumoniae . Based ocroarray . nuc, mecA, (SCCmec)-orfX, vanA, vanB, ddl, and tuf genes to screen for MRSA in nasal swabs [nuc versus tuf) and detection of mecA, aacA, ermA, ermC, tetM, and tetK as well as Panton-Valentine leukocidin (PVL) for the rapid detection and characterization of staphylococci directly from positive blood culture bottles [At present, the DNA microarray technology is mostly used in the routine detection of antimicrobial resistance of TB and HIV –66. The al swabs , and the bottles . gyrA, gyrB, and parE within Salmonella typhi [ gyrA within Campylobacter jejuni and Campylobacter coli [The description of cooccurring single nucleotide polymorphism (SNP) mutations in antimicrobial resistance associated genes allows for targeted resistance testing. For example, unequivocally genetic studies have proven that there are 5 different mutations in quinolone resistance-determining region (QRDR) ofla typhi . Similarter coli . gyrA, gyrB, and parE of Salmonella Typhi and Salmonella Paratyphi A [ gyrA of C. jejuni and C. coli [Rapid simultaneous detection of cooccurring single nucleotide polymorphism (SNP) mutations in antimicrobial resistance associated genes remains, however, challenging. Most molecular assays such as qPCR and pyrosequencing lack the capability to simultaneously detect cooccurring single nucleotide polymorphism (SNP) mutations in different genes in a given specimen . Howeveratyphi A . Further C. coli . In comp C. coli –76.Near whole genome sequencing (WGS) or next generation sequencing (NGS) allows for the assessment of bacterial genomes within several hours. A variety of different technological solutions have been introduced, including laser printer sized benchtop devices like 454 GS Junior , MiSeq , and Ion Torrent PGM . In a previous analysis, the MiSeq (Illumina) system scored best regarding both throughput per run and error rates, while both the 454 GS Junior (Roche) and the Ion Torrent PGM (Life Technologies) systems were prone to homopolymer-associated indel errors .Result interpretation of whole bacterial genomes is based on either allelic comparisons  or singlβ-lactamases , plasmid-mediated AmpCs , quinolone resistance , aminoglycoside resistance , or carbapenemases  [NGS allows for resistance identification by the presence of the underlying mechanism rather than just in pharmacodynamic terms , so it mblaNDM) . M. tuberculosis, for which rapid identification or exclusion of resistance determinants is of high relevance for the therapeutic approach. Ion Torrent full-gene sequencing with consecutive complete genetic analysis within 5 days  or extensive-drug resistance (XDR)n 5 days  allowed  the USA . Similar the USA . In a diand pncA . Furtherand pncA . So it m E. coli isolates expressing an ESBL phenotype and transferring their cefotaxime resistance marker at high frequency in laboratory conjugation experiments [However, NGS-based resistance testing is not restricted to mycobacteria. Recently, NGS was used to identify transmissible plasmids in multidrug-resistanteriments . High-theriments . Howevereriments .Current obstacles to a routine use of NGS technologies in diagnostic microbiology and resistance testing comprise costs and scarcely available user-friendly bioinformatics platforms . Neverth in situ hybridization) is a cheap and convenient option for the identification and resistance testing of bacterial pathogens. Traditional FISH is based on specific hybridization of short, usually 18–25 bases long, fluorescent-labelled, single-stranded oligonucleotide probes to ribosomal RNA (rRNA) of the target organism with subsequent analysis under the fluorescence microscope, usually allowing for the identification of microbes at genus or species level. In principle, each kind of intracellular RNA can be hybridized with FISH probes. However, rRNA is particularly well suited as a FISH target, because ribosomes are numerous in a protein-synthesizing cell, thus allowing for a boostering of fluorescence intensity [FISH  probes or probes containing locked nucleic acids (LNA) instead of simple single-stranded DNA probes. PNA-FISH technology reduces nonspecific probe attachment due to the electrically neutral backbone of the oligonucleotides and is recommendable for routine diagnostics due to a higher degree of standardization. However, patent-protected PNA probes are expensive, although they are well suited for the diagnostic routine setting .FISH is particularly suitable for the detection of resistance determinants if two prerequisites are guaranteed. Ribosomally mediated resistance, for example, affecting antibiotic drugs like macrolide or linezolid, is well suited, because ribosomal RNA copies are numerous in living cells, allowing for bright fluorescence signals. Further, FISH can be successfully applied if only one or few variable bases provide resistance, so there is no need for a large number of probes in the probe panel. Helicobacter pylori. Therefore, FISH-based resistance testing was early evaluated for this indication [ H. pylori is basically mediated by three point mutations in the ribosomal 23S rRNA [These prerequisites are fulfilled in case of clarithromycin resistance testing indication . Clarith23S rRNA  which ca23S rRNA  , is an example of a non-rRNA-based FISH protocol for detecting a particular resistance determinant using the probe 5′-GAC-CGG-AGC-TAG-CAA-GCG-3′ [First successful attempts of FISH-based resistance testing were described for non-rRNA-based resistance mechanisms as well. FISH-based detection of blaA-GCG-3′ . However in situ amplification of the respective gene as in case of the rolling circle amplification (RCA) FISH [ mecA gene in Methicillin resistant Staphylococcus aureus (MRSA) based on the mecA-probes MR-1 5′-AAG-GAG-GAT-ATT-GAT-GAA-AAA-GA-3′ and MR-2 5′-GGA-AGA-AAA-ATA-TTA-TTT-CCA-AAG-AAA-A-3′ [Further progression of FISH technology comprises signal-amplified, catalyzed reported deposition (CARD) FISH; doubly labeled oligonucleotide probe- (DOPE-) based FISH; combinatorial labelling and spectral imaging (CLASI) FISH; and the combination of FISH with other diagnostic approaches as well as FISH procedures for gene identification, requiringCA) FISH . RCA-FISAAA-A-3′ .FISH-based detection of resistance determinants is a promising diagnostic approach due to its rapidity, convenience, and cost effectiveness. The associated rapid detection of antimicrobial resistance may lead to early resistance-adapted optimization of antimicrobial therapy with associated benefits for the patient's health. The main advantage of FISH is its potential use for resistance testing directly from primary material including tissue with low effort. So FISH can also be applied in resource-limited settings where expensive technologies are not available . In contHowever, so far, FISH is restricted to very few indications for which protocols have been described. As a further drawback, standardization of FISH-based resistance testing is widely missing. If applied from primary sample materials like tissue, tissue autofluorescence has to be considered, requiring considerable experience to interpret such diagnostic results. To reduce potential interpretation errors, FISH from tissue further requires counterstaining with a pan-eubacterial FISH probe and nonspecific DNA staining, for example, with DAPI , to confirm the presence of nucleic acids of the detected pathogens as recently demanded .Given all these limitations, FISH for resistance testing will presumably stay a bridging technology until amplification-based technologies will be available as easy-to-apply and cost-efficient benchtop systems on the market.β-lactamases [β-lactamases in a proof-of-principle analysis [Nonnucleotide probes labelled with reporter and quencher molecules, allowing for fluorescence energy transfer (FRET), can be used to detect enzymatic resistance mechanisms as described for ctamases . After eanalysis  and broaMatrix-assisted laser desorption ionization time-of-flight mass spectrometry- (MALDI-TOF MS-) based intact cell mass spectrometry (ICMS) has recently advanced to the standard method for species identification for cultured bacteria and fungi , 110–114 Salmonella enterica ssp. enterica serovar Typhi from other less virulent Salmonella enterica ssp. enterica serotypes [ Campylobacter jejuni MLST-ST22 and ST45 from other MLST sequence types [ Rhodococcus erythropolis [ Pseudomonas putida [ Neisseria menigitidis [MALDI-TOF MS-based intact cell mass spectrometry (ICMS) is potentially able to characterize strains at the subspecies level and could act as useful tool for taxonomy and epidemiology , 138. Foerotypes , to distce types  or to pehropolis , Pseudoms putida , or Neisigitidis . Staphylococcus aureus (MSSA) from methicillin resistant Staphylococcus aureus (MRSA) [ S. aureus (VISA) strains and vancomycin-susceptible S. aureus strains, as well as between SCCmec types IV and V isolates and SCCmec types I–III isolates [ S. aureus lacking or artificially harboring SCCmec could not be distinguished in a mass range from 2000 to 15000 m/z [The first approaches to associate MS fingerprints with susceptibility patterns were designed to differentiate methicillin susceptibles (MRSA) –148. Thes (MRSA) , 150. A isolates . Further5000 m/z , whereas5000 m/z . vanB positive vancomycin-resistant Enterococcus faecium (VRE) and vancomycin-susceptible E. faecium using ICMS fingerprinting is feasible [One study from New Zealand showed that the discrimination offeasible , but thefeasible . Clostridium difficile demonstrated a sufficient discriminatory power of MALDI-TOF MS spectra analysis to recognize the PCR ribotypes 001, 027, and 126/078 [Other studies on 126/078 . Phylopr Klebsiella pneumoniae ST258 (expressing KPC carbapenemase) and E. coli ST131, ST69, ST405, and ST393 (expressing ESBL) [Compared to Gram-positive bacteria, Gram-negative bacteria are particularly problematic because their resistance genes are often encoded on plasmids, which can be easily exchanged with other Gram-negative bacteria even across species boundaries . But somng ESBL)  belong t E. coli strains [ E. coli B2 ST131 and D  from other E. coli strains [Similar phyloproteomic analysis has been successfully demonstrated to discriminate between different subsets of strains . Couplin strains , 118. Bacteroides fragilis infections is the possibility that some strains express a high-potential metallo-β-lactamase encoded by the gene cfiA [ B. fragilis is subdivided into two divisions (I and II) and usually only isolates of division II harbor cfiA. Recently, two independent studies identified a set of biomarkers or precisely shifts in biomarker masses that help to distinguish both divisions using MALDI-TOF MS coupled with a cluster algorithm [One likely problem in the calculated treatment ofene cfiA . The miclgorithm , 120. Candida and Aspergillus species. They also comprise important reserve antimicrobial agents especially in the case of infections with azole-resistant strains, for example, Aspergillus species. Due to the increasing use of echinocandins in the treatment of fungal infections, the prevalence of echinocandin-resistant isolates caused by mutations in the fks1-3 (hypersensitive for the immunosuppressant FK560) genes increases [Echinocandins, namely, anidulafungin, caspofungin, and micafungin, are the treatment of choice for invasive and systemic infections withncreases . Thus, r Candida albicans was shown by Marinach and coworkers [ Candida cells were incubated for 24 hours in liquid medium containing different concentrations of fluconazole. After harvesting and acid extraction of the Candida cell pellets, the supernatants were spotted on a MALDI-TOF target plate and mass spectra were recorded. Comparable to the estimation of minimal inhibitory concentrations (MIC), the so-called minimal profile changing concentration (MPCC), the lowest concentration of fluconazole at which changes in the mass spectrum were recordable, was estimated by comparing the mass spectra of the particular suspensions of the fluconazole dilution series. Remarkably, MPCC differed only in one dilution step from the MIC and therewith it is a comparably sufficient parameter reflecting antimicrobial susceptibility [In a pioneer study, the feasibility of MALDI-TOF MS-based testing to estimate fluconazole susceptibility ofoworkers . During tibility . C. albicans, Candida glabrata, Candida parapsilosis, Candida krusei, Aspergillus fumigatus, and Aspergillus flavus for echinocandin MICs that are due to mutations in fks1 and, in the case of C. glabrata, also in fks2 [de Carolis and coworkers adapted this procedure to test in fks2 . Additio in fks2 .This procedure was further optimized by Vella and coworkers . They re S. aureus (MSSA) and methicillin resistant S. aureus (MRSA) [ P. aeruginosa to three antibiotics of different classes with different modes of action: meropenem (β-lactam antibiotic), tobramycin (aminoglycoside), and ciprofloxacin (fluoroquinolone) [The successful application of mass spectrometry (MS) in the detection of antimicrobial resistance has also opened a door for the entry of another quantitative proteomics approach known as SILAC into the era of rapid detection of antibiotic resistance. This approach is based on the principle that proteins are made up of amino acids. Hence, cells grown in media supplemented with amino acids incorporate these amino acids into their cellular proteome . In addis (MRSA) . Also, iinolone) . In bothβ-lactamase assay (MSBL) is not based on the analysis of the bacterial proteome. The MSBL is based on the direct mass spectrometric detection of β-lactamase metabolites [β-lactam antibiotic. This suspension is incubated for 1 to 3 hours. After centrifugation, the supernatants are analyzed by MALDI-TOF MS. Specific peaks (mass shifts) for intact and hydrolyzed β-lactams indicate functional presence of β-lactamases. It was demonstrated that the MSBL delivers results within 2.5 hours for bacteria inactivating ampicillin, piperacillin, cefotaxime, ceftazidime, ertapenem, imipenem, and meropenem [ Acinetobacter baumannii, and Pseudomonas spp. was detectable [In contrast to the aforementioned mass spectrometric assays, the mass spectrometric abolites –131. Thetectable , 130.With a total turn-around-time after positive primary bacterial culture of circa 4 hours, this method is significantly faster than culture-based susceptibility testing –131.KPC-2-10) [ gyrA and parC point mutations, which are associated with quinolone resistance in A. baumannii [PCR/electrospray ionization-mass spectrometry (PCR/ESI MS) combines, nucleic acid amplification with mass spectrometric analysis of the amplicons, which are brought into a gas phase using electrospray ionization. The major advantage of this technique is its high multiplexing capacity that enables the parallel detection of a wide panel of resistance genes. It was demonstrated that PCR/ESI MS is able to accurately detect nine different KPC carbapenemases (blaPC-2-10)  as well aumannii . M. tuberculosis; on the other hand, multidrug-resistant tuberculosis (MDR-TB) strains must be detected. PCR/ESI MS-based assays have been developed to facilitate NTM species identification and parallel detection of resistance genes associated with rifampicin, isoniazid, ethambutol, and fluoroquinolone resistance in TB and NTM [Also because of its high multiplexing capacity, PCR/ESI MS is a suitable tool for simultaneous (sub)species identification and resistance gene detection, which is of particular importance for the treatment of mycobacterial infections. On the one hand, it is necessary to distinguish nontuberculosis mycobacteria (NTM) from and NTM . Moreove and NTM –164.The high sensitivity of PCR/ESI MS in the detection of hard-to-culture or even nonculturable bacteria makes it a reliable method for the direct detection of pathogens in hardly acquirable samples like heart valves  as well Another method that was also adapted for the rapid detection of ganciclovir resistance in HCMV  by Zürcher and coworkers is single nucleotide primer extension  followed by matrix-assisted laser desorption/ionization time-of-flight analysis (PEX/MALDI-TOF) . In geneFor the primer extension reaction, the reverse PEX primer (5′-CTT-GCC-GTT-CTC-CAA-C-3′) was added in high concentration. The 3′-end of the primer is located directly at the site of mutation  to be detected. The extension reaction catalyzed by a DNA polymerase is terminated in the case of a wild-type allele just after one nucleotide complementary to the mutated nucleotide and in the case of a mutant after two nucleotides by a didesoxynucleotide (ddNTP). Because of the molecular weight difference in consequence of the varying mass increase of the PEX primer, mutant and wild type can be discriminated using MALDI-TOF MS .According to current standards, HCMV resistance testing is performed using Sanger sequencing . By moniblaTEM genes, a set of seven internal primers have been designed to bind near the three codons of Ambler's positions in such a way that the masses of all possible reactions products are maximally distant from each other and are easy to distinguish in the mass spectrum. All primers are used in one multiplex reaction. Thus it is feasible to detect different types of TEM-type ESBL in one reaction [A comparable test setup was designed to detect TEM-type ESBL in Enterobacteriaceae . Conversreaction . N. gonorrhoeae [ Helicobacter pylori [ M. tuberculosis [Other minisequencing protocols have been established to detect fluoroquinolone resistance related SNPs inorrhoeae , clarithr pylori , and rifrculosis .Mass spectrometry-based comparative sequence analysis (MSCSA) was initially established by Honisch and coworkers  for the genotyping of bacteria using mass spectrometric fingerprinting of the standard multilocus sequence typing (MLST) loci .The MSCSA principle was adapted to facilitate the detection of mutations in the UL97 gene to detect ganciclovir resistance of human cytomegalovirus (HCMV) .HCMV reactivation occurs frequently in consequence of immune suppression especially after stem cell and solid organ transplantation . Thus, H in vitro transcribed in two separate reactions using T7 and SP6 RNA polymerase followed by cytosine or uracil specific RNaseA cleavage of plus and minus strand RNA transcripts. After this, all four obtained RNaseA cleavage products are transferred to a SpectroCHIP array . MALDI-TOF mass spectra are recorded and in silico compared to calculated MS spectra of reference sequences. Based on the obtained data, the UL97 sequence can be assembled and thereby the presence of a ganciclovir resistance associated single nucleotide polymorphism can be detected [These UL97 single nucleotide polymorphisms are detected by MSCSA as follows: after DNA isolation from EDTA-plasma samples, the 3′-region of the UL97 is amplified in two amplicons using T7-promotor-tagged forward primers and SP6-tagged reverse primers. Both amplicons aredetected . Due to  S. aureus (VRSA) as well as vancomycin-resistant enterococci (VRE) means the detection of Van-A, Van-B, and rarely Van-C [To solve the increasing problem of a worldwide rising prevalence of infections due to multidrug- or even pan-drug-resistant bacteria, medical microbiology has to establish a new generation of rapid resistance testing assays. The key features of these new assays should be significant reduction of turn-around-time  and a hily Van-C .β-lactamases (ESBLs), carbapenemases, aminoglycoside-blocking 16S rRNA methylases, and many other mechanisms associated with several hundreds of gene variants/mutations [In contrast to this situation in Gram-positive bacteria, multidrug resistance in Gram-negative bacteria is due to the expression of extended-spectrum utations –8. The mBut rapid resistance testing is only one key to the solution of this problem, especially because the multiplexing capacities of the individual assays are limited and the costs are too high. Thus, resistance surveillance programs are and have been established at different levels: hospital-wide, regional, and international. For example, some hospitals introduced a general ESBL screening in analogy to the MRSA screening in high-risk groups. In recent years, various studies were carried out to identify the ESBL-transmission rate in maximum care hospitals and in households with ESBL-colonized individuals. The studies showed that the ESBL-transmission rate of 1.5% to 4.5% is relatively low if compliance with standard hygiene measures is guaranteed , 181. InAs recently predicted, next generation sequencing (NGS) with its high multiplexing capacity will soon be part of routine diagnostics, more and more replacing cultural approaches as an accurate and cheap procedure in routine clinical microbiology practice. This will include sequence-based resistance testing and additional detection of particular virulence factors, making culture unnecessary on the intermediate or long term . The genβ-lactamase assay (MSBL) as well as adaptations to other anti-microbiota classes will expectantly advance to helpful tools of the diagnostic microbiologist. Finally, the combination of both nucleic acid amplification and mass spectrometric analysis, for example, in PCR/ESI MS assays with its high multiplexing capacity, has the potential to enter routine diagnostic in the coming years.Besides NGS, mass spectrometry will be the second key technique in rapid medical microbiology. The integration of subtype specific mass spectra databases in MS associated software packages will enable the identification of high-virulent strains within very short time periods. The mass spectrometric   in situ hybridization (FISH) might be an option for such settings [Nevertheless, these highly sophisticated and expensive diagnostic solutions will hardly be available in resource-limited countries, for example, in the sub-Saharan tropics, where multidrug resistance is nevertheless on the rise . Cheap asettings  until MAsettings , 189. In


G-quadruplexes are four-stranded structures formed in guanine-rich nucleotide sequences. Several functional roles of DNA G-quadruplexes have so far been investigated, where their putative functional roles during DNA replication and transcription have been suggested. A necessary condition for G-quadruplex formation is the presence of four regions of tandem guanines called G-runs and three nucleotide subsequences called loops that connect G-runs. A simple computational way to detect potential G-quadruplex regions in a given genomic sequence is pattern matching with regular expression. Although many putative G-quadruplex motifs can be found in most genomes by the regular expression-based approach, the majority of these sequences are unlikely to form G-quadruplexes because they are unstable as compared with canonical double helix structures.Here we present elaborate computational models for representing DNA G-quadruplex motifs using hidden Markov models (HMMs). Use of HMMs enables us to evaluate G-quadruplex motifs quantitatively by a probabilistic measure. In addition, the parameters of HMMs can be trained by using experimentally verified data. Computational experiments in discriminating between positive and negative G-quadruplex sequences as well as reducing putative G-quadruplexes in the human genome were carried out, indicating that HMM-based models can discern bona fide G-quadruplex structures well and one of them has the possibility of reducing false positive G-quadruplexes predicted by existing regular expression-based methods. Furthermore, our results show that one of our models can be specialized to detect G-quadruplex sequences whose functional roles are expected to be involved in DNA transcription.http://tcs.cira.kyoto-u.ac.jp/~ykato/program/g4hmm/.The HMM-based method along with the conventional pattern matching approach can contribute to reducing costly and laborious wet-lab experiments to perform functional analysis on a given set of potential G-quadruplexes of interest. The C++ and Perl programs are available at  G-quadruplex (G4), Z-DNA, cruciform and triplex. Recent advances in providing in vitro evidence of these specific structures develop the hypothesis that these structures are considered to have some functional roles in living cells


In particular, the characteristic increase in cardiac arrhythmias with age is prevented in ilk and mys heterozygous flies with nearly identical genetic background, and they live longer, in line with previous findings in Caenorhabditis elegans for ilk and in Drosophila for mys. Consistent with these findings, we observed elevated β1-integrin protein levels in old compared with young wild-type flies, and cardiac-specific overexpression of mys in young flies causes aging-like heart dysfunction. Moreover, moderate cardiac-specific knockdown of integrin-linked kinase (ILK)/integrin pathway-associated genes also prevented the decline in cardiac performance with age. In contrast, strong cardiac knockdown of ilk or ILK-associated genes can severely compromise cardiac integrity, including cardiomyocyte adhesion and overall heart function. These data suggest that ilk/mys function is necessary for establishing and maintaining normal heart structure and function, and appropriate fine-tuning of this pathway can retard the age-dependent decline in cardiac performance and extend lifespan. Thus, ILK/integrin-associated signaling emerges as an important and conserved genetic mechanism in longevity, and as a new means to improve age-dependent cardiac performance, in addition to its vital role in maintaining cardiac integrity.Cardiac performance decreases with age, which is a major risk factor for cardiovascular disease and mortality in the aging human population, but the molecular mechanisms underlying cardiac aging are still poorly understood. Investigating the role of integrin-linked kinase ( It is well known that the heart undergoes many age-related functional and structural changes  model system with a heart  extends lifespan in C. elegans. Interestingly, heterozygous Drosophila mutants for β1-integrin (mys) also have an increased lifespan . Their activation affects cytoskeletal remodeling and other intracellular signaling pathways  show extended lifespan compared with their wCS controls  have embryonic muscle phenotypes similar to ilk mutants  flies age, the heart rhythm becomes progressively less regular, as exemplified by increases in AI  and myocardial stiffening.As is observed in human cardiac aging , but rather the cardiac aging phenotype observed in wild-type flies  and the weaker cardiomyocyte-specific GMH5 , which was visualized by GFP-tagged myosin heavy chain A/MYO-3  significantly extends lifespan, similar to ilk KD  and comparable to long-lived ilk and mys heterozygotes , and talin . All three heterozygotes failed to show an increase in AI or decrease in diastolic diameter at old ages , ILK-GFP;ilk54 : UAS-parvinRNAi (#11670), UAS-pinchRNAi (#52538), UAS-mysRNAi (#29619), UAS-talinRNAi (#40399) and UAS-paxillinRNAi (#107789). UAS-ilkRNAi  was kindly given by F. Schnorrer. The effectiveness of ilk, mys, parvin, and talin RNAi lines had been tested in Schnorrer et al.  and then on larger scale (200–250 flies). Data were analyzed using Prism 5.0 .C. elegans strain RW1596: myo-3 (st386)V; stEx30 [myo-3p::gfp::myo-3 + rol-6]  method.The et al., et al., Flies were anesthetized with fly nap  and dissected as previously described -treated hemolymph to arrest contraction. Prior to indentation, probes were calibrated via thermal noise method in MFP-3D Bio software (Asylum Research). Nanoindentation was performed on an MFP-3D Bio Atomic Force Microscope (Asylum Research) mounted on a Ti-U fluorescent inverted microscope  with 120 pN nm−1 silicon nitride cantilevers with premounted, 2-μm radius borosilicate glass spheres . All indentation curves were analyzed to calculate myocardial stiffness using previously published, automated software custom-written in MATLAB . All data were checked prior to analysis using the D’Agostino & Pearson omnibus normality test to determine whether the data violated the assumption of a Gaussian distribution. We employed a one-way analysis of variance (ANOVA) when comparing more than two groups (e.g., Fig.


The established interaction between multiple myeloma cells and bone marrow microenvironment components provides malignant cells with various survival, growth and drug resistance signals. As a new concept, identification of miRNAs and their related gene/protein targets, signaling molecules and pathways in the context of bone marrow microenvironment will help understanding more deeply the pathogenesis of the disease and possible mechanisms underlying environment-induced drug resistance. Recent studies suggest that bone marrow stromal cells can modulate some miRNAs  in multiple myeloma cells through direct adhesion, cytokine secretion or transfer of miRNA-containing exosomes, however; the specific miRNA targets are not clear. In spite of a remarkable progress in understanding myeloma biology and therapy, the disease persists to be hard to treat. This review will discuss the most recent findings on miRNAs expression and function in the context of bone marrow microenvironment highlighting the miRNAs as potential therapeutic targets in multiple myeloma. MYC, TP53, PTEN) and have proved to significantly influence MM tumor in vitro and in vivo[To survive and expand, multiple myeloma (MM) tumor cells have to be in a dynamic interaction with their stromal environment ,2. The pd in vivo-12. HowemiRs belong to a class of small non-coding RNAs (22–25 nt. in length) which repress the expression of their target genes at translational level by binding to 3′UTR through partial sequence complementarity . Recent It is now understood that miRs mediate substantial functions in tumor microenvironment  or lymph node stroma (HK cell line) ,26. UsinIn a more recent study, Emanuela et al. also demonstrated that HMCLs with low miR-21 expression (INA-6) displayed a high expression of miR-21 when adhered to BMSCs. miR-21 inhibition significantly decreased viability and clonogenic growth of MM cells but in stroma-free conditions . Of noteRoccaro et al. reported a miR signature in MM cells indicating downregulation of miR-15a/16-1 . SpecifiDLEU2, a regulatory gene which is frequently deleted in CLL leading to repression of miR-15-a/16  [Later on, Hao et al. also observed that BMSCs maintained survival of MM cells and protected them from bortezomib-induced apoptosis through suppressing miR-15a in MM cells . Of noteoptosis) . It remaoptosis) . Furtheroptosis)  or osteooptosis) ,34  might play role in stroma-mediated drug resistance of MM cells but the specific targets and the underlying mechanisms are not clear yet. Moreover, the possibility of involvement of other miRs, their potential targets, and involved signaling pathways, warrants more in-depth research. As a matter of fact, reinforcing our knowledge of miRs expression and function in MM cell-BMME interaction will help us to find potential drug targets to overcome CAM-DR, which has been suggested to build the intrinsic (de novo) drug resistance and contribute to acquired drug resistance over time in MM patients [Research on biology and function of miRs tends to achieve a hot spot in the field of MM therapy, with some evidence to introduce miRs as promising therapeutic targets in MM . Howeverpatients -41.Akt: Also known as protein kinase B is a serine/threonine-specific protein kinase; BMSC: Bone marrow stromal cell; BTG: B cell translocation gene; CAM-DR: Cell adhesion-mediated drug resistance; ICAM-1: Intercellular cell adhesion molecule-1; JAK/STAT: Janus kinase/Signal transducer and activator of transcription; MAPK: mitogen activated protein kinase; MDM2: Minute double minute 2; mTOR: Mammalian target of Rapamycin; NFκB: Nuclear factor kappa-light-chain-enhancer of activated B cells; PI3K: Phosphatidylinositol-4,5-bisphosphate 3-kinase; PTEN: Phosphatase and tensin homolog; Ras: A member of small GTPase family; RhoB: Ras homolog gene family member B; VCAM-1: Vascular cell adhesion molecule-1; VLA4: Very late antigen 4.The authors declare that they have no competing interest.JA participated in the design and drafted the review, LQ provided critical revision, HC designed, supervised the study, participated in drafting the review and provided critical revision; All authors read and approved the final manuscript.


MiR-302-367 is a cluster of polycistronic microRNAs that are exclusively expressed in embryonic stem (ES) cells. The miR-302-367 promoter is functional duringembryonic development but is turned off in later stages. Motivated by the cancer stemcell hypothesis, we explored the potential expression of miR-302 in brain tumor cell lines. In the present experimental study, we have tried to expandour knowledge on the expression pattern and functionality of miR302 cluster by quantifying its expression in a series of glioma  and medulloblastoma (DAOY) cell lines. To further assess the functionality of miR-302 in these celllines, we cloned its promoter core region upstream of the enhanced green fluorescentprotein (EGFP) or luciferase encoding genes. Our data demonstrated a very low expression of miR-302 in glioma cell lines,compared with that of embryonal carcinoma cell line NT2 being used as a positivecontrol. The expression of miR-302 promoter-EGFP construct in the aforementionedcell lines demonstrated GFP expression in a rare subpopulation of the cells. Serumdeprivation led to the generation of tumorospheres, enrichment of miR-302 positivecells and upregulation of a number of pluripotency genes.Taken together, our data suggest that miR-302 could potentially be used asa novel putative cancer stem cell marker to identify and target cancer stem cells withintumor tissues. Based on the cancer stem cell (CSC) hypothesis,tumors arise from a unique subset of tumor cells,exhibiting properties similar to stem cells. Such alink between cancer and stem cells is supportedby the observations that many altered pathways incancer cells normally contribute to the stemnessproperty of embryonic and adult stem cells .A CSC iMicroRNAs  are a unique class ofnon-coding RNAs involved in diverse physiologicaland developmental processes including proliferation,differentiation, and apoptosis . MiRNAs Recent efforts to define ESC-specific miRNAshave led to the discovery of several miRNA clusterswhich are expressed in undifferentiated ESCsand are turned off upon the induction of differentiation.The cluster of hsa-miR-302-367 is locatedon chromosome 4 and consists of nine members co-transcribed in a poly-cistronic manner. MiR-302Recently, miR-302 has been implicated in reprogramming and tumoIn this experimental study, a ~1200bp genomicsegment corresponding to the human miR-302-367promoter was amplified from HEK cells, using thefollowing primers containing the BglII and HindIIIrestriction sites (underlined letters) respectively.Forward: 5´ATTTAGATCTCAAGAGTAACACATCTGG3´Reverse: 5´TATTAAGCTTCCCAAAGATTCGTGTTC3´The amplified product was cloned either in thepEGFP-N1 vector replacing the CMV promoter orin the pGL3 vector replacing the SV40 promoter.7TGC vector  was dige2 atmosphere. All cell lines were transfected withFugene HD transfection reagent according to the manufacturer’s instructions.The expression of green fluorescent protein (GFP) and luciferase, as indicators for promoter activityof mir-302s, were monitored by fluorescent microscopyor luminometry  and huminometry , 24-48 hLuciferase activity was measured by a luciferasereporter assay system . Briefly,the cells transfected with pGL3-pmiR werewashed and lysed prior to luciferase assay .All expTM Universal RT microRNA),according to the manufacturer’s instructions. RealtimePCR was performed with SYBR green mastermix, Universal RT  and micro-RNA LNATM primer sets, and analyzed with anABI 7500 real-time PCR system. To quantify theexpression level of pluripotency genes, a carefullydesigned set of primers . The U6 snRNA gene wasused as an internal control. The locked nucleicacid (LNA) primers for U6 and miR-302s weremanufactured by Exiqon (Denmark). Briefly, twomicrograms of total RNA was used for RT reaction,using a cDNA Reverse Transcription Kit(MiRCURY LNA primers  were emp primers .10RQ=Log10 2−Δ(ΔCT). ALog10RQ=0 corresponds to no expression change,while a Log10RQ=1 means 10 times elevation inexpression level compared to the internal control(GAPDH for protein-coding genes and U6 for microRNAs).All reactions were performed in duplicateor triplicate. Group-wise comparison and statisticalanalysis of the relative expression results of real-timePCR were carried out by REST 2008 Relative ExpressionSoftware Tool 2008 . Excel 2007 and GraphPadInstat3 were used to plot the charts. Student t test andANOVA were used to analyze the significance of differencesamong different groups.Fold changes in the expression levels were calculatedwith the formula LogThe expression of miR-302 members  in brain tumor cell lines1321N1, DAOY, A172 and U87MG was evaluatedby means of real-time RT-PCR, employing commerciallyavailable LNA primer pairs for specificamplification of each member. The embryonalcarcinoma cell line NT2 was used as a positivecontrol to optimize the amplification of miR-302members. As shown in Due to the very low expression of miR-302sin the glioblastoma cell lines, we constructedan expression vector in which the open-readingframe of the GFP had been cloned under the controlof miR-302 promoter . While mTo quantitate the data, we used another constructin which the miR-302 promoter wasplaced upstream of the open-reading frame ofthe luciferase gene. As shown in Using the luciferase assay, it was possible tolook at the expression level of miR-302s in glioblastomaand medulloblastoma cell lines underdifferent cell culture conditions. Interestingly,while U87MG cells infected with miR302-367promoter-GFP/SV40 promoter-DsRed lentiviralvectors  showed bThe colony formation was not restricted to theserum deprived glioma cell lines as the same phenomenonwas also observed in the medulloblastomacell line DAOY . To furtFurthermore, the expression levels were alsocompared for the main transcription factorsinvolved in self-renewal and pluripotency ofstem cells, namely OCT4, Sox2 and Nanog.Except for Nanog, the expression of otherfactors was upregulated in DAOY colonies. Among OThere is accumulating evidence on the reexpressionof embryonic genes in tumor cells, 2. IdenIn the present study, we examined the potentialexpression and function of miR-302s,an ES-specific microRNA cluster, in four differentbrain tumor cell lines. The cluster ofmiR-302 is the most abundantly expressedset of miRNA in undifferentiated ESCs andits expression is sharply turned off upon theinduction of differentiation . Indeed,Our data showed a very low expression ofmembers of the miR-302s cluster in gliomacell lines in comparison with the embryoniccarcinoma cell line NT2. Our data is in agreementwith a similar finding by Lavon et al.-29, 31, Initially, we used BMSCs (an adult stem cell)as a negative control, but we discovered a low,but significant, miR-302 expression in this cellline. Based on this observation, it seems thatthere is a subpopulation of pluripotent cellswithin these heterogeneous cell populations.This is in accordance with previous reports,suggesting the existence of such a subpopulationwithin BMSCs . InteresThe miR-302 promoter-Luc assay generateda more quantitative data, compared with themiR-302 promoter-GFP assay, in decipheringthe functional activity of miR-302 promoter inthe cell lines employed. Furthermore, it confirmedthe data produced by real-time PCR.This confirmation was necessary to rule outnon-specific amplification of related sequences.While the promoter of miR-302-367 clusteris exclusively functional in pluripotent cells,the ES-specific expression of other membersof this family  has not been elucidated.Using microRNA as a CSC, or more generallyas a tumor marker, has several advantagescompared with mRNA or protein markers.Firstly, due to their small sizes, they are verystable molecules under harsh conditions. Thisproperty of microRNAs could make them idealmarkers in different clinical samples includingserum , urine  and formFor detection of miR-302s, we used lockednucleic acid (LNA) primers, which providedhigh level of specificity for the detection ofmiRNAs. As expected for a positive control,we detected a high expression level of miR-302s in the NT2 cell line. While all membersof the miR-302 cluster were transcribed undera common promoter, the level of expressionvaried among them. This could be due to thevariation in the efficiency of different primersto specifically amplify individual members ordue to the innate differences in the stability ofmature members after being generated .Altogether, we used serum deprivation toenrich putative CSCs from several brain tumors,as described previously by others .Our dat


Understanding the structure and dynamics of cortical connectivity is vital to understanding cortical function. Experimental data strongly suggest that local recurrent connectivity in the cortex is significantly non-random, exhibiting above-chance bidirectionality, an overrepresentation of certain triangular motifs, and a heavy-tailed distribution of synaptic efficacies . AdditioHere we present a spiking network model of a Layer V-like cortical slice culture (panel B) that, via the interactions of a few simple biologically motivated plasticity mechanisms, qualitatively reproduces many of these non-random effects, such as synaptic weight (panel A) and triangular motif distribution (panel C) Additionally, it reproduced experimentally observed synaptic growth and efficacy dynamics . These p


Immunotherapy is rapidly becoming one of the pillars of anti-cancer therapy. GBM has not received similar clinical successes as of yet, which may be attributed to its relative inaccessibility (the blood-brain barrier (BBB)), its poor immunogenicity, few characterized cancer antigens, or any of the many other immune mechanisms known to be hampered. Focused ultrasound (FUS) is emerging as a promising treatment approach. The effects of FUS on the tissue are not merely thermal. Mounting evidence suggests that in addition to thermal ablation, FUS induces mechanical acoustic cavitation and immunomodulation plays a key role in boosting the host anti-tumor immune responses. We separately discuss the different pertinent immunosuppressive mechanisms harnessed by GBM and the immunomodulatory effects of FUS. The effect of FUS and microbubbles in disrupting the BBB and introducing antigens and drugs to the tumor milieu is discussed. The FUS-induced pro-inflammatory cytokines secretion and stress response, the FUS-induced change in the intra-tumoral immune-cells populations, the FUS-induced augmentation of dendritic cells activity, and the FUS-induced increased cytotoxic cells potency are all discussed. We next attempt at offering a conceptual synopsis of the synergistic treatment of GBM utilizing FUS and immunotherapy. In conclusion, it is increasingly apparent that no single treatment modality will triumph on GBM. The reviewed FUS-induced immunomodulation effects can be harnessed to current and developing immunotherapy approaches. Together, these may overcome GBM-induced immune-evasion and generate a clinically relevant anti-tumor immune response.Patients with glioblastoma multiforme (GBM) exhibit a deficient anti-tumor immune response. Both arms of the immune system were shown to be hampered in GBM, namely the local cellular immunity mediated by the Th O6-methylguanine-DNA methyltransferase (MGMT) is a key repair enzyme that contributes to the resistance of tumors to alkylating agents (carmustine or temozolomide). MGMT promoter methylation silences the gene, thereby increasing the susceptibility of the tumor cells to these agents .–47.26–47+ or CD8+ co-receptors molecules  , platelet-derived growth factor receptor (PDGF-R), and PI3-kinase/PTEN cascades , 14. An H1 differentiation) and increased local concentrations of anti-inflammatory cytokines and mediators (those driving to the TH2 differentiation) drive both the tissue and immune-elements that manage to reach the tumor site into an inactive or dysfunctional state .–47.26–47The addition of microbubbles (MB) to FUS can help generate more local heating in the area of focus , but at Several preclinical studies have demonstrated the feasibility of BBB disruption by FUS and MB for the administration of chemotherapeutics in the treatment of glioma. Aryal et al.  demonstr+CD8+ CTLs in the tumor region. IL-12 administration triggered a profound increase in all TIL populations, including CD3+CD4+ T helper cells, CTLs, and CD4+CD25+ Treg. The combined FUS-BBB opening with IL-12 administration produced the most significant IL-12 increase, CTL increase and CTL/Treg ratio increase, thus contributing to the most significant suppression of tumor progression and increased animal survival. These reports provided evidence that FUS-mediated BBB opening can enhance immune-modulating agent delivery to the brain.Chen et al.  reported+ T helper cells) endocytose the HSP-tumor peptide complex and present the chaperoned peptides directly to tumor-specific CTLs. This evokes potent cellular immune responses against tumor cells [HSPs are intracellular molecular chaperones, able to bind tumor peptide antigens and enhance tumor cell immunogenicity –79. Antior cells –79. Autoor cells . Becauseor cells –79.HIFU was shown to upregulate the expression of HSP70 both in vitro and ex vitro , 31, 42.+), expressing perforin and granzyme B molecules, indicative of a cytotoxic effector function [FUS was shown to enrich the TILs population in immune-potent pro-inflammatory potent anti-tumor effector cells. In human breast cancer specimens collected 1–2 weeks after FUS treatment , a signifunction . A randofunction .+ T cells, CD4+ T cells, a higher CD4+/CD8+ ratio, or NK cell stimulation [This TILs enrichment phenomenon is not limited to breast cancer. It was shown in patients treated with FUS suffering posterior uveal melanoma , pancreamulation  were obsmulation , 35. RefFUS was shown to enhance the infiltration capabilities of dendritic cells (DCs) , 18 as wA portion of the immunomodulatory effects of FUS noted in different reports are lacking exact molecular mechanisms as of yet, collectively termed as resistance to tumor re-challenge. Some overlap with CTLs potentiation is suspected and hence these mechanisms are discussed together. Zhang et al.  reportedSeveral reports of potentiated CTLs effector activity and potency after HIFU support this mechanism Table . IncreasIn an attempt to direct hypothesis-based approach to FUS-induced immunomodulation in GBM, the key established mechanisms hampered in GBM and the key immune-modulating effects of FUS discussed in this review are presented in Fig. Future research still needs to be done to both dissect the different FUS-induced molecular and immunological mechanisms at play. Further research is needed to optimize the FUS treatment method, find other combinational therapies, defining how to most effectively use it in combination with immunotherapy .No single treatment modality will cure GBM. It is increasingly apparent that surgery alone, HIFU ablation, chemotherapy, or immunotherapy will not solely triumph. We reviewed the FUS-induced immunomodulation effect, which can be harnessed to the current and developing immunotherapies approaches. Together, these modalities may overcome GBM-induced immune-evasion and generate a clinically relevant anti-tumor immune response. Further study to the synergistic collaboration of different therapeutic approaches and the elaborated molecular immune interplay will shed light on this formidable challenge.


To determine the prevalence of patient-reported joint pain among patients with human immunodeficiency virus (HIV)/chronic hepatitis C virus (HCV) coinfection, chronic HCV monoinfection, and HIV monoinfection followed in hepatology and infectious disease outpatient practices.Standardized interviews were performed among 79 HIV/HCV-coinfected, 93 HCV-monoinfected, and 30 HIV-monoinfected patients in a cross-sectional study within hepatology and infectious disease clinics at three centers. The Multi-Dimensional Health Assessment Questionnaire was used to ascertain joint pain and associated symptoms. Information on potential risk factors for joint pain was obtained during the interview and by chart review. Logistic regression was used to determine adjusted odds ratios (aORs) with 95% confidence intervals (CIs) of joint pain associated with risk factors of interest among chronic HCV-infected and HIV-infected patients.Joint pain was more commonly reported in HCV-monoinfected than HIV/HCV-coinfected  and HIV-monoinfected  patients. A previous diagnosis of arthritis and current smoking were risk factors for joint pain among HCV-infected patients  and HIV-infected  patients.Patient-reported joint pain was prevalent among all three groups, but more common among chronic HCV-monoinfected than either HIV/HCV-coinfected or HIV-monoinfected patients. A prior diagnosis of arthritis and current smoking were risk factors for patient-reported joint pain among both HCV-infected and HIV-infected patients.The online version of this article (doi:10.1186/s12891-015-0552-z) contains supplementary material, which is available to authorized users. Hepatitis C virus (HCV) infection is the most common blood-borne infection in the United States and is a leading cause of advanced liver disease. Over 4 million people in the U.S. and more than 170 million people worldwide have been infected with HCV infection . Many exRheumatologists are frequently faced with managing chronic HCV-infected patients with joint pain. However, few published studies have evaluated: 1) the prevalence of patient-reported joint pain among patients with chronic HCV in the U.S., 2) how joint symptoms among patients with chronic HCV affect quality of life and functional ability, or 3) whether coinfection with chronic HCV and human immunodeficiency virus (HIV) affects the prevalence of patient-reported joint pain. HIV infection has also been associated with rheumatic conditions, including reactive arthritis, psoriatic arthritis, and arthralgias and myalgias without identifiable inflammatory disease . While HThe primary objective of this study was to determine the prevalence of patient-reported joint pain  among patients with HIV/chronic HCV coinfection, chronic HCV monoinfection, and HIV monoinfection followed in hepatology and infectious disease clinics. We hypothesized that there would be a higher prevalence of arthralgias in HIV/HCV-coinfected patients compared to those who have chronic HCV or HIV alone. Our second objective was to examine potential risk factors for patient-reported joint pain among chronic HCV-infected  and HIV-infected  patients.We performed a cross-sectional study among patients with HIV/chronic HCV coinfection, chronic HCV monoinfection, and HIV monoinfection. Consecutive patients with chronic HCV and/or HIV infections were recruited in hepatology and infectious disease clinics at three tertiary care medical centers within Philadelphia . The PPMC infectious disease outpatient practice specializes in viral hepatitis care; hepatologists and infectious disease physicians see patients concurrently in the HUP viral hepatitis clinic; and the PVAMC outpatient infectious disease and hepatology clinics are located within the same practice space. Patients were enrolled between November 2008 and March 2012.Participants were eligible for inclusion if they were between 18 and 80 years of age and had documentation of HIV (HIV antibody- or RNA-positive) and/or chronic HCV infection (HCV RNA-positive) in their medical record. Exclusion criteria included the inability to either speak or understand English or to provide informed consent.Participants were asked to complete an interviewer-administered questionnaire that ascertained the presence or absence of patient-reported joint pain over the one week prior to the study visit, the duration of joint pain, the joints affected, and previous diagnoses of joint disorders or arthritis. Among patients reporting joint pain, an interviewer administered the Multi-Dimensional Health Assessment Questionnaire   given thMedical records were also reviewed to abstract age at study visit, sex, race, height and body weight, analgesic medication use , medical comorbidities , HIV-related data , HCV-related data , relevant social history , and most recently recorded laboratory results of total bilirubin, asparate aminotransferase (AST), alanine aminotransferase (ALT), albumin, international normalized ratio (INR), white blood cell count, hemoglobin, and platelet count.2), AST, ALT, current HCV therapy, use of analgesic medications, HCV genotype (in chronic HCV-infected patients only), and CD4 cell count (in HIV-infected patients only). Since HCV treatment has been associated with arthralgias, we performed a sensitivity analysis in which we excluded patients currently utilizing this therapy and repeated the analyses above.The prevalence of joint pain was determined and expressed as a point estimate (percent) and 95% confidence interval (CI) within each group. Descriptive statistics were used to compare characteristics between groups . Differences between groups were determined using Chi-square or Fisher’s exact tests, when appropriate, for categorical data, and Wilcoxon rank-sum tests for continuous data. Logistic regression was used to determine adjusted odds ratios (aORs) with 95% CIs of joint pain associated with risk factors of interest among chronic HCV-infected  and HIV-infected  patients. Variables evaluated as risk factors for joint pain included age, sex, race, history of arthritis, history of anxiety disorder or depression, obesity . The characteristics of each group are presented in Table Over half of the patients reporting joint pain also self-reported depression and anxiety on the MD-HAQ. Depression was reported by 50% of HCV-monoinfected, 80% of HIV-monoinfected, and 64% HIV/HCV-coinfected patients. Similarly, anxiety was reported by 51% of HCV-monoinfected, 67% of HIV-monoinfected, and 77% of coinfected patients.Joint pain was more commonly reported among HCV-monoinfected than coinfected  or HIV-monoinfected patients . After adjustment for age and sex, HCV-monoinfected patients were 1.9-fold more likely to report joint pain compared to coinfected patients  and 2.4-fold more likely to report arthralgias compared to HIV-monoinfected patients . No differences in the prevalence of joint pain were observed between coinfected and HIV-monoinfected patients . HIV/HCV coinfection remained unassociated with arthralgias compared to HIV monoinfection after adjustment for age and sex . A sensitivity analysis excluding chronic HCV-infected patients currently on HCV therapy (n = 22) revealed similar proportions of chronic HCV-infected persons reporting joint pain (data not shown).Among all three groups, fingers, knees, and back were the most frequently cited areas of joint pain Figure . The disMD-HAQ results among participants reporting joint pain are reported in Table A previous diagnosis of arthritis  and current smoking  were risk factors for self-reported joint pain among chronic HCV-infected patients Table . These aThis cross-sectional study demonstrated that joint pain was commonly reported and associated with diminished functional status and emotional well-being among patients with chronic HCV and/or HIV infection. The prevalence of joint pain was significantly higher among HCV-monoinfected than HIV/HCV-coinfected and HIV-monoinfected patients. Over two-thirds of patients with chronic HCV monoinfection reported joint pain, while approximately half of coinfected and HIV-monoinfected patients reported arthralgias.Our study extends results of prior cross-sectional analyses, which have reported that 67-81% of chronic HCV-infected patients complain of musculoskeletal pain ,24. TsuiThe etiology of joint pain in chronic HCV infection remains unclear. Immune activation, direct viral particle deposition in the synovium, and the high prevalence of concomitant mood disorders might be important contributors to the arthralgias commonly reported by chronic HCV-infected patients ,27,28.The differences in the prevalence of joint pain among the three study groups raise important questions for further study. Hypothesized explanations for these findings include differences in unmeasured environmental exposures among the groups or potentially a decrease in local inflammation related to immune dysfunction in HIV infection, resulting in a decreased effect of chronic HCV in the HIV/HCV-coinfected patients ,30 AdditWe found that current smoking was strongly associated with joint pain. Smoking has been associated with musculoskeletal pain and the development of rheumatoid arthritis  In addit7We found that nearly half of patients with chronic HCV infection (both HCV-monoinfected and HIV/HCV-coinfected) and joint pain self-reported 20 or more symptoms which has previously been reported to indicate a fibromyalgia diagnosis . FibromyOur study was limited by its cross-sectional design, use of convenience sampling, our inability to perform physical examinations and obtain imaging studies to rule out osteoarthritis, and the relatively small sample. In particular, there is the possibility that patients with joint pain may have been more likely to complete the survey. Additionally, we were unable to determine the specific etiologies for the joint pain. While many patients reported a diagnosis of arthritis, we did not confirm these diagnoses on physical examination or further testing. Furthermore, some patients were prescribed analgesic medications, and these patients might have less commonly self-reported joint pain. Finally, our study evaluated patient-reported joint complaints present within the one week prior to the survey date. Thus, there may have been patients with intermittent joint pain who were not identified in this study. However, use of the one week time frame limits the risk of recall bias and is the time period specified on the MD-HAQ for assessment of disease activity in rheumatoid arthritis.Among patients with HIV and/or chronic HCV infections, joint pain was prevalent. HCV-monoinfected patients more frequently reported arthralgias compared to HIV/HCV-coinfected or HIV-monoinfected persons. These results suggest that joint pain remains a major health concern and a determinant of health-related quality of life among these patients. Providers should seek to address modifiable risk factors for joint pain  in such persons. Future studies should determine the etiologies and strategies for management of joint pain, including fibromyalgia, in patients with chronic HCV and HIV infection, as well as the mechanisms for modulation of joint symptoms in HIV/HCV-coinfected patients.Additional file 1: Table S1.Laboratory results of human immunodeficiency virus (HIV)/chronic hepatitis C virus (HCV)-coinfected, chronic HCV-monoinfected, and HIV-monoinfected participants.