We recently described a new autoinflammatory syndrome associated with lymphedema (AISLE), and a frameshift mutation in the MDFIC gene was identified by homozygosity mapping and targeted sequencing using DNA samples of a consanguineous Turkish family as the cause of this syndrome. The same mutation in the MDFIC gene was identified in an Italian patient with similar clinical findings. Clinical findings of these patients are characterized by recurrent attacks of fever, erythematous/urticarial rash with hyperesthesia, myalgia, serositis, chylous serosal effusions, edema/lymphedema on the face and extremities. These patients gradually developed a permanent lymphedema in the same areas affected during attacks. On the other hand, there is limited information about the functions of MDFIC gene, which include transcriptional regulator interacting with cellular transcription factors, including such as axin and wnt/b-catenin signaling pathway. Although, its expression has been documented in immune cells, no clear function has been described in immune mediated or inflammatory conditions.We evaluated the clinical course and laboratory investigations of the patients. We investigated the gene expression patterns in peripheral blood mononuclear cells in one of the patients and his parents following LPS stimulation to understand involved pathways in MDFIC-related inflammatory changes using an Illumina gene expression kit. We also evaluated pathological specimens obtained from the same patient during surgical resection of the giant scrotal tissue affected from lymphedema. Detailed immunohistochemical analyses were done to define the characteristics of lymphatics in involved tissues.LPS stimulation resulted in a significant deviation in gene expression pattern in the AISLE patient compared to the expression patterns of one of his parents and un-related healthy controls. Expression of CCL8, IFITM3, IFIT1, IFIT3, IFIT2, ISG15, OAS3 genes showed more than two times decrease following LPS stimulation, and these gene expression changes were interpreted as a negative interferon signature. Pathological examinations revealed lower than expected number of lymphatic vessels as well as rare enlarged lymhatics in scrotal tissues with lymphedema. Regarding two of the Turkish patients’ treatments, no clear response was observed to monoclonal anti-IL-1 beta canakinumab antibody. However, both patients are doing well with daily anakinra injections. The first patient experienced no adverse event during and following two operations for giant scrotal lymphedema, and a normal recovery period was observed.Gene expression findings indicating a negative interferon response following LPS stimulation need to be confirmed in other two patients and cell line models. Current immunological and pathological findings suggest an interferon-signature mediated inflammatory response defect, which may affect lymphatic endothelial cells and their survival, leading to gradual decrease in the number of lymphatic vessels and development of lymphedema. Also differential response to anakinra but not canakinumab may suggest possible contribution of IL-1 alpha to the pathogenesis of AISLE.


Livestock grazing can be used as a key management tool for maintaining healthy ecosystems. However, the effectiveness of using grazing to modify habitat for species of conservation concern depends on how the grazing regime is implemented. Timing of grazing is one grazing regime component that is less understood than grazing intensity and grazer identity, but is predicted to have important implications for plant and higher trophic level responses. We experimentally assessed how timing of cattle grazing affected plant and arthropod communities in high-elevation grasslands of southwest Montana to better evaluate its use as a tool for multi-trophic level management. We manipulated timing of grazing, with one grazing treatment beginning in mid-June and the other in mid-July, in two experiments conducted in different grassland habitat types  in 2011 and 2012. In the upland grassland experiment, we found that both early and late grazing treatments reduced forb biomass, whereas graminoid biomass was only reduced with late grazing. Grazing earlier in the growing season versus later did not result in greater recovery of graminoid or forb biomass as expected. In addition, the density of the most ubiquitous grassland arthropod order (Hemiptera) was reduced by both grazing treatments in upland grasslands. A comparison of end-of-season plant responses to grazing in upland versus wet meadow grasslands revealed that grazing reduced graminoid biomass in the wet meadow and forb biomass in the upland, irrespective of timing of grazing. Both grazing treatments also reduced end-of-season total arthropod and Hemiptera densities and Hemiptera biomass in both grassland habitat types. Our results indicate that both early and late season herbivory affect many plant and arthropod characteristics in a similar manner, but grazing earlier may negatively impact species of conservation concern requiring forage earlier in the growing season. Grazing is a key process in grasslands that has far reaching effects on plant and animal diversity Understanding the role of grazing in species conservation requires a thorough assessment of the various components of the grazing regime. Grazing intensity and grazer identity are key components that have received the most attention Altering timing of grazing within a short growing season may have large effects on multiple trophic levels if grazing time periods coincide with key life history stages of organisms in the ecological community  In addition to uncertainties with respect to timing of grazing, little is known about how grazing regimes will affect plant and arthropod communities among different grassland habitat types that form the broader vegetation mosaic. Wet meadow and upland grassland habitats often differ in soil moisture, soil type, and major limiting resources In an effort to quantify the effects of timing of grazing on plants and arthropods in high-elevation grasslands, we experimentally manipulated timing of cattle grazing over the course of two growing seasons in two grassland habitat types. We asked: (1) how do ungrazed wet meadow and upland grasslands differ in terms of plant, arthropod, and soil moisture characteristics, (2) how does timing of grazing affect plant and arthropod communities in upland grasslands, and (3) what is the range of possible plant and arthropod responses to timing of grazing across wet meadow and upland grasslands? We hypothesized that ungrazed wet meadow grasslands would have greater soil moisture than ungrazed upland grasslands due to the closer proximity of ground water to the soil surface in wet meadows This field study was conducted in collaboration with the US Fish and Wildlife Service and The Nature Conservancy; all permissions for site access were granted and no permits were required. This study did not involve any endangered or protected species.We conducted grazing experiments in 2011 and 2012 in the Centennial Valley of southwest Montana  on grasslands leased or owned by Red Rock Lakes National Wildlife Refuge (RRL) and The Nature Conservancy. Climate in the Centennial Valley is characterized by long, cold winters and short, mild summers with highly variable annual precipitation Deschampsia cespitosa), clustered field sedge (Carex praegracilis), and mat muhly (Muhlenbergia richardsonis), while Baltic rush  is found in wetter areas Iris missouriensis), common dandelion , and darkthroat shooting star (Dodecatheon pulchellum), among others Deschampsia cespitosa), clustered field sedge (Carex praegracilis), basin wildrye (Leymus cinereus), Kentucky bluegrass (Poa pratensis) and Sandberg bluegrass (Poa secunda) Lupinus argenteus), rosy pussytoes (Antennaria rosea), and common yarrow (Achillea millefolium) are the most common Numenius americanus), sandhill crane (Grus canadensis), savannah sparrow (Passerculus sandwichensis), western-meadowlark (Sturnella neglecta), and other migratory birds Our 2011 grazing experiment was conducted in wet meadow grasslands, while our 2012 experiment took place in upland grasslands. Wet meadow grasslands occupy over 2,800 hectares at RRL, and vegetation in this habitat is dominated by a dense layer of graminoids and low forb canopy cover unpubl. data). Additionally, since the 1950s, 90% of grazing has been initiated after July 10th due to concern over nest trampling of protected avian species (e.g. long-billed curlew and sandhill cranes). Although the long-term grazing history of the specific sites used in this study was unavailable because they were privately owned until 2008, most grazing units  owned or leased by RRL are grazed by cattle on a 3-year rest-rotation Current management allows grazing to occur on many of the grasslands owned or leased by RRL, with grazing intensities ranging from 0.31 to 0.85 AUM/acre  between 1994 and 2006  and traditional late graze (starting mid-July). We also established ungrazed, control plots . In 2011, our grazing experiment was conducted at a large spatial scale, using c. 38 hectare (∼1120 m by 340 m) experimental plots in heterogeneous, sub-irrigated wet meadow grasslands. We observed a high degree of patchiness in grazing perhaps due to the large amount of heterogeneity in plant community structure and soil moisture in wet meadows. In 2012, we used smaller experimental plots  in nearby homogeneous, non-irrigated upland grasslands. Experimental blocks containing control, early-graze, and late-graze plots were established in both years. We had three blocks in 2011, with each block sized ∼114 hectares (∼1120 m by 1016 m), and four blocks in 2012, each block sized ∼6 hectares (∼405 m by 150 m). Within each block, plots were randomly selected for one of the three treatments. Due to trespass grazing in early July of 2012, two plots were removed from analyses (one control and one early-grazed), thus reducing the number of replicates for control and early-grazed treatments from four to three. In both years, early- and late-graze treatment plots were grazed by cattle for equal durations (grazed for 2 weeks) and at equal intensities (0.9 AUM/acre).Vegetation and arthropods were collected concurrently within each plot at six sampling events between mid-June and early September in 2011 and four sampling events between early June and late August in 2012. Sampling event timing and number of events differed between years due to a shorter growing season in 2012 than in 2011. On each sampling event in 2011, ten sampling points were randomly selected for sampling out of 25 sampling points randomly established in each plot. On each sampling event in 2012, ten sampling points that were randomly established east or west of a centrally located north-south transect within each plot were sampled. Since cattle severely trampled the area near water tanks, we created a 30 m buffer zone near the water tanks where no sampling occurred. In both years, we avoided potential “edge effects” between treatments by not sampling in 10 m buffer zones between neighboring plots.2 quadrat in 2011 and to 5 cm in a 0.25 m2 quadrat in 2012. On each sampling event, we sampled in different ordinal directions from sampling points to avoid clipping the same vegetation. Quadrats were located at least 1 m away from previously sampled quadrats. Total plant biomass included both live and dead vegetation that represented the previous and current year of growth, with dead vegetation being included because it represents an important structural habitat for arthropods and birds 2 quadrat At each sampling point (N = 10 per plot), above-ground plant biomass was quantified by clipping vegetation to ground level in a 0.03 m2 plastic barrel that was quickly placed over the collection area, minimizing arthropod escape and restricting the sample to a known area. We then used an inverted leaf blower/vacuum sampler  to extract arthropods by moving the sampler evenly across the vegetation in the enclosed area In both years, prior to sampling vegetation at each sampling point, we sampled arthropods using an enclosed 0.25 mWe measured soil moisture in each grassland habitat type to determine how local-scale water availability influenced plant and arthropod responses to grazing treatments. In the wet meadow grassland (2011), we measured soil moisture at each of the 25 sampling points in each plot three times throughout the summer. In the upland grassland (2012), we measured soil moisture at each of the ten sampling points in each plot on all four sampling events. Three soil moisture readings were taken within 30 cm of each other using an Aquaterr 300-T soil probe at a depth of 15 cm. All readings were within 60 cm of the sampling point.In the wet meadow grassland experiment (2011), we did not detect strong grazing effects because of large variability in soil moisture and plant biomass within experimental plots resulting from sub-irrigation and visible heterogeneity in cattle grazing. Additionally, the discrepancy between the large plot sizes relative to the small quadrats used for plant sampling in 2011 may not have adequately captured the mean effects of the grazing treatments. We therefore restricted our in-depth analysis of timing of grazing effects throughout the growing season to the upland grassland experiment conducted in 2012. In contrast, we used both years of data to examine the range of possible plant and arthropod responses to timing of grazing across grassland habitat types (wet meadow vs. upland grassland). By comparing cumulative (late August =  end-of-season) responses, grassland communities had a longer timeframe to develop patterns and reflect grazing treatment differences between grassland habitat types.To quantify grassland habitat type differences without the added complexity of grazing, we compared plant, arthropod, and soil moisture metrics from ungrazed plots in wet meadow and upland grasslands. We focused on comparing end-of-season characteristics because this time period represented the cumulative effects over most of the growing season. We first compared mean graminoid biomass, forb biomass, total arthropod density, and total arthropod biomass in each grassland habitat type using paired t-tests.Histograms of a cumulative soil moisture metric (range 0–200%) were generated to depict end-of-season soil moisture for each grassland habitat type. This cumulative metric, based on the summed average soil moisture values from all August sampling events, was used to better represent moisture conditions in plots at the end of the growing season. To compare soil moisture between grassland habitat type, we limited the sampling points to N = 10 by randomly subsetting ten sampling points in the wet meadow (out of 25 possible) on each sampling event to match N = 10 in the upland grassland. Due to non-normality of distributions, we conducted a Kolmogorov-Smirnov two-sample test to examine inter-habitat differences in soil moisture.To examine relationships between plant and arthropod variables across habitat types, we calculated Pearson correlation coefficients for end-of-season plant biomass and arthropod density in each grassland habitat type. We also used Pearson correlation coefficients to examine finer-scale relationships between microsite cumulative soil moisture and end-of-season (1) graminoid biomass, (2) forb biomass, (3) total arthropod density, and (4) total arthropod biomass.post-hoc one-way ANOVAs and Tukey's Honestly Significant Difference (HSD) multiple comparisons were used to determine treatment differences within each sampling event, as well as sampling event differences within each grazing treatment. We did not adjust alpha levels with a Bonferroni correction because doing so may lead to a higher probability of a type II error and a lack of a standard alpha across studies To determine the effects of timing of grazing on plant and arthropod characteristics in the upland grassland, we used a repeated-measures analysis of variance (rmANOVA). Treatment and sampling event (sampling event as the repeated measure) were included as independent variables, as well as the interaction term between them. When significance in main effects or interactions were detected (p<0.05), Because patterns in sweep net sample results were similar to vacuum sample results, we only report the vacuum sample results because they can be expressed on a per area basis. All data were analyzed using R version 2.15.2 Our two study years varied in terms of grassland habitat type, but also in accumulated precipitation , Hymenoptera (23%), and Araneae (10%) across all sampling events. The remaining 10% was composed of Coleoptera, Diptera, Orthoptera and six additional low-density orders. Patterns with respect to total arthropod biomass were similar, with the majority of arthropod biomass consisting of Hemiptera (40%), Hymenoptera (19%), Araneae (16%), and Orthoptera (14%).2; Average Hemiptera density differed significantly across grazing treatments, but these effects depended on the sampling event . HemipteThere was no effect of grazing on average body size  of Hemiptera. However, there was a marginally significant effect of grazing on Hemiptera total biomass  was reduced by both grazing treatments. While both grazing initiation dates shared similarities in responses, our results suggest that early grazing has the potential to negatively affect grassland birds that are reliant on forbs and Hemiptera as a main forage source in June. Our results also suggest that the influence of grazers on plant communities may depend on both grassland habitat type and plant functional group, but climatic variation is likely to influence grazing outcomes.We found that the ungrazed wet meadow grassland had greater graminoid and forb biomass, as well as total arthropod density, than the ungrazed upland grassland. We expected such differences due to the influence of soil moisture in wet meadows resulting from spring flooding and closer proximity to ground water Many plant characteristics were temporarily reduced by both grazing treatments, as expected. However, plant height, but not plant biomass, was greater with early grazing than late grazing at the end of the season. Several explanations may help elucidate why plant biomass did not recover to a greater degree with early grazing than late grazing, despite the increased time available for regrowth. First, a plant's ability to regrow after grazing can vary with local conditions, such as the availability of nutrients and moisture We found no significant differences in graminoid biomass at the end of the season between early and late grazing, as well as between grazed and control plots. However, the 40% reduction in mean graminoid biomass in both early- and late-grazed plots  suggestsGeneralizations about effects of grazers on arthropods are elusive With the exception of Hemiptera, our results showed that arthropod orders were unaffected by grazing across the growing season in upland grasslands. While some studies that manipulated grazing intensity concur with our results e.g., , many otRegardless of timing of grazing, we found that end-of-season plant biomass was often reduced by grazing, but the effect depended on grassland habitat type and plant functional group. End-of-season forb biomass was unaffected by grazing in the wet meadow grassland, while end-of-season graminoid biomass was unaffected by grazing in the upland grassland. A possible reason for these differences in responses is likely related to the competitive abilities of the two functional groups. Mueggler found that forb growth benefited more from increased soil moisture than grass growth and suggested that forbs grazed during a wet year would be damaged less than those grazed during a dry year across the growing season with the rmANOVA, the effect size analysis revealed there were still cumulative negative effects of grazing on several arthropod groups at the end of the season, which has important implications for arthropods' ability to recover from a grazing event.We found that some arthropod orders were unaffected by grazing , while grazing reduced many other end-of-season arthropod densities in both study years, regardless of timing of grazing or grassland habitat type. End-of-season Hemiptera density was reduced for both grazing treatments in both grassland habitat types, while Araneae density was reduced in the wet meadow grassland. While we did not find as many negative effects of grazing on arthropods Different plant and arthropod responses in 2011 vs. 2012 may be due to differences in grassland habitat type, but as previously stated, may also be due to interannual variation in climate, such as accumulated precipitation, timing of snowmelt, and length of growing season. It is also possible there were other unmeasured ecological drivers that varied between our two study sites and contributed to the observed responses of plants and arthropods to grazing, such as differences in fine-scale topography Our results demonstrate that the effects of timing of grazing on plant and arthropod communities are not uniform throughout the growing season and such temporal shifts have important implications for species conservation. First, initiating grazing earlier in the growing season in upland grasslands may impact bird species of conservation concern that rely on forbs and Hemiptera as a food source in June. Other studies have similarly suggested that temporal variation in bird forage may have important implications for post-fledgling survival Secondly, several plant community variables were affected by timing of grazing and such alterations in vegetation structure have important implications for nesting habitat for bird species of conservation concern. Early grazing in the upland grassland resulted in 17% taller plant height at the end of the season compared to late grazing. Since residual plant height persists into the next growing season, this information could be used to inform management of grassland birds that require specific vegetation heights for fulfilling life history requirements Proper planning of grazing regimes may aid in reducing possible negative effects of early season grazing on higher trophic levels. Early season grazing could take place outside of core bird nesting and foraging areas in the future, which may help mediate potential early-season impacts on higher trophic levels. One of the most beneficial grazing strategies may include a matrix of grazing plots, with varying grazing initiation dates and years of rest between grazing periods at the landscape level. Doing so would aid certain areas of the grassland in recovery from grazing at any point during the growing season, and provide the necessary structure and forage availability for a broad suite of species with differing life history requirements. A better understanding of how timing of grazing affects plants and arthropods will be useful for conservation grazing management in similar high-elevation grassland systems and in particular, for determining how potential changes in forage availability will affect species conservation.


Tumor heterogeneity is a major challenge and the root cause of resistance to treatment. Still, the standard diagnostic approach relies on the analysis of a single tumor sample from a local or metastatic site that is obtained at a given time point. Due to intratumoral heterogeneity and selection of subpopulations in diverse lesions this will provide only a limited characterization of the makeup of the disease. On the other hand, recent developments of nucleic acid sequence analysis allows to use minimally invasive serial blood samples to assess the mutational status and altered gene expression patterns for real time monitoring in individual patients. Here, we focus on cell-free circulating tumor-specific mutant DNA and RNA (including mRNA and non-coding RNA), as well as current limitations and challenges associated with circulating nucleic acids biomarkers. Yet, we rely on few standard diagnostic tumor biopsies for the characterization of a given cancer. These specimens will provide only a partial characterization of the overall makeup of the dynamic systemic disease cancer represents with intratumoral and interlesional heterogeneity as well as emerging host responses SETD2, PTEN and KDMSC). Additionally, this study demonstrated extensive heterogeneous mutational profiles in 26 out of 30 tumor samples from four renal cell carcinoma patients A striking example that illustrates intratumoral heterogeneity was recently described for kidney cancer specimen that revealed distinct expression of an autoinhibitory domain of the mTOR kinase and multiple tumor-suppressor genes  Here we will discuss cell-free circulating tumor-specific mutant DNA and RNA , i.e. fragments of DNA found in the cell-free blood compartment was first described in 1948 It is believed that ctDNAs are results of apoptosis. Nucleosomes play essential roles in the fragmentation of DNA during programmed cell death and a recent study developed a genome-wide nucleosome map that showed ctDNA fragments bearing footprint of transcription factors in specific tissues 4ctDNA detection methods have improved substantially during the past few decades. In the early 1990s, recovery of ctDNA was performed by conventional polymerase chain reaction, followed by Sanger sequencing. However, recovery of ctDNA was often inconsistent, and was considered inferior to other biomarkers, including circulating tumor cells (CTCs) and cancer-related protein markers . The main obstacle in the detection of ctDNA is the relatively low abundance per milliliter of blood examined. Conventional methods of PCR detection and Pyrosequencing have their lower limit of detection at 10% of ctDNA copies in the bulk of background normal DNA . SimilarMore recent methods using droplet digital PCR PCR-based assays do carry limitations related to their detection methods. The numbers of ctDNA that can be detected in one assay are limited. The number of fluorescence acquisition channels available often dictates the number of multiplex-droplet PCR amplification and probe-hybridization reactions. BEAMing is labor-intensive and requires both Streptavidin bead emulsion PCR and flow cytometry, thus, decreasing productivity and the possibility for high-throughput analyses. Also, only known targeted mutations are measured in BEAMing or ddPCR analysis. This also generates a challenge in situations where the amount of template DNA is limited and multiple mutations may be emerging.EGFR, BRAF or KRAS. However, initial efforts to utilize shot-gun approaches with whole-exome sequencing to identify and measure ctDNA were difficult due to ctDNAs being fragmented and degraded in the circulations. This further complicates the validation of variant calling in extensively fragmented DNA samples Genome wide approaches to assess global ctDNA in the circulation have gained significant attention. This is because only a fraction of patients has known cancer-related driver mutations, i.e. 5ctDNA are found at a relatively high concentration in the peripheral circulation in patients with metastatic cancer, compared with localized disease 6Conventionally, cancer-related protein markers have been used to monitor patients with limited sets of cancers for recurrent disease, i.e. CA-125 in ovarian cancer, AFP for hepatocellular carcinoma, carcinoembryonic antigen (CEA) for colorectal adenocarcinoma, or lactate dehydrogenase (LDH) for malignant melanoma. Unlike germ-cell tumors where cancer-related protein markers are highly sensitive and specific to cancer activities, the majority of cancer-related proteins, i.e. LDH, remain only screening tools for cancer recurrence without adequate specificity.ctDNA are more abundant in the circulation among metastatic cancers than early-staged disease, and the prevalence of ctDNA detected in patients with no radiographic evidence of metastasis varies between 49–78%, compared with 86–100% in metastatic disease Alternative method to monitor disease activity is through the detection of unique sets of single nucleotide point mutations specific to the patient as indicators of disease activity. Also, identification of a patient's specific somatic chromosomal translocation through high-throughput sequencing,  or through next-gen, matched-pair sequencing analysis have recently been established. 7KRAS in patients with non-small cell lung cancers. This correlated with the presence of KRAS mutations in tumors and with poorer prognosis for overall survival KRAS ctDNA, the higher concentration of ctDNA correlated with a poorer survival rate, independent of ECOG performance status, and CEA level Earlier studies used restriction fragment-length polymorphism and polymerase chain reaction (RFLP-PCR) assays on circulating DNA to selectively detect circulating mutant 86-methyl-guanine-methyl-transferase (MGMT) promoter methylation from ctDNA in glioblastoma multiforme (GBM) patients. This would determine potential benefits from adjuvant alkylating chemotherapy such as temozolomide or dacarbazine, in addition to standard post-operative adjuvant radiation MGMT methylation using methyl-BEAMing and bisulfite-pyrosequencing techniques in metastatic colorectal cancers demonstrated 86% agreement of MGMT methylation status the tumor and ctDNA analyses with the most methylated allele in the tissues presented in the circulation. Additionally, MGMT methylation status in ctDNA was associated with improved median PFS  Predictive biomarkers that can guide treatment decision have been sought after to identify subsets of patients who would be “exceptional responders” to specific cancer therapies, or individuals who would benefit from alternative treatment modalities. An example of ctDNA as a potential predictive biomarker is the measurement of OctDNA can also be incorporated into prospective clinical studies to identify predictive markers of response to cancer therapy with stratifications based on the underlying somatic mutation that will render subjects susceptible to specific targeted therapies.  or indicate emerging resistant subclones.9Several studies have utilized ctDNAs as markers of metastatic disease activities to monitor disease response and overall disease burden. In one study, a total of 30 out of 52 patients with metastatic breast cancers were found to have somatic variants in their tumors, either by targeted gene sequencing, or whole-genome paired-end sequencing. Compared with CTCs and CA 15–3, 97% of patients had measurable ctDNA, compared with 78% for CA 15–3, and 87% for CTCs. The trend of serial ctDNA levels appeared to correlate with radiographic response to therapy. A comparison showed fluctuations of CTCs that are not informative when the number of CTCs was below 5 cells/ml, and CA 15–3 changes in response to cancer treatment were only small.Application of ctDNA for treatment monitoring and surveillance could be useful in certain malignancies where there is no optimal method of screening and surveillance, such as pancreatic cancer, or ovarian cancers. Pereira et.al 10While ctDNA monitoring could offer potential improvements in non-invasive cancer treatment monitoring, there are inherent limitations related to ctDNA tumor markers. ctDNAs demonstrates a strong correlation with tumor burden but are not always detectable in peripheral blood. Most studies have shown an approximately 70–80% concordance between tumor somatic mutation and the presence of ctDNA in the circulation ctDNA quantification is highly dependent on pre-analytical specimen handling. While it is possible to recover ctDNA at a comparable concentration between 2–4 h and 24 h processing time The source of ctDNA should also be standardized, either from serum or plasma. Prior studies While ctDNA could be useful in the early detection of cancer recurrence, a potential major limitation is the lack of a consensus on the next step of management following detection of ctDNA in individuals without radiographic evidence of cancer recurrence or relapse. A great example has been CA-125, a protein biomarker for ovarian cancer, in the MRCOV05/EORTC 55955 trial Similarly, lead-time bias is another major challenge in early cancer screening tools, as previously mentioned in yearly low-dose CT scan for lung cancer screening, and routine PSA monitoring in prostate cancer 1111.1Circulating messenger RNAs (mRNA) in human cancer patients were first described in the 1990s in patients with different type of cancers, i.e. gastric cancer, pancreatic cancer 11.2Non-coding DNA sequences are actively transcribed into non-coding RNAs consisting of long non-coding RNAs (lncRNA), microRNAs (miRNA), short interfering RNAs (siRNAs), and piwi-interacting RNAs (piRNA), among other lncRNA species. Unlike mRNA, the function of non-coding RNA is the regulation of gene expression. The vast majority of observations in the field of circulating RNAs involve miRNAs and lncRNAs, however as increasing RNA sequencing data is being generated, it is becoming clear that piRNAs and snoRNAs in human plasma are gaining importance.11.3http://pirnabank.ibab.ac.in/stats.html) there are more than 32,000 unique piRNAs. In addition to their role in maintaining the integrity of germ line DNA, piRNAs are found to be deregulated in cancer. PiRNAs are single stranded 26–31 nucleotide long RNAs which can repress transposons and target mRNAs, mediated by binding to PIWI proteins. PIWI proteins belong to a subfamiliy of Argaunate proteins. piRNA biogenesis is Dicer and Drosha independent 11.4snRNA and snoRNA consist of large number of non-coding RNA species of 60–300nucleotide long that were transcribed from intervening sequences of protein-coding genes (a.k.a.host genes). snRNAs are important in RNA-RNA remodeling and spliceosomes assembly. snoRNAs involve in post-transcriptional modification of ribosomal RNA and play integral roles in formation of small nucleolar ribonucleoprotein particles (snoRNP), which are important cellular regulation and homeostasis. There are two major classes of snoRNA, the first one is box C/D snoRNA, a.k.a. SNORDs (contains box C (RUGAUGA) and D (CUGA) motif), and box H/ACA snoRNA, a.k.a. SNORAs, (contains box H (ANANNA) motif and ACA elements)  are highly conserved short strands of non-coding RNA, derived from hairpin precursor transcripts KRAS in colon cancer cell lines leads to decreased Ago2 secretion in exosomes and Ago2 knockdown resulted in decreased secretion of let-7a and miR-100 in exosomes whilst cellular levels of the respective miRs remained unchanged compared to control cells. miRNAs are dysregulated in cancer and play crucial roles in cell proliferation, apoptosis, metastasis, angiogenesis and tumor-stroma interactions A systematic expression analysis of 217 mammalian miRNAs from 334 samples, including multiple human cancers revealed extensive diversity in miRNA expression across cancers, and a large amount of diagnostic information encoded in a relatively small number of miRNA. More than half of the miRNA (129 out of 217) had lower expression levels in tumors compared to normal tissues, irrespective of cell types miRNAs are present and stable in the peripheral circulation. The first report on miRNA expression in the circulation in 2008 described detection of four placenta-associated miRNAs  in maternal plasma during pregnancy, after which the level decreased following delivery The vast majority of research on circulating miRNA signatures in oncology is focused on diagnostics The origin of cf-miRNA is heterogeneous. miR-21 is a good example to illustrate this point. Although the release of miR-21 into the circulation is correlated with a multitude of cancer types, it is also highly expressed in activated T-cells and associated with inflammation and wound healing Cancer progression and systemic drug therapy involve many organ systems and are not limited to the primary tumor. This makes cf-miRNA attractive biomarkers for cancer progression and drug efficacy monitoring. For instance, in serum obtained pre-surgically from patients with early stage colorectal cancers, a panel of 6 circulating miRNAs can predict cancer recurrence 12Human serum contains ribonucleases (RNase) that originate from leukocytes and the pancreas and catalyze the cleavage of bonds between ribonucleotides. Levels of serum RNases are elevated in patients with cancer One explanation for the circulating RNAs' stability is encapsulation by protective membrane bound vesicles. These vesicles consist of a lipid bilayer membrane surrounding a small cytosol and are separated into three types: exosomes, microvesicles , and apoptotic bodies (ABs). Each vesicle type can originate from normal or cancerous cells, transfer molecular cargo to both neighboring and distant cells, and modulate cellular behaviors involved in physiology and pathology Exosomes were first identified as vesicles with 5′nucleotidase activity in 1981 by Trams et al. Interestingly, bovine miRNAs were detected in the human plasma exosomes. However their origin remains to be elucidated since it is unknown whether dietary miRNAs can enter the human circulation through the gastrointestinal system. Microvesicles are larger vesicles (50 to 1000 nm) created through direct budding from the plasma membrane and contain metalloproteases in addition to lipids, cytokines, growth factors, membrane receptors and nucleic acids which exosomes also carry Despite the protection provided by extracellular vesicles against RNA degradation, miRNA in plasma can pass through 0.22 μm filters and remain in the supernatant after ultracentrifugation, indicating the non-vesicular origin of a portion of extracellular miRNA 13There are multitudes of commercial RNA isolation kits available that serve their purpose adequately The gold standard for RNA quantitation is qRT-PCR and this applies to circulating RNAs as well. The required input for this assay is as low as a few nanograms of RNA which makes qRT-PCR attractive for low abundant cf-RNA detection. Whether based on Taqman, Locked-Nucleic-Acid or Sybr-Green technology, overall RNA-specific qPCR is sensitive, the specificity of the assay is high and results are obtained within a day. A relatively novel technology called Droplet Digital PCR  is described above and can also be applied to RNA. This analysis enables highly reproducible quantitation of low abundant RNAs. The limitations of RNA-specific qPCR are the low throughput, lack of suitable housekeeping gene normalizers and the inability of miRNA discovery.Broad gene expression arrays allow for higher throughput, as they can include several hundreds of target RNAs in a single assay. Arrays are either based on qPCR or hybridization technologies and are commercially offered by ABI, Agilent, Affymetrix, Exicon, Nanostring, Toray, MiRXES and Illumina among many other companies. These assays require 30–100 ng RNA input. It has been reported that qRT-PCR-based arrays performed better than hybridization platforms with respect to limits of miRNA detection For RNA discovery beyond detection of known target genes, RNA sequencing is necessary. However, cDNA library preparation may introduce sample bias. Deep sequencing with the use of small RNA-cDNA libraries is suitable for shorter RNAs, however for adequate mRNA and lncRNA transcript discovery, longer read sequencing may be more suitable.Recently, next-generation sequencing (NGS) has emerged as an unbiased alternative option with greater dynamic range of detection, increased sensitivity and reproducibility. NGS platform could overcome fundamental problems with array-based platform that rely on hybridization of RNAs to the pre-specified probes and rendered small dynamic range of detection as well as limitation in discovery of new ncRNA species. It is also important to realize that cross-comparison of ncRNA, especially miRNA, between different platforms remains problematic due to (i) enrichment of ncRNA species that is below or exceed the detection limit (ii) amplification bias and (iii) false positive detection from non-full length RNA sequencing. For example, NanoString miRNA detection platform that utilize solution-based hybridization and fluorescent-based barcode digital counting system showed only moderate correlation Validation of results obtained by any of the aforementioned methods is necessary and this is usually done with qPCR. Although collection of larger sample numbers are achieved in multi-institutional studies, acquiring robust data is usually problematic in this setting due to technical differences in blood processing, RNA isolation and quantitation methods. Strict methodological standardization must be applied to generate informative circulating RNA data.14Circulating cell-free RNA has a major potential as a cancer biomarker. A number of RNA species are deregulated as a result of the uncontrolled cell proliferation, stromal remodeling and immune regulation that define cancer. Distinct alteration in circulating RNA reflects dysregulation of cancer immunity, cell growth, proliferation and stromal interaction. Given the systemic nature of cancer, its biology should be studied in the context of the host response, which makes cf-RNAs suitable complementary tools. Besides the non-invasive nature of blood sampling, liquid biopsies allow for serial sample collection at different time points relative to treatments. This is particularly valuable with respect to the promising cancer immune therapy research that relies on the host response.15Circulating biomarkers development is a fledgling but rapidly growing field in cancer research. Circulating biomarkers will continue to evolve with ongoing improvements in detection limits, decreasing the amount of nucleic acids template, expanding the number genes available for analysis and reduction of the operating cost and time. An overall estimation of tumor characteristic with a snapshot of circulating nucleic acids is no doubt going to support treatment decisions and monitoring of cancer due to the dynamic nature of the disease and its heterogeneity. However, the major challenge in biomarker discovery is its validation in prospective clinical studies to assess their impact. Finally, until each of them is thoroughly validated and compared with standardized assessment for treatment response (i.e. RECIST criteria) and overcome problems of standardization, tumor-liquid biopsy discrepancies and lead-time bias, circulating biomarkers are still experimental and represent an interesting set of research tools.


Background. Natalizumab treatment is frequently discontinued and replaced by alternative medication in multiple sclerosis (MS) patients having a high risk of progressive multifocal leukoencephalopathy (PML). Case Presentation. We report a PML case that was missed on magnetic resonance imaging (MRI) at the time Natalizumab treatment was discontinued. The patient subsequently developed a PML-immune reconstitution inflammatory syndrome after the initiation of Fingolimod treatment, suggesting that immune reconstitution may occur even during Fingolimod induced lymphopenia. Conclusion. This report highlights the need for strict drug surveillance using MRI of Natalizumab-associated MS patients at the time of drug discontinuation and beyond. This is important with respect to pharmacovigilance purposes not only for Natalizumab, but also for alternative drugs used after Natalizumab discontinuation. Progressive multifocal leukoencephalopathy (PML) is a serious adverse event in multiple sclerosis (MS) patients treated with Natalizumab  . Many paHere we report a patient with an initially missed diagnosis of Natalizumab-associated PML at the time of drug discontinuation, who developed PML-immune reconstitution inflammatory syndrome (IRIS) after initiating Fingolimod treatment.A 52-year-old relapsing-remitting MS patient switched to Natalizumab treatment in 2011, due to a breakthrough of MS disease activity while being treated with Glatiramer . Due to a positive STRATIFY JCV antibody test (Unilabs) in the spring of 2013, Natalizumab was discontinued and Fingolimod initiated. At that time (22-04-2013), brain MRI showed a new, small lesion in the cortical grey matter of the right precentral gyrus, which, according to recently published PML imaging characteristics , 10, canCerebrospinal fluid multiplex quantitative polymerase chain reaction performed at the US National Institute of Health Laboratory of Molecular Medicine and Neuroscience on 11 September 2013 did not reveal JCV DNA copies. However, radiological characteristics and the disease course make alternative diagnoses very unlikely.Lymphocyte counts were measured before, during, and after Fingolimod treatment see , suggestNo cases of PML have been described in patients using Fingolimod monotherapy most likely because it clearly differs from Natalizumab in pharmacodynamic immune mechanisms of action and subsequently its safety profile. This case study documenting the development of PML-IRIS during Fingolimod treatment after Natalizumab discontinuation backs up our conviction that a suspicion of a possible Fingolimod-associated PML in this setting has to be analysed very carefully. Current Natalizumab surveillance programs use MRI as a sensitive screening tool for PML detection preferably in the presymptomatic/asymptomatic stage, which is associated with a better prognosis , 10. OurOur case may also imply that the PML-IRIS phenomenon seems to start independently of the usage of Fingolimod, suggesting that lymphocytic cellular host-defence against JCV reactivation might not be  impaired during Fingolimod treatment ; FingoliRebound disease activity after cessation of Natalizumab is well recognized and breakthroughs have been reported even in patients who switched to Fingolimod , 14. Our


PEDF is a secreted glycoprotein that is widely expressed by multiple organs. Numerous functional contributions have been attributed to PEDF with antiangiogenic, antitumor, anti-inflammatory, and neurotrophic properties among the most prominent. The discovery that null mutations in the PEDF gene results in Osteogenesis Imperfecta Type VI, a rare autosomal recessive bone disease characterized by multiple fractures, highlights a critical developmental function for this protein. This ultra-rare orphan disease has provided biological insights into previous studies that noted PEDF’s effects on various stem cell populations. In addition to bone development, PEDF modulates resident stem cell populations in the brain, muscle, and eye. Functional effects on human embryonic stem cells have also been demonstrated. An overview of recent advances in our understanding by which PEDF regulates stem cells and their potential clinical applications will be evaluated in this review. Serpinf1, is located on chromosome 17p13 and is widely expressed throughout many tissue sites [Pigment epithelium derived factor (PEDF) is a 50kDa secreted glycoprotein consisting of 418 amino acids that was first identified and isolated from the conditioned medium of cultured human fetal retinal pigment epithelium cells . A membeue sites ,4. The hue sites , but theue sites . In the Osteogenesis imperfecta (OI) type VI is an autosomal recessive disease with marked defects in bone mineralization and multiple fractures ,11. OI VSerpinf1, and encodes for PEDF [Serpinf1 have been reported, which result in complete absence of circulating PEDF [Serpinf1 gene cause retention or degradation of PEDF within cellular compartments and results in significantly diminished PEDF secretion with clinical manifestations resembling the null PEDF state [The causative single gene defect in OI type VI is for PEDF ,14–16. Sing PEDF ,14. In aDF state . In thesDF state –21. StudDF state –25. Thisin vivo transplantation also led to massive tumor growth suggesting that PEDF has context specific effects [Tombran-Tink and colleagues first identified PEDF in 1991 as a neuronal differentiating factor that could induce retinoblastoma (Rb) cells to adopt morphological and neuronal markers of a more terminally differentiated state ,26,27. T effects . This is effects . Many fu effects –33. Struvs. progenitor cell functional motifs at the current time are not considered the same region. Given the role of Wnt signaling in stem cell biology, future studies will need to be conducted to evaluate the effects on stem cell biology by different peptide fragments of PEDF.Based on these earlier discoveries regarding its function as a non-inhibitory Serpin, structure-function relationships were further characterized . Two dis1F0-ATP synthase, has been proposed to exert anti-angiogenic and anti-tumorigenic activity by inhibiting cell surface ATP synthase activity [Another interpretation of the diverse biological activities of PEDF stems from the multitude of cellular receptors proposed for PEDF and their cell type-specific localization. PNPLA2-PEDF-R, patatin-like phospholipase domain-containing 2 (PNPLA2), binds to 44-mer PEDF and promotes neuronal survival and differentiation by stimulating neuronal surface lipid metabolism ,39. Whetactivity . Additioactivity . Bindingin vivo by PEDF, these effects do not indicate that PEDF is unnecessary for regenerative processes such as wound repair. Rather, PEDF has specific temporal effects on the different stages of wound repair that are necessary for resolution of the wound-healing process [Ma and colleagues first reported that PEDF is an endogenous antagonist of low density lipoprotein receptor-related protein 6 (LRP6), and blocks Wnt ligand-mediated effects in retinal cells . LRP6 is process . This is process . At the in vivo are consistent with activation of Wnt signaling. We next turn our attention to the role of PEDF in stem cell biology and address potential therapeutic roles for PEDF involving regenerative potential.Work from our lab and others corroborate multiple functional outcomes that are consistent with a predominately Wnt inhibitory effect for PEDF. Among these is the inhibition of cellular proliferation and fibrogenic responses in the pancreas and liver that are consistent with previously reported effects of Wnt signaling on tissue fibrosis ,37,45–47Stem cells are undifferentiated cells that are capable of self-renewal and differentiation into mature, specialized cell types . BroadlyPEDF has been implicated as necessary for maintenance of ESCs in the undifferentiated state and for proliferation/differentiation of multiple types of tissue resident stem cells . In humaIn contrast to ESCs; adult stem cells are partially differentiated cells that are capable of differentiating into several cellular types of the tissue from which they originate. Studies have provided evidence that adult stem cells are indeed multipotent and not simply progenitors of different types of cells of their original tissue ,62. In tMesenchymal stem cells (MSCs) are a heterogeneous collection of multipotent progenitor cells, capable of differentiating into mesodermal cells such as osteoblasts, adipocytes, chondrocytes, or myocytes. Multiple proteins, hormones, and growth factors influence the lineage specification of MSCs to different cellular lineages from MSCs . Prior tMSCs are responsible for bone development and depend on signaling pathways such as Wnt signaling to induce osteoblast differentiation . ActivatConfirmatory studies from other groups demonstrated that PEDF could direct MSCs to the osteoblast lineage and regulate other genes involved in osteogenesis ,21. NiyiThe mechanisms by which PEDF promote mineralization of the bone matrix are becoming better understood. One mechanism appears to be that PEDF suppresses factors that inhibit mineralization such as sclerostin  and matrix constituents such as extracellular phosphoglycoprotein (MEPE). PEDF also enhanced β-catenin levels, the nuclear transcription factor for terminal Wnt signaling activation ,21. In oin vivo [Other mesoderm-derived tissue sites are also PEDF responsive. Skeletal muscle consists of multinucleated contractile muscle cells, called myofibers. The adult stem cells are satellite cells, which are mitotically quiescent and found in the basal lamina of myofibers. Satellite cells represent a pool of cells that can undergo proliferation in response to injury to form new myoblasts. Myoblasts then migrate to the site of injury, fully differentiate, and fuse to form new myofibers. Recently, PEDF and a synthetic peptide derived from PEDF (Ser93-Leu112) were shown to induce satellite cell proliferation and muscle regeneration in vivo . In a roin vivo . These dSelf-renewal of neural stem cells (NSCs) involves maintenance and proliferation of undifferentiated cells in response to signals contained in the local environmental niche. An overview of the stem cell niche identifies NSCs in two main regions of the adult brain: the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone of the dentate gyrus in the hippocampus ,72. At lIn vitro, PEDF or drugs that increase PEDF cause neurospheres to proliferate [in vivo whereas senescent NSCs appear unresponsive to PEDF.Experimental evidence demonstrates that PEDF supports the renewal of stem cells in the brain. PEDF injection into the ventricles stimulated Type B cell proliferation, which did not occur with injection of the PEDF C-terminus (aa195–400). Thus, stem cell properties reside in the N terminus of the PEDF protein. In contrast, PEDF depletion reduced NSC self-renewal . In vitrliferate ,77. Thesliferate . Interesliferate . Thus, PThese functional effects on neurogenesis by PEDF occur in conjunction with activation of developmental programs. For instance, Notch signaling promotes the self-renewal capacity of NSCs and maintains NSCs in an undifferentiated state . PEDF enIn vitro, PEDF significantly increased the number of large hippocampal neurospheres and impeded the differentiation of neurospheres, thereby maintaining multipotency. Neurosphere proliferation, however, occurred only in the presence of epidermal growth factor or fibroblast growth factor (FGF)-2, indicating that PEDF promotes an environment that is permissive for progenitor cell proliferation but does not induce stem cell proliferation alone [Clinically used drugs in neurodegenerative disorders modulate PEDF levels. For instance, PEDF increased stem cell populations in the hippocampus after injection of memantine, an NMDA receptor antagonist used in Alzheimer’s disease. Memantine injection led to strong PEDF expression in the adult hippocampus . In vitron alone . These din vivo through anti-angiogenic and pro-apoptotic effects [PEDF’s role in neurogenesis and neuronal differentiation may have therapeutic implications. Secretion of PEDF by somatic stem cells derived from the umbilical cord promotes neuronal maturation, pointing to the context specific effects of PEDF . In the  effects ,93. Althet al. transduced PEDF expressing MSCs through adenoviral vectors in vitro before sub-retinal transplantation into rats and showed that MSCs adopt RPE-like characteristics and promote the rescue of photoreceptor cells. Their findings indicated a role for PEDF in maintaining the RPE layer [Age-related macular regeneration (AMD) is a leading cause of blindness in the elderly population. AMD is characterized by degeneration and loss of retinal pigmented epithelial (RPE) cells and photoreceptor cells in the macular region . Recent PE layer . Other gPE layer . RemovalPE layer . PEDF, thttps://www.clinicaltrials.gov) and human neural stem cells  to protect photoreceptors in Retinitis Pigmentosa and dry AMD, respectively, are ongoing but newer studies using induced pluripotent stem cells (iPSCs) may be more efficacious. Here, iPSCs differentiated to RPE cells have been injected into models of macular dystrophy and retinal degeneration to assess effects on cellular function and photoreceptor integrity [These studies have clinical relevance. Clinical trials using human MSCs . LSCs are located between cornea and conjunctiva, and their asymmetric division generates transient amplifying cells that renew the corneal epithelium . Culturein vitro . In a coin vitro . Anotherin vitro .Identification of PEDF null mutations as the cause of OI Type VI has defined its importance as a regulator of bone development. A number of common clinical disorders are characterized by an osteodystrophy and alterations in PEDF levels. Whether PEDF regulates bone homeostasis in these diseases warrants further investigation. Further, the PEDF null state in humans has permitted a re-examination of PEDF’s previously described effects on stem cell biology through the framework of developmental pathways.


Brain oscillations reflect changes in electrical potentials summated across neuronal populations. Low- and high-frequency rhythms have different modulation patterns. Slower rhythms are spatially broad, while faster rhythms are more local. From this observation, we hypothesized that low- and high-frequency oscillations reflect white- and gray-matter communications, respectively, and synchronization between low-frequency phase with high-frequency amplitude represents a mechanism enabling distributed brain-networks to coordinate local processing. Testing this common understanding, we selectively disrupted white or gray matter connections to human cortex while recording surface field potentials. Counter to our original hypotheses, we found that cortex consists of independent oscillatory-units (IOUs) that maintain their own complex endogenous rhythm structure. IOUs are differentially modulated by white and gray matter connections. White-matter connections maintain topographical anatomic heterogeneity  and gray-matter connections segregate cortical synchronization patterns . Modulation of distinct oscillatory modules enables the functional diversity necessary for complex processing in the human brain. Rhythmic brain activity is routinely measured for neuroscientific inquiry and for clinical evaluation of disease. This oscillatory activity reflects changes in cortical potentials caused by a summation of firing in neuronal populations. Systematic changes in cortical oscillations accurately reflect cognitive operations and correlate with activity in neuronal populations  to a region of neocortex and recording local field potentials. First, we tested the hypothesis that low- and mid-frequency rhythms, notably μ/β rhythms between 8 and 23 Hz, predominantly arise from white-matter circuitry modulating local cortical activity  and did not alter phase/amplitude coupling. Disruption of surrounding gray matter connections did not significantly alter oscillatory power but substantively increased cross-frequency phase/amplitude coupling at a synchronized phase. When white matter and gray matter lesions were combined at the same site , endogenous oscillations and complex oscillatory cross-frequency relationships  were maintained, or enhanced. These oscillations persisted virtually unaltered for up to 10 min or longer even after cortex was removed. These findings suggest that cortex consists of independent oscillatory-units (IOUs) that maintain their own complex endogenous rhythm structure without the need for external neural input. IOUs are differentially modulated by their white and gray matter connections, where white-matter connections maintain topographical heterogeneity , and gray-matter connections segregate cortical synchronization patterns . Modulation of these distinct oscillatory modules allows for the functional diversity necessary for complex signal processing in the human brain.in vivo prior to their removal. The experimental surgical manipulations were performed on cortical regions that were planned for resection and there was no alteration in the standard-of-care of the surgical procedure. Experimental manipulations did not pose any significant additional risk to the human subject. Although valuable, animal data has inherent limitations because interspecies differences have been documented for cortical innervation, anatomy and pathology .Neurosurgical access to pathological tissues frequently requires resection of normal neocortex at the surface. This resection of normal tissue is necessary to allow for a direct corridor to access the pathology. In other specific clinical circumstances, surrounding normal tissue is resected along with pathological tissue. To take advantage of this unique opportunity, cortical oscillations were recorded from the sites of planned resection while performing selective white and gray matter transection  Figures . SelectiTen patients undergoing surgical treatment for intractable epilepsy or brain tumors participated in this study, which received ethical approval by the Washington University School of Medicine Department of Neurosurgery Ethical Review Board Committee and the Human Research and Protection Organization Institutional Review Board at Washington University. Before inclusion, all patients gave written informed consent. Neurosurgical access to pathological tissues often requires resection of normal neocortex at the surface. This resection of normal tissue allows for a direct corridor necessary to access the pathology. In other specific clinical circumstances, surrounding normal tissue is resected along with pathological tissue. Clinical decision making and initial selection of experimental candidates were performed independently by separate individuals . For each subject, the patient-tailored clinical operative strategy was created prior to and independently of experimental plans; hence, experimental protocols had no bearing or effects on the clinical care of each patient, and the risk of harm to patients was not increased by the experiments. Patients only qualified for the study if they were receiving a corticotomy and resection of cortical tissue  were used to acquire ECoG signals. For two subjects 64 platinum electrodes were used. Four-contact electrodes were used for the remainder of experiments. Electrodes were made of platinum, each 4 mm in diameter with 2.3 mm exposed to the cortical surface and spaced apart by 1 cm. Recordings were made between electrodes along the same gyrus.Each subject underwent a standard craniotomy under general sevoflurane anesthesia. Standard neurosurgical and neuroanesthesia practices were maintained to ensure similar craniotomy conditions. After removal of the cranial window and dural incision, the selected gyrus was exposed, ECoG electrodes were placed on the selected gyrus Figure  and locain vivo ECoG data were collected from cortical tissue under general anesthesia. Two distant cortical electrodes were selected as ground and reference contacts and subject's left lower extremity was grounded. All surgical manipulation and operating room activity were halted during recordings. Prior to any recording, exquisite care was taken to ensure excellent contact between the pia and electrode surfaces. Adequate signal voltage recordings and signal-to-noise ratios were confirmed by an online BCI2000 graphics user interface. For each recording, 6 min epochs were collected with minimal operating room activity.Intraoperative top; Supplementary Figure Human ECoG was recorded before and after white and gray matter disruptions. Selective white matter transections eliminated white matter inputs underneath cortical tissue while maintaining surrounding gray matter inputs. Meanwhile, gray matter transections eliminated superficial gray matter inputs/outputs to the cortex of interest while preserving white matter connections. Experiments were only performed on tissues that were to be resected from a clinical standpoint and experimental plans had no bearing on clinical plans and decisions. Pre-procedural magnetic resonance imaging was evaluated for recording site tissue quality, distance to pathology and thickness of cortex. All transections were performed by a board-certified neurological surgeon subspecialized in epilepsy and brain-tumor surgery. Prior to experiments reported here, surgical transections were optimized and perfected to ensure to consistent lesions. All transections were directly visualized with expanded-field surgical telescopes (3.5X magnification) and complete transections were confirmed following each experiment. Most subjects received both white and gray matter disruptions in a sequential order. For some subjects, operative plan allowed only for use of one electrode. Three selective lesions were performed: 1) white matter transection, (2) gray matter transection or (3) combined gray/white transections . White matter transection selectively and completely disrupted the gray-white junction under the electrode of interest without disrupting white matter under adjacent cortex and without disrupting surrounding gray matter. The precision of selective transections were confirmed post-hoc after experimental procedures. After baseline recording, a needle was used to puncture the pia matter adjacent to electrode #2 at four corners (1–4) surrounding the electrode . Four pinholes were created in the pia surrounding electrode #3. A blunt needle was bent at the tip to allow for subpial vertical transections similarly as described  which can identify white and gray matter similarly to traditional gross pathology specimens Figure . AccuratImmediately after white and gray matter transections, the pia was carefully incised and the disconnected cortex was placed into a custom chamber with room-temperature Lactated Ringer's solution . A 4-contact electrode was placed over the correctly-oriented cortex with the cortex under electrode #2 and the remaining electrodes in buffer. Electrodes 3 and 4 served as reference and ground, respectively. ECoG of freshly explanted tissue was recorded within 2 min of explanation. Operating room noise was measured by placing two 4-contract electrode strips into room temperature lactated ringers solution. After designating two of the eight electrodes and reference and ground, ECoG noise data were collected with equivalent operating room equipment and activity.in vivo recordings, the signal at each remaining electrode were rereferenced to a distant frontal cortical electrode to minimize common sources of noise from the signals. Ex vivo explant data was re-referenced to the remaining adjacent electrode. Operating room noise measurements were referenced similarly. All re-referenced ECoG voltages were then processed through digital 0.5 Hz high-pass and 60-Hz-notch band-stop digital butterworth-filters.All signal processing scripts were custom written in MATLAB, unless otherwise noted. Signals from every electrode were visually inspected and those electrodes identified as having predominately poor signal-to-noise characteristics (amplitude greater than 10x that of the majority of electrodes in the array) were excluded from further analysis. Less than 5% of data acquired were excluded due to poor signal-to-noise characteristics. Data were also excluded if there were incomplete transections , intraoperative inconsistencies or incomplete data. For Spectral analysis was done using the Welch's power spectrum density estimate method Welch, . Welch'sMutual information is a quantification of the information gained about a random variable X from measurement of a second variable Y  is the marginal probability density of time series X or probability that X = x in system X, H(X) is the marginal entropy of system X, and H(X|Y) is the information of X gained by measurement of Y. To assess effects of lesions on functional connectivity, mutual information of band-passed signal voltage magnitudes  and comparing magnitudes of change. A directional general change was considered significant when the number of experiments showing specific changes in mutual information exceeded the number expected by chance .Phase-amplitude coupling signal analyses was performed to study how the phases of low frequency oscillations modulate the amplitude of high frequency oscillations within a single region of cortex, similarly as previously described  is the individual raw phase amplitude modulation time series, A(t) is the analytic amplitude time series , and ϕ(t) is the phase time series.NORM is the normalized modulation index Z-score, MRAW is the mean of z(t), μ is mean of surrogate lengths and σ is their standard deviation.Where Mp = 0.0494, Z = 3.9). The low frequency phase at phase amplitude coupling represents the mean phase at every instantaneous amplitude-phase complex. A phase of zero represented a peak. Each mean phase was displayed on a rose plot and distributions of mean phases were plotted histograms to show “preferred” phase, or phase that is tied to maximum amplitude of high-frequency power. Kurtosis  were performed on balanced groups. Data from 7 of the subjects  allowed for accurate comparisons for balanced n-way ANOVA statistics. Two-factor interactions were calculated due to limited factor combinations. In order to control for inter-subject and inter-electrode variability, data were normalized to pre-lesion data. When significant effects were measured, planned s Sidak, :where m is the number of frequency band comparisons.Binomial probability of changes in power and mutual information vs. chance were calculated. The binomial probability of observing k successes in N trials was:P-values, after multiple comparison correction, degrees of freedom and F- and T-statistics are reported in the manuscript or supporting materials. Statistical values with p < 0.05 indicated statistical significance.Differences in preferred phase of PAC were assessed visually by plotting distribution of phase before and after transections on histograms and assessed statistically by comparing concentration parameters, κ, with circular statistics κ-test, which employs Bartlett's test  which can identify white and gray matter and also by detailed histological examination Figures .in vivo human cortical transections, it was imperative to confirm that the experimental tissue did not show abnormalities and were not affected by the pathology that the surgery was intended to address . To reduce the impact of disease on experimental results, candidates were only selected for this study if the site of each experimental recording would be performed a significant distance away from the pathological region. All of the operations required the unavoidable surgical resection of normal brain tissue in order to access the pathological region. Thus, the region of abnormality  was quite distant from the region tested in these experiments. Despite the notable distance from pathology, detailed radiological, and histological examinations of recording sites were performed. These experiments demonstrated that recordings were not performed on abnormal tissue. First, high-resolution magnetic resonance imaging of the brain was performed on each subject and T1-, T2-, and diffusion-weighted sequences were directly evaluated by a neuroradiologist for causes and consequences of epilepsy and other pathology. Radiographic examination of the recorded gray matter tissue showed no abnormal tissue in 10/10 subjects  = 1.59]. Post hoc Tukey's honestly significant difference analysis after ANOVA showed white matter lesions reduced PSD in 100% of δ-band frequencies but only 46% of γ-band frequencies tested. Analysis of band-passed power frequencies showed that white-matter disruption reduced PSD in all frequency bands . Finally, white matter transection reduced PSD in all subjects for δ, β, and γ frequency band . Each of these analyses showed that white matter transection reduced power in low and high frequency domains suggesting that white matter circuits contribute to power of cortical oscillations.Historically, lower frequency rhythms are thought to represent longer range circuits such as thalamocortical interactions  = 58.6], it had no significant effect on frequency-dependent PSD  or on PSD of canonical frequency bands . Hence, white matter transection reduced PSD in lower frequency power but also reduced gamma power as well. However, transections of surrounding gray matter had no significant effects on the power of both low- and high-frequency oscillations. This can be interpreted to suggest that gray matter circuits between cortex does not contribute to the power of local oscillationsAlthough gray matter disruption had a main effect on PSD  but not white matter disruption  significantly affected frequency-dependent PAC  = 1.02]. However, rose plots demonstrated that gray matter disruption synchronized phase for PAC, especially for β-γ resulting in a more strongly peaked distribution after gray matter disruption  vs. baseline  and reduced broad-band PSD in all experiments . Despite disruption of all input and output circuits, functionally-disconnected cortex had significantly more frequency-dependent cortical oscillations than experimental noise . In post-hoc analysis, PSDs of functionally disconnected cortex were significantly different from noise at all frequencies between 1 and 100 Hz. When binned into canonical frequency bands, functionally-disconnected cortex also had cortical oscillation PSD greater than noise in all bands . To further control for the potential effect of volume conduction from adjacent connected tissue, ECoG measurements were then recorded from extracted cortical brain tissue. Physical explant (and devascularization) led to a frequency-dependent reduction in power when compared to functional disconnection . Post hoc analysis showed all frequencies had population means significantly different from functional disconnection. When binned into frequency bands, explant's PSD was different from baseline in the θ band (pt = 0.04). Despite physical extraction, cortical oscillations from explanted cortex had significantly greater frequency-dependent PSD than noise  and, in post-hoc comparisons, PSD was greater than noise for β-θ frequencies. Explant's PSD was also greater than noise in all frequencies when binned into canonical frequency bands . Functional isolation of the cortex led to highly variable changes in functional connectivity resulting in insignificant shared information  and long range (white matter) connections had on the oscillatory behavior of cortex by their independent transection, we further characterized true local rhythms by combining these transections to see what rhythmic behavior remained after a full functional disconnection. We hypothesized that cortex, devoid of inputs, was still capable of generating cortical oscillations. Functional disconnection of cortex had a significant negative effect on PSD  in vitro  = 2.90] and when compared to sham . Post hoc comparisons showed that PAC was greater than baseline for β frequencies (17–25 Hz). Furthermore, post-hoc analysis showed that β frequencies in functionally disconnected cortex had significantly greater PAC than the 1 Hz δ frequency. Sham had no significant effect on frequency-dependent PAC . Similar to the gray matter disruption, complete functional disconnection of cortex synchronized phase of PAC . PAC was observed in 1/5 explanted specimens (Supplementary Figure Pharmacologically-induced cortical PAC has been observed in slice models in vitro slices (Kristiansen and Courtois, Selective lesions, functional disconnection and ultimately full explant demonstrate that mesoscale regions of cortex can independently generate complex oscillations and oscillatory relationships. Several previous studies support the existence of these IOUs. Amzica et al. observed cortical oscillations after disrupting deep circuits in anesthetized cats (Amzica and Steriade, For white matter lesions, the bands that show the most notable increases in NMI are also similar to those with the most reduced power. This relationship could represent relatively increased input from the lesioned area's cortical neighbor through gray matter connections. Numerous prior studies support this finding that white matter connections facilitate spatial heterogeneity. Amzica et al. showed that disconnection of intracortical synaptic linkages in anesthetized cats disrupts synchronization of slow oscillations (Amzica and Steriade, increase in shared information at the gamma band. The finding that information increases between separated regions at first seems counterintuitive, given that high-frequency rhythms are thought to represent local cortical circuits (Manning et al., It is also notable that when a cortical site's gray matter is circumferentially separated that there is an The mid-twentieth-century was notable for a debate over cortical vs. subcortical origins for rhythmic electrical activity of the brain (Kristiansen and Courtois, There are several limitations and assumptions in this study that deserve attention. First, volume conduction presents problems for the interpretation of functional connectivity measures between signals recorded from adjacent regions of cortex (Buzsaki et al., It is well-known that the cytoarchitecture of cortex varies across the human brain (Amunts et al., in vivo transections in the human brain demonstrates that mesoscale regions of cortex are independently capable of maintaining complex oscillatory behavior and the alteration of this activity and its cortical heterogeneity is differentially modulated by white and gray matter mediated circuits. This mechanism offers added insight into how the brain establishes functional diversity between adjacent cortical regions.In conclusion, AH, DB, and EL conceived and designed the experiments; AH and EL performed the experiments; AH, DK, NL, and SD analyzed the data; EL and DB supervised the research. All authors wrote the manuscript.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. The reviewer KS and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.


Drugs that simultaneously target multiple proteins often improve efficacy, particularly in the treatment of complex diseases such as cancers and central nervous system disorders. Many approaches have been proposed to identify the potential targets of a drug. Recently, we have introduced Space-Related Pharmamotif (SRPmotif) method to recognize the proteins that share similar binding environments. In addition, compounds with similar topology may bind to similar proteins and have similar protein-compound interactions. However, few studies have focused on exploring the relationships between binding environments and protein-compound interactions, which is important for understanding molecular binding mechanisms and helpful to be used in discovering drug repurposing.50 of 56 and 25 flavonoid-kinase inhibitions are less than 10 μM and 1 μM, respectively. Furthermore, these experimental results suggest that these flavonoids can be used as anticancer compounds, such as oral and colorectal cancer drugs.In this study, we propose a new concept of "Homopharma", combining similar binding environments and protein-compound interaction profiles, to explore the molecular binding mechanisms and drug repurposing. A Homopharma consists of a set of proteins which have the conserved binding environment and a set of compounds that share similar structures and functional groups. These proteins and compounds present conserved interactions and similar physicochemical properties. Therefore, these compounds are often able to inhibit the proteins in a Homopharma. Our experimental results show that the proteins and compounds in a Homopharma often have similar protein-compound interactions, comprising conserved specific residues and functional sites. Based on the Homopharma concept, we selected four flavonoid derivatives and 32 human protein kinases for enzymatic profiling. Among these 128 bioassays, the ICThe experimental results show that the Homopharma is useful for identifying key binding environments of proteins and compounds and discovering new inhibitory effects. We believe that the Homopharma concept can have the potential for understanding molecular binding mechanisms and providing new clues for drug development. Developing a new drug is difficult and takes on average of 13 years as well as US$1.8 billion -4. Some One challenge of targeted polypharmacology is to identify the molecular targets that bind the compound. To recognize the proteins with similar binding sites of a given protein sequence or structure, the computational approaches of searching the protein sequence or structure databases are usually utilized -21. As tin vitro enzymatic profiling. The experimental results identified that the IC50 values of 56 and 25 flavonoid-kinase inhibitions are less than 10 μM and 1 μM, respectively. Some novel protein-compound interactions also suggest that these flavonoids could be used as anticancer compounds such as oral and colorectal cancer drugs. These results show that the concept Homopharma is not only useful for identifying potential targets of compounds, but can also reveal the key binding environment. Furthermore, it would be helpful for discovering the new usages for existing drugs. We believe that this approach can be further applied to understand molecular binding mechanisms and take a new direction on drug discovery.To address this issue, we proposed a new concept of "Homopharma" to describe similar binding environments and the relationships of interactions between proteins and compounds. A Homopharma is a set of proteins which have the conserved sub-binding environment at the protein-compound interfaces and a set of compounds with similar topology. Our results demonstrated that protein-compound complexes of a Homopharma perform similar protein-compound interactions and comprise conserved specific residues and important functional sites. According to the Homopharma concept, four similar flavonoid derivatives were tested against 32 human protein kinases using In this study, we propose a new concept "Homopharma" to describe similar binding environment and the relationships of interactions between proteins and compounds. Proteins sharing similar binding environments can usually be targeted by a set of compound with similar topology. We considered that a Homopharma comprises of a set of proteins which have the conserved sub-binding environment at the protein-compound interfaces and a set of compounds with similar topology. For a complex structure, the proteins with similar pharma-interfaces are first recognized by SRPmotif. The complexes with significant compound topology similarity are reserved. Then, the protein-compound interaction similarity scores are measured. Consequently, the proteins and compounds which share similar protein-compound interactions are considered as a Homopharma. Figure SRPmotif is utilized to rapidly search potential targets having similar pharma-interfaces Figure . A pharmiGEMDOCK [A compound would bind to proteins having similar binding environments; as well as proteins often interact with compounds of similar topology. These protein-compound interactions provide the opportunities to explore the molecular binding mechanisms. After identifications of candidate complexes, similarities between all crystal ligands of complexes are calculated by atom pairs (AP) . The striGEMDOCK . DiffereAfter estimations of the protein-compound interaction profile, conserved interaction similarity scores between protein-compound interactions are evaluated. To measure the interacting conservation, the conservation weight of each position of multiple sequence alignment (MSA) is given as:i interacting with compounds with the interaction type k . The interaction similarity score between two protein-compound interactions (A and B) is defined as following:where N is the number of compound members of the pharma-interface and i Aand i Wis the interaction and the conservation weight in position i of the MSA, respectively.where Consequently, the protein-compound interactions are clustered by two-way hierarchical clustering. These interactions are divided into several Homopharma groups, which consist of similar binding environment, compound topology and interactions.To verify the Homopharma concept, protein-compound complexes were obtained from PDB. The complexes are eliminated when compounds are metal ions, surface compounds, cofactors, and small compounds (heavy atoms < 6). Two protein-compound pairs are grouped according to the same proteins or compounds. The protein-compound groups are filtered by number of complexes below 50% of all possible protein-compound combinations. Two groups which overlap with more than 50% protein or compounds are merged. Finally, 176 protein-compound groups that contain 1,325 complex structures including 672 proteins and 471 compounds are used as validation dataset.A protein-compound complex having significant compound similarity is considered as a candidate of a Homopharma. In order to decide the threshold of compound topology similarity, we collected 3,151 crystal complex structures of 957 proteins. The distribution demonstrates the relationships between percentage and compound topology similarity  curves were estimated to compare protein similarity, compound similarity and interaction similarity  complex  as the query complex. Due to specific expression of herpes thymidine kinase (TK) in herpesvirus-infection cells, TK becomes an antiviral target of current drugs, such as acyclovir and vidarabine. The identified pharma-interface contains five conserved pharma-motifs. Furthermore, the clustering of interaction profiles demonstrates protein-compound interactions can be separated into four Homopharma groups.The proteins and compounds in a Homopharma would share conserved interactions and similar physicochemical properties. Therefore, these compounds are often able to inhibit these proteins in a Homopharma. Figure The complexes in a Homopharma present similar binding modes and have the conserved interacting residues Figures  and 3C. Flavonoids are a class of plant secondary metabolites and have more than 5,000 kinds of derivatives. Flavonoid derivatives have been crystallized within more than 300 protein complex structures, such as protein kinases. Protein kinases play important roles in cell growth and signal transduction ,40. ProtIn this study, we use serine/threonine-protein kinase Pim-1 (PIM1) and quercetin complex  to identify pharma-interface and Homopharma groups. After recognitions of 975 protein-compound complexes by four pharma-motifs, six Homopharma groups are identified according to the interaction similarity Figures . The conHomopharma 4 also performs the conserved interactions to hinge motif Figure . HoweveriGEMDOCK; (2) interaction profiles were analyzed from the docking poses that have the lowest energy; (3) interaction similarity scores were measured to the PIM1-quercetin complex; (4) the kinases of the same Homopharma group have the priority with selection. Most kinases were selected from Homopharma 2, 4, and 5. Furthermore, some kinases which are either not considered potential targets or not searched by pharma-interface were also collected. Eventually, 32 protein kinases were chosen  using the "HotSpot" assay platform [These 32 tested kinases were divided into two Homopharma groups and the superimposed structures of two groups are presented in Figure 50 ≤ 10 μM) by in vitro enzymatic profiling. Among these 56 inhibitions, the IC50 values of 25 bioassays are less than 1 μM and these flavonoids can be considered as potential anticancer compounds. These results show that Homopharma is not only useful to identify potential targets of compounds, but also can reveal the key binding environments. We believe that the Homopharma concept is useful for understanding molecular binding mechanisms and providing new opportunities for drug repurposing.In this study, we propose a new concept of "Homopharma", combining similar binding environments and protein-compound interaction profiles, to explore the molecular binding mechanisms and drug repurposing. A Homopharma consists of a set of proteins sharing conserved binding environment and a set of compounds which have similar structures and functional groups. In a Homopharma, the compounds are often able to inhibit these proteins because their interactions and physicochemical properties are often consensus. Experimental results show that protein-compound complexes of a Homopharma often perform similar interactions in which formed by conserved binding residues (protein sites) and similar important functional groups (compound sites). According to the Homopharma concept, we successfully discovered 56 flavonoid-kinase inhibitions (ICThe authors declare that they have no competing interests.YYC, CTL, and JMY conceived and designed the experiments. YYC, JHT, KHL, and JMY implemented the materials/analysis programs. YYC, JHT, KHL, CTL, KCH, and JMY performed the experiments and analysed the data. YYC, JHT, KHL, CTL, and JMY wrote the paper.Click here for file


Our current HBV antiviral drugs are highly effective in suppressing viral replication, but they cannot cure the infection that is present in >2 billion people worldwide.2 The immune system of most of these people is capable of effectively controlling the acute phase of HBV infection. However, a proportion of people with HBV infection have persistent viral replication and chronic inflammation that predisposes them to cirrhosis and hepatocellular carcinoma (HCC).The treatment for hepatitis C virus (HCV) infection has been revolutionized by the introduction of highly potent antiviral drugs that can successfully cure most patients.2 The replicative episomal form of HBV DNA, called covalently closed circular (ccc) DNA, prevents current therapies, including antiviral drugs, from being curative. These drugs need to be taken indefinitely to prevent viral relapse.3A vaccine has been available since the early 1980s that is highly effective in preventing HBV infection, but it has no therapeutic efficacy for the 360 million people who are chronically infected with HBV and have persistent disease.3 All of these therapies primarily target the virus itself and indeed many, if not most of our anti-infective agents rely on interfering with microbial proteins or the microbial genome to interrupt the ability of the pathogens to replicate.Strengthened by the advent of HCV curative treatments, there is a huge interest in developing curative therapies for HBV infection. Potential new therapies for chronic hepatitis B include direct-acting antivirals, such as viral assembly inhibitors, gene silencing approaches and viral entry inhibitors.3 However, to date none of these treatments have shown great efficacy in curing HBV infection.A less explored avenue for the treatment of chronic infections, which may offer enormous potential, is targeting host cell factors that modulate cell signalling, innate or adaptive immune responses. Interferon therapy is an established method of modulating host responses to HBV infection and toll-like receptor agonists  are currently being tested.5 If this success translates to efficacy in clinical trials, infection can be eliminated along with the nidus for HCC development. Such a therapeutic intervention to combat chronic HBV infection has not been explored before. We discovered that gene-targeted mice lacking specific cellular IAPs (c-IAP1 and c-IAP2) were able to quickly and efficiently eliminate HBV-infected cells without causing overt collateral damage.4Recently, we developed a completely novel approach of inducing death of HBV-infected hepatocytes to eliminate the viral reservoir and cure infection in a small animal model.5IAPs are central critical regulators of a large number of cell-signalling pathways involved in the immune response, but also in regulating survival and cell death signalling downstream of death receptors. Our discoveries, potentially, had immediate therapeutic implications because IAP antagonists were already in clinical trials for the treatment of cancers. Birinapant is an example of an IAP antagonist currently being investigated in cancer clinical trials. We found that it effectively antagonised IAPs in hepatocytes and it promoted TNF-dependent elimination of HBV and cured infection in preclinical models.7 In contrast, other studies have suggested that TNF may abrogate HBV replication by deregulating hepatocyte nuclear factors.8 Recent work implicated a role for TNF and IFNgamma, produced by T cells, in promoting non-cytolytic control of chronic HBV infection by diminishing the pool of cccDNA.9HBV is considered a non-cytopathic virus and it may utilise diverse mechanisms to abrogate TNF-mediated antiviral responses to infection. Indeed, TNF siganling is hijacked by HBV to enhance NF-κB transcriptional activity, and promote cell survival and activation to facilitate viral replication.10 We observed no marked changes in c-IAP1 and XIAP levels in the liver during the first weeks after induction of HBV infection in our animal studies.4 Assessment of mouse c-IAP2 protein levels was not possible in these studies as there was no reliable antibody against this protein. Regardless of whether the levels of IAPs change during HBV infection, we found that c-IAP1 and c-IAP2 prevent the clearance of HBV infection.4 The therapeutic implications of this discovery were immediately tangible because IAP antagonists were already in clinical trials for other indications.TNF-mediated cell survival signalling is tightly regulated by IAPs, which function as ubiquitin E3 ligases via their RING domain.11 Multiple clinical cancer trials validated the therapeutic applicability of Smac mimetics to induce targeted TNF-mediated death of tumour cells.These drugs mimic the activity of an endogenous inhibitor of IAP function called Smac/Diablo. The small-molecule compounds called Smac mimetics were designed to mimic the inhibition of IAPs, antagonise their function and induce TNF-dependent cell death.5 In addition, the ability of the immune system to localise production of TNF at the site of infection may mitigate collateral damage.In a completely novel approach, we used the Smac mimetic birinapant to reroute the signalling activity of endogenous TNF away from NF-κB activation and towards cell death induction in HBV-infected hepatocytes. We exploited the vulnerability created by HBV – a dependence on TNF/NF-κB and we harnessed the activity of endogenous TNF to kill cells in the absence IAPs. In our immunocompetent mouse model of chronic HBV infection, the Smac mimetic birinapant promoted TNF-mediated apoptosis of infected hepatocytes. Moreover, our study showed that birinapant preferentially killed infected cells over uninfected cells due to the vulnerability created by the virus.6 TNF and other pro-inflammatory cytokines in turn increase stability of the HBV X protein and they increase NF-κB signalling, which is essential for HBV replication.7 But the molecular details and mechanism of how HBV replication affects cell signalling and regulates induction of apoptosis or necroptosis warrant further investigations.Our Smac mimetic therapeutic approach takes advantage of an Achilles heel in HBV’s attempt to utilise TNF to promote host cell survival and NF-κB activity. HBV X protein was shown to upregulate TNF expression.12 We would speculate that additionally Smac mimetics could promote killing of the reactivated HIV-infected cells.Another virus that has hijacked NF-κB signalling is HIV. In this case too much or too little NF-κB activity can cause problems for the host. HIV requires NF-κB for viral replication, but in the absence of sufficient. NF-κB activity HIV can adopt a quiescent latent integrated form. Latently infected cells are a dormant viral reservoir of HIV that can reseed the viral pool as soon as conventional antiviral therapy is stopped, thus preventing HIV cure. If all latently infected cells could be reactivated and killed, then the viral reservoir could be eliminated. It was recently shown that Smac mimetic-mediated depletion of c-IAP1, which is also a repressor of non-canonical NF-κB signalling, could activate transcriptional activity and reverse HIV-1 latency.5Our work demonstrated that we are able to beat the HBV virus at its own game. In a completely novel approach, we were able to promote TNF-mediated apoptosis of HBV-infected cells by taking advantage of the viruses addiction to TNF.