Dendroctonus rufipennis) outbreaks are rapidly spreading throughout subalpine forests of the Rocky Mountains, raising concerns that altered fuel structures may increase the ecological severity of wildfires. Although many recent studies have found no conclusive link between beetle outbreaks and increased fire size or canopy mortality, few studies have addressed whether these combined disturbances produce compounded effects on short-term vegetation recovery. We tested for an effect of spruce beetle outbreak severity on vegetation recovery in the West Fork Complex fire in southwestern Colorado, USA, where much of the burn area had been affected by severe spruce beetle outbreaks in the decade prior to the fire. Vegetation recovery was assessed using the Landsat-derived Normalized Difference Vegetation Index (NDVI) two years after the fire, which occurred in 2013. Beetle outbreak severity, defined as the basal area of beetle-killed trees within Landsat pixels, was estimated using vegetation index differences (dVIs) derived from pre-outbreak and post-outbreak Landsat images. Of the seven dVIs tested, the change in Normalized Difference Moisture Index (dNDMI) was most strongly correlated with field measurements of beetle-killed basal area (R2 = 0.66). dNDMI was included as an explanatory variable in sequential autoregressive (SAR) models of NDVI2015. Models also included pre-disturbance NDVI, topography, and weather conditions at the time of burning as covariates. SAR results showed a significant correlation between NDVI2015 and dNDMI, with more severe spruce beetle outbreaks corresponding to reduced post-fire vegetation cover. The correlation was stronger for models which were limited to locations in the red stage of outbreak (outbreak ≤ 5 years old at the time of fire) than for models of gray-stage locations (outbreak > 5 years old at the time of fire). These results indicate that vegetation recovery processes may be negatively impacted by severe spruce beetle outbreaks occurring within a decade of stand-replacing wildfire.Spruce beetle ( Climate-related disturbances in North American forests have been increasing in frequency and extent in recent decades . In the Dendroctonus ponderosae), spruce beetle (Dendroctonus rufipennis), western balsam bark beetle (Dryocoetes confusus), Douglas-fir beetle (Dendroctonus pseudotsugae), and pinyon Ips beetle (Ips confusus) [One of the most pressing research questions related to multiple disturbances in western forest systems is whether severe bark beetle outbreaks can increase the ecological severity of subsequent wildfires –10. Millonfusus) . These ionfusus) ,13. The onfusus) ,15. Spruonfusus) .Picea engelmannii). P. engelmannii typically co-occurs with subalpine fir (Abies lasiocarpa) at elevations ranging from about 2,850–3,500 m a.s.l. [In the southern Rockies (southern Wyoming to northern New Mexico), the primary spruce beetle host species is Engelmann spruce  after fires have damaged physical evidence of beetle activity . AlthougAn additional challenge in assessing the relationship between outbreak severity and fire impacts is that a number of contingent factors may affect the nature of the disturbance interaction. These factors may complicate the effect of beetle outbreaks on fuel structures, alter the effect of fuel structure on fire behavior and burning intensity, or may affect vegetation recovery independently from fuel structure. First, the fuel structure of beetle-killed stands changes with time following the initial outbreak. Older beetle-killed stands contain greater amounts of downed woody material and ladder fuels from sapling regeneration, which allow faster surface spread and increase the probability of fire spreading to the crown . HoweverSeveral recent studies have assessed impacts of beetle outbreaks on fire severity, and many have found no evidence of a conclusive link between disturbances –43,46–52To determine whether spruce beetle outbreak severity shows an effect on short-term vegetation recovery from fire, we used the Landsat-derived Normalized Difference Vegetation Index (NDVI) to assess understory vegetation recovery two years after a large, high-severity wildfire. NDVI provides an indicator of grass and herbaceous cover in early recovery stages ,45,56. WThe West Fork Complex fire burned from June 5 –July 6, 2013. The complex consisted of three lightning-caused wildfires: Papoose , West Fork , and Windy Pass 573 ha). A total of over 44,000 hectares of subalpine spruce/fir forest in the Rio Grande National Forest, San Juan National Forest, and private lands northeast of Pagosa Springs, Colorado, were burned . Fire sp3 ha. A tP. engelmannii and A. lasiocarpa, with some lodgepole pine (Pinus contorta), quaking aspen (Populus tremuloides), and Douglas fir (Pseudotsuga menziesii). Elevation ranges from ~2700–4000 m a.s.l. with steep slopes and rugged topography. Mean temperatures range from -7.80°C in January to 11.50°C in July, and annual precipitation is 95 cm . Significant spruce beetle activity within the burn perimeter was first detected by ADS in 2004 . The outbreak was very severe, affecting more than 80% of spruce/fir forest within the study area by the time of the 2013 fire.The burn area was dominated by Processing steps for Landsat images and other explanatory variables are outlined in 2 ≥ 0.86.Although the images were pre-processed to eliminate atmospheric biases between image dates, slight band differences between Landsat 7 ETM+ and Landsat 8 OLI may result in systematic biases between images collected with the different sensor types. In order to eliminate this bias when comparing images, we applied a normalization technique to the 2015 image using pseudo-invariant features (PIFs). We manually selected 40 PIFs as single pixels representing non-vegetative features where reflectance should be constant between image dates . A linear regression calculation between each image band was used to adjust the 2015 image according to the method described by Schott et al. . In all We assessed the ability of Landsat-derived VIs to approximate spruce beetle severity using field measurements of beetle-caused spruce mortality. In August of 2015, we collected measurements in 58 unburned, beetle-affected spruce/fir plots within ~5 km of the West Fork Complex burn perimeter see . FifteenP. engelmannii trees with evidence of recent beetle activity within each 20 x 20 m plot area, and converted these measurements to total basal area. Basal area of beetle-killed trees within plot areas (400 m2) was our selected metric of beetle outbreak severity, and was assumed to estimate total change in canopy cover from pre-outbreak to post-outbreak. Our metric of beetle severity is therefore an absolute value of beetle-killed P. engelmannii basal area per 400 m2 (20 x 20 m plot area) rather than a percentage of total canopy. Standing dead trees with no evidence of beetle activity were small in diameter, and we assumed that these trees did not significantly affect the spectral changes resulting from beetle outbreak.We used a handheld GPS to place plots within ~3 m of the center of a 2 x 2 Landsat pixel grid (60 x 60 m). We measured diameter at breast height (DBH) of all dead We tested seven VIs which have been shown to respond to canopy disturbance: the Normalized Difference Moisture Index (NDMI) ,61, Norm2002). We compared these image differences to field measurements of beetle-caused overstory mortality by calculating the means of dVI values extracted from the 2 x 2 (60 x 60 m) pixel grid area surrounding field plot centers. We used mean values to account for potential spatial inaccuracies in the GPS location of the plot and overlay with the Landsat grid. Relationships between dVIs and plot-level values of basal area of beetle-killed trees were assessed using ordinary least squares (OLS) regression. The dVI which yielded the highest OLS R2 value was assumed to be the best indicator of beetle outbreak severity, and the difference in the selected index from 2002 to 2012 (VI2012 –VI2002) was included as an explanatory variable in post-fire NDVI models.For each VI, we calculated a multi-date image difference by subtracting 2002 pre-disturbance values from the 2015 value . TPI is a numeric indicator of slope position, with higher values representing locations closer to ridgetops and lower values representing valley bottoms . Aspect NDVI models included variables accounting for daily weather conditions over the two-week burn period. This was done using daily burn perimeter maps, which we obtained from the USGS Geospatial Multi-agency Coordination (GeoMAC) Wildland Fire Support service . Each ofWe determined outbreak stage using the earliest year of spruce beetle detection from ADS data. Using annual ADS extents for all years since 1994, we determined that 2004 was the earliest year when significant spruce beetle activity was mapped within the study area. Polygon areas with detection years 2004–2008 were classified as gray-stage, while polygons with detection years 2009–2013 were classified as red-stage . These aExplanatory variable layers were clipped to areas of spruce/fir forest cover type which burned at high severity. The spruce/fir forest cover mask layer was derived from the Southwest Regional Gap Assessment (SWReGAP) land cover classification , using cThe effects of spatial autocorrelation must be taken into account when assessing patterns in a contagious disturbance such as wildfire, because spatial dependence in either the response or the explanatory variables can violate assumptions of observation independence and inflate parameter values ,73. Vari R 3.3.1  using th R 3.3.1 .2015. The best-fitting model based on Akaike’s Information Criterion (AIC) value was used to select the scale for each variable to be included in the final multivariate model. This scale selection process ensures that the explanatory power of each variable is maximized in the final multivariate model [To further account for the spatially connected nature of wildfire, we considered how variable scale may affect relationships between vegetation recovery patterns and explanatory variables. Wildfire is a rapid-spreading, contagious process, and fire behavior is likely to be influenced by topographic and fuel characteristics over a broader area than that covered by a 30-m Landsat pixel. Because fire behavior influences the degree of fuel consumption and burning intensity across the landscape, and can ultimately influence patterns of vegetation recovery , we expete model ,78.After determining the best-fitting scales for explanatory variables, we used a stepwise selection procedure to select a model from a full set of explanatory variables. This full model can be described by the following equation:We selected the combination of variables which minimized AIC value for both the red and gray stage. Relative importance of each variable to the final model was determined by removing variables from the final selected model and calculating the change in AIC (ΔAIC).2 values indicated that dNDMI was the index most strongly correlated to field-measured basal area of beetle-killed spruce  are correlated with lower vegetation cover in 2015. This relationship is negative in gray-stage models.Univariate relationships between explanatory variables and NDVI2002 is the strongest single-variable predictor in both red and gray-stage models, based on AIC value. All topographic variables are significant in both subsets, while weather variables are not consistently significant. Topographic variables were selected at greater spatial scales, either at 150 m or 300 m. In the gray-stage subset, all topographic variables predicted NDVI2015 more accurately than dNDMI. In the red-stage subset, dNDMI was a more accurate predictor than either slope or TPI, or any weather variable.NDVI2015, and included all explanatory variables except air temperature and wind speed. The top-performing model for red-stage locations explained 68% of variance in NDVI2015 and included all explanatory variables except humidity and wind speed. Variable importance calculations showed that NDVI2002 is by far the most important variable in both red and gray-stage models  than in the top-performing gray-stage model (β = 0.25 ± 0.015), indicating that dNDMI has a greater influence on NDVI2015 in the red-stage model.Parameter estimates for the top-performing multivariate SAR models revealed variable relationships similar to those determined by univariate models . dNDMI i2015 matched that of the multivariate relationship in red-stage models but was reversed in gray-stage models, possibly indicating that the compounded disturbance effect becomes less significant with increasing time between disturbances. However, when all relevant variables were accounted for there was a consistently negative relationship between beetle outbreak severity and NDVI2015 in both stages. Although dNDMI was a less significant predictor of NDVI2015 than NDVI2002 (according to ΔAIC), the significant correlation between the indicator of beetle severity and NDVI2015 suggests the presence of a compounded disturbance effect on the rate and trajectory of vegetation recovery.Because dNDMI is negatively correlated with spruce beetle outbreak severity, the results of multivariate SAR models indicate that recovery of NDVI in the West Fork Complex fire was negatively correlated with the severity of spruce beetle outbreaks which occurred in the decade or so prior to the fire. The direction of the univariate relationship between dNDMI and NDVIOur results add a new dimension of understanding to those of recent studies which have found no correlation between outbreak severity and subsequent fire severity when accounting for differences in outbreak stage, burning conditions, or topography . Most ofThis difference in linked vs. compounded disturbance effects can be seen when comparing the results of our study to the findings of Andrus et al. , which aP. engelmannii and A. lasiocarpa are shade-tolerant species, meaning that mature stands become stocked with ladder fuels which incur a high probability of active crown fire [Effects of spruce beetle outbreak on regeneration processes may be a more significant ecological impact than effects to canopy loss, due to the typical high severity of fires in subalpine forests. High canopy mortality is expected in subalpine systems because climatic conditions typically make fires infrequent, and the long interval between fire results in dense fuel stocking . P. engeown fire . They arown fire ,79. Becaown fire ,80, it iown fire .Past studies have assessed pre-fire beetle mortality in the field after fire has occurred, which requires close examination of all trees within a field plot for larval galleries beneath the bark. This is a time-consuming process, and may also be prone to underestimation of mortality when the bark and wood surface have been damaged by fire . DiffereWe observed that spruce cover was high in most of our study area and that high abundance of subalpine fir was restricted to flat valley bottoms, which made up a low proportion of the total area. Our severity quantification method was therefore focused on spruce-dominated stands . Consideration should be taken in applying the dNDMI severity quantification method to areas with more mixed forest communities, as it is possible that growth in secondary species between image dates could cause dNDMI to underestimate spruce mortality . In our 2002 in models of NDVI2015 indicates that some factors relating to site greenness are unaltered by fire. Differences between pre-disturbance and post-disturbance NDVI may be the result of alteration of soils and microclimate which affect the ability of understory species to re-establish.Previous wildfire recovery studies have shown that NDVI tends to increase rapidly in the two years following fire occurrence ,45. NDVIIt is important to note that understory recovery is not necessarily an indicator of overstory regeneration . However2015 in red and gray stages reveals that in the gray stage, the effect of beetle severity is mediated to a greater extent by other explanatory variables. This difference may indicate that the effect of beetle-caused canopy mortality on fire impacts diminishes over time. This may be due to the fact that fine surface fuels decompose or are lost from the site after the initial outbreak [Comparing univariate to multivariate relationships between beetle outbreak severity and NDVIoutbreak . Canopy outbreak ,85.Multivariate models indicated that outbreak severity has a significant influence on post-fire recovery, but did not have a greater effect than topography or pre-disturbance NDVI. Topography is important in influencing fire behavior and micro-climate conditions which can promote or impede vegetation recovery ,86. The Weather within daily burn perimeters did not play a significant role in predicting NDVI recovery in our models. This result is not unexpected due to the coarse resolution of the data, where the entirety of a daily burn area was attributed with a single value of mean air temperature, humidity, and wind speed. Weather factors at the time of burning certainly play a role in the spread of fire and consumption of vegetation and litter, but in this study weather did not appear to have a strongly significant influence on soil alteration and post-fire recovery. This may be due to the coarse scale of weather data applied to daily burn extents, or because the majority of the study area burned under extreme conditions beyond a threshold where weather may have become more significant.Warming climates are resulting in a shift toward large, high-impact wildfires occurring at greater frequency throughout western North America , and theImpacts of severe beetle outbreak on vegetation recovery also create additional need for enhanced post-fire restoration efforts in areas where outbreak was known to have occurred prior to burning. Our model results indicate that these efforts should prioritize high-elevation, steep, south-facing slopes, as these topographic factors also show a significant effect on vegetation recovery. Restoration of ground vegetation mitigates flooding hazards, prevents soil erosion, and mitigates rising soil temperatures and evapotranspiration potential . FacilitAlthough many studies have tried to determine whether bark beetles lead to larger, more frequent, or more severe wildfires, there have been a number of limitations to determining the true ecological impacts of these overlapping disturbances. Our study quantified pre-fire beetle impacts using the Landsat-derived dNDMI, which likely provides a more accurate measure of beetle severity compared to studies focused on post-fire field measurements. We also used Landsat-based measurements of NDVI recovery to address how beetle-fire interactions may result in compounded effects on surface fuels and soil recovery, and found more conclusive evidence supporting a compounded disturbance interaction compared to studies which have assessed fire severity as a metric of canopy mortality. Future research should focus on long-term examinations of recovery dynamics following wildfires in beetle-killed forests, which will be important for improving understanding of how compounded disturbance interactions from bark beetles and wildfire will affect future forest communities. Additionally, future high-severity fires in beetle-killed spruce forests will need to be studied to determine whether the compounded effects observed in the West Fork Complex are consistent across geographic areas. Although many recent studies have concluded that there is no evidence of a link between beetle outbreaks and increased fire severity, our results indicate that the combined disturbances may result in compounded effects on vegetation recovery.


Bruton’s tyrosine kinase (BTK) is a critical terminal enzyme in the B-cell antigen receptor (BCR) pathway. BTK activation has been implicated in the pathogenesis of certain B-cell malignancies. Targeting this pathway has emerged as a novel target in B-cell malignancies, of which ibrutinib is the first-in-class agent. A few other BTK inhibitors (BTKi) are also under development . While the predominant action of BTKi is the blockade of B-cell receptor pathway within malignant B-cells, increasing the knowledge of off-target effects as well as a potential role for B-cells in proliferation of solid malignancies is expanding the indication of BTKi into non-hematological malignancies. In addition to the expansion of the role of BTKi monotherapy, combination therapy strategies utilizing ibrutinib with established regimens and combination with modern immunotherapy compounds are being explored. Bruton’s Tyrosine Kinase (BTK) is a critical terminal kinase enzyme in the B-cell antigen receptor (BCR) signaling pathway. The BTK gene is located on the X chromosome Xq21.33-Xq22) and was first described in the setting of X-linked agammaglobulinemia, a disorder characterized by a lack of maturation and development of B lymphocytes and gammaglobulins due to a mutation in BTK .33-Xq22 . Subsequ50) of BTK of 0.5 nM [Bruton’s Tyrosine Kinase is a member of the Tec tyrosine kinase family and is involved in BCR signaling . BCR actf 0.5 nM . Sequencf 0.5 nM .Chronic activation of the B-cell Receptor pathway is a key driver of B-cell non-Hodgkin lymphomas and hence, downstream blockade of this pathway utilizing BTK inhibitors (BTKi) in these diseases has proven very effective to date. Chronic lymphocytic leukemia (CLL) was the first malignancy where ibrutinib has become a critical component of standard therapy . IbrutinWhile the predominant mechanism of action of BTKi is blockade of the B-cell Receptor pathway within malignant B-cells, increasing knowledge of off-target effects as well as a potential role for B-cells in proliferation of solid malignancies is expanding the indication of BTKi beyond B-cell malignancies. Research has extended into the sphere of non-hematological malignancies where BTKi are currently being explored. These other potential targets are not altogether unexpected with BTKi given the established effect on multiple kinases central to cancer growth. There is precedent for such repurposing in other malignancies. The prototype example of this is imatinib, a drug originally developed for the breakpoint cluster region/Abelson murine leukemia viral oncogene homolog 1 (BCR-ABL)—driven chronic myeloid leukemia, being found to be active against mast/stem cell growth factor receptor (KIT) driven gastrointestinal stromal tumors (GIST) . In addiIbrutinib activity has been explored in several tumor types either as a single agent or in combination with other compounds. Clinical and pre-clinical data by tumor type will be discussed by tumor type and current clinical trials are summarized in EGFR) mutated non-small cell lung cancer (NSCLC) comprises approximately 15–20% of non-small cell lung cancer in white populations with higher rates in the order of 22–62% reported in Asian populations [T790M resistance mutation (more than 50% of cases) [EGFR-mutated NSCLC. It has been shown to have high activity against EGFR mutated (L858R) cells, with moderate activity to T790M mutated cells [EGFR kinases through formation of a covalent bond with Cys797 similar to other irreversible EGFR TKIs such as gefitinib and erlotinib [EGFR mutant cell lines was similar to that of erlotinib in one comparative study, but also with preserved activity against T790M mutated cell lines which were not sensitive to erlotinib [EGFR expressing lung cancer cells. Tumor progression was slowed by ibrutinib in xenograft mouse models, however not to same extent as seen with gefitinib [T790M mutation mouse model. A combination of ibrutinib and trametinib (a BRAF inhibitor) showed promise in cell line research, however did not translate to a benefit in xenograft mouse models [EGFR-mutant NSCLC . Its future role in this subtype of lung cancer is unclear given the encouraging data of osimertinib in this clinical cohort [Epidermal growth factor receptor  ,24. Ibrued cells ,26. Subsed cells . A more rlotinib . This inefitinib . Ibrutine models . One rese models . Based ol cohort .Breast cancer is the most common cause of cancer death in women globally . It is aA novel isoform of BTK (BTK-C) has also been described as expressed in several human breast cancer cell lines and tumor tissues at relatively low levels, yet still providing an important function in protecting breast cancer cells from apoptosis . One stuMetastatic gastric cancer and esophageal cancer is associated with a poor prognosis despite the availability of several cytotoxic regimens. Approximately 15% of Western patients with adenocarcinoma overexpress HER2, with trastuzumab being an option to incorporate into first line treatment in this setting . There iBTK over-expression has been described in gastric carcinoma tissues and gastric carcinoma cells . In compMYC amplified esophageal tumor lines and this genetic dependency could be demonstrated with ibrutinib. Ibrutinib elicited G1 cell cycle arrest and apoptosis in both MYC and HER2 amplified tumors, suggesting this drug could be used to treat biomarker-selected groups of patients with esophageal cancer. MYC amplification is described in 32% of esophageal adenocarcinomas and 23% of squamous cell carcinomas [To identify new biomarker directed therapeutic approaches in esophageal cancer Chong et al. integrated genomic profiles of 17 esophageal tumor-derived cell lines with drug sensitivity data from small molecule inhibitor profiling . Sensitircinomas . This rePancreatic cancer is a highly aggressive malignancy, with pancreatic ductal adenocarcinoma (PDAC) accounting for 85% of cases. It is associated with a high mortality rate and is the fourth leading cause of cancer deaths in Europe . BTK sigSox-2 and BCL-XL genes. Cell viability testing was performed after treatment with cisplatin alone, ibrutinib alone or a combination of both. Synergistic effects of the combination of ibrutinib plus cisplatin were demonstrated on serous and clear cell types of ovarian cancers. The synergistic effect suggested ibrutinib potentially sensitizes cancer cells to platinum, and it was postulated that each drug also had independent activity. The authors of the study concluded that ibrutinib may be a candidate drug to attempt to overcome platinum resistance in clear cell and malignant cystadenocarcinoma, via an on-target BTK effect.Ovarian cancer is the second most common gynecological malignancy in developed countries . PlatinuAs with other adenocarcinomas, BTK expression has also been described in prostate cancer cells, with the BTK-C isoform predominantly expressed . In prosGlioblastoma multiforme (GBM) is the most common primary brain tumor, with currently no effective targeted therapies. Chemoradiotherapy is central to the management of this disease, however, prognosis remains poor, with limited cytotoxic choices due to the necessity of blood-brain barrier penetration of any therapeutic. The prognostic significance of BTK expression in glioma has been studied, with high expression of BTK proposed as a novel prognostic marker . BioinfoCurrent treatment options for metastatic renal cell carcinoma (RCC) include molecularly targeted agents or immunotherapy. Molecularly targeted agents include drugs that predominantly focus on vascular endothelial growth factor (VEGF) pathways—such as sorafenib, sunitinib, pazopanib, axitinib and cabozantinib or agents that target the mechanistic target of rapamycin (mTOR) pathway such as everolimus and temsirolimus . EstabliIbrutinib has entered clinical care for multiple lymphoid malignancies, and the toxicity is well established. Other next-in-class BTKi have similar adverse event profiles. Unlike the majority of multi-target tyrosine kinase inhibitors, ibrutinib causes minimal rash, only modest gastrointestinal and rheumatological toxicities, with infection, myelosuppression, cardiac arrhythmias, and anti-platelet activity the predominant toxicities. These side effects are proving to be manageable.In lymphoid malignancies, BTKi have been safely combined with multiple different chemotherapeutic regimens and monoclonal antibodies such as rituximab and ofatumumab ,57,58.The known off-target effects of BTKi, in addition to the role of inhibiting host immune B-cells in the tumor microenvironment married with the attractive side effect profile, and knowledge of safe use with multiple other therapies provide good rationale for novel combinations in solid tumors.As mentioned above, ibrutinib synergizes with chemotherapy in pre-clinical models of pancreatic, ovarian, and neurological cancers. It also potentially synergizes with other agents such as checkpoint inhibitors—such as PD-1 inhibitors. In healthy cells, PD-1 blockade inhibits B-cell receptor mediated signaling, and more understanding of this interaction in malignant cells is required .The tumor microenvironment is a complex process where many tumors actively suppress various immune mechanisms to protect the tumor. Tec family kinases play a role in regulating the tumor microenvironment.Ibrutinib inhibits (ITK), an enzyme important for the survival of Th2 T cells and therefore might have immunomodulatory effects in addition to its BTK activity by shifting the balance between Th1 and Th2 T cells . The shiEmerging data from various mouse models of cancer suggest that ibrutinib may have immunological effects by affecting myeloid-derived suppressor cells. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous group of immature myeloid cells and expansion has been linked to loss of immune effector cell function and reduced efficacy of immune-based cancer therapies . MDSCs cCombination of Ibrutinib and anti-PD-L1 antibody suppressed tumor growth in mouse models of lymphoma that are intrinsically insensitive to ibrutinib . The comBTK inhibitors are an established therapy in the treatment of numerous lymphoid malignancies. However, pre-clinical studies with BTKi in non-B-cell cancers show efficacy due to numerous mechanisms. Some effects are attributed to BTK signaling pathways, such as the inflammatory stroma of pancreatic cancer previously described and have furthered our understanding and knowledge of these processes. In several tumor subtypes such as lung, breast and esophageal, BTKi efficacy appears to be via other intracellular signals with a corresponding cysteine residue in the ATP binding site demonstrating that ibrutinib is not entirely specific in its binding to BTK. Relative binding affinity of ibrutinib to signals within 10 other kinase pathways  has been described . The posThe growing evidence of BTKi activity on signaling pathways beyond the original intended clinical application of BTK signal blockade highlights the importance of ongoing thorough laboratory research in testing of compounds to evaluate their full clinical potential. Pursuit of such research beyond drug registration for one class of diseases is necessary to ensure potential targets are identified and exploited. Such drug development has been shown to lead to clinically meaningful repurposing of established therapies. Haura and Rix describe potential methods to achieve this . They prThe data demonstrating BTKi activity in non-hematological malignancies has been predominantly pre-clinical to date with only a small number of clinical studies published. Given the mechanisms of action vary in the varying tumor subtypes, future evaluation of BTKi in solid malignancies will require tumor specific trials, as the rationale of combinations will differ. Ibrutinib is the most established of the BTKi, and next-in-class novel BTK inhibitors continue to develop. These include acalabrutinib (ACP 196), tirabrutinib (ONO/GS 4059), zanubrutinib (BGB 3111), spebrutinib  and CGI-1746. These compounds have different affinities and hence likely different actions than those demonstrated with ibrutinib. Acalabrutinib, a second generation BTK inhibitor, has been granted accelerated FDA approval based on preliminary results of a phase II trial in mantle cell lymphoma (NCT02213926). Little is known about the diversity of action of these novel BTKi on other intracellular signaling pathways. Based on the knowledge of ibrutinib, these newer agents will also require ongoing studies.Ibrutinib has demonstrated encouraging preclinical activity in a wide range of non-hematological malignancies. Some of these effects relate directly to BTK signaling, furthering our understanding of this complicated pathway and its multitude of effects in cell physiology and pathology. Yet many studies have proven BTKi activity in a multitude of other kinase pathways reliant on the cysteine residue binding and necessary for cancer cell survival. This knowledge exposes potential avenues for expanding the clinical utility of BTKi and provides a lesson for development of other novel agents that may have valuable hidden efficacy signals. Research driven by scientific, rather than commercial interests is desperately required to allow the full potential of new therapeutics to be achieved.


Asthma is a chronic inflammatory immune disorder of the airways affecting one in ten children in westernized countries. The geographical disparity combined with a generational rise in prevalence, emphasizes that changing environmental exposures play a significant role in the etiology of this disease. The microflora hypothesis suggests that early life exposures are disrupting the composition of the microbiota and consequently, promoting immune dysregulation in the form of hypersensitivity disorders. Animal model research supports a role of the microbiota in asthma and atopic disease development. Further, these model systems have identified an early life critical window, during which gut microbial dysbiosis is most influential in promoting hypersensitivity disorders. Until recently this critical window had not been characterized in humans, but now studies suggest that the ideal time to use microbes as preventative treatments or diagnostics for asthma in humans is within the first 100 days of life. This review outlines the major mouse-model and human studies leading to characterization of the early life critical window, emphasizing studies analyzing the intestinal and airway microbiotas in asthma and atopic disease. This research has promising future implications regarding childhood immune health, as ultimately it may be possible to therapeutically administer specific microbes in early life to prevent the development of asthma in children. Recent evidence supports a role of the intestinal microbiome in the development of childhood asthma and atopic disease. Animal model studies have made significant advancements in the quest to understand the gut-lung axis; identifying large-scale shifts in gut microbial compositions in asthma and allergy-induced mice and manipulating the intestinal microbiome with antibiotics, which enhanced the severity of these diseases –3. HowevAllergic asthma is an immunoglobulin E (IgE)-mediated chronic inflammatory disease of the airways . Other mSome of the most striking data related to asthma prevalence comes from the United States, where it was reported from 1999 to 2009, that the proportion of people diagnosed with asthma increased by 15% . In otheORMDL3 and SMAD3) [The underlying cause of asthma is a complex product of genetic and environmental factors resulting in significant heterogeneity of the disease. Parental history of asthma increases the likelihood of developing this disease, however assessment of this factor alone is not enough to confirm a person’s risk of asthma development –16. Therd SMAD3) . Thus itd SMAD3) .In addition to the within-generation rise in the prevalence of asthma, there is also an inverse relationship between the incidence of infectious diseases and hypersensitivity diseases, where a high incidence of infectious diseases appears to protect against allergic and autoimmune diseases . FurtherThis concept of decreased microbial exposure in modern or more urban societies has become a booming research area in the etiology of immune dysregulation. One particular arm of asthma etiology in particular, focuses on factors associated with improved health and hygiene; for example, increased antibiotic exposure, and household size , 27–35. The hygiene hypothesis proposes that a lack of early life exposure to microbes alters early life immune system priming and, consequently, increases susceptibility to atopic diseases . StrachaThe microflora hypothesis extends Strachan’s hygiene hypothesis by emphasizing the role of microbes residing in and on the human body (collectively known as the human microbiota) . OriginaThe gut-lung axis attempts to mechanistically define how microbes in the gut might influence immune function in the lung . One potThus it is possible that interactions with specific gut microbes, via their corresponding PAMPs, could result in varying phenotypic changes in DCs, with downstream effects on lymphocyte priming/homing and ultimately, shifts in anti-inflammatory responses in the airways . In a hoAnother area of gut-lung axis research involves microbial-derived metabolites, such as short chain fatty acids (SCFAs). SCFAs are direct by-products of bacterial fermentation of carbohydrates and are key energy sources for many host tissues and gut bacterial species . There aCXCL16, which in turn decreased accumulation of invariant natural killer T (iNKT) cells (prominent in the pathogenesis of asthma) in the colon [It is also possible that the intestinal microbiota is linked to lung immunity through microbe-mediated epigenetic modification. Distinct whole blood DNA methylation patterns were associated with two major bacterial phyla, either Firmicutes or Bacteroidetes and pathway analysis revealed differential methylation (associated with a high or low Firmicute/Bacteroidetes ratio) among genes enriched in functional networks such as cardiovascular disease, inflammatory responses, obesity, and lipid metabolism . Furtherhe colon . Thus ulLactobacillus showed that protection from allergic responses is mediated by specific bacterial species [Lactobacillus reuteri resulted in decreased airway hyper responsiveness, while treatment with Lactobacillus salivarius had no effect on the allergic symptoms of the mice [Bifidobacterium longum, Bifidobacterium breve and L. salivarius [L. salivarius had no effect, and both Bifidobacterium species increased Peyer’s patch and splenic Foxp3+ T-reg cells in infant mice [B. longum introduced in the perinatal period resulted in T-reg cell induction in adult mice and protected against allergic airway inflammation in OVA-sensitized mice [B. longum suggests the presence of an early life window in which microbial-driven immune changes are most effective.Mechanistic studies analyzing how the intestinal microbiome is involved in asthma and atopic disease are typically conducted in mouse models of allergic inflammation. GF mice lacking a microbiota show increased allergic responses, including increased lymphocyte and eosinophil inflammation in the airways, accompanied by increased Th-2 cytokines and elevated IgE production . However species . Supplemthe mice . This splivarius . L. saliant mice . Howeverzed mice . NotablyAntibiotics  have also been shown to increase airway inflammation in mouse models of experimental asthma , 64, 65.Helicobacter pylori resulted in protection against OVA and HDM-induced airway hyper responsiveness [Age is the main driver of compositional and functional differences in the intestinal microbiota , 66. Thusiveness . Howeversiveness . As notesiveness .Fig. 1DeRussell et al. demonstrate the effects of early life antibiotic exposure in mice , 3. ThisMouse studies have made substantial mechanistic strides in microbiome-atopic disease research. In parallel, improvements in DNA sequencing technology over the past decade have made analyzing the microbiomes of humans much more feasible. With these sequencing improvements, a similar ‘critical window’ in which microbial alterations can be associated with the development of asthma and atopic disease is also becoming more apparent in humans Fig. b. Using Faecalibacterium, Lachnospira, Rothia, and Veillonella (FLVR), in the 3-month fecal microbiota, which were associated with atopy and wheezing at 1-year of age among 319 infants enrolled in the Canadian Healthy Infant Longitudinal Development (CHILD) Study [However more recent studies have identified shifts in specific bacterial taxa in early life, rather than global compositional changes, that are associated with increased risk of asthma later in life. In fact, our group identified decreases in the abundances of four bacterial genera, D) Study . Atopy aD) Study . FurtherD) Study .Lachnospira remained decreased in the 3-month fecal microbiota while one particular bacterial species, Clostridium neonatale, was increased in asthmatics at this time-point [Lachnospira to C. neonatale (L/C) and using quartile analysis, showed that children with the lowest L/C ratio (quartile 1) were 15 times more likely to be diagnosed with preschool-age asthma than children in the other L/C quartiles [Since the CHILD Study is a longitudinal cohort, our group was able to conduct a follow-up study on this same cohort when they reached 4-years of age and could be diagnosed with preschool-age asthma . We founme-point . DemonstNature Medicine was able to distinguish asthmatic and atopic children by their neonatal (35 days post birth) intestinal microbiome compositions [Notably however, additional bacterial taxa as well as other microbes (e.g. fungi) have been associated with asthma and atopic disease development in children , 70. In ositions . Childreositions . Thus, eE. coli, to the lung was shown in a TLR4-dependent manner to induce γδ-T cells, decrease activation of lung DCs, and abrogate Th-2 cytokine production to confer protection of mice from allergic airway inflammation [Though the intestinal microbiome is one of the most populated regions of the human body, recent research supports a role of the airway microbiome in asthma and atopic disease pathogenesis. In an OVA-induced mouse model of asthma, administration of a common gut pathogen, ammation .Klebsiella species [In humans, airway microbial dysbiosis has been associated with increased risk of asthma , 73. 16S species . However species .− T-regs in a programmed death ligand 1 (PD-L1)-mediated manner [Streptococcus in the nasopharyngeal (NP) microbiome with chronic wheezing at ages 5 and 10 years [Continuing the early life theme, Gollwitzer et al. provide evidence of a 2-week window in which shifts in the airway microbiota are associated with decreased responsiveness to aeroallergens and the induction of Heliosd manner . If PD-Ld manner . Further10 years . Interes10 years . Thus thIn conclusion, the current literature suggests a role of the microbiome in asthma and atopic disease development, with particular emphasis on early life dysbiosis. Notably, recent studies have identified shifts in specific bacterial genera and species, which could ultimately be applied as probiotic interventions prior to the development of asthma. These probiotic interventions could be given directly to the baby in early infancy once all safety concerns have been addressed. Another option for colonizing the infant is through maternal exposure to these microbes either before or after delivery. Prior to establishing these probiotic regimens however, shifts in early life gut and airway microbes could also be applied as microbe-based diagnostics to identify children at the highest risk of developing asthma and related allergic diseases.Before any of these preventative or diagnostic techniques can be applied, future research should focus on validating the current findings in additional longitudinal human cohorts and improving humanized microbiome mouse models of airway and lung inflammation to mechanistically characterize the microbe-immune cell interactions promoting or protecting against asthma and atopic disease. Additionally, targeted-metabolomic and shotgun metagenomic sequencing strategies using stool, urine, and potentially breast-milk samples in human cohorts will better characterize the functional roles of these specific taxa in infant immune development.Further, to better elucidate this early life critical window in humans, additional longitudinal cohorts should begin stool sample collection beginning at birth and continuing up to age 1 year (with at least bi-weekly collection points within the first 3-montths of life). Additionally, the collection of additional biological samples (namely blood and urine) during the first 3-months of life (though this is not often feasible) would be ideal to determine whether these gut microbial alterations occur prior to immune-dysregulation or vice versa.Moreover, although this review focuses on the bacterial microbiome in asthma, there are many other microbial organisms  that also play key roles in host physiology and immune development , 76–79.


In vitro genetic modification can be used to overexpress or knock down certain genes and allow for reversal of the aging phenotype. Pharmaceutical administration is another approach that allows for manipulation of signaling pathways related to cell proliferation and cell senescence. Since the stem cell niche can contribute to the age-related decline in stem cell function, rejuvenation strategies also include optimization of extracellular factors. Overall, improving the intrinsic properties of aging stem cells as well as the surrounding environment allows these cells to adopt a phenotype similar to their younger counterparts.Ischemic heart disease affects a majority of people, especially elderly patients. Recent studies have utilized autologous adult stem/progenitor cells as a treatment option to heal cardiac tissue after myocardial infarction. However, donor cells from aging patients are more likely to be in a senescent stage. Rejuvenation is required to reverse the damage levied by aging and promote a youthful phenotype. This review aims to discuss current strategies that are effective in rejuvenating aging cardiac stem cells and represent novel therapeutic methods to treat the aging heart. Recent literature mainly focuses on three approaches that aim to reverse cardiac aging: genetic modification, pharmaceutical administration, and optimization of extracellular factors.  Cardiovascular disease is the leading cause of mortality in the United States , and its2 or H2O2, whose accumulation promotes senescence, DNA mutations, inflammation, and cell death pathways [Obstacles to the success of stem cell-based clinical therapies include the poor survival of donor cells along with the age-related loss of stem cell regenerative capacity. More than 90% of transplanted mesenchymal stem cells (MSCs) die within the first few days . Aging lpathways . ROS canpathways . TherefoThis review is designed to highlight current work in the field of rejuvenation of aging cardiac stem cells. Studies in this field have focused on three different approaches, summarized in In vitro genetic modification of aging cardiac stem cells to enhance proliferation, survival, and commitment is an effective strategy to enhance stem cell function and ensure improved cardiac output. Pim-1, a conserved serine/threonine protein kinase [n kinase  with itsn kinase . Pim-1 kn kinase . Recent n kinase . Geneticn kinase . A recenn kinase . In addin kinase . These nκB pathway  [A variety of pathways  includinn (mTOR) , a majorn (mTOR) . mTOR's n (mTOR)  and brinn (mTOR) . A recenn (mTOR) . The comn (mTOR) . As withn (mTOR) .β-catenin pathway / pathway  as a potntiation . The WNTelopment .Ink4a, a cyclin-dependent kinase inhibitor that leads to cell arrest by activating Rb. The expression of p16Ink4a is also known to increase with aging  is another method to improve therapeutic effectiveness of myocardial repair. CoPP is an inducer of heme oxygenase-1 (HO-1), which induces cellular protection. A previous study has shown that preconditioning of hCSCs with CoPP increases the resistance of these cells to oxidative stress-induced apoptosis via the activation of ERK/NRF2 signal pathways . Knockdoitioning .in vitro while being treated with pharmaceutical agents. However, a potential limitation of this strategy is that pharmaceutical agents can have a wide variety of effects, and therefore, a balance between side effects and benefits must be reached before the drugs can be used in a clinical setting. Perhaps, a combination of pharmacological treatment and genetic modification can be employed to rejuvenate aging stem cells, allowing for a more comprehensive approach.Pharmaceutical administration is a valid methodology for rejuvenation. Similar to the genetic modification technique, stem/progenitor cells can be extracted from a patient and cultured The host environment plays a very important role in the outcome of cell therapy; allogenic cells from young, healthy donors have been used to overcome age-related stem cell dysfunction . FurtherManipulation of the Notch pathway, in which ligands such as Delta interact with the Notch receptor , can be in vivo [Another circulating factor implicated in cardiac disease is insulin-like growth factor 1 (IGF-1). IGF-1 is important in the recovery process of the heart because of its subsequent activation of PI3K/Akt signaling , which ain vivo . A studyin vivo .in vitro and wielded beneficial effects on age-associated pathologies in mice [Many other cytokines and growth factors have been implicated as potential candidates for rejuvenation. Macrophage migration inhibitory factor (MIF)  is a cyt in mice . An undeα-induced TWIST expression (κB and antiapoptotic proteins Bcl-2 and Blc-xL. Hypoxia-preconditioned MSC transplantation leads to enhanced angiogenesis and vascularization as compared to normal MSCs. Since 90% of grafted cells die within the first few days of transplantation, this protective effect of hypoxia is crucial to protect transplanted cells [Oxygen levels of stem cell niches are known to play a role in stem cell quiescence; changing oxygen concentrations can affect the survival and proliferation of stem cells when used for myocardial therapy. It has been shown that expansion of hCSCs under hypoxic conditions leads to greater engraftment and functional benefit after implantation into infarcted hearts of mice . Furtherpression , which ied cells .The identification of rejuvenating growth factors and environmental conditions opens new prospects to reverse the effects of cardiac aging. This strategy allows for preconditioning of the stem cells in order to optimize the extracellular environment before transplantation into the postinfarct heart. Altering systemic expression of factors that promote stem cell activity and culturing cells in hypoxic conditions are powerful methods of rejuvenation. However, the stem cell niche is very complex; therefore, further research is required before this strategy can lead to clinical translation.Other engineering strategies are being developed to rejuvenate the senescent heart. Two novel stem cell products have been engineered: CardioChimeras and CardioClusters. CardioChimeras are formed by fusing together CPCs with MSCs. Combined administration of hMSCs and human c-kit-positive CPCs into an infarcted heart has significantly improved myocardial structure and function . This tein vitro genetic modification, pharmaceutical administration, and optimization of extracellular factors. Recent studies show promising results of the ability of these techniques to rejuvenate the aging heart. However, more understanding of the combinatorial effects of these interventions and fine-tuning of these techniques, depicted in This review discusses the current strategies for the rejuvenation of aging stem cells in order to improve therapeutic techniques for myocardial repair. Cardiac stem cells have an innate ability to rejuvenate the myocardium; however, the aging population of patients has a compromised stem cell population in terms of functional capacity and regenerative potential . Therefo


Immunological reactions may have a role in subgroups of patients suffering from psychiatric disorders. Possible markers for such subgroups may be autoantibodies of currently unknown nature. If identified, they could indicate which patients that would benefit from immunomodulatory treatment in addition to standard interventions. Modern proteomic methods allow analyses of antibody binding to thousands of different human proteins, facilitating the identification of currently undiscovered autoantibodies.We have explored the association between any seroreactivity in plasma samples from first episode psychosis patients against more than 2000 randomly chosen protein fragments derived from human proteins, and the development of disorders characterized by chronic or relapsing psychotic symptoms. Plasma from 53 patients and 41 non-psychotic controls were assessed; the clinical course of the patients were followed for a mean duration of 7 years. The plasma samples were analyzed for IgG reactivity to 2304 fragments (approx 100 a.a. residues in length) of human proteins using a multiplexed afﬁnity proteomic technique, and positive hits validated for binding in two additional assays.Thirty patients were diagnosed with schizophrenia, delusional disorder, schizoaffective disorder, bipolar disorder or a long-term unspeciﬁed nonorganic psychosis during follow-up, while 23 patients achieved complete remission. Eight patient samples showed autoreactivity to the N-terminal fragment of the PAGE protein family (PAGE2B/PAGE2/PAGE5), whereas no such autoreactivity was seen among the controls. PAGE autoreactivity was associated with a signiﬁcantly increased risk of being diagnosed with schizophrenia during follow-up (odds ratio 6.7). An antisera raised against the N-terminal fragment stained an unknown extracellular target in human cortical brain tissue .We are currently investigating the identity of this target. In addition, two other putative new autoantibodies found primarily among the patients, and rarely in the controls, will be discussed at the meeting.Our ﬁndings suggest that autoreactivity to the N-terminal portion of the PAGE protein family is associated with schizophrenia in a subset of patients with ﬁrst-episode psychosis. In addition, we propose that searching for novel autoantibodies in an unbiased way may be feasible using state-of-the-art proteomic methods, and can yield useful biological markers for immune involvement in subgroups of individuals diagnosed with psychiatric disorders.


Bioprosthetic aortic valve degeneration may present as acute, severe aortic regurgitation and cardiogenic shock. Such patients may be unsuitable for emergency valve replacement surgery due to excessive risk of operative mortality but could be treatable with transfemoral valve-in-valve transcatheter aortic valve implantation (TAVI). There is a paucity of data regarding the feasibility of valve-in-valve TAVI in patients presenting with cardiogenic shock due to acute aortic insufficiency from stentless bioprosthetic valve degeneration. We present one such case, highlighting the unique aspects of valve-in-valve TAVI for this challenging patient subset. Acute severe aortic regurgitation is an emergency necessitating immediate intervention due to the inability of the left ventricle to adapt to the sudden increase in preload and left ventricular diastolic pressure, which ultimately results in pulmonary edema and cardiogenic shock. Pharmacologic preload and postload reduction can temporize clinical deterioration, but only valve replacement can abort the inevitable hemodynamic collapse. We present a case of successful valve-in-valve transcatheter aortic valve implantation (TAVI) for a structurally deteriorated Medtronic Freestyle stentless bioprosthesis in a patient presenting with severe aortic regurgitation and cardiogenic shock. Due to clinical presentation in extremis and presence of stentless bioprosthetic degeneration, the case presented unique challenges that highlight important aspects of preprocedural care, heart team collaboration, imaging guidance, and implantation technique.The patient is a 72-year-old female with a history of diabetes mellitus, hypertension, and bicuspid aortic valve status post aortic valve and root replacement using a 23 mm Freestyle  aortic valve and root bioprosthesis implanted by root inclusion method 12 years prior to admission. She was transferred from an outside hospital with acute-onset chest pain and shortness of breath. She denied fever and chills. Physical examination was notable for blood pressure 100/30 mmHg and hypoxia. Grade 3/6 systolic and diastolic murmurs were heard at the right 2nd intercostal space. Subsequently, she developed pulmonary edema and shock necessitating mechanical ventilation and vasopressor support. A transthoracic echocardiogram suggested flail aortic valve leaflet , and traDue to rapid deterioration of the patient, the heart team was immediately organized. Two cardiac surgeons deemed her unsuitable for redo surgical aortic valve replacement . Therefore, valve-in-valve TAVI was considered. After review of the operative report and manufacturer product information, the inner diameter of the Freestyle valve was confirmed to be 20 mm. A 23 mm S3 valve  was selected. Images from a remote contrast-enhanced CT scan suggested absence of peripheral arterial calcification and adequate caliber of iliofemoral arteries for large-bore sheath insertion.2  was used to guide the procedure. The etiology of valve deterioration was confirmed to be leaflet flail; no vegetations were identified. Arterial and venous access was obtained followed by placement of a transvenous pacemaker. Aortography was performed , followe2 .Several large, multicenter registries have demonstrated that valve-in-valve TAVI is safe and effective in stable patients with degenerated aortic bioprostheses –4. In anstented bioprostheses, valve-in-valve TAVI for acute, severe aortic insufficiency and cardiogenic shock due to stentless bioprosthetic degeneration represents a particularly challenging TAVI subset, and published data are limited to a few case reports. Bagur and colleagues [In contrast to more stable patients with failed lleagues  treated lleagues  treated lleagues  reportedacute, severe aortic insufficiency and cardiogenic shock presents unique technical challenges. Preprocedural stabilization in the crucial moments prior to TAVI must include airway intubation and hemodynamic support. Pharmacologic vasopressor support may be necessary in the short term to increase central aortic pressure and perfuse vital organs. Intra-aortic balloon pump and transaortic axial flow pumps ) are generally contraindicated. Extracorporeal membrane oxygenation may be effective as a temporizing measure, as was reported by Duncan et al. [Emergency valve-in-valve TAVI in a patient with n et al. , but an n et al. . Therefostented aortic prostheses, stentless prostheses do not have radiopaque markings to fluroscopically guide transcatheter valve implantation depth. The exception may be if there is heavy calcification in the body of the prosthesis. Importantly, the sewing ring may be more ventricular than the calcium landmarks in the body of the prosthesis, so a combination of TEE and aortography, with one or two pigtail catheters to locate the sewing ring relative to the root, may be useful [In contrast to e useful . For stae useful –12. Howee useful . In the We present a challenging case of emergency valve-in-valve TAVI in a stentless bioprosthesis for a patient presenting with severe aortic regurgitation and cardiogenic shock using a balloon-expandable transcatheter valve. This case adds to the experience of the few reported cases of valve-in-valve TAVI for this uniquely challenging patient subset. Prompt diagnosis, preprocedural airway and hemodynamic stabilization, immediate heart team collaboration, and skillful use of invasive and noninvasive imaging were all instrumental in achieving a successful outcome.


Electrospinning has been used for the fabrication of extracellular matrix (ECM)-mimicking fibrous scaffolds for several decades. Electrospun fibrous scaffolds provide nanoscale/microscale fibrous structures with interconnecting pores, resembling natural ECM in tissues, and showing a high potential to facilitate the formation of artificial functional tissues. In this review, we summarize the fundamental principles of electrospinning processes for generating complex fibrous scaffold geometries that are similar in structural complexity to the ECM of living tissues. Moreover, several approaches for the formation of three-dimensional fibrous scaffolds arranged in hierarchical structures for tissue engineering are also presented. Tissue engineering is an emerging multidisciplinary field that aims to regenerate damaged or lost tissues/organs of living organisms using a combination of cells and scaffolds ,2. TheseElectrospinning has been used for the fabrication of ECM-mimicking fibrous scaffolds for several decades ,11,12. EAs with other biocompatible materials used to mimic native tissues, electrospun fibrous scaffolds provide nanoscale/microscale fibrous structures with interconnecting pores, resembling the natural ECM in tissues, and showing a high potential to facilitate the formation of artificial functional tissues . For exaTo date, the scientific community has investigated a considerable number of fabricated structures of electrospun fibers and their corresponding functions for tissue engineering, including the generation of skin, bone, muscle, cartilage, and blood vessels ,27,28,29Improving our understanding of the structural features of tissues is critical for selecting appropriate materials for reconstructing damaged tissues. Within the last several decades, many studies have aimed to fabricate fibrous nanoscale/microscale structures by applying different electrospinning process parameters . ElectroTissues in the human body also have unique ECM structures that are found in specific tissues  exhibiting anisotropic fibrous structures. From a structural viewpoint, structures that can mimic these features are believed to be necessary for precisely guiding cell growth and tissue regeneration, which lead to the recovery of their natural tissue function. For this purpose, electrospun fibrous scaffolds with various alignments have shown an outstanding ability to guide cell morphology and affect cell function when compared with other types of random fibrous scaffolds, both in vitro and in vivo. For example, Gnavi et al. demonstrated that aligned fibrous scaffolds provide contact guidance to cultured nerve cells, resulting in the alignment and elongation of cells along the contacted fiber direction . Lee et Although numerous fibrous scaffolds with ECM-mimicking structures have been introduced to date, most studies of fibrous scaffolds have verified the effects of unitary structures, such as fiber scale, porosity, and orientation. However, most of the native tissues in humans are composed of hierarchical structures and are not unitary structures. For example, bone tissue has a hierarchical organization over various scales ranging from microscale to nanoscale-structured components . TendonsDual extrusion electrospinning employs two extrusion processes that function simultaneously. This technique enables the differential control of spatial geometry depending on extrusion conditions, e.g., polymeric concentrations and independent solvents. This permits nanofibrous/microfibrous structures to be combined in a single scaffold and the distribution of electrospun fibers to be controlled. Leverson et al. investigated the cellular response using hybrid scaffolds of mixed nanoscale/microscale fibers fabricated by dual extrusion electrospinning, in which one syringe creates nanoscale fibers, and the other generates microscale fibers . Park etFibrous scaffolds can also be fabricated by temperature-assisted electrospinning, using additional temperature-controlled equipment to improve cell permeability. Cryogenic electrospinning uses ice crystals throughout the process of depositing fibrous layers in order to prevent the formation of highly compacted fibrous scaffolds during the electrospinning process. Scaffold porosity can be adjusted from 10–500 μm, depending on various controllable factors such as size and the amount of ice crystals. The ice crystals are then removed through the freeze-drying of the fibrous scaffolds, leaving large void spaces. This technique is a promising technique for developing fibrous scaffolds, permitting cell infiltration into these void spaces. Bulysheva et al. have used cryogenic electrospinning to generate fibrous scaffolds that are capable of inducing an in-growth of fibroblasts and epithelial cells into scaffolds . Similar3 air/h/m scaffolds) for rotator cuff tendon tissue engineering [Alternatively, melt electrospinning is another temperature-assisted electrospinning approach using a higher temperature. Melt electrospinning uses a polymer melt instead of a polymer solution, permitting the controlled fibrous deposition of 3D scaffolds with programmable porosity and alignment. In principle, the polymer is placed in a syringe that can be heated to a desirable temperature (~400 °C) and extruded using air pressure. This technique overcomes the disadvantages of conventional electrospinning, e.g., toxic solvents are not required . This laineering . They shFibrous scaffolds generated using electrospinning are deposited on a conductive metallic collector in a randomly oriented fibrous direction due to their chaotic whipping nature in the electrified liquid jet. These 2D features may be problematic to the broad application of such scaffolds for tissue engineering applications. Zhang et al. utilised a unique patterned collector and fabricated different microscale architectures and macroscale 3D tubular structures . The res−12 and 10−15 s for picosecond and femtosecond lasers, respectively) [For many years, researchers have attempted to fabricate scaffolds with a hierarchical fibrous structure by generating a nanoscale/microscale structure on prefabricated fibrous scaffolds using chemical modification methods, such as ultraviolet (UV) irradiation ,55. Howectively) ,57. The ctively) . In addictively) ,60. Recectively) . They dectively) . They dectively) .Another simple way to create additional patterns on electrospun fibers is nanoimprinting lithography (NIL). Nandakumar et al. utilised nanoimprinting lithography on electrospun fibers at physiological temperatures . They prThe fibrous scaffolds produced by conventional electrospinning are usually two-dimensional rather than three-dimensional, hampering cell infiltration. Although several approaches have been explored to increase the porosity of electrospun fibrous scaffolds to overcome cell ingrowth, these approaches do not produce 3D scaffolds with respect to thickness. Several approaches to form 3D fibrous scaffolds using different electrospinning processes have been developed . A technique using liquid reservoirs as collectors has attracted attention as a method for preparing 3D fibrous scaffolds. The utilization of liquids with low surface tension, such as water, ethanol, and methanol, causes the extruded fibers to sink during electrospinning, and overcomes the effects of fiber bonding. This results in the loosening of fibrous layers of scaffolds with higher internal porosity. The choice of the liquid to use in the reservoir should be considered according to surface tension and hydrophilicity. For example, Chen et al. fabricated 3D fibrous scaffolds incorporating fibrous morphologies and interconnected pore structures using liquid-collecting electrospinning. In their study, a bath containing diluted alcohol solution was used as the liquid-based collector . During 4 as a gas foaming agent [4 solution, and bubbles were then formed/generated on the 2D fibrous scaffolds, creating a more open scaffold. Researchers demonstrated that a higher concentration of gas foaming agent significantly increased the thickness of the prepared 2D fibrous scaffolds. In addition, cells successfully infiltrated and grew throughout the gas foamed 3D fibrous scaffolds. A similar technique was reported by Hwang et al. involving the use of a gas foaming/salt leaching technique [Three-dimensional fibrous scaffolds using gas foaming provide viable alternatives to open the pores between fibrous networks with a minimal application of force. The gas foaming technique for prepared 2D fibrous scaffolds usually involves three steps: 2D fibrous scaffold/gas solution formation, gas bubble nucleation/growth, and 2D fibrous scaffold expansion to 3D fibrous scaffolds. Jiang et al. reported on the feasibility of this process using prepared 2D fibrous scaffolds and NaBHng agent . The preechnique . BrieflySelf-assembly is a process in which a disordered arrangement forms an ordered system as a consequence of specific, local interactions among the components themselves. Self-assembly of the fibrous scaffolds was investigated for several types of polymers, including polyethylene oxide (PEO), polyacrylonitrile (PAN), PCL, and polyvinyl alcohol. Liang et al. utilised precursor polymer solutions (PEO dissolved in water) and fabricated self-assembled fibrous structures with honeycomb patterns . They exBased on the hierarchical architecture of soft tissues, such as tendon and ligaments, the fabrication of multifilament yarn scaffolds has been highlighted for tissue engineering applications . Yarns aHydrogel-integrated fibrous scaffolds may also be used to mimic the native tissue environment. An advantage of combining fibrous scaffolds with hydrogels is that cells can easily migrate through 3D environments, such as the 3D environment of native ECM. Indeed, hydrogel-integrated fibrous scaffolds allow for cellular contact guidance within 3D environments, which cannot be accomplished with separate hydrogel or electrospun fiber systems. Sadat-Shojai et al. attempted to reconstruct the structure using electrospun fibers as the inner layer, and a hydrogel as the 3D structure . BrieflyDespite extensive studies, it is still not possible to fabricate highly organised fibrous scaffolds with controlled uniformity and architecture using conventional electrospinning generated by the chaotic whipping of liquid jets. NFE is a relatively new method in the field of electrospinning, and has recently been actively applied by researchers. NFE uses a short distance between the spinneret and collector (less than 3 mm) to prevent the bending instability and split in ejected fibers. This method stabilizes the region of the ejected polymer jet to control fiber deposition and enable the production of 3D structures. For example, Lu et al. introduced an NFE method to fabricate 3D microstructures of fibrous scaffolds  by balanAlternatively, an electrohydrodynamic printing process was developed to deposit the electrohydrodynamically printed fibers into customised patterns by controlling the X–Y motion stage . BrieflyFattahi et al. designed 3D fibrous scaffolds in combination with 3D printing technology and NFE . This apThe native tissue has complex structures with somewhat unique arrangements and architectures of fibrous shapes, and ECM-mimicking materials have been of great interest to scientists, particularly in the field of tissue engineering. Electrospinning offers advantages for the preparation of fibrous scaffolds resembling the fibrillar architecture of the ECM in native tissues, yielding materials with major advantages in tissue engineering. There are many methods for fabricating 2D and 3D fibrous scaffolds, which show structural characteristics similar to those of the native ECM. In this review, we discussed recent advances in the fabrication of fibrous scaffolds with desired geometries and architectures using several electrospinning techniques. This review provided an overview of the fundamental principles of the electrospinning process for generating complex fibrous scaffold geometries similar to the structural complexity of the ECM in living tissue. As our understanding of the origins of these features increases, we can begin to design, fabricate, and apply this knowledge in the biomedical field.Although there are still many challenges to overcome, electrospinning shows enormous potential in the fabrication of fibrous scaffolds with controllable geometric/architectural structures, enabling researchers to design and develop novel fibrous scaffolds that more closely mimic the structural environment of the native ECM. In the development of “ECM-mimicking materials,” the objective is to improve our understanding of native complex structures and prepare highly efficient biomedical scaffolds for tissue engineering. Further studies of fibrous scaffolds are ongoing, and will be useful for achieving efficient tissue engineering.


Rieske non-heme iron oxygenases, which have a Rieske-type [2Fe–2S] cluster and a non-heme catalytic iron center, are an important family of oxidoreductases involved mainly in regio- and stereoselective transformation of a wide array of aromatic hydrocarbons. Though present in all domains of life, the most widely studied Rieske non-heme iron oxygenases are found in mesophilic bacteria. The present study explores the potential for isolating novel Rieske non-heme iron oxygenases from thermophilic sources. Browsing the entire bacterial genome database led to the identification of 45 homologs from thermophilic bacteria distributed mainly among Chloroflexi, Deinococcus–Thermus and Firmicutes. Thermostability, measured according to the aliphatic index, showed higher values for certain homologs compared with their mesophilic relatives. Prediction of substrate preferences indicated that a wide array of aromatic hydrocarbons could be transformed by most of the identified oxygenase homologs. Further identification of putative genes encoding components of a functional oxygenase system opens up the possibility of reconstituting functional thermophilic Rieske non-heme iron oxygenase systems with novel properties.The online version of this article (doi:10.1186/s13568-016-0318-5) contains supplementary material, which is available to authorized users. Pseudomonas sp., the family has since garnered a great deal of attention for two major reasons. First, ROs are key enzymes responsible for the initial attack on otherwise inert aromatic nuclei, thereby making them targets of a cascade of downstream enzymes, leading to their complete mineralization  constitute a large family of oxidoreductase enzymes involved primarily in the oxygenation of various aromatic compounds. Although Gibson et al.  first deox) have also been investigated in certain plant species, such as Arabidopsis thaliana, Zea mays, Pisum sativum, Oryza sativa, Physcomitrella patens, Amaranthus tricolor, Ocimum basilicum and Spinacia oleracea  proteins (such as ferredoxin and reductase) transfer electrons from reduced nucleotides, such as NAD(P)H, to the terminal oxygenase component ,which in turn catalyzes the di- or mono-oxygenation of the aromatic nucleus of the substrate  based on their phylogenetic distribution, substrate preferences and mode of attack on aromatic nuclei , as these are the functional prerequisites of ROs  for genes putatively encoding ET components (both ferredoxin and reductase) using a set of queries . Among 45 non-redundant sequences (Table Alicyclobacillus acidoterrestris ATCC 49025 (GenBank: EPZ42375) was found to be truncated at the N-terminal end and was therefore excluded from further analysis. Analysis of the distribution of the remaining candidate ROs among both thermophiles and mesophiles revealed that they were present mainly among thermophilic strains belonging to the phyla Chloroflexi, Deinococcus–Thermus, Firmicutes and Thermotogae  showing values as high as 90 . Several putative genes . All observations discussed thus far concern the α-subunits of ROox genes from Proteobacteria or Actinobacteria and further evolved separately. The role of Firmicutes in the evolution of ROs in thermophiles cannot be ruled out, as several RO homologs were found among mesophilic strains of Firmicutes  vicinal to those encoding the terminal oxygenase(s) are responsible for the proper functioning of the ROs. Similar observations have previously been made in The existence of unexplored microbial diversity, together with the availability of whole genomes, represents a large pool for future industrial catalysts. Thermostable ROs may be attractive candidates for carrying out efficient biotransformation at elevated temperatures. Apart from enhancing our understanding of the distribution of ROs in nature, the present study may aid in designing new bioremediation strategies or industrial biosynthetic processes. Based on the information provided here, functional RO systems can be reconstituted from each organism by cloning both terminal oxygenase and ET genes into suitable vectors and performing biotransformation assays using the predicted substrates Fig. . On the


SDH subunit genes . SDH loss of function is generally seen only in GIST lacking RTK mutations, and SDH-deficient GIST respond poorly to imatinib therapy.Most gastrointestinal stromal tumors (GISTs) harbor mutually exclusive gain of function mutations in the receptor tyrosine kinase (RTK) KIT (70–80%) or in the related receptor PDGFRA (~10%). These GISTs generally respond well to therapy with the RTK inhibitor imatinib mesylate (IM), although initial response is genotype-dependent. An alternate mechanism leading to GIST oncogenesis is deficiency in the succinate dehydrogenase (SDH) enzyme complex resulting from genetic or epigenetic inactivation of one of the four Tumor and normal DNA from a GIST case carrying the IM-resistant PDGFRA D842V mutation was analyzed by whole exome sequencing (WES) to identify additional potential targets for therapy. The tumors analyzed were separate recurrences following progression on imatinib, sunitinib, and the experimental PDGFRA inhibitor crenolanib. Tumor sections from the GIST case and a panel of ~75 additional GISTs were subjected to immunohistochemistry (IHC) for the SDHB subunit.SDHB subunit gene  was identified in both tumors. Sanger sequencing confirmed the presence of this inactivating mutation, and IHC for the SDHB subunit demonstrated that these tumors were SDH-deficient. IHC for the SDHB subunit across a panel of ~75 GIST cases failed to detect SDH deficiency in other GISTs with RTK mutations.Surprisingly, a somatic, loss of function mutation in exon 4 of the PDGFRA mutant GIST exhibiting SDH-deficiency. A brief discussion of the relevant GIST literature is included.This is the first reported case of a The online version of this article (doi:10.1186/s12885-017-3499-7) contains supplementary material, which is available to authorized users. KIT exon 11 are generally IM-sensitive while GISTs harboring KIT exon 9 mutations require IM dose-escalation. The most common PDGFRA mutation, the D842V mutation in exon 18 encoding the kinase activation loop, confers primary clinical resistance to IM ral PGLs . They idp.W557_V5delinsF aal blood . Subsequ in GIST . SDHB IHtion p.C1Yfs*42. He report . Histoloividuals .PDGFRA mutant GIST that also exhibited SDH deficiency. The tumors we examined in our case report were separate recurrences approximately 3 years after resection of the primary gastric tumor. Both tumors had a heterozygous PDGFRA D842V mutation , so the SDH inactivation may have been a later event. Interestingly, the patient also progressed rapidly (~3 months) on the investigational PDGFRA inhibitor crenolanib, which has been shown in biochemical and cellular models to be ~100–150-fold more potent than IM against the D842V mutation [Our literature search did not identify other examples of ant GIST , 10, altant GIST . Theoretmutation . It is tKIT or PDGFRA supports the clinical diagnosis of GIST and can be used to guide therapy, it does not preclude the possibility of the presence of SDHX gene mutations in these tumors, suggesting that the inclusion of these genes may be warranted in routine testing for GISTs. As detailed above, reports that clearly document GISTs with kinase and SDHX mutations are rare but not non-existent. Most documented cases are either CSS patients with both GIST and PGL and/or PCC tumors, or carry the germline SDHX mutations that predispose to the development of these tumors. Our case is unique in that both oncogenic mutations (PDGFRA and SDHB) were somatic and there was no germline involvement. Patients with germline SDHX mutations require long-term screening for disease detection and management [SDHX mutations along with a KIT or PDGFRA mutation, standard IM therapy may still be effective. Anecdotally, in the CSS case described by Gasparotto et al. [KIT mutation led to significant response and tumor stabilization for the patient. Further investigation is required to understand the biological and clinical consequences of SDH deficiency in GISTs with activating KIT or PDGFRA gene mutations. While molecularly targeted treatment options tailored to SDH deficiency in GIST are limited, a recently completed phase 2 clinical trial of linsitinib, an inhibitor of the IGF1 receptor that is over-expressed in RTK-wild type GIST, indicated that the drug provided clinical benefit in 45% of patients, albeit without RECIST response [SDHC seen in the CT/pediatric subset, suggests the potential of demethylating agents such as decitabine for treating these GISTs [Although the identification of an activating mutation in either nagement  as well o et al. , IM trearesponse . As mentresponse , 67, posresponse . Finallyse GISTs , 47.PDGFRA mutation. Oncogenic mutations in GIST are generally mutually exclusive; however documented exceptions exist which may have diagnostic and therapeutic implications.We report a unique case of an SDH-deficient GIST case with an activating


Over the past couple of decades, measuring immunological parameters has become widespread in studies of ecology and evolution. A combination of different immunological indices is useful for quantifying different parts of the immune system and comprehensively assessing immune function. Running multiple immune assays usually requires samples to be repeatedly thawed and re-frozen. There is some evidence that repeated freezing and thawing can affect assay results, but this has never been comprehensively studied in some common ecological immunology assays. We tested the effect of multiple  freeze-thaw cycles on the results of four commonly used immunological assays: haemolysis-haemagglutination titres, haptoglobin concentration, bacterial killing capacity and total immunoglobulins (IgY). We tested five different bird species from four different bird orders , and we included both captive and free-living individuals. In addition, we tested for haptoglobin concentrations and the haemolysis-haemagglutination assay if re-analysing samples 1 year apart led to different results. For the haemolysis-haemagglutination assay we also tested two different sources of rabbit blood, and we compared untreated microtitre plates with plates that were “blocked” to prevent nonspecific interactions between the plate and assay reagents.Repeated freezing and thawing of plasma had no effect on lysis titres, haptoglobin concentrations, bacterial killing capacity, or total immunoglobulin levels. Agglutination titres were unaffected by up to five cycles but were lower after ten freeze-thaw cycles. For the haemolysis-haemagglutination assay and haptoglobin concentrations, re-analysing samples 1 year apart yielded highly correlated data. For the haemolysis-haemagglutination assay, the source of rabbit blood did not influence the results, and the untreated vs. blocked plates differed slightly overall, but at the individual level assay results were highly correlated. Using different rabbit blood sources or different types of microtitre plates yielded highly correlated data.Our data suggest that repeated freeze-thaw cycles do not impair assay results to the point of influencing ecological or evolutionary conclusions. Plasma samples can be safely stored in one tube and thawed repeatedly for different assays. Nevertheless, we recommend consistent treatment of samples in terms of freeze-thaw cycles or other laboratory treatments to minimize the potential for introducing a systematic bias. In the mid 1990’s, evolutionary ecologists began to consider that the costs and benefits associated with the immune system might lead to important trade-offs between defences against diseases and other behavioural and physiological processes that impact individual fitness . Since tAlthough multiple immune assays are potentially available when testing innate and adaptive immune function , 9, 11, Running multiple immune assays on the same individual often results in samples that experience multiple freeze-thaw cycles. Freeze-thaw cycles can degrade samples by altering chemical composition and molecular function . For exaHere, we present a comprehensive study testing if repeated freeze-thaw cycles influence the results of four immune assays  frequently used in studies of ecology and evolution. We used samples from five bird species representing four different orders. Furthermore, we also systematically examined several other methodological concerns. With haemolysis-haemagglutination titres and haptoglobin concentrations, we also explored the relationships between results from duplicate intra-sample analyses when the assays were separated by a year. Lastly, we tested if lysis and agglutination titres depended on the source of rabbit red blood cells (used as an antigen-rich particulate) or on microtitre plate characteristics.Corvus monedula) from a study colony in Sweden during the 2015 breeding season, adult free-living Common Blackbirds (Turdus merula) and Ruffs (Philomachus pugnax) from Portugal between February and May 2015, adult Homing pigeons (Columba livia f. domestica) from a captive population in The Netherlands in the summer of 2015 ). Ultimately, antibody levels are calculated as the mean of the duplicates minus the mean of the blanks and are transformed based on the plate standards to correct for variation among plates.We quantified the total level of antibodies  in plasma by means of an enzyme-linked immunosorbent assay (ELISA) following the protocol described by Sköld-Chiriac et al.  that hasIn addition to analysing the series of freeze-thaw cycles under standard assay conditions with a range of species, we conducted additional tests concerning haptoglobin concentrations and lysis and agglutination titres using surplus plasma collected only from pigeons . First, we quantified haptoglobin concentrations and lysis and agglutination titres in 32 pigeon samples that were collected over the course of 2013. Using the standard procedures, we analysed these samples first in the summer of 2014 and analysed them again in the summer of 2015; samples were stored at −20 °C. Second, we concurrently quantified only agglutination in 61 samples from pigeons  using tTo test for the effects of freeze-thaw cycles on the immune parameters, we used linear mixed models (function lme of package nlme)  in R verFor the other methodological analyses, we used paired comparisons to test group differences and correlations to evaluate patterns among individuals. Parametric and non-parametric tests gave qualitatively similar results; here we report the parametric test statistics.p = 0.262). Species differed in terms of haptoglobin concentrations, lysis titres and agglutination titres . Across samples, haptoglobin concentrations were correlated between the 2 years . Raw absorbance values  also showed between-year differences  and correlations across samples . Re-analysis of samples after 1 year of storage resulted in significantly lower lysis titres . Across samples, lysis titres were correlated between the 2 years . Finally, re-analysis of samples after 1 year of storage lead to similar agglutination titres . Across samples, agglutination titres were not correlated between the 2 years . The assay standard  did not differ between assay years , and within- and among-assay-plate variation was consistently low .Re-analysis of samples after 1 year of storage resulted in significantly lower haptoglobin (Hp) concentrations . Across samples, agglutination titres using the two sources were highly correlated .The comparison of the two different sources of rabbit blood revealed no effect on agglutination titres . Across samples, lysis titres arising under the two plate conditions were highly correlated . Blocked plates resulted in significantly higher agglutination titres compared to unblocked plates . Across samples, agglutination titres arising under the two plate conditions were highly correlated .Blocked microtitre plates did not differ significantly from unblocked plates in terms of lysis titres  probably underlies the resistance of agglutination to freezing and thawing [Since storage time in a freezer might also impact the results of immunological assays, we investigated this possibility with haptoglobin concentrations and for lysis and agglutination titres, albeit in a less detailed manner than with our freeze-thaw analyses. Re-analysis of samples after 1 year of storage at −20 °C, generally resulted in values that were significantly correlated with values from the previous year. Such correlations were found for haptoglobin and lysis, but with both of these indices, 1 year of storage resulted in overall declines. In the case of haptoglobin, the between-year differences were not simply a by-product of between-year differences in the standard curves used to calculate concentrations, since raw absorbance values also showed between-year differences and correlations across samples. The correlations we observed suggest that biological patterns at the individual level should be discernible even if samples have aged somewhat. Inclusion of sample age or a factor related to analysis batch in statistical analyses could help to account for the decline due to storage time. However, lab analyses of samples that differ both in age and another factor (e.g. source population identity) that is both of study interest  thawing , 44. The thawing . The absTests aimed at the origin of the rabbit red blood cells and at a characteristic of the assay plates, two other potential methodological sources of variation in the haemolysis-haemagglutination assay, also provided new insights into this assay. The two types of rabbit blood did not differ in agglutination titres (lysis was absent in these assay bouts) and sample values were highly correlated, suggesting that researchers working in different regions of the world and with different accessibility to markets for biological products, should be able to compare assay results, if all else is equal. Assay plates might also differ in terms of non-specific binding depending on manufacturer . To systematically gain insight into possible effects of non-specific interactions between antibodies, the red blood cell targets, and the plastic plates, we compared normal plastic assay plates to ones that had been blocked with powdered milk. As would be expected, blocking had no impact on lysis; however, blocked plates had higher agglutination titres on average. Blocked plates probably reduce natural antibodies from binding to plastic, and instead allow these antibodies to bind to the rabbit red blood cells and drive agglutination. Effects of blocking are an unlikely source of variation within an individual study or lab . But blocking and potentially other plate characteristics, could influence the results of agglutination and complicate comparisons among studies, particularly if absolute levels (and not intra-study patterns) are being compared.To conclude, our data suggest that several commonly used ecoimmunological assays produce consistent results even in the face of several factors that could potentially introduce variation or otherwise influence results. Most notably, repeated freeze-thaw cycles do not impair assay results or study conclusions. Thus, plasma (or serum) samples can be safely stored in one tube and thawed (at least 5–10 times) as needed for different assays. Still, best laboratory practices dictate pre-aliquoting samples per assay whenever logistically feasible. When this measure is not achievable, freeze-thaw cycles, assay order, and other assay parameters should be standardized to the greatest extent possible.