Diabrotica virgifera virgifera LeConte) (WCR) is a major insect pest of corn (Zea mays L.) in the United States (US) and is highly adaptable to multiple management tactics. A low level of WCR field-evolved resistance to pyrethroid insecticides has been confirmed in the US western Corn Belt by laboratory dose-response bioassays. Further investigation has identified detoxification enzymes as a potential part of the WCR resistance mechanism, which could affect the performance of insecticides that are structurally related to pyrethroids, such as organophosphates. Thus, the responses of pyrethroid-resistant and -susceptible WCR populations to the commonly used pyrethroid bifenthrin and organophosphate dimethoate were compared in active ingredient bioassays. Results revealed a relatively low level of WCR resistance to both active ingredients. Therefore, a simulated aerial application bioassay technique was developed to evaluate how the estimated resistance levels would affect performance of registered rates of formulated products. The simulated aerial application technique confirmed pyrethroid resistance to formulated rates of bifenthrin whereas formulated dimethoate provided optimal control. Results suggest that the relationship between levels of resistance observed in dose-response bioassays and actual efficacy of formulated product needs to be further explored to understand the practical implications of resistance.The western corn rootworm ( Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae) is a major pest of corn Zea mays L. in the United States (US) and has adapted over time to many management tactics2. Broadcast application of organochlorine soil insecticides starting in the late 1940’s became rapidly ineffective with control issues apparent by late 1950’s3. Aerial applications of carbamates and organophosphates selected adult WCR for significant levels of resistance in the 1990’s6. Resistance to transgenic corn producing rootworm-specific Cry toxins from the soil bacterium Bacillus thuringiensis (Bt) was initially reported for Cry3Bb1 in 2009, mCry3a in 2011, and Cry34/35Ab1 in 201610. Furthermore, the cultural practice of corn rotation with a nonhost crop was circumvented in areas of the US eastern Corn Belt by WCR oviposition in nonhost crops indicating the evolution of behavioral resistance12. These events collectively have made WCR management exceedingly difficult.The western corn rootworm (WCR) 12. However, this intensive system of continuous corn production facilitates build-up of WCR densities over time making WCR management an annual challenge14. Consequently, aerial applications of pyrethroid and organophosphate insecticides are often used to reduce WCR densities and complement other management tactics15. In this system, other corn pests like western bean cutworm Striacosta albicosta (Smith) and two-spotted spider-mite Tetranychus urticae Koch are also managed with the same insecticide classes used to manage rootworms, so WCR adults can be exposed to aerial applications as nontarget insects as well18. These practices have placed annual selection pressure on WCR populations which has led to field-evolved resistance to pyrethroids in southwestern areas of Nebraska and Kansas13.Although crop rotation is the most recommended tactic to manage the WCR, continuous corn (consecutive planting of corn for ≥ two years) is a common agronomic practice in the US western Corn Belt because under irrigation, continuous corn production is often the most profitable, and corn demand is high for confined livestock and ethanol production13. Furthermore, cross-resistance and synergism studies performed with field collected bifenthrin-resistant populations suggested that multiple mechanisms of resistance could be involved such as target-site insensitivity and higher activity of detoxification enzymes14. Although the levels of bifenthrin resistance found were relatively low, resistance levels were highest in the adult stage14. Despite having laboratory bioassay data documenting resistance, the efficacy of formulated bifenthrin commonly used in aerial applications to control WCR adults had not been formally evaluated.Laboratory bioassays conducted with WCR populations collected across the US Corn Belt have shown that populations from west of the Missouri River were consistently more tolerant to the active ingredient bifenthrin than eastern populations19. Some parameters such as spray carrier volumes, droplet size distribution, crop canopy, and environmental conditions often influence the coverage and uniformity of insecticide deposition which can confound field trial results24. Carrying out consistent field trials to evaluate the efficacy of insecticide aerial applications on adult rootworms is even more challenging considering the potential interaction of population density and pest movement that may occur in the field27. Thus, methods that minimize factors influencing aerial application performance are necessary to evaluate the impact of WCR pyrethroid resistance on the efficacy of recommended foliar insecticides.Field efficacy of foliar insecticides depends on a combination of several factors such as target susceptibility, exposure, and application technique efficiency. For example, aerial application of insecticides can provide uneven coverage in the corn canopy resulting in insect sublethal exposure and reduced control33. Thus, the likely involvement of detoxification enzymes in the mechanism of WCR bifenthrin-resistance could impact organophosphate efficacy, which needs further investigation14. The past use of organophosphates (e.g. dimethoate) may also contribute to selection of WCR adults with increased detoxification enzyme activity. Resistance to both insecticide classes would greatly reduce alternative insecticide options and impact the integration of these compounds with other management tactics. Indoxacarb represents an insecticide class i.e., the oxadiazines, with unique mode of action that was recently registered by the US Environmental Protection Agency (EPA) for use to control WCR adults in field corn35. This product was reported to reduce adult WCR populations in the field and may provide a tool to help manage WCR pyrethroid resistance36. However, the susceptibility of pyrethroid-resistant WCR to the indoxacarb active ingredient needs to be evaluated.Pyrethroid and organophosphate classes contain many of the insecticides recommended for WCR control. However, enhanced metabolism can confer cross-resistance to structurally related insecticides, which has been observed between pyrethroids and organophosphates for different insect species14 and to optimize WCR resistance management and integrated pest management (IPM) programs, the objectives of this study were: (1) to develop a method of simulated aerial application that uniformly reproduces insecticide field deposition; (2) use the simulated aerial application method to assess the performance of bifenthrin and dimethoate commercial formulations against pyrethroid-resistant WCR populations; and (3) to conduct dose-response lab bioassays to estimate and compare the susceptibility of pyrethroid-resistant WCR populations to bifenthrin, dimethoate and indoxacarb active ingredients.Therefore, as part of a larger project to characterize WCR pyrethroid resistance13. The Saunders Co. field was located at the University of Nebraska Eastern Nebraska Research and Extension Center, which is surrounded by a large area of continuous corn that had not received insecticide aerial applications for over ten years. Bifenthrin had only been used in that area for soil applications in a few small-plot trials. In 2016, two field populations (R-Field1 and R-Field2) previously confirmed to have field-evolved resistance to bifenthrin13 were collected from commercial fields 18 Km apart in Keith County, NE. Each field was in continuous corn production plus annual soil- and aerial bifenthrin applications had been made for at least five consecutive years prior to this study. A third bifenthrin-resistant population tested in 2017 bioassays (R-Lab) was collected from Perkins County, NE in 2014 and then reared in a non-diapause background for nine generations under adult selection with a pre-established bifenthrin diagnostic concentration (LC99)13. Populations were maintained in the Department of Entomology, University of Nebraska-Lincoln, Lincoln, NE using standard lab rearing procedures and temperature profiles to facilitate egg diapause development and termination9.Six WCR adult populations were tested during this study. Two non-diapausing populations purchased in 2016 and 2017 from Crop Characteristics, Inc., Farmington, MN (S-Lab1) and French Agricultural Research, Lamberton, MN (S-Lab2), and one field population collected from Saunders County, NE (S-Field) in 2016 were used as pyrethroid-susceptible controls given their high susceptibility to bifenthrin measured in prior bioassays37. The spray solution consisted of water and a 90% non-ionic surfactant  at 0.25% v/v. At the 18.7 L/ha application rate, a total of 36 nozzles were used, where nozzles were deflected 15 degrees downward and operated at 317 kPa at an airspeed of 225 Km/h. At the 46.8 L/ha rate, a total of 68 nozzles were used with nozzles deflected at 30 degrees and operated at 413 kPa and at an airspeed of 209 Km/h.A series of aerial spray applications were performed in a late season cornfield (R5) located just south of Snook, TX  in order to document deposition characteristics that would be expected under typical aerial application conditions. Applications were made using an airplane  equipped with 40° 10-orifice flat fan nozzles at spray application rates of 18.7 and 46.8 L/haDuring the applications wind speeds were steady at 4 m/s with a temperature of 30 °C and relative humidity of 75%. Water sensitive papers (WSP)  were placed in the field canopy at three locations denoted as Top, Middle, and Bottom. The Top position was located near the uppermost fully extended leaf with the Bottom near the lowermost extended leaf and the Middle located midway between Top and Bottom positions. WSPs were attached to plants spaced 2 m apart covering a full 20 m application swath using binder clips such that the surface was horizontal to the ground. Applications were performed in two sampling lines (blocks), each with ten WSP (replicates) for each canopy and application rate treatment combination.39. Spray deposition rate (L/ha) and droplet size diameters at which 10, 50, and 90% of the total spray volume is contained in droplets of the specified size or less  were measured and reported40.After each spray pass replication, WSPs were collected and stored in labeled film negative sleeves. WSPs were digitally scanned at 600 dpi (EPSON Perfection V600) and analyzed for droplet size characteristics using Dropletscan , an image processing software package designed for WSP analysis42. Droplet size distribution data collected at Middle corn canopy position were targeted since this would include the major feeding and activity zone for WCR adults in the field43. Tap water solutions of Brigade 2EC  were prepared using the highest label rate recommended for rootworm control (112.1 g ai/ha) at the lowest and highest recommended carrier volume rates (18.7 and 46.8 L/ha respectively). The application of the insecticide solutions was then performed in the wind tunnel with a TT110015 ground spray nozzle  at different spray pressures until targeted droplet size parameters were achieved. Droplet size distribution data were evaluated by a Sympatec Helos/Vario KR laser diffraction system  placed at 0.3 m from the nozzle tip to ensure full spray atomization prior to measurement. The system was equipped with an R7 lens that detects droplets in a range from 9 to 3700 μm. Nozzles were attached to an actuator and traversed vertically at a constant speed (0.2 m/s) to ensure that the entire spray plume crossed the laser diffraction system. Volumetric droplet size distribution parameters DV0.1, DV0.5, and DV0.9 were reported. The wind tunnel insecticide applications and respective droplet size distribution measurements were carried out with three replications under controlled conditions of 20 ± 1 °C and 60-70% relative air humidity. Once the best spray pressure for the TT110015 ground spray nozzle was found, the timed output volume per nozzle was measured three times with a graduated glass cylinder . A customized multi-nozzle research track spray chamber 44 was then calibrated to simulate aerial applications of commercial insecticides recommended for adult WCR control. Two TT110015 nozzles spaced 0.76 m apart and 0.56 m above the target were used and the travel speed calculated according to the formula46:In 2016, a low-speed wind tunnel at the Pesticide Application Technology Laboratory  was used to identify ground application nozzles and operating pressures that could effectively simulate the spray deposition collected from the cornfield aerial application. This system has been used for droplet sizing of different nozzle designs, tank solutions and pesticide application scenariosThe aerial application simulation previously described was used to evaluate the performance of two commercial insecticides recommended for adult WCR control. Replicated experiments were repeated twice within a two-year period (2016/2017) in the research track spray chamber available in the PAT-Lab, North Platte, NE. The pyrethroid Brigade 2EC  was tested at the lowest and highest label rates recommended for rootworm control, 36.8 and 112.1 g a.i./ha respectively. The organophosphate Dimethoate 4EC  was tested at the lowest label rate of 369.9 g a.i./ha, which has been the common recommendation to provide WCR adult control in the US western Corn Belt. Insecticide solutions were prepared in tap water at the lowest and highest carrier volumes recommended for aerial application rates .The aerial spray coverage obtained in the cornfield at the Middle position was reproduced on 100 mm-diameter ×15 mm-height Petri dishes  that were pre-labeled and evenly distributed in the spray chamber to receive the insecticide applied to internal surfaces of dish bottoms and lids. Treatments were combinations of insecticide rate, carrier volume rate and WCR population. A total of four Petri dishes were used as replicates for each treatment, which were sprayed separately during four different rounds. Treated dishes were left opened for 30 min to ensure complete drying, then closed and transferred to cardboard boxes where they were kept in darkness at 23 ± 1 °C for a maximum of 16 h until use for WCR bioassays.1 generations of 2016 field-collected beetles already under lab rearing procedure were used. In both years, each Petri dish was infested with a group of 20 mixed-age beetles of even sex ratio. Active beetles were collected from rearing cages with a mouth aspirator and each group placed individually in 15 ml centrifuge tubes . Beetles were anesthetized (1.5 minute inside a −20 °C freezer) and transferred to treated dishes. Four untreated dishes were used as controls for each WCR population tested and infested dishes were maintained under laboratory conditions of 23 ± 1 °C and 13 ± 1 h photophase. Mortality of beetles at each treatment combination was recorded after 24 h. Insects that did not respond to prodding or were unable to walk consistently when placed ventral side down were considered dead.The WCR bioassays were conducted at the University of Nebraska-Lincoln, Lincoln, NE. In 2016, field-collected beetles were provisioned with ears of sweet corn for one week prior to testing whereas in 2017, the FAnalytical standards of bifenthrin, dimethoate and indoxacarb were used. Bifenthrin 98% was obtained from Chem Service Inc., West Chester, PA (Cat. No. N-11203-100MG/CAS: 82657-04-3). Dimethoate 99.5% and indoxacarb ≥95.0% were purchased from Sigma-Aldrich Corp., St. Louis, MO . The insecticides were dissolved and diluted in acetone ≥99.9% supplied by Sigma-Aldrich Corp (Cat. No. 650501/CAS: 67-64-1).1 generation of beetles that were field-collected in 2016 started to emerge. The vial bioassay method previously adapted for WCR13 was used. The evaluation time and procedure were modified for this study as described below. Wheaton™ Glass 20 ml scintillation vials  were treated with 500 μl each of increasing concentrations of bifenthrin, dimethoate and indoxacarb diluted in acetone. For control, vials were treated with acetone only. The number of insecticide concentrations used to test each population varied from 5 to 7 depending on the number of insects available at the time. Insecticide concentrations were replicated three times for each WCR population tested. Vials were homogeneously coated internally and allowed to dry under a fume hood by rolling for 30 minutes at room temperature on a commercial roller machine . Each treated vial was infested with a group of ten 48h-old adults of even sex ratio. Vial caps were loosely closed to allow air exchange inside the vials while preventing beetle escape. After 24 h, one kernel of sweet corn was added to each vial to allow beetle feeding and at 48 h after infestation the mortality of beetles was recorded. Insects that did not respond to prodding or were unable to walk consistently when placed ventral side down were considered dead. All treatments were maintained at 23 ± 1 °C and 13 ± 1 h photophase.The susceptibility of adult WCR to insecticide active ingredients was estimated in 2017 when the FCorn canopy spray deposition rate and droplet size parameters reported from WSPs were subjected to analysis of variance in SAS 9.4 software  at significance level α = 0.05. The treatment design was a factorial arrangement with spray application rates (18.7 and 46.8 L/ha) and corn canopy position  as factors in a randomized complete block experimental design (RCBD) where application lines were considered blocks.47. The corrected proportion mortality that has a continuous distribution within the restricted interval of  was analyzed with a Beta-binomial distribution using a logit link function with a generalized mixed model in SAS 9.4 software49. A completely randomized experimental design and factorial treatment design were used. Multiple comparison of treatment means was performed using Fisher’s least significant difference procedure at significance level α = 0.05.To analyze the performance of commercial insecticides on adult rootworm populations under simulated aerial application conditions, insect mortality data collected from each treatment combination was corrected by Abbott’s formula for mean mortality in the respective untreated controls52. The probit procedure also estimated a Pearson goodness-of-fit chi-square value (χ2) testing the null hypothesis that the expected regression model adequately fits the data. Resistance ratios (RR50) with correspondent 95% confidence intervals (95%CI) were calculated by dividing the estimated LC50s of resistant populations by the estimated LC50 of each susceptible population of reference53.The susceptibility of adult WCR populations to insecticide active ingredients was evaluated by analyzing the relationship between insecticide concentrations tested and mortality responses obtained. Data were corrected by Abbot’s formula for natural control mortality and analyzed with a probit link function with Normal distribution in POLOPlus-PC software (LeOra Software LLC)F2,113 = 0.11; p = 0.8999). Therefore, the interaction term was removed from the model. Application rate influenced spray deposition rate , whereas corn canopy position did not . The average deposition rate across the three corn canopy positions  was within a 2.9–15.7 L/ha 95%CI for the aerial application performed at 18.7 L/ha, whereas the one performed at 46.8 L/ha resulted in average deposition values within a 7.3–20.1 L/ha 95%CI. The average deposition rates at Middle canopy position  were 10.9 and 16.5 L/ha for the lowest and highest aerial application rates evaluated, respectively.The spray deposition rates and droplet size characteristics obtained by aerial applications across corn canopies were considerably variable Fig. . The WSP0.5 was not influenced by the interaction between corn canopy and application rate , therefore the interaction term was removed from the model. Corn canopy alone influenced the DV0.5 of the spray deposition , whereas application rate did not . Estimated 95%CI for droplets deposited on the Top canopy position had greater DV0.5 (334–382 μm) when compared to Middle (299–346 μm) and Bottom (291–339 μm) canopy positions. The interaction between corn canopy position and application rate did not influence the DV0.1  or the DV0.9  of the spray deposition. Furthermore, both DV0.1 and DV0.9 were not affected by either application rate  or corn canopy position .The droplet size distribution reported in the WSP analysis indicated that the DVThe wind tunnel test conducted in 2016 identified combinations of TT110015 nozzle and operating pressures compatible with the spray deposition data collected at Middle canopy position of corn , so this factor was excluded from the statistical model used. In both years, bifenthrin efficacy was significantly affected by the interaction of WCR populations and insecticide rates  and 100% mortality in all treatments during the second run.Overall, there were no significant two- and three-way interaction effects of carrier volume on the performance of bifenthrin and dimethoate rates tested  exhibited reduced susceptibility to both bifenthrin and dimethoate active ingredients. Confidence intervals of resistance ratios (RRs) estimated for pyrethroid-resistant WCR populations overlapped for each insecticide tested and were different than those estimated for pyrethroid-susceptible populations (Table Probit regressions were obtained for WCR populations exposed to bifenthrin, dimethoate and indoxacarb active ingredients Table . Values 56. Although the wind tunnel calibration was performed with only one rate of formulated bifenthrin, droplet size deposition obtained could practically be used for other rates and insecticides. Previous research has shown that nozzle type has more influence on spray droplet size distribution than active ingredient and carrier solution24. Therefore, the droplet size distribution data collected worked as a useful baseline for the insecticide treatments tested in this study.The method of aerial application simulation used in this study accurately reproduced aerial application parameters such as the combination of spray carrier volumes and droplet size distribution compatible to what is deposited on corn leaves in a field situation. This allowed a more realistic representation of aerial application than could be obtained with more traditional spray techniques often used in laboratory studies14. Conversely, the lowest label rate of formulated dimethoate provided optimal control of both pyrethroid-resistant and -susceptible WCR populations under simulated aerial application conditions despite the low level of dimethoate resistance that had been revealed when pyrethroid-resistant populations were bioassayed with active ingredients. Results suggest that the observed shift in dimethoate susceptibility of pyrethroid-resistant WCR populations did not reach a level that would lead to “practical resistance” defined as field-evolved resistance that reduces field efficacy of a pesticide with practical consequences for pest control57. This same phenomenon has been reported in other insect pest systems suggesting that the relationship between levels of resistance confirmed in dose-response bioassays and actual efficacy of formulated product in the field needs to be explored in order to understand the practical impact of resistance61.When used to evaluate the efficacy of formulated bifenthrin against WCR populations, the aerial application simulation consistently captured differences in performance among the rates tested. The significant difference in mortality between pyrethroid-resistant and control populations at both the lowest and highest label rates of formulated bifenthrin confirmed resistance to bifenthrin revealed in active ingredient bioassays and was consistent with anecdotal reports of reduced WCR adult control received from farmers and local crop consultants19. Variable levels of WCR exposure to insecticide applications could potentially lead to greater survival of WCR beetles in the field than measured in the aerial application simulation method, which may contribute to evolution of insecticide resistance and increase resistance levels measured in the lab over time63.The simulated aerial application method used was a conservative approach since it tested one model of spray deposition compatible with mid-canopy of corn plants of a given stage and beetles could not escape from treated surfaces. In the field there is a considerable interaction of environmental conditions, beetle movement behavior, and different levels of insecticide coverage within the corn canopy that could lead to differential adult exposure to insecticides. In fact, the spray deposition we collected in the field was fairly variable confirming the uneven canopy coverage previously observed for aerial applications65. However, compliance with management strategies depends on several social-economic aspects such as compatibility with growers’ tradition and past experiences, technology complexity, visibility of results, as well as technology cost and associated profitability66. Price and convenience often drive growers’ choice of pest management practices, which may lead to continued use of a specific insecticide68. The increase in off-patent generic bifenthrin formulations has lowered pricing of this insecticide often leading to grower preference over other products and frequent inclusion in tank mixtures with other pesticides. Also, regulatory action such as the Food Quality Protection Act has led to reevaluation and cancellation of many insecticides uses in agriculture69 so fewer modes of action remain available to manage WCR. Collectively, these factors probably have facilitated ongoing selection pressure and contributed to the evolution of WCR resistance to pyrethroids. Educational and incentive programs often fail to consider sociopolitical perspectives for effective resistance management and could be better improved by considering the farmers perception, the potential support of networked communities, and the compatibility between industry interests and federal policies70.Effective insecticide resistance management strategies include the integration of different control strategies and rotation of insecticide modes of action to reduce selection pressure and potential evolution of resistance35 provides a different mode of action that may be useful in WCR pyrethroid-resistance management programs. In this study, LC50’s from dose-response bioassays performed with indoxacarb active ingredient were lower for pyrethroid-resistant than -susceptible WCR populations. Also, calculated resistance ratios were <1.00, which suggests that pyrethroid-resistant WCR populations tested were more sensitive to indoxacarb than pyrethroid-susceptible populations. Indoxacarb is considered a pro-insecticide that needs bioactivation by esterase/amidase enzymes present in the target to become a more toxic compound71. Therefore, insecticide resistance mechanisms that involve increased activity of hydrolytic enzymes can result in a higher activation rate of indoxacarb in a negative cross-resistance relationship73 which could be further explored to manage pyrethroid-resistant WCR populations with enhanced metabolism. Our bioassay results suggest that the previously reported likely involvement of enhanced metabolism as part of the WCR pyrethroid-resistance mechanism14 may be contributing to increased susceptibility to indoxacarb.The recent EPA registration of indoxacarb for adult rootworm control in field corn33, dimethoate and other organophosphate insecticides should be used with caution in areas where WCR pyrethroid-resistance has been confirmed. In the western Corn Belt, WCR resistance evolution to rootworm-active Bt traits has increased the importance of aerially-applied insecticides and crop rotation as tactics to manage densities and mitigate resistance74. To develop more sustainable WCR management strategies, additional field research and modeling is needed to determine the biological and economical value of short- and long-term best management practices and specifically the optimal role of insecticides in the system. Because of the highly adaptable nature of WCR populations to selection pressure we reinforce the importance of using insect resistance management within an IPM framework to delay the evolution of WCR resistance and prolong the efficacy of formulated insecticide products and plant-incorporated traits.In summary, we conclude that the simulated aerial application method used to evaluate the performance of formulated insecticides in corn effectively confirmed bifenthrin resistance and could be useful to evaluate the efficacy of various aerially-applied insecticides for other pest insects. Many of the insecticides still used in the US western Corn Belt belong to the pyrethroid class, so WCR resistance to bifenthrin and potential cross-resistance with other compounds significantly restricts control options in affected areas. Dimethoate and indoxacarb could be useful compounds to manage bifenthrin WCR resistance using rotation-based approaches. However, because of potential cross-resistance between structurally related pyrethroids and organophosphates


We sought to identify candidate biomarkers for early brain metastasis (BM) recurrence in patients who underwent craniotomy followed by adjuvant stereotactic radiosurgery. RNA sequencing was performed on eight resected brain metastasis tissue samples and revealed B-cell related genes to be highly expressed in patients who did not experience a distant brain failure and had prolonged overall survival. To translate the findings from RNA sequencing data, we performed immunohistochemistry to stain for B and T cell markers from formalin-fixed parffin-embedded tissue blocks on 13 patients. CD138 expressing plasma cells were identified and quantitatively assessed for each tumor sample. Patients’ tumor tissues that expressed high levels of CD138 plasma cells (N = 4) had a statistically significant improvement in OS compared to low levels of CD138 (N = 9) (p = 0.01). Although these findings are preliminary, the significance of CD138 expressing plasma cells within BM specimens should be investigated in a larger cohort. Immunologic markers based on resection cavity analysis could be predictive for determining patient outcomes following cavity-directed SRS. Prognosis can vary considerably depending on factors such as the primary site of disease, histology, burden of systemic disease, and intracranial disease6. A standard treatment option for limited brain metastases from solid tumor primaries is stereotactic radiosurgery (SRS), which allows for rapid treatment of brain metastases with preservation of cognition, but it is an expensive modality compared to its alternative, whole brain radiation therapy (WBRT) and often requires a specialized center10.Approximately 170,000 patients in the United States are diagnosed with brain metastasis (BM) every year12. Methods of scoring clinical and histopathologic factors have attempted to predict intracranial failure, but these have been met with modest successes, predominantly because of the heterogeneity of the brain metastasis population15. Developing an accurate biomarker that can help predict for improved DBF or OS in patients with brain metastases treated with SRS would be a valuable tool.One limitation of SRS is the potential need and expense of additional treatments for distant brain failure (DBF)16.Specific patterns of immune cells within the tumor microenvironment are associated with improved outcome in patients with many types of cancers, regardless of the type of therapy administered17. Furthermore, the Immunoscore, which is based on an automated calculation of the CD3/CD8 ratio among TILs, has been shown to have independent prognostic significance in patients with brain metastases18. How TILs influence the response of brain metastasis to SRS is not known.Tumor infiltrating lymphocytes (TILs) of variable density can be observed in brain metastases and are typically composed of various cell types with a higher fraction of T cells than B cells. The presence of TILs, CD3 and CD8 T cells in specific, has been associated with improved survival as compared to patients with only sparse or scattered TIL infiltrationTo identify potential immune biomarkers that could be predictive of outcomes in brain metastasis patients, we performed a retrospective study and tissue analysis of patients who have undergone surgical resection of a brain metastasis followed by SRS. In the initial group of patients, we used microarray to identify immune signatures from resected brain metastasis tissue. We then expanded this microarray profile and sought to characterize immune cells within the brain metastasis tissue with immunohistochemistry associated with favorable outcomes.This study was approved by the Wake Forest Institutional Review Board IRB00008427. Patients signed informed consent for advanced tissue tumor banking prior to analysis. We selected 8 patients with new brain metastases from various primary tumors who underwent craniotomy and adjuvant SRS. These samples were frozen brain metastasis tissues were collected from patients who consented to the Wake Forest Brain Tumor Center of Excellence Tumor Tissue Bank. These samples were analyzed with RNA sequencing. Six of the original 8 patients had their formalin-fixed paraffin-embedded (FFPE) tissue also evaluated by immunohistochemistry (IHC) and 7 additional patients with available FFPE tissues who had craniotomy and received adjuvant SRS were also included in the study. All methods were performed in accordance with institutional policies, particularly for genomic analysis. A Clinical Laboratory Improvement Amendments certified laboratory was utilized for immunohistochemistry analysis of FFPE tissue with a board certified pathologist (SQ) reviewing the slides.et al.19. The targeting of the resection cavity of the metastasis was previously described by Jensen et al.20.SRS was performed on the Leksell Gamma Knife Model C  prior to May 2009, and Perfexion after May 2009. Same day headframe fixation was used for immobilization. Patients underwent a contrast-enhanced stereotactic magnetic resonance imaging scan of the brain with headframe in place. The GammaPlan Treatment Planning System  was used to develop the treatment plan. Median marginal dose prescribed was 18–22 Gy and was generally prescribed to the 50% isodose line. The dose selected was based on guidelines previously described by Shaw After adjuvant SRS, patients were followed with repeat MRI approximately 1-2 months later, and then every 3 month basis for 2 years. Distant brain failures (DBF) were determined to be new metastases that developed outside of the prior SRS volume.21, and gene counts determined using featureCounts software22. Differential gene expression was analyzed using the DESeq2 algorithm23. Significant genes were defined as p < 0.05 after adjustment for false discovery (Benjamini-Hochberg). Genes and samples were hierarchically clustered using Pearson correlation as the distance metric and visualized by heatmap analysis  with genomic DNA removal. RNA integrity (RIN) was determined by electrophoretic tracing using an Agilent Bioanalyzer. RNAseq libraries were constructed from samples (RIN > 7.0) using the Illumina TruSeq Stranded Total RNA kit with Ribo-Zero rRNA depletion. Indexed libraries were sequenced on an Illumina NextSeq 500 DNA sequencer using 150 × 150-nt paired end reads, generating > 40 million reads per sample (12 samples per flow cell) with > 80% of sequences achieving > Q30 Phred quality scores. Quality of raw sequencing reads were assessed by FASTQC analysis (Babraham Bioinformatics). Sequence reads were aligned using the STAR sequence alignerAfter initial RNA sequencing was performed in a CLIA certified laboratory, IHC was performed on a larger cohort of patients for proteins of interest, 13 in total, including 6 of the previous patients who underwent RNA Seq to correlate the findings from RNA sequencing. Two of the patients in the initial genomic sampling were excluded from IHC analysis because these patients did not meet the criteria of craniotomy followed by adjuvant SRS.Cases with corresponding formalin-fixed paraffin-embedded tissue were selected for immunohistochemical testing. IHC stains were performed according to validated procedures using DAKO Link 48 autostainer . The tissue was stained for CD20 (DAKO), CD3 (DAKO), CD138-expressing plasma cells (DAKO), Programmed cell death protein-1 (PD1) (Spring Bioscience) and Program death ligand-1 (PDL-1) (Spring Bioscience). Immunohistochemistry stains for T and B cell lymphocyte markers were chosen due to a genomic analysis of a smaller group of heterogeneous patients that revealed high expression of lymphocyte genomic products. The stains were scored on a scale of 0–3 by a board-certified pathologist who was blinded to the immune signature profiles. The number of cells expressing CD138 was quantitatively assessed per 40x high power field. The difference in assessment methods was due to the pattern of distribution of CD138, which had patchy infiltration whereas the other markers showed diffuse infiltration.24. Brain metastasis velocity (BMV) was calculated for each patien as described by Farris et al.15. Kaplan-Meier estimates, and competing risk with subdistribution hazards and figures were calculated using version 3.4.0 software .Patient outcomes were calculated from time of adjuvant SRS to event using competing risks estimates as previously described by McTyreHLA-DQA2, IGKJ4, IGKV1-16, IGHG4, IGHJ3, IGHV1-18, IGHV1-24, IGHV1-46, IGHV4-28, IGHV4-31, IGHV4-34, IGHV4-39 and IGHV4-55, exhibited higher expression compared to patients who experienced a DBF at 58 months post-treatment  11.1–37.5]. Histologies represented were melanoma (N = 2), Breast (N = 4), non-small cell lung cancer (N = 6), and small cell lung cancer (N = 1)  and 4 patients had CD138 cell counts of greater than 140 (range 143–247). Figure Of the patients with high expression of CD138, no deaths have been observed, no local failures, and one DBF has been recorded. All patients with high expression of CD138 were still alive. Of these patients, 3 had NSCLC and 1 had breast cancer. The median OS in patients with low expression of CD138 was 14.7 months vs not reached for high expression of CD138 (Log Rank p = 0.01) (Fig. 26. However, the infiltration of CD138 plasma cells in solid tumors has been associated with longer survival in patients with ovarian, gastroesophageal, NSCLC, and colon cancers27. These effects might be augmented by the localization within tumor, which would enable high concentrations of antibody to accumulate locally. Antibodies could opsonize tumor antigens, thereby facilitating antigen presentation and broadening of T-cell responses30. Moreover, antibodies could mediate direct antitumor effects by binding to and disrupting the function of their cognate antigens, activating the complement pathway, and/or triggering antibody-dependent cellular cytotoxicity31.For patients who underwent craniotomy followed by adjuvant SRS for brain metastases, high levels of CD138 lymphocytes within craniotomy samples were associated with improved survival compared to patients with low levels of CD138 lymphocytes. CD138 is a plasma cell marker and a proteoglycan, syndecan 1, which is expressed by solid tumor cells and plasma cells. When expressed by tumor cells, CD138 has been associated with metastasis and poor survivalversus upfront WBRT. Several statistical models have been developed in order to help triage patients, but the ability of validate them has been somewhat questionable32. A major issue with validation of predictive models for brain metastases has been the biological heterogeneity of brain metastases and the fact that brain metastases of different primary tumors have distinct natural histories due to variations in systemic disease burden and control33. The discovery of the immunotype changes that drive these biological differences will hopefully help to improve the predictability of brain metastasis outcomes moving forward.Unlike biomarkers that predict for brain metastasis-specific response to a systemic agent, a biomarker with the capability of predicting SRS outcomes in patients with brain metastases from different primary tumors would have significant clinical potential given the current dilemma by which patients are selected for upfront SRS This study is limited by a small sample size, retrospective nature, and requires further validation in a large cohort of patients prior to utilizing CD138 as a biomarker for clinical practice. However, the identification of an immune marker within resected brain metastasis tissue that translates into improved survival outcomes is an exciting finding and warrants further investigation.Patients with high levels of CD138 expressing plasma cells may have improved OS compared to patients with low levels of CD138 with a trend towards fewer intracranial failures. The results are hypothesis generating and CD138 expression should be investigated in a larger cohort of patients with resected brain metastasis tissue.


Reduced expression of FZP in the reproductive stage increases the extent of higher order branching of the panicle, resulting in increased grain number. Genotype analysis of this gene in cultivars from the publicly available National Institute of Agrobiological Sciences (NIAS) Core Collection demonstrated that the extent of higher order branching, especially in the upper panicle, was increased in those cultivars carrying the FZP allele associated with qSrn7. Furthermore, chromosome segment substitution lines resulting from a cross between Koshihikari and Kasalath, the latter of which carries qSrn7/FZP, also showed that upper panicle higher order branching and grain yield were increased by qSrn7/FZP. Our findings indicate that qSrn7/FZP influences panicle branching pattern and is thus useful in the breeding of high-yield rice varieties.Rice grain number directly affects crop yield. Identifying alleles that improve panicle architecture would greatly aid the development of high-yield varieties. Here, we show that the quantitative trait locus  Rice (Oryza sativa L.) is a vital crop worldwide and sustains more than three billion people3. The panicle is a pivotal organ in determining rice grain yield, and its size and architecture are key targets in selective breeding for improved rice grain yield and quality5. The rice panicle is comprised of several organs, namely the rachis (the main axis), primary rachis branches (PBs), and secondary rachis branches (SBs), which include additional higher-order rachis branches  reduces panicle development, resulting in fewer panicle rachis branches8, and lax (lax panicle) inhibits rachis branching10. Although these mutants are useful for elucidating the regulatory mechanisms of panicle development, their applicability in agriculture is limited because they have abnormal plant architecture. By contrast, quantitative traits found in various rice cultivars are influenced by multiple alleles derived from the accumulation of natural mutations. Several of these alleles are associated with grain yield. A natural allele of DENSE AND ERECT PANICLE 1 (DEP1) was shown to influence panicle architecture, resulting in reduced panicle internode length and increased grain number11, whereas Grain Number 1a (GN1a)12 and WEALTHY FARMER’S PANICLE (WFP)13 were shown to significantly increase PB number, grain number, and yield. These past studies have examined terminal phenotypes such as grain number and panicle length; however, panicle structure as determined by its branching pattern has not been characterized in detail, such as through comprehensive analysis of several organs.Many genes have been identified that are associated with rice panicle development. Through mutant characterization, it was shown that 14. High-yield modern rice varieties have higher grain filling rate in upper areas of the panicle16. The rice panicle exhibits a recursive structure in which higher-order rachis branches radiate from lower-order rachis branches, which in turn radiate from the rachis. Therefore, knowledge of how the order of each rachis branch is affected by a particular gene will greatly assist in efforts to alter the architecture of the rachis for optimal yield. Furthermore, identifying a novel quantitative trait locus (QTL) that regulates these organs would assist in breeding new rice varieties with increased yields.In the rice panicle, flower opening and grain filling occur in a sequential developmental process, which is initiated in the spikelet furthest from the rachis axis and proceeds inward. As a result, the position of a spikelet within a panicle determines the timing of both flower fertilization and grain development. Panicle size and branching pattern are thought to determine grain shape and quality directlyqSrn7, which regulates a novel panicle branching pattern in rice. Analysis of chromosome segment substitution lines (CSSLs) carrying qSrn7 showed that grain number and yield increased in conjunction with changing panicle structure and branching pattern. These results describe the mechanism by which qSrn7 influences panicle structure, and suggest that qSrn7 may be used in efforts to generate rice varieties with high grain yield.Here, we describe the identification and characterization of a major QTL, To identify genes that influence rice grain yield, we selected two rice varieties that display clear differences in grain number per panicle, namely Koshihikari, a typical Japonica rice variety, and ST-5, from the Stocked rice collections of Togo field and Nagoya University-5. Koshihikari and ST-5 have approximately 182 and 394 grains in the main panicle, respectively Fig. . The panTo define the panicle structure that results in the increased grain number of ST-5, we schematically described the panicle branching pattern by detailing the number of secondary rachillae (SRs) and SBs on each PB Fig. . Each PB2 population derived from a cross between Koshihikari and ST-5. We detected one major QTL with a log10 odds (LOD) score of >3 on chromosome 7 (LOD score = 13.6), which was denoted as qSrn7 (QTL for secondary rachis branching on chromosome 7). A total of 1700 F3 individuals derived from heterozygous Koshihikari and ST-5 F2 plants were screened for recombination within the target region defined by simple sequence repeat (SSR) markers RM22060 and RM22156 , there are eight predicted open reading frames (ORFs) in this region , which encodes an AP2/ERF-type transcription factor that regulates spikelet number in the panicle18. We hypothesized that the phenotype of increased SBs in ST-5 panicles was caused by weak FZP activity. Therefore, we performed an allelism test between ST-5 and fzp plants. The panicles of the resulting F1 hybrids displayed a ST-5 panicle phenotype, indicating that genetic complementation of fzp had occurred  and Kasalath carrying qSrn7/FZP. The progeny of the cross are herein referred to as KKSLs. SB number in the upper regions of the panicle was higher in Kasalath and the KKSLs (KKSL_221 and KKSL_222) than in Koshihikari  launched a breeding program for New Plant Type (NPT) rice to increase the yields of modern indica cultivars by incorporating genetic material from tropical japonica landraces21. The NPT breeding program attempted to boost yield by simultaneously selecting for increased panicle size (sink) and photosynthetic capacity (source)21. The NPT cultivar successfully generated large panicle phenotypes but had lower yields than modern indica cultivars, because it did not exhibit the desired combinations of sink and source traits that are characteristic of high-yielding varieties23. This result suggests that physiologically optimizing panicle architecture for grain filling and yield will probably involve managing a highly interactive network or trait complex. Thus, analyses that consider complex panicle phenotypes are of great importance20. Furthermore, as changes to panicle branching pattern and structure are associated with increases in grain yield15, it is important to characterize how a particular gene affects the panicle branching pattern.Panicle architecture is the major target in efforts to improve crop yield in rice. Efforts to improve panicle grain number would benefit both from focusing on previously characterized genes that influence branching pattern and on identifying additional genes that influence panicle traits. To the best of our knowledge, genes that affect panicle branching pattern had not hitherto been identified. Therefore, we used a rice variety with increased panicle grain number to identify and characterize a gene involved in panicle branching pattern.qSrn7 revealed that the locus contains FZP and its maize ortholog BRANCHED SILKLESS 1 (BD1)24, which encodes an AP2/ERF-type transcription factor. Furthermore, we showed that qSrn7 in the existing variety ST-5 and in CSSLs using in this study  produced more SBs with attached filled grains . QTLs were identified using Haley–Knott regression and the significance threshold was set using 1,000 permutations. SSR markers used in positional cloning are listed in Supplementary Table QTL analysis was performed with 94 FFZP, the coding sequence of FZP was amplified from Koshihikari cDNA using a forward primer (5′-GCAAGCTTTCAATGGGAGAGGAAGCTGAATGG-3′) containing a HindIII site and a reverse primer (5′-GCACTAGTATGAACACTCGAGGCAGCGGCAGT-3′) containing a SpeI site. The fragment was cloned into the HindIII–SpeI sites of pUbi::nos-pCAMBIA1380 and introduced into Nipponbare, a japonica variety, by Agrobacterium tumefaciens (EHA105)-mediated transformation according to the methods of Ozawa et al.28. Transformed cells and plants were selected by hygromycin resistance, and regenerated seedlings were grown to maturity in pots under greenhouse conditions. More than ten independent T0 plants were isolated and five plants were analyzed. As a control, transformants containing an empty vector were included, and five of the resulting control plants were included in each analysis.For antisense expression of et al.29 with some modifications and was treated with DNase I. First-strand complementary DNA (cDNA) was synthesized from 1 μg of total RNA using a QuantiTect Reverse Transcription Kit (Qiagen). Quantitative real-time PCR was performed using the QuantiTect SYBR Green PCR Kit (Qiagen) and a LightCycler System . Transcript levels were measured in three independent biological replicates. The UBQ gene from rice was used as an internal standard for normalizing variations in cDNA concentration. The primer sets used for PCR were as follows: FZP primers, 5′-CACATTGGCTCGTACGGTC-3′ and 5′-GAGAAGAGGAAGTCGTGG-3′; UBQ primers, 5′-AGAAGGAGTCCACCCTCCACC-3′ and 5′-GCATCCAGCACAGTAAAACACG-3′.Total RNA from various organs was prepared as described by Sambrook 30. Digoxigenin-labeled antisense RNA probes for qSrn7/FZP transcript were prepared as described by Komatsu et al.17. Hybridization and immunological detection of the digoxigenin-labeled probes were performed according to a previously described method31 with some modifications.Tissues were fixed with 4% (w/v) paraformaldehyde and 0.25% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.2) overnight at 4 °C and embedded in SCEM compound . The cut surface was covered with an adhesive film  and frozen sections (8–14 μm) were prepared with a cryostat  according to a previously described methodDataset 1


Intravenous (IV) zanamivir could be a suitable alternative for the treatment of severe influenza A(H1N1)pdm09 infection in patients who are unable to take oral or inhaled medication, for example, those on mechanical ventilation and extracorporeal membrane oxygenation (ECMO). However, data on the clinical outcomes of such patients is limited.We report the clinical outcomes of four patients who were admitted at the intensive care unit during the 2017–2018 influenza season with severe sepsis (SOFA score > 11) and acute respiratory distress syndrome requiring ECMO and mechanical ventilation. Two patients were immune-compromised. The A(H1N1)pdm09 genome was confirmed by polymerase chain reaction (PCR) on nasopharyngeal specimen swabs prior to administration of IV zanamivir at a dose of 600 mg twice daily. Weekly qualitative PCR analysis was done to monitor viral clearance, with zanamivir treatment being discontinued upon receipt of negative results. In addition, the patients were managed for concomitant multidrug-resistant bacterial infections, with infection resolution confirmed with blood cultures.The median time for zanamivir treatment was 10 days (IQR 10–17). The clinical outcome was favourable with all four patients surviving and improving clinically. All four patients achieved viral clearance of A(H1N1)pdm09 genome, and resolution of multidrug-resistant bacterial infections.IV zanamivir could be a good therapeutic option in patients with severe influenza A(H1N1)pdm09 infection who are unable to take oral or aerosolised antiviral medication. We recommend prospective randomized control trials to support this hypothesis. Influenza due to the 2009 pandemic A/H1N1 virus, abbreviated as A(H1N1)pdm09, a viral disease of public health concern, causes between three to 5 million cases globally, frequent hospitalizations and over 650,000 deaths annually .Oseltamivir is the first-line treatment for A(H1N1)pdm09 infection and is administered orally . HoweverOn 28 February 2019, the Committee for Medicinal Products for Human Use (CHMP) adopted a positive opinion, authorizing the marketing of IV zanamivir under exceptional circumstances , 11. It In this case series, we report the clinical evolution and survival outcome of the largest number of patients who, to the best of our knowledge, underwent ECMO support and were treated with IV zanamivir. Another case report described a single patient who suffered severe respiratory insufficiency was treated with IV zanamivir but was not prescribed ECMO .During the influenza season in the years 2017–2018, four patients  (aged from 37 to 59 years) were admitted to our ICU for ECMO respiratory support due to severe ARDS related to A(H1N1)pdm09 infection. All patients were transferred from peripheral ICUs for ECMO treatment, after a median hospital length of stay of 9 days (range: 1–10 days). Two patients were immune-compromised; patient #1 due to relapsing Hodgkin lymphoma and patient #2 due to multiple myeloma. The other patients did not suffer from any significant underlying medical conditions.At ICU admission, all four patients had a sequential organ failure assessment (SOFA) score greater than 11, predicting a mortality rate higher than 50%, while the median acute physiology and chronic health evaluation (APACHE) score was 20 (range: 18–22).® FilmArray® Respiratory Panel, BioMèrieux diagnostics, Italy), using the procedure previously reported [B. pertussis, C. pneumoniae, and M. pneumoniae). In summary, the test pouch contains all dry reagents required for specimen extraction, first-stage multiplex PCR, and individual second-stage real-time PCRs. Operatively, the pouch was placed in the FilmArray® instrument, and a pre-programmed run was initiated. Results were generated using amplification and melting curve data. Negative controls were certified by the manufacturer before the Food and Drug Administration (FDA) and Conformité Européenne (CE) approval of the test. All precautions have been used during the procedure to avoid pre-analytical contamination. The test pouch is a sealed, non-reusable item. Negative controls were run periodically to evaluate the performance of the PCR test using sterile collection media. Weekly nasopharyngeal swabs were taken for PCR qualitative analysis to detect viral clearance and were reported as positive or negative indicating presence or absence of the virus in the swab specimen, respectively. No other viruses were detected in this study and virus isolation was not attempted.The A(H1N1)pdm09 genome was detected through multiplex polymerase chain reaction (PCR) performed on nasopharyngeal swabs  obtained by gas analysis from arterial blood samples. Table Figure The median time of ECMO support was 7 days (IQR 6–8 days), while the median time spent under mechanical ventilation was 15 days (IQR 8–21). The median length of stay in the ICU was 20 days (IQR 14–28). The median duration of antiviral therapy was 10 days (IQR 10–17). During treatment, no adverse reactions or side effects were reported. Even though the expected survival of the patients was less than 50% at the time of admission as predicted by SOFA score, clinical conditions progressively improved and all four patients survived.Acinetobacter baumannii (Ab) pneumonia (susceptible only to colistin) and carbapenem-resistant Klebsiella pneumoniae (CR-Kp) and treated with IV colistin and ceftazidime/avibactam for 7 days. Patient #2 had CR-Kp pneumonia and was treated with a two-week course of IV/aerosolized colistin plus meropenem. Patient #3 suffered from CR-Kp pneumonia and secondary bacterial infection and was treated with IV colistin plus double carbapenem (ertapenem and meropenem for 7 days). Patient #4 suffered from XDR-Ab pneumonia with secondary bacteremia and was treated with IV/aerosolized colistin, tigecycline, and rifampicin for 12 days.The clinical conditions of the four patients progressively improved with IV zanamivir and adjuvant therapies despite superimposed multi-drug resistant infections. As summarised in Table All four patients demonstrated both clinical and radiological evidence of resolution of bacterial lung complications. Bloodstream bacterial infection resolution, as defined by no growth of bacteria in the follow-up blood cultures, performed after 5 and 4 days from the previous diagnosis, was confirmed in patients #3 and #4, respectively. Bloodstream infections were determined by detections of bacteria on 3–4 sets of blood cultures, collected from peripheral puncture of one forearm vein of patients under sterile conditions, at the time of fever spikes, chills, leucocytosis and/or septic shock . To exclDespite having severe life-threatening illness because the expected survival of the patients was less than 50% at the time of admission as predicted by SOFA score requiring life support treatment with ECMO, and despite subsequent MDR bacterial superinfections, all four patients at the end of this study survived. This survival could be attributed to the effects of IV zanamivir, although we cannot underestimate the role of the multidisciplinary proactive treatment approach  employed in the management of the patients. Moreover, it should be noticed that none of the four cases was treated with zanamivir alone because all received oseltamivir in addition to zanamivir. Therefore, the latter drug contributed to the treatment effect. Although the two drugs were administered separately, they may also have had an additive or synergistic effect in preventing the emergence of A(H1N1)pdm09 isolates resistant to one or the other drug, or treating pre-existing resistant isolates more effectively. Indeed, virological potency of antiviral combination therapy for the treatment of influenza was higher than that of oseltamivir monotherapy, although this did not translate into greater clinical efficacy . MoreoveZanamivir proved effective particularly in one patient who was diagnosed with severe pneumonia, impaired gastrointestinal peristalsis and drug malabsorption, requiring life support measures such as deep sedation, neuromuscular blockade and prone positioning . The autSome studies have demonstrated the advantage of IV zanamivir over oral oseltamivir. In one study involving mutated viral strains of influenza H1N1 (H274Y mutation), patients who received oseltamivir had prolonged viral shedding and delayed resolution of their symptoms, unlike their counterparts who received zanamivir . This shA large retrospective report by Chan-Tack et al. described the characteristics of 364 patients who received IV zanamivir, retrieved from the Food and Drug Administration (FDA)’s Emergency Investigational New Drug application database . Among tPatient #1 and #2 were immune-compromised. Although this partially explains their prolonged treatments because of virus persistence, it does not seem to offer a similar explanation for patient #3 and #4 who did not have any underlying medical conditions, yet received treatments for relatively prolonged periods of time ranging from 13 to 15 days. In the case of virus persistence and treatment prolongation, virus isolation and running genotypic tests for potential antiviral resistance on neuraminidase as well as molecular monitoring and phenotypic assays are recommended. Indeed, drug-resistance to oseltamivir and zanamivir has been reported in some patients with influenza  However,In conclusion, the findings in our study suggest that IV zanamivir is a good therapeutic option in patients with severe influenza, who are unable to take an oral or aerosolised drug such as those on ECMO and mechanical ventilation. We recommend prospective randomized trials to support this hypothesis as well as pharmacokinetic evaluations and molecular characterisation of influenza viruses to help delineate better prevention and treatment strategies for possible resistant strains.


To validate the utility of the chemokine ligand 12 (CXCL12) as prognostic marker in patients with localized and metastatic germ cell tumors (GCT).CXCL12 expression was analyzed on a tissue microarray consisting of 750 tissue cores of different histological tumor components, Germ cell neoplasia in situ (GCNIS) and adjacent normal tissue of 263 testicular cancer patients using a semi-quantitative score. The association between CXCL12 expression and recurrence-free survival (RFS) as well as overall survival (OS) was assessed using Kaplan-Meier curves with log-rank tests.p = 0.270). In contrast, post chemotherapy RFS of patients with metastatic non-seminoma and positive CXCL12 expression was significantly shorter compared to CXCL12 negative patients (p = 0.003). OS differences were not statistically different between patients with CXCL12 positive or negative tumors for either localized or metastatic disease.CXCL12 expression was absent in all seminomas but was found in 52 of 99 (52.5%) non-seminomas. Follow-up was available for 260 patients of which 36 (13.8%) recurred. In patients with stage 1 non-seminoma GCT, CXCL12 expression was not associated with higher risk of disease recurrence (CXCL12 is almost exclusively expressed in non-seminoma. Pure seminoma, GCNIS and adjacent normal testicular tissue are CXCL12 negative. Our analysis suggests that patients with metastatic disease and a CXCL12-positive non-seminoma are at higher risk for disease recurrence after first-line chemotherapy and might thus be candidates for more intensive treatment and/or closer follow-up. Testicular germ cell tumors (GCT) represent the most common solid neoplasm of men in their third and fourth decade . SeventyRecently Gilbert et al.  were ablA consecutive series of patients with surgically resected testicular GCT between 2000 and 2014 was evaluated. Patients with bilateral testicular tumors, extra-gonadal primary or missing follow-up data were excluded from the analysis. Clinical characteristics , histology of the primary tumor, staging characteristics according to the tumor, nodes and metastases system (TNM) of the American Joint Committee on Cancer (AJCC) and to the International Germ Cell Cancer Collaborative Group (IGCCCG) and blood-based tumor markers , beta-human chorionic gonadotropin (b-hCG) and lactate dehydrogenase (LDH)) as well as follow-up information were extracted from the medical charts. The study was approved by the local ethics committee (KEK StV. 25–2008).A tissue microarray (TMA) was constructed of formalin-fixed, paraffin-embedded tumor tissue of all included patients. If more than one tumor component was present in one patient all different GCT components were separately punched and included on the TMA. Tumors were classified by an experienced uropathologist (PKB) specialized in GCT pathology according to the 2016 WHO Classification . Each tuSections of 3 μm thickness of the TMA blocks were mounted on glass slides , deparaffinized, rehydrated and stained with hematoxylin and eosin using standard histological techniques. CXCL12 immunohistochemistry was performed using the monoclonal CXCL12 antibody . Peripheral neuronal tissue served as internal positive control for CXCL12 staining. The stained slides were digitalized and evaluated using imaging software . Two investigators  evaluated the CXCL12 staining of each spot on the TMA. A patient’s tumor was classified as being positive if any of its components showed either moderate or strong CXCL12 expression and negative if CXCL12 expression was either absent or weak  and overall survival (OS) were calculated from time of diagnosis to first recurrence after initial management or death respectively. RFS and OS of patients with CXCL12 positive and negative tumors were compared using Kaplan-Meier curves and the log-rank test. Statistical analysis was performed using IBM SPSS Statistics . The results for continuous normally distributed variables are expressed as means ± standard deviation (SD). Continuous non-normally distributed variables are presented as median and interquartile ranges (IQR). The results for categorical variables are presented as percentage. A The mean age at diagnosis was 26 years and patients were followed for a median of 64 months Table . At initp < 0.001) and b-HCG values  compared to CXCL12 negative patients  or time to death (p = 0.17) . There was even a trend for a shorter time to recurrence in patients with high CXCL12 expression.In patients with localized non-seminoma, CXCL12 expression was not significantly associated with time to disease recurrence (17) Fig. . Also afp = 0.66). In contrast, patients with metastatic non-seminoma and higher CXCL12 expression showed a significantly shorter time to disease recurrence (p = 0.03), whereas time to death was not significantly different (p = 0.21) compared to CXCL12 negative patients.In patients with metastatic non-seminoma CXCL12 expression was not associated with worse IGCCCG risk groups  –16. CXCLThe role of CXCL12 in GCT is controversial. In contrast to most solid cancers Gilbert et al. found that CXCL12 expression is a protective factor in patients with stage 1 non-seminoma [For metastatic GCTs, the IGCCCG classification is currently recommended to counsel patients with metastatic GCT regarding their risk of disease recurrence and survival and to guide treatment for these patients . This clThe present analysis is limited by its retrospective design, single center approach and relatively small sample size. A further limitation of our study is that we were not able to perform a meaningful multivariate cox regression analysis due to the limited number of events. Additionally, multiple testing may lead to type I errors, which might overestimate the described associations between CXCL12 expression and oncologic outcomes. Thus, our analyses should rather be considered exploratory. A larger dataset including lymph vascular invasion status or IGCCCG classification is needed to reliably assess a potential independent prognostic impact of CXCL12 expression in localised and metastatic disease respectively. Especially the planned IGCCCG update might be an opportunity to elucidate whether CXCL12 would ultimately possess the potential to predict oncologic outcomes besides the IGCCCG classification .CXCL12 is exclusively expressed in non-seminoma and absent in seminoma, normal tissue or GCNIS. We were not able to show an association between CXCL12 and lower recurrence rates in stage I non-seminoma patients. However, positive CXCL12 expression was associated with shorter RFS after first-line chemotherapy in patients with metastatic GCT and thus might help to identify patients at a higher risk for disease recurrence.


NHE-1 activation drives Na+ into cardiomyocytes in exchange for H+ with its exchange rate intensified upon reperfusion during the resuscitation effort. Na+ accumulates in the cytosol driving Ca2+ entry through the Na+-Ca2+ exchanger, eventually causing cytosolic and mitochondrial Ca2+ overload and worsening myocardial injury by compromising mitochondrial bioenergetic function. We have reported clinically relevant myocardial effects elicited by NHE-1 inhibitors given during resuscitation in animal models of ventricular fibrillation (VF). These effects include: (a) preservation of left ventricular distensibility enabling hemodynamically more effective chest compressions, (b) return of cardiac activity with greater electrical stability reducing post-resuscitation episodes of VF, (c) less post-resuscitation myocardial dysfunction, and (d) attenuation of adverse myocardial effects of epinephrine; all contributing to improved survival in animal models. Mechanistically, NHE-1 inhibition reduces adverse effects stemming from Na+–driven cytosolic and mitochondrial Ca2+ overload. We believe the preclinical work herein discussed provides a persuasive rationale for examining the potential role of NHE-1 inhibitors for cardiac resuscitation in humans.Out-of-hospital sudden cardiac arrest is a major public health problem with an overall survival of less than 5%. Upon cardiac arrest, cessation of coronary blood flow rapidly leads to intense myocardial ischemia and activation of the sarcolemmal Na Out-of-hospital sudden cardiac arrest is a major public health problem worldwide with close to 360,000 cases assessed every year by Emergency Medical Services (EMS) in the United States. In approximately half of these cases, cardiopulmonary resuscitation (CPR) is attempted, and of these only 9% survive the episode and return to their pre-arrest condition with adequate neurological function . Efforts2+ overload followed by eventual opening of the mitochondrial permeability transition pore and collapse of the proton-motive force required for ATP synthesis [The crucial first step in the resuscitation effort is the restoration of cardiac activity, which depends largely on the ability of CPR to generate sufficient coronary blood flow to ameliorate myocardial ischemia and establish the metabolic conditions required for the return of an electrically organized and mechanically effective cardiac activity. However, conventional CPR is unable to generate more than 10% to 20% of the coronary blood flow ,4, constynthesis ,8,9,10.We have conducted preclinical research on various animal models of cardiac arrest focusing for the past two decades on the hypothesis that resuscitation outcomes can be improved by interventions able to attenuate myocardial reperfusion injury. We have examined various strategies ,25,26,27The present review is focused on our work exploring the myocardial effects of NHE-1 inhibitors during cardiac resuscitation discussing mechanisms and physiological effects that we propose are relevant for clinical translation.+-H+ exchange and the remaining 315 amino acids forming a cytoplasmic domain containing various regulatory sites  and intensifies the Na+-H+ exchange along with the corresponding cytosolic Na+ entry [+ from the cytosol during ischemia is limited consequent to inhibition of the Na+-K+ pump [+ accumulation in the cytosol [+ may enter the cytosol through Na+ channels and the Na+-HCO3− co-transporter  [Left ventricular distensibility is important for adequate preload. During cardiac resuscitation, left ventricular distensibility gradually declines compromising the ability of cardiac compression to generate forward blood flow . Our NHE < 0.05) .The preceding observations supported the concept that a more distensible left ventricle would allow a larger volume of blood to fill the left ventricular cavity before each chest compression resulting in a large volume of blood to be ejected during compression. This effect would be expected to enhance the hemodynamic efficacy of chest compression and—in turn—explain the increased coronary perfusion pressure and higher resuscitability observed in the swine model . To furtWe conducted two series of experiments in which rats were subjected to 10 min of untreated VF followed by 8 min of chest compression before attempting defibrillation with the compression depth adjusted to maintain an aortic diastolic pressure between 26 and 28 mmHg in the first series and between 36 and 38 mmHg in the second series. Within each series, rats were randomized to receive a bolus of cariporide (3 mg/kg) or NaCl 0.9% (vehicle-control) before starting chest compression.In rats that received cariporide, higher cardiac output and higher organ blood flows (including heart and brain) were observed for a given compression depth . Thus, aLeveraging on this blood flow effect, we anticipated a positive interaction between NHE-1 inhibitors and vasopressor agents; i.e., for a higher blood flow generated by chest compressions in the presence of an NHE-1 inhibitor, the same increase in peripheral vascular resistance elicited by a particular vasopressor agent would be expected to generate a higher blood pressure and a higher coronary perfusion pressure aiding successful resuscitation. This effect, however, presumes that NHE-1 inhibitors lack or have minimal vasodilatory effects. We examined this interaction in our rat model of VF and chest compression . We condin situ the descending aorta with a Krebs-Henseleit solution at a flow rate titrated to generate an aortic pressure between 30 and 35 mmHg. This flow corresponded to ~20 mL/min. In addition, a 0.9% NaCl solution containing cariporide was infused and compared to NaCl control. Bolus administration of either epinephrine or vasopressin elicited—as expected—a transient aortic pressure increase of magnitude close to 80 mmHg. The effect was the same in the presence or absence of cariporide when vasopressin was administered but of smaller but not statistically significant magnitude when epinephrine was administered . As shown in 2+ overload adversely affects myocardial function by compromising mitochondrial bioenergetic function  and by precluding relaxation of cardiomyocytes. This last effect results from the alluded cytosolic Ca2+ overload and also from the inability of the energy-requiring sarcoplasmic reticulum Ca2+-ATPase to pump Ca2+ from the cytosol into the sarcoplasmic reticulum as part of the normal cardiomyocyte Ca2+ cycling. Altogether, these effects explain the left ventricular wall thickening with reduction in cavity size  during VF and post resuscitation left ventricular systolic and diastolic dysfunction. In a series of experiments, discussed below, we documented the actions of NHE-1 inhibitors attenuating these abnormalities and the corresponding functional effects. We first used our rat model of VF and closed-chest resuscitation to examine the effects of NHE-1 inhibition and Na+ channel blockade—interventions collectively referred to as “Na+-limiting interventions”—on intracellular Na+ content, mitochondrial Ca2+ content, cardiac function, and plasma levels of cardio-specific troponin I (cTnI) [+-limiting intervention immediately before starting chest compression or vehicle control. The Na+-limiting interventions included the NHE-1 inhibitor AVE4454B (1 mg/kg), lidocaine (5 mg/kg), and the combination of AVE4454B and lidocaine.The cytosolic and mitochondrial CaI (cTnI) . Measure+ entry attenuated increases in cytosolic Na+ and mitochondrial Ca2+ overload during chest compression and the post-resuscitation period. This effect was associated with attenuation of post-resuscitation cTnI increase with the level inversely proportional to cardiac work. In a previous study using an isolated perfused rat heart model we reported that VF contributed independently to left ventricular Na+ overload and speculated that the fibrillatory activity—by opening of Na+ channels—could have been the contributing mechanism [As shown in echanism .We also used an open-chest pig model of electrically-induced VF and extracorporeal circulation to study the effects of inhibiting NHE-1 on myocardial energy metabolism under conditions of controlled coronary perfusion pressure . For thiAs shown in 2+ overload—and supportive of the concept that preservation of left ventricular distensibility during resuscitation is likely to stem from lesser cytosolic and mitochondrial Ca2+ overload, correspondingly impacting cardiomyocyte relaxation and energy generation.These energy effects are consistent with NHE-1 inhibition protecting mitochondrial bioenergetic function—probably as a result of limiting mitochondrial Cac, which is a mitochondrial protein responsible for the transfer of electrons from complex III to complex IV of the electron transport chain. Its release to the plasma is associated with the severity of myocardial injury after cardiac arrest [+-H+ exchange upon buffering of the extracellular pH with the adverse myocardial effect consequent to intensification of the Na+–driven cytosolic and mitochondrial Ca2+ overload. The excessive lactatemia is best explained by acceleration of the glycolytic pathway after buffer administration and attenuation of the intracellular acidosis given the pH-dependency of phosphofructokinase activity, which is the rate-limit enzyme of the glycolytic pathway [An additional consideration particularly relevant to cardiac resuscitation is the effect that changes in extracellular pH have on reperfusion injury after NHE-1 activation. We recently reported in our rat model if VF and closed-chest resuscitation a profound detrimental myocardial effect caused by the systemic administration of a buffer solution immediately after return of spontaneous circulation . The bufc arrest ,59. Buff pathway ,61,62. C pathway ,64.Only a handful of studies—sponsored by pharmaceutical companies—have been conducted examining the effects of NHE-1 inhibition in humans. These studies, however, have examined conditions other than cardiac arrest including effects in patients undergoing coronary interventions for acute myocardial infarction ,66,67 anWe propose that the preclinical findings herein discussed are relevant to cardiac resuscitation in humans given the highly phylogenetically conserved nature of NHE-1, its physiological role, and its pathophysiological significance during ischemia and reperfusion. Functionally, preservation of left ventricular distensibility during CPR, greater electrical stability upon return of spontaneous circulation, attenuation of post-resuscitation myocardial dysfunction, and amelioration of the adverse effects or epinephrine are all highly desirable effects expected to improve outcome from cardiac arrest. Clinical studies determining the extent to which these effects can be translated to humans during cardiac resuscitation are eagerly awaited.