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Abstract Background The aim of this study is comparison the effectiveness of stereotactic, hypofractionated and conventional radiotherapy assessed by the tumor volume changes of paraganglioma located in the head and neck region concerning fractional and total doses. Methods We analyzed 76 patients after radiotherapy due to paraganglioma who were assigned to 3 groups considering fractional (≤2 Gy, 3–5.5 Gy, ≥6 Gy) and total (≤20 Gy, 21–40 Gy, >40 Gy) doses. The volumes of irradiated tumors were measured and compared based on diagnostic images performed before and after the treatment. Results The mean tumor volume after the treatment with the lowest fractional dose (≤2 Gy) was decreased by 14.4 cm3. In patients treated with higher fractional doses (>2 Gy), the mean tumor volumes decreased by less than 1 cm3 for hypofractionated and stereotactic radiotherapy. 15.9 cm3 reduction of the mean tumor volume after the treatment with the highest RT total dose (>40 Gy) was stated. In patients treated with total doses ≤20 Gy and 21–40 Gy, the mean tumor volume was stable and reduced by 1.15 cm3, respectively. The analysis demonstrates a statistically significant (p 40 Gy.

Nowadays there is a concern to improve the quality of education by including an interdisciplinary approach of concepts and their integration in the curriculum of scientific disciplines. The development of microbial fuel cells as a potential alternative for production of renewable energies gives undergraduate students the challenge of integrating interdisciplinary concepts in a hot topic of global interest as alternative energies. We present a laboratory experiment that has been part of a third-year undergraduate course in biology where students gained experience in assembling microbial fuel cells and the understanding of how they work. In this process, the students could integrate biological, biochemical, and electric concepts. In addition, the acquisition of manual skills and experimental design decisions are important for the development of future professionals.

Young almond (Prunus amygdalus) orchards replanted where old orchards of stone fruits (Prunus sp.) have been removed are subject to physical, chemical, and biotic stressors. Among biotic challenges, for example, is almond/stone fruit replant disease (ARD; formally known as Prunus replant disease), which specifically suppresses the growth and yields of successive almond and other stone fruit plantings and is caused, in part, by a soil microbial complex. During four orchard trials representing different almond replant practices and scenarios in the San Joaquin Valley in California, we examined the impacts of phosphorus (P) fertilization on the growth of replanted almond. During all trials, P was applied to tree root zones just after replanting, and the impact was assessed according to trunk cross-sectional area (TCSA) growth for 2 years. Expt. 1 was performed where a previous almond orchard was cleared using whole orchard recycling (i.e., the old orchard was “chipped” and then turned into the soil). The land was replanted without preplant soil fumigation. We tested separate fertilizer treatments based on various P, nitrogen, micronutrient, and “complete” formulations. Expt. 2 was also performed where an old almond orchard was recycled, but the soil was preplant-fumigated before replanting. Here, we tested only P fertilization. Expts. 3 and 4 were conducted where an old peach (Prunus persica) orchard was removed. Here, P and nitrogen fertilizer treatments were tested among additional factors, including preplant soil fumigation (Expts. 3, 4) and whole orchard recycling chips (Expt. 4). During all four trials, P fertilization (P at 2.2 to 2.6 oz/tree within a few weeks after planting) significantly increased TCSA growth. The growth benefit was nuanced, however, by almond cultivar, date of replanting, rootstock, and other site-specific factors. Although P fertilization did not match the benefit of preplant soil fumigation for the management of ARD, our data indicated that P fertilization can improve the growth of young almond orchards in diverse replant settings with or without preplant soil fumigation and should be considered by California almond producers as a general best management practice.

Background: Human immunodeficiency virus (HIV) drug resistance is a major cause of treatment failure in children and adolescents infected with the virus. Objectives: The objectives of the study are to investigate HIV drug resistance (HIVDR) in patients who attended a referral care center in Argentina over a 15-year period and to compare mutational patterns between HIV-1 pol sequences characterized as B or BF recombinants. Methods: Individual resistance-associated mutations (RAMs) (to protease and reverse transcriptase inhibitors) were identified according to IAS-USA guidelines in 374 HIV-1-infected children and adolescents. HIV-1 subtype was characterized by phylogenetic and recombination analysis using MEGA5.1 and Simplot. Poisson linear regression was used to model the dynamics of the RAMs over time. Results: The prevalence of RAMs to protease inhibitors (R2 = 0.52, p = 0.0012) and nucleoside reverse transcriptase inhibitors (R2 = 0.30, p = 0.0225) decreased over time. HIVDR to non-nucleoside reverse transcriptase inhibitors remained moderate to high, ranging between 33% and 76%. BF recombinants showed a higher frequency of thymidine analog mutation 1 RAMs profile and I54V mutation. Conclusion: In Argentina, HIVDR observed in children and adolescents has decreased over the past 15 years, regardless of the viral subtype.

Management choices during the pullet phase can affect behavior, welfare, and health later in life, but few studies have evaluated the pullet phase, particularly in extensive housing systems. This study was a 2 × 2 factorial randomized complete block design (RCBD) with two strains and two stocking densities. The Lohmann LB-Lite and Lohmann LSL-Lite were housed on the floor at high-stocking density (619–670 cm2/bird) and low-stocking density (1249–1352 cm2/bird), which changed with age from 2 to 16 weeks of age (WOA). Bird-based measures of appearance, blood parameters, organ measurements, and production values were evaluated. Stocking density alone affected (p < 0.05) only relative bursal weight (% of body weight)—3.32% in the low-density versus 3.08% in the high-density group. High-stocking density was correlated with decreased uniformity (high—89.33 ± 0.24%; low—90.41 ± 0.24; p < 0.02) and worse feather coverage in the brown strain. High-stocking density was correlated with greater uniformity (High—90.39 ± 0.24%; Low—88.47 ± 0.24%; p < 0.001) and better feather coverage in the white strain. This study’s feed conversion ratio (FCR) was improved by 0.07 in the low-stocking density for both strains. The remaining parameters were affected by strain and age only. Thus, while stocking density effects vary slightly depending on the strain used, cage-free pullets had limited negative effects at both the high and low-stocking densities tested in this study; there were few to no changes in the numerous bird-based welfare parameters tested.

Abstract Lung cancers bearing oncogenic EML4-ALK fusions respond to targeted tyrosine kinase inhibitors (TKIs; e.g., alectinib), with variation in the degree of shrinkage and duration of treatment (DOT). However, factors that control this response are not well understood. While the contribution of the immune system in mediating the response to immunotherapy has been extensively investigated, less is known regarding the contribution of immunity to TKI therapeutic responses. We previously demonstrated a positive association of a TKI-induced interferon gamma (IFNγ) transcriptional response with DOT in EGFR-mutant lung cancers. Herein, we used three murine models of EML4-ALK lung cancer to test the role for host immunity in the alectinib therapeutic response. The cell lines (EA1, EA2, EA3) were propagated orthotopically in the lungs of immunocompetent and immunodeficient mice and treated with alectinib. Tumor volumes were serially measured by μCT and immune cell content was measured by flow cytometry and multispectral immunofluorescence. Transcriptional responses to alectinib were assessed by RNAseq and secreted chemokines were measured by ELISA. All cell lines were similarly sensitive to alectinib in vitro and as orthotopic tumors in immunocompetent mice, exhibited durable shrinkage. However, in immunodeficient mice, all tumor models rapidly progressed on TKI therapy. In immunocompetent mice, EA2 tumors exhibited a complete response, whereas EA1 and EA3 tumors retained residual disease that rapidly progressed upon termination of TKI treatment. Prior to treatment, EA2 tumors had greater numbers of CD8+ T cells and fewer neutrophils compared to EA1 tumors. Also, RNAseq of cancer cells recovered from untreated tumors revealed elevated levels of CXCL9 and 10 in EA2 tumors, and higher levels of CXCL1 and 2 in EA1 tumors. Analysis of pre-treatment patient biopsies from ALK+ tumors revealed an association of neutrophil content with shorter time to progression. Combined, these data support a role for adaptive immunity in durability of TKI responses and demonstrate that the immune cell composition of the tumor microenvironment is predictive of response to alectinib therapy.

Conventional broiler diets include synthetic methionine to optimize fast muscle growth. Recently, a conventional synthetic methionine-rich diet was compared to alternative diet regimens providing natural sources of methionine. Broilers fed diets with natural methionine sources grew at a slightly slower rate. From this study, we hypothesized that the difference in a growth rate would be reflected in features of the breast muscle from broilers fed the alternative diet. We hypothesized that white striping of pectoralis major muscle would be reduced in slower growing broilers fed the alternative diet regimen with natural methionine. We also hypothesized that there would be associated differences in gene expression for cell differentiation and pathology markers. Broilers fed a conventional corn/soy diet regimen with synthetic methionine were compared to those fed roasted cowpea and sunflower seed meal (60% corn/soy, 20% sunflower seed meal, and 20% roasted cowpea) and no synthetic methionine. Overall broiler growth, muscle gene expression, and muscle collagen content data were compared. Expression analyses of combinations of MYOD1, PPARG, COL1A2, TRIM63, SOD1, PTGS2, and CD36 genes were used to examine differentiation and inflammation in the pectoralis muscles. The group fed an alternative diet gained less weight than those fed the control diet in the starter and grower phases but not in the finisher phase. Ultimately, the conventional diet resulted in a greater final weight for the broilers. However, mean white striping scores for the pectoralis major muscles were greater in the conventional control diet regimen. Gene expression results indicated greater expression of PPARG, PTGS2, and CD36 in the muscle of broilers fed the control diet. These data associate white striping with fat deposition and inflammation. Thus, whether due to differences in feed intake, growth rate, or actual compositional differences, the alternative diet with natural methionine sources seemed to curtail amounts of white striping in broiler muscle. More studies are necessary to further discern the effect of growth rate and natural methionine sources on white striping.

Next-generation sequencing (NGS) of amplicons is used in a wide variety of contexts. In many cases, NGS amplicon sequencing remains overly expensive and inflexible, with library preparation strategies relying upon the fusion of locus-specific primers to full-length adapter sequences with a single identifying sequence or ligating adapters onto PCR products. In Adapterama I, we presented universal stubs and primers to produce thousands of unique index combinations and a modifiable system for incorporating them into Illumina libraries. Here, we describe multiple ways to use the Adapterama system and other approaches for amplicon sequencing on Illumina instruments. In the variant we use most frequently for large-scale projects, we fuse partial adapter sequences (TruSeq or Nextera) onto the 5' end of locus-specific PCR primers with variable-length tag sequences between the adapter and locus-specific sequences. These fusion primers can be used combinatorially to amplify samples within a 96-well plate (8 forward primers + 12 reverse primers yield 8 × 12 = 96 combinations), and the resulting amplicons can be pooled. The initial PCR products then serve as template for a second round of PCR with dual-indexed iTru or iNext primers (also used combinatorially) to make full-length libraries. The resulting quadruple-indexed amplicons have diversity at most base positions and can be pooled with any standard Illumina library for sequencing. The number of sequencing reads from the amplicon pools can be adjusted, facilitating deep sequencing when required or reducing sequencing costs per sample to an economically trivial amount when deep coverage is not needed. We demonstrate the utility and versatility of our approaches with results from six projects using different implementations of our protocols. Thus, we show that these methods facilitate amplicon library construction for Illumina instruments at reduced cost with increased flexibility. A simple web page to design fusion primers compatible with iTru primers is available at: http://baddna.uga.edu/tools-taggi.html. A fast and easy to use program to demultiplex amplicon pools with internal indexes is available at: https://github.com/lefeverde/Mr_Demuxy.

Background: The aim of this study is comparison the effectiveness of stereotactic, hypofractionated and conventional radiotherapy assessed by the tumor volume changes of paraganglioma located in the head and neck region concerning fractional and total doses. Methods: We analyzed 76 patients after radiotherapy due to paraganglioma who were assigned to 3 groups considering fractional (≤2 Gy, 3–5.5 Gy, ≥6 Gy) and total (≤20 Gy, 21–40 Gy, >40 Gy) doses. The volumes of irradiated tumors were measured and compared based on diagnostic images performed before and after the treatment. Results: The mean tumor volume after the treatment with the lowest fractional dose (≤2 Gy) was decreased by 14.4 cm3. In patients treated with higher fractional doses (>2 Gy), the mean tumor volumes decreased by less than 1 cm3 for hypofractionated and stereotactic radiotherapy. 15.9 cm3 reduction of the mean tumor volume after the treatment with the highest RT total dose (>40 Gy) was stated. In patients treated with total doses ≤20 Gy and 21–40 Gy, the mean tumor volume was stable and reduced by 1.15 cm3, respectively. The analysis demonstrates a statistically significant (p < 0.05) treatment advantage in patients after the lowest fractional and highest total doses. Conclusion: The reduction of the tumor's volume was reported after conventional and unconventional radiotherapy. The most significant depletion of the paraganglioma volume was noted after a factional dose ≤2 Gy and a total dose >40 Gy. [ABSTRACT FROM AUTHOR]

Background Pulmonary endarterectomy (PEA) for chronic thromboembolic pulmonary hypertension improves resting hemodynamics and right ventricular (RV) function. Because exercise tolerance frequently remains impaired, RV function may not have completely normalized after PEA. Therefore, we performed a detailed invasive hemodynamic study to investigate the effect of PEA on RV function during exercise. Methods and Results In this prospective study, all consenting patients with chronic thromboembolic pulmonary hypertension eligible for surgery and able to perform cycle ergometry underwent cardiac magnetic resonance imaging, a maximal cardiopulmonary exercise test, and a submaximal invasive cardiopulmonary exercise test before and 6 months after PEA. Hemodynamic assessment and analysis of RV pressure curves using the single‐beat method was used to determine load‐independent RV contractility (end systolic elastance), RV afterload (arterial elastance), RV–arterial coupling (end systolic elastance–arterial elastance), and stroke volume both at rest and during exercise. RV rest‐to‐exercise responses were compared before and after PEA using 2‐way repeated‐measures analysis of variance with Bonferroni post hoc correction. A total of 19 patients with chronic thromboembolic pulmonary hypertension completed the entire study protocol. Resting hemodynamics improved significantly after PEA. The RV exertional stroke volume response improved 6 months after PEA (79±32 at rest versus 102±28 mL during exercise; P

IntroductionIn vivo, cancer cells respond to signals from the tumor microenvironment resulting in changes in expression of proteins that promote tumor progression and suppress anti-tumor immunity. This study employed an orthotopic immunocompetent model of lung cancer to define pathways that are altered in cancer cells recovered from tumors compared to cells grown in culture.MethodsStudies used four murine cell lines implanted into the lungs of syngeneic mice. Cancer cells were recovered using FACS, and transcriptional changes compared to cells grown in culture were determined by RNA-seq.ResultsChanges in interferon response, antigen presentation and cytokine signaling were observed in all tumors. In addition, we observed induction of the complement pathway. We previously demonstrated that activation of complement is critical for tumor progression in this model. Complement can play both a pro-tumorigenic role through production of anaphylatoxins, and an anti-tumorigenic role by promoting complement-mediated cell killing of cancer cells. While complement proteins are produced by the liver, expression of complement proteins by cancer cells has been described. Silencing cancer cell-specific C3 inhibited tumor growth In vivo. We hypothesized that induction of complement regulatory proteins was critical for blocking the anti-tumor effects of complement activation. Silencing complement regulatory proteins also inhibited tumor growth, with different regulatory proteins acting in a cell-specific manner.DiscussionBased on these data we propose that localized induction of complement in cancer cells is a common feature of lung tumors that promotes tumor progression, with induction of complement regulatory proteins protecting cells from complement mediated-cell killing.

Melatonin has been reported to cause myocardial electrophysiological changes and prevent ventricular tachycardia or fibrillation (VT/VF) in ischemia and reperfusion. We sought to identify electrophysiological targets responsible for the melatonin antiarrhythmic action and to explore whether melatonin receptor-dependent pathways or its antioxidative properties are essential for these effects. Ischemia was induced in anesthetized rats given a placebo, melatonin, and/or luzindole (MT1/MT2 melatonin receptor blocker), and epicardial mapping with reperfusion VT/VFs assessment was performed. The oxidative stress assessment and Western blotting analysis were performed in the explanted hearts. Transmembrane potentials and ionic currents were recorded in cardiomyocytes with melatonin and/or luzindole application. Melatonin reduced reperfusion VT/VF incidence associated with local activation time in logistic regression analysis. Melatonin prevented ischemia-related conduction slowing and did not change the total connexin43 (Cx43) level or oxidative stress markers, but it increased the content of a phosphorylated Cx43 variant (P-Cx43368). Luzindole abolished the melatonin antiarrhythmic effect, slowed conduction, decreased total Cx43, protein kinase Cε and P-Cx43368 levels, and the IK1 current, and caused resting membrane potential (RMP) depolarization. Neither melatonin nor luzindole modified INa current. Thus, the antiarrhythmic effect of melatonin was mediated by the receptor-dependent enhancement of impulse conduction, which was associated with Cx43 phosphorylation and maintaining the RMP level.

Abstract Tyrosine kinase inhibitors (TKIs) targeting EGFR-mutant lung cancers promote a range of tumor regression responses to yield variable residual disease, a likely incubator for acquired resistance. Herein, rapid transcriptional responses induced by TKIs early in treatment that associate with the range of patient responses were explored. RNAseq was performed on EGFR mutant cell lines treated in vitro with osimertinib and on tumor biopsies of eight EGFR mutant lung cancer patients before and after 2 weeks of TKI treatment. Data were evaluated for gene expression programs altered upon TKI treatment. Chemokine and cytokine expression were measured by ELISA and quantitative RT-PCR. IκB Kinase (IKK) and JAK-STAT pathway dependence was tested with pharmacologic and molecular inhibitors. Tumor sections were stained for the T-cell marker CD3. Osimertinib stimulated dynamic, yet wide-ranging interferon (IFN) program regulation in EGFR mutant cell lines. IL6 and CXCL10 induction varied markedly among the EGFR mutant cell lines and was sensitive to IKK and JAK-STAT inhibitors. Analysis of matched patient biopsy pairs revealed marked, yet varied enrichment of IFN transcriptional programs, effector immune cell signatures and T-cell content in treated tumors that positively correlated with time to progression in the patients. EGFR-specific TKIs induce wide-ranging IFN response program activation originating within the cancer cell. The strong association of IFN program induction and duration of clinical response indicates that the TKI-induced IFN program instructs variable recruitment and participation of immune cells in the overall therapeutic response.

Abstract Cardiac development is a dynamic process, temporally and spatially. When disturbed, it leads to congenital cardiac anomalies that affect approximately 1% of live births. Genetic variants in several loci lead to anomalies, with the transcription factor NKX2-5 being one of the largest. However, there are also non-genetic factors that influence cardiac malformations. We examined the hypothesis that hyperoxia may be beneficial and can rescue genetic cardiac anomalies induced by an Nkx2-5 mutation. Intermittent mild hyperoxia (40% PO2) was applied for 10 h per day to normal wild-type female mice mated with heterozygous Nkx2-5 mutant males from gestational day 8.5 to birth. Hyperoxia therapy reduced excessive trabeculation in Nkx2-5 mutant mice compared to normoxic conditions (ratio of trabecular layer relative to compact layer area, normoxia 1.84 ± 0.07 vs. hyperoxia 1.51 ± 0.04) and frequency of muscular ventricular septal defects per heart (1.53 ± 0.32 vs. 0.68 ± 0.15); however, the incidence of membranous ventricular septal defects in Nkx2-5 mutant hearts was not changed. Nkx2-5 mutant embryonic hearts showed defective coronary vessel organization, which was improved by intermittent mild hyperoxia. The results of our study showed that mild gestational hyperoxia therapy rescued genetic cardiac malformation induced by Nkx2-5 mutation in part.

Abstract Background Segment length in cine (SLICE) strain analysis on standard cardiovascular magnetic resonance (CMR) cine images was recently validated against gold standard myocardial tagging. The present study aims to explore predictive value of SLICE for cardiac resynchronization therapy (CRT) response. Methods and results Fifty-seven patients with heart failure and left bundle branch block (LBBB) were prospectively enrolled in this multi-center study and underwent CMR examination before CRT implantation. Circumferential strains of the septal and lateral wall were measured by SLICE on short-axis cine images. In addition, timing and strain pattern parameters were assessed. After twelve months, CRT response was quantified by the echocardiographic change in left ventricular (LV) end-systolic volume (LVESV). In contrast to timing parameters, strain pattern parameters being systolic rebound stretch of the septum (SRSsep), systolic stretch index (SSIsep-lat), and internal stretch factor (ISFsep-lat) all correlated significantly with LVESV change (R − 0.56; R − 0.53; and R − 0.58, respectively). Of all strain parameters, end-systolic septal strain (ESSsep) showed strongest correlation with LVESV change (R − 0.63). Multivariable analysis showed ESSsep to be independently related to LVESV change together with age and QRSAREA. Conclusion The practicable SLICE strain technique may help the clinician to estimate potential benefit from CRT by analyzing standard CMR cine images without the need for commercial software. Of all strain parameters, end-systolic septal strain (ESSsep) demonstrates the strongest correlation with reverse remodeling after CRT. This parameter may be of special interest in patients with non-strict LBBB morphology for whom CRT benefit is doubted.

BACKGROUND: In recent years, laparoscopic inguinal hernia repair has become one of the elective techniques, attributing the advantages of minimally invasive procedures to it. However, the high costs related to the need for hospitalization and materials make them a limitation at the time of its indication. OBJECTIVE: Evaluate the feasibility of performing this procedure in an outpatient surgery center and the cost analysis of an outpatient procedure regarding the same in the setting of hospitalization. Retrospective cohort study of feasibility and minimization cost. METHODS: A retrospective analysis was carried out on a prospective database in which all patients were included in those who underwent laparoscopic inguinal hernioplasty on an outpatient basis between August 2015 and June 2018. Feasibility is expressed as the percentage of patients who were referred from the outpatient surgery unit. A cost minimization study was conducted taking the average cost of performing an ambulatory procedure versus the same procedure requiring a day of hospitalization. This work has been reported in line with the CHEERS criteria. RESULTS: 116 patients were operated as outpatients, of which 109 were men (93.96%). The median age was 56.5 years (RIQ 19). 102 patients (87.93%) were operated on due to bilateral inguinal hernia, and 14 of them (12.07%) due to recurrent unilateral hernia, adding a total of 218 inguinal hernioplasties. The mean operative time in bilateral interventions was 112 minutes (DS 24) and in the unilateral recurrences it was 79 minutes (DS 13). 114 patients were discharged from the outpatient unit with 98.3% feasibility. The average postoperative stay was 2.53 h (DS 1). The average cost of ambulatory inguinal hernioplasty was $17725.1 vs $27297.3 in hospitalization. The same implies a cost reduction of 35%. CONCLUSIONS: Laparoscopic inguinal hernioplasty is a feasible and safe technique to perform on an outpatient basis. It provides a significant reduction in the costs of the procedure.

Background The success of pulmonary endarterectomy (PEA) for chronic thromboembolic pulmonary hypertension (CTEPH) is usually evaluated by performing a right heart catheterisation (RHC). Here, we investigate whether residual pulmonary hypertension (PH) can be sufficiently excluded without the need for a RHC, by making use of early post-operative haemodynamics, or N-terminal pro-brain natriuretic peptide (NT-proBNP), cardiopulmonary exercise testing (CPET) and transthoracic echocardiography (TTE) 6 months after PEA. Methods In an observational analysis, residual PH after PEA measured by RHC was related to haemodynamic data from the post-operative intensive care unit time and data from a 6-month follow-up assessment including NT-proBNP, TTE and CPET. After dichotomisation and univariate analysis, sensitivity, specificity, positive predictive value, negative predictive value (NPV) and likelihood ratios were calculated. Results Thirty-six out of 92 included patients had residual PH 6 months after PEA (39%). Correlation between early post-operative and 6-month follow-up mean pulmonary artery pressure was moderate (Spearman rho 0.465, p300 ng·L−1 had insufficient NPV (0.71) to exclude residual PH. Probability for PH on TTE had a moderate NPV (0.74) for residual PH. Peak oxygen consumption (V′O2)