OsMDHAR), as well as its metabolites (ascorbate and glutathione) and uptake of nutrients , while addition of Si reversed Buta-induced alterations. Buta stimulated the expression of Si channel and efflux transporter genes- Lsi1 and Lsi2 while the addition of Si further greatly induced their expression under Buta toxicity. Buta increased free proline accumulation by inducing the activity of Δ1-pyrroline-5-carboxylate synthetase (P5CS) and decreasing proline dehydrogenase (PDH) activity, while Si reversed these effects caused by Buta. Our results suggest that Si-governed mitigation of Buta toxicity is linked with favorable modifications in energy flux parameters of photosynthesis and leaf and root anatomy, up-regulation of Si channel and transporter genes, ascorbate-glutathione cycle and nutrient uptake, and lowering in oxidative stress. We additionally demonstrate that NO might have a crucial role in these responses.Reckless use of herbicides like butachlor (Buta) in the fields represents a serious threat to crop plants, and hence to their productivity. Silicon (Si) is well known for its implication in the alleviation of the effects of abiotic stresses; however, its role in mitigating Buta toxicity is not yet known. Therefore, this study was carried out to explore the role of Si (10 µM) in regulating Buta (4 µM) toxicity in rice seedlings. Buta reduced growth and photosynthesis, altered nitric oxide (NO) level and leaf and root anatomy, inhibited enzyme activities of the ascorbate-glutathione cycle (while transcripts of associated enzymes, increased except  Butachlor, a chloroacetanilide herbicide, , is commonly used in rice fields3. In water, the half-life of Buta ranges from 1.65–2.48 days while in soil 2.67–5.33 days4. Approximately 6.75 ×106 kg Buta are used annually in India5. Besides controlling weeds, the extensive use of herbicides may also affect other crop plants and thus may negatively impact their productivity. Herbicides indeed negatively affect seed germination, hormone synthesis, cell division, cell permeability, nutrient uptake and their assimilation, as well as lipid and protein metabolism11. García-Garijoet al.12 have observed that imazamox, a herbicide, drastically decreased root nodule biomass in Phaseolus vulgaris and Vicia sativa, thereby affecting their nitrogen balance. Herbicides including Buta generally inhibit growth of weeds by affecting the biosynthesis of photosynthetic pigments14 and either electron carriers or enzymes partaking in photosynthesis17. Due to their negative interactions with metabolic processes, herbicides including Buta have been reported to cause oxidative stress by inducing the accumulation of reactive oxygen species (ROS) that enhanced lipid peroxidation, and thus, disturbed the fluidity of membranes20. In response to herbicide-created oxidative stress, challenged plants respond by differential activation of antioxidant defense system, and sometimes the magnitude of their activation is not synchronized with oxidative stress, consequently leading to declined biomass production. Further, Burns et al.21 reported that in Avena fatua L. glutathione-related proteins are essential for herbicide resistance. Wang et al.22 have noticed severe negative impact of nicosulfuron herbicide in the ascorbate-glutathione cycle in Zea mays L. Herbicides being persistent and toxic in nature pose serious consequences on plant anatomy, physiology and biochemistry and they can impact, via the food chains, human health too. Therefore, investigations on their ecotoxicology and management strategies are currently highly sought-after.Rice is a major cereal crop of the world. However, the invasion invasion of weeds is one of the major issues in the rice cultivated areas. To control weeds in the rice fields, several herbicides are being used at larger scale. Among the different herbicides, butachlor (hereafter referred to as Buta) has been extensively used in India for the control of weedsth most abundant element in nature and the 2nd most abundant element in the earth crust after oxygen24. The application of Si shows many beneficial effects in grasses, among which an increased mechanical strength25. Previous studies showed that Si could improve plant growth and yield by promoting photosynthesis, mineral uptake and antioxidant capacities against environmental constraints28. Besides, Si also increases the resistance against diseases and pathogens, metal toxicity, salinity, drought, etc.33. Therefore, over the past years; a clear role of Si in plant biology has emerged. Si is a quasi-essential element controlling growth and development of crops: its effects are latent in the absence of (a)biotic stresses. Indeed, its prophylactic properties are visible upon exogenous constraints. However, the role of Si in mitigating herbicide toxicity like Buta is, to the best of our knowledge, still not elucidated.Silicon (Si) is the 8Therefore, this study was aimed to explore the role of Si in regulating Buta toxicity in rice seedlings by evaluating growth, photosynthesis, leaf and root anatomical characteristics, oxidative stress, the ascorbate-glutathione cycle, proline metabolism and the regulation of nutrients. Our study has important physiological and ecotoxicological implications and paves the way to follow-up studies on the role of Si under herbicide toxicity in important crops.Oryza sativa L.var. Narendra) seeds were bought from the market. Rice seedlings were grown in Petri dishes in a growth chamber23  at 28 °C with a daylength of 12 h, relative humidity of 60–70% under 300 µmol photons m−2 s−1 PFD (photon flux density) for 25 days. Half-strength Hoagland solution was provided regularly to the growing seedlings.Rice  is based on its dose response curve. It significantly reduced growth (in the terms of fresh weight) of rice seedlings and represents LC18. Further, it is an environmentally relevant dose. Sodium silicate salt was used as source of silicon while Buta (EC 50%) was purchased from the market. The samples prepared were: control , Si (10 µM), Buta (4 µM) and Si (10 µM) + Buta (4 µM). In the case of Si and Buta combination, seedlings were pretreated with Si for 24 h. Separate treatments of 60 ml each were carried out in plastic pots. Five homogeneously-sized rice seedlings were placed in each plastic pot. Then these plastic pots containing the rice seedlings were again kept in the growth chamber for 7 days. Finally, seedlings were harvested from each pot and used for the analysis of various parameters. The experiment was performed in triplicates.After 25 days of growth (seedlings having three well developed leaves), uniformly sized seedlings were selected. Thereafter, treatments of Si (10 µM) and butachlor  alone and in combinations were given. Our previous study showed that 10 µM of silicon stimulates maximum growth of rice seedlingset al.33. Fresh leaves (25 mg) from each sample were extracted in 5 ml of 80% (v/v) acetone for measurement of total chlorophyll and carotenoids content. Lichtenthaler34 protocol was used to calculate total chlorophyll and carotenoids in each sample. The amount of protein in each sample was estimated as per the protocol given by Bradford35.Length, fresh and dry weights of both shoot and root were measured as explained in Tripathi a fluorescence transient in dark-adapted leaves. The following parameters of PS II photochemistry were calculated as per the given formulae of Strasser et al.36. Subsequently different parameters like yields or flux ratios-Phi_P0 , Phi_E0 (quantum yield of electron transport) and Psi_0 (probability that a trapped exciton moves an electron into the electron transport chain beyond QA), specific energy fluxes per reaction center (RC) i.e. ABS/RC, electron transport flux/RC, TR0/RC, trapped energy flux per RC, absorbance flux per RC ET0/RC, and DI0/RC, dissipated energy flux per RC and performance index (PIABS) were determined.FluorPen FP 100, Photon System Instruments, Czech Republic was used to study chlorophyll et al.33.Anatomical changes in root and leaf were studied as described in detail in Tripathi 2¯ formed from hydroxylamine in the presence of superoxide radical  was used for measurement of O2•¯ in each sample as explained by Elstner and Heupel37. The absorbance of colored aqueous solution was recorded at 530 nm. Further, the amount of superoxide radicals was determined by the standard curve prepared with sodium nitrite. The quantity of hydrogen peroxide (H2O2) was measured by the protocol given by Velikova et al.38. Similarly the standard curve made with a solution of H2O2 was used to estimate the amount of peroxide radicals in each sample. The lipid peroxidation (LPO) malondialdehyde (MDA) content was determined as explained by Heath and Packer39.The amount of NOIn vivo localizations of O2 – radical, H2O2, LPO and membrane disruption (MD) was carried out by nitro blue tetrazolium (NBT), 3,3′-diaminobenzidine (DAB), Schiff’s reagent and Evans blue respectively as given by Castro-Mercado et al.40, Thordal-Christensen et al.41, Pompella et al.42 and Schutzendübel et al.43. Stained leaves were photographed with a Nikon camera, while stained roots were photographed with an Olympus compound dark-field microscope using a digital camera.et al.44 using a fluorescence microscope .Nitric oxide (NO) was detected using the fluorescent dye 4,5-diaminofluorescein diacetate (DAF-2DA), as explained by Xie 45 method was used for preparation of plant samples for the cell viability analysis. Briefly leaves were homogenized in phosphate buffer saline (PBS) and centrifuged. 0.5 mL of phosphate buffer saline was added to the pellet so as to completely dissolve the pellet containing 106cells. After fixing in 70% ethanol these cells were kept for incubation at 4 °C for 30 min. Further, cells were washed with 1X phosphate buffer saline and kept for centrifugation at 1200 rpm for 15 min. Supernatant was discarded and the pellet was re-suspended in 200 mL PBS, stained and observed as per the method described in Vishwakarma et al.46.Pozarowski and Darzynkiewiczet al.47. The amount of these enzymes was calculated using UV-visible spectrophotometer  at room temperature.Enzyme extract for ascorbate peroxidase (APX) glutathione reductase (GR) monodehydroascorbate reductase (MDHAR) and dehydroascorbate reductase (DHAR) were prepared as described in Singh 49 was used to quantify APX activity in each sample. One unit (U) of enzyme activity is defined as 1 nmol ascorbate oxidized min−1. Schaedle and Bassham4 gave the protocol to determine GR activity in different treatments. One unit (U) of enzyme activity is defined as 1 nmol NADPH oxidized min−1. MDHAR and DHAR activity was measured as per the method of Hossain et al.50 and Nakano and Asada48 respectively. Subsequently, one unit (U) of enzyme activity for MDHAR is defined as 1 nmol NADPH oxidized min−1. One unit (U) of enzyme activity for DHAR is defined as 1 nmol DHA reduced min−1. The detailed procedures for these enzymes are given in Singh et al.47.The procedure described by Nakano and Asadaet al.51 and Brehe and Burch52, respectively, given in detail by Singh et al.47.AsA, DHA, GSH and GSSG were measured as per the procedures of Gossett TM plant total RNA Isolation kit (Sigma Life Sciences). The RNA concentration and purity were measured with the Nano drop and the ratio of 260/280 (1.8–2) was maintained. 1 μg of total RNA was used for the synthesis of cDNA using Quanti Tect Reverse Transcription Kit (QIAGEN). PCR conditions and primer details for quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) are given in Table −ΔΔCT method of Livak and Schmittgen53. All treatments were analysed in triplicates for the housekeeping gene and all the genes of interest.Liquid nitrogen was used to grind frozen plant tissue (approx. 100 mg) samples in a pre chilled mortar with a pestle. RNA was extracted from the plant tissues according to the procedure given by Spectrumet al.54. Toluene was taken as blank and the absorbance of the samples was measured at 520 nm. A standard curve was plotted to determine the amount of proline in each treatment. The activities of proline dehydrogenase (PDH) and Δ1-pyrroline-5-carboxylate synthetase (P5CS) in rice seedlings were measured as NADH formation and NADPH oxidation according to the methods of Reno and Splittstoesser55 and Garcia-Rios et al.56, respectively. The procedures are described in detail in Singh et al.57.Free proline amount in leaves and roots was estimated as per the method of Bates et al.33 using an atomic absorption spectrometer.Determination of macronutrients  and micronutrient (Fe), and Si in control and treated seedlings was carried out essentially as previously described in Tripathi p < 0.05 significance level. The values presented are the means of three independent experiments (n = 3).Duncan’s multiple range test (DMRT) was used to measure the significance of differences between the control and the treatment mean values at The growth of rice seedlings was significantly decreased by Buta. The decline in growth was 19% in shoot and 26% in root fresh weight, respectively as compared to the control. Similarly, shoot and root dry weight and length were also decreased by Buta. Inhibition in root growth was more under Buta treatment than other growth parameters. On the other hand, supplementation of Si ameliorated Buta-caused reduced growth, as the fresh weight of the shoot and root was comparable to the control , hydrogen peroxide (H2O2) and MDA contents in shoot and root of rice seedlings are presented in Fig. p < 0.05) O2•− by 53 and 63% in shoots and roots, respectively with respect to the control. Similarly, H2O2 increased by 52% in shoots and 76% in roots compared to the control. Si supplementation significantly (p < 0.05) decreased O2•− and H2O2 contents under Buta toxicity  accelerated lipid peroxidation (MDA formation) as the value increased by 34 and 36% in shoots and roots, respectively, as compared to the control  lowered MDA content as compared to the amount recorded under Buta toxicity alone  and membrane damage (MD) are shown in Fig. Lsi1 and Lsi2, are shown in Fig. Lsi1) and efflux transporter (Lsi2) though stimulated by Buta , were greatly enhanced by the addition of Si under Buta toxicity. Under Si+Buta combination, the relative expression of Lsi1 and Lsi2 was enhanced by 379 and 193%, respectively. Si alone also stimulated the expression of Lsi1 and Lsi2  and even more than Buta treatment alone  further stimulated the activity of APX, GR, MDHAR and DHAR, even in comparison to their respective controls  was drastically decreased. However, Si addition together with Buta greatly increased the expression of OsAPX, OsGR, OsMDHAR and OsDHAR genes by 207, 185, 311 and 719%, respectively  reduced the levels of AsA and GSH which results into a decline in ratios of AsA/DHA and GSH/GSSG. Under Buta treatment, ratios of AsA/DHA and GSH/GSSG in shoots and roots were 6.29 and 4.77 and 6.31 and 6.09, respectively. However, the addition of Si with Buta improved AsA and GSH which results into higher ratios of AsA/DHA and GSH/GSSG as they were 9.80 and 7.97 and 11.07 and 9.90 in shoots and roots, respectively. These results indicate that under Buta toxicity, the addition of Si re-establishes the redox status of AsA and GSH and hence protects the cell against oxidative stress.The results relating to the redox status of AsA and GSH are shown in Table The results show that in leaves and roots free proline accumulation  and activity of P5CS enzyme  was stimulated by Buta, while Si addition caused lowering in these physiological parameters  and PIABS after treatment with Buta indicated alterations in the photochemistry of PS II. These changes resulted into a significant decrease in PIABS. Similarly to our results, Pan et al.60 reported that an increase in Buta concentration resulted in a decrease in Phi_P0, Phi_E0, Psi_0 and PIABS and an increase in DI0/RC. Recently, alterations in chlorophyll a fluorescence were also noticed in herbicide-challenged plants17. However, Si supplementation improved the values of flux ratios, energy fluxes and PIABS with respect to the control due to improvement in photosynthetic pigments  on chlorophyll a fluorescence parameters. These results suggest Si can protect rice seedlings against Buta toxicity too by protecting PSII photochemistry.The photochemistry of PSII is a reflection of photosynthetic efficiency. To verify it, the fast chlorophyll ity Fig. . In the 62. Therefore, smaller stomata size would be less efficient in transpiration and gas exchange. Hence, reduction in the photosynthetic pigments , which act in a coordinated manner in order to metabolize H2O2 and decide its fate, i.e. whether it will act as a signaling or damaging molecule. Besides its role in controlling H2O2 level, this cycle also maintains the redox buffer of the cell by regulating the level of glutathione and ascorbate and their redox ratios, which help in sensing and signaling of stress in order to modulate cellular compartment specific changes by altering the expression of metabolism- and stress-related genes80. Our results show that Buta stress significantly (p < 0.05) inhibited the activities of APX, GR, MDHAR and DHAR and the amount of ascorbate and glutathione which resulted into a great decline in ratios of AsA/DHA and GSH/GSSG , except Na (Table 84.Being crucial for enzymes, vitamins, pigments and other biomolecules, mineral nutrients play a vital role in the growth and development of plants and provide biochemical and mechanical strengths by maintaining the structural integrity of the cellNa Table . HoweverNa Table . These rNa Table . Since tHerein, we reported for the first time that Si supplementation alleviated Buta toxicity in rice seedlings. Si-mediated alleviation of Buta toxicity was due to adaptive changes in leaf and root anatomical characteristics, up-regulation of Si channel and efflux transporter genes and the ascorbate-glutathione cycle. These changes reduced oxidative stress and impacted nutrient regulation. In Si-mediated amelioration of Buta toxicity, NO has a role in promoting the above-mentioned favorable changes in rice seedlings. These results are valuable from an agronomical point of view, as they will inspire the use of sustainable agricultural practices relying on Si fertilization to sustain crop productivity at herbicide-polluted sites, and thus, this study can open newer avenues for research in this direction.Supplementary Information Table S1.


We describe the complete mitochondrial genome of Phiaris dolosana. It is 15,562 bp in length, and contains 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), 2 ribosomal RNA genes (rRNAs), and a 612 bp D-Loop. All PCGs start with ATN codon except for COI gene, which uses CGA as the initiation codon. Nine of 13 PCGs use a typical stop codon of TAA, the rest use incomplete stop codon of T or TA. Phylogenetic analysis of P. dolosana with other 17 leaf rollers is conducted with neighbor-joining method, the result is consistent with the conventional classification. Phiaris Hübner, [1825] 1816 is a member of the family Tortricidae, and closely related to the type-genus of Olethreutinae, Oletherutes Hübner, 1822. It is difficult to clearly separate these two genera based on morphological and genital characters, and there are also controversies regarding the classification status of Phiaris and some related species  is a widely distributed species in Russian, Japan, and mainland China. The specimen used for the study was collected from Huoditang , Shaanxi Province, China and preserved in alcohol at Herbarium of Northwest university, China (registered no. NWU.L2018320).Genus The mitochondrial genes were extracted from the alcohol-soaked muscle tissue with a DNA Extraction Kit  and sequenced bidirectionally. The raw data were spliced by Novoplasty-NOVOplasty 3.0 to form a typical circular DNA molecule , 22 transfer RNAs (tRNAs), 2 ribosomal RNAs (rRNAs) and a 612 bp D-Loop. Nucleotide composition of the complete mitochondrial genome includes A (40.6%), T (40.0%), C (11.7%), G (7.8%), and the content of A + T is 80.5% that is distinctly biased. All PCGs start with ATN codon, except for cytochrome oxidase subunit I (COI), with CGA initiation codon. Nine of 13 PCGs have stop codon TAA, and the remaining PCGs contain T or TA stop codon. Seven overlaps at gene junctions were identified – that between ND1 and tRNA-Leu is the longest with 8 bp. Eighteen gene gap regions were also found in a total of 160 bp, and length of the gaps is between 1 and 56 bp. Based on the complete mitochondrial genome from 18 species in Tortricidae, with the outgroup of Amata formosae in Erebidae, we generated a neighbor-joining phylogenetic tree (P. dolosana shows a relationship with some species of Olethreutini than the members of other tribes in Olethreutinae. Such a clustered classification profile is consistent with that generated based on conventional taxonomic methods.The complete mitochondrial genome of tic tree , illustr


Background: Donor human milk (DHM) bank practices, such as pasteurization and pooling according to postpartum age of milk donations and number of donors included in a pool may impact the resulting concentration of bioactive components of DHM.Aims: We determined the impact of Holder pasteurization, postpartum milk age, and pool donor number (number of donors included in a pool) on resulting concentrations of total immunoglobulin A  and insulin (an important hormone for gut maturation).We also documented inter-relationships between these bioactive components and macronutrients in DHM pools.Methods: Pre and post-pasteurization aliquots of 128 DHM samples were obtained from the Rocky Mountain Children's Foundation Mother's Milk Bank . Macronutrients were measured via mid-infrared spectroscopy. Total IgA was measured via customized immunoassay in skim milk and insulin was measured via chemiluminescent immunoassay.Results: Mean post-pasteurization total IgA concentration was 0.23 ± 0.10 (range: 0.04–0.65) mg/mL a 17.9% decrease due to pasteurization (n = 126). Mean post-pasteurization DHM insulin concentration was 7.0 ± 4.6 (range: 3–40) μU/mL, a decrease of 13.6% due to pasteurization (n = 128). The average DHM pool postpartum milk age was not associated with total IgA or insulin concentrations, but pool donor number was associated with bioactive components. Pools with only one donor had lower total IgA and lower insulin concentrations than pools with at least 2 donors (p < 0.05). Increasing the number of donors in a pool decreased the variability in total IgA and insulin concentrations (p < 0.04).Conclusion: Increasing the number of donors included in DHM pools may help optimize bioactive components in DHM received by premature infants. These results help inform milk banking practices to decrease compositional variability in produced DHM pools. The American Academy of Pediatrics recommends pasteurized donor human milk (DHM) as the best option to feed a premature infant when mother's own milk is unavailable . The HumHuman milk composition is dynamic, changing over the course of a feed, over the course of a day, and over the course of lactation. For these reasons, DHM banks routinely pool milk donations from multiple donors in order to limit extreme variation. Criteria used to select individual donations for pooling are not standardized and vary among milk banks. Currently, milk postpartum age (age of the infant at the time milk was expressed) is not typically taken into account when pooling individual donations. Furthermore, milk banks each have different acceptance criteria for milk age.Upon pooling, DHM is aliquoted and pasteurized using Holder Pasteurization (62.5°C for 30 min and rapid cooling). At this stage, bottles are frozen and distributed. It is well-documented that Holder Pasteurization reduces the concentration of various bioactive components of HM. However, the degree to which individual bioactive components are degraded is widely variable, and not consistently reported in the literature (IgA)  . HM totaIgA) , 8. SecrIgA) , 7. TotaIgA) , 9. AddiIgA) , 9, 10.HM insulin plays important roles in infant intestinal maturation , may impBecause IgA represents a significant component of total HM protein, and because both HM insulin and total fat have been reported to correlate with maternal BMI –19, we aGiven the importance of these bioactive components of HM, we aimed to characterize the mean and variability of total IgA and insulin concentrations in HMBANA-produced DHM pools and investigate the impact of Holder Pasteurization and relationships between milk postpartum age and number of donors included in a pool.This research received designation as “Not Human Subject Research” by the Colorado Multiple Institution Review Board.Pre- and post-pasteurization samples from 128 DHM pools were obtained from the Colorado Mothers' Milk Bank . This milk bank selects individual donations to pool based on the expiration date of donated milk. Milk donations are considered to “expire” 12 months post expression date. DHM pools are then aliquoted, pasteurized in glass bottles via Holder Pasteurization and frozen for distribution.Preterm milk pools included only human milk expressed by a mother who delivered at or before 36 weeks gestation, within the first 4 weeks postpartum and up until her infant's corrected age is 40 weeks. Pool donor number was defined as the number of milk donors represented in each pool. Pool postpartum age was defined as the unweighted mean of the postpartum ages of all the donations included in any given pool as previously described .2SO4) was added to each well.Total IgA concentrations of pre- and post-pasteurization samples were measured using an adapted version of a commercially available immunoassay . A flat bottom 96 well Maxisorp plate was coated with a 1:100 dilution of anti-human IgA (Bethyl Lab A80–102A) in 0.05M carbonate-bicarbonate: 100 uL per well, overnight at 4°C. The following day, 200 uL of 1% bovine serum albumin (BSA) in PBS was added to each well to block the plate for 30 min at room temperature. Skim milk was generated by spinning samples at 10,000 g for 10 min at 4°C. Skim milk samples were diluted 1:5000 in 1% BSA in PBS and then incubated on the plate for 2 h at room temperature. Assay controls were prepared from a serial dilution of control serum (Bethyl Lab RS10–110) in 1% BSA in PBS. Secondary antibody (Bethyl Lab A80–102P) was added at a concentration of 1:100,000 in 1% BSA in PBS and incubated for 1 h at room temperature: 100 uL per well. Following the 1 h incubation time, 50 uL of stop solution  were formerly assessed using the Miris Human Milk Analyzer  as previously reported . Insulint-tests.Comparison of milk composition between Preterm vs. Term DHM pools and by pool donor number was conducted using non-parametric tests. For comparison by pool donor number, DHM pools were categorized as consisting of donations from 1, 2, or ≥3 donors. A comparison of milk composition between pre-pasteurized and post-pasteurized samples was conducted using paired Simple linear regression was used to determine if the pool Postpartum Age was related to milk composition, and if any individual milk components were inter-correlated. Log transformation was used on any non-linear variables. The normality of these regression's residuals was established to ensure model assumptions were satisfied. The Brown–Forsythe test was performed to test whether variation in log DHM Total IgA and Insulin concentrations differed by pool donor number. Analyses were performed using JMP Pro 14 .Out of the 128 DHM pools, 5 (3.9%) of the pools were “Preterm,” and 4 (3.1%) of the pools were “Dairy Free” meaning donors completely omitted dairy products from their diet. Data regarding pool donor number (number of donors in each pool), pool volume, and milk age have been previously reported . Of the Two of the 128 post-pasteurization samples were inconclusive for total IgA analysis due to high %CV, giving a final sample size for post-pasteurization total IgA analysis of 126. Two of the term post-pasteurization samples were not included in the final data analysis in order to not bias results, as both were outliers over five standard deviations from the IgA mean (1.575 and 1.212 mg/mL). Excluding these data points did not change the nature of reported relationships.n = 128) vs. 0.23 ± 0.09 mg/mL in post-pasteurization samples (n = 126). The total IgA concentrations in post-pasteurization samples decreased on average 0.08 mg/mL. The range in decrease was −0.61 to + 0.19 mg/mL), or 17.9% .The DHM total post-pasteurization IgA concentrations were not correlated with pool postpartum milk age. DHM total post-pasteurization IgA concentrations did differ by pool donor number: DHM pools with one donor had a lower total IgA concentration than pools with at least two donors  to 7.0 ± 4.6 μU/mL (range: 3–40 μU/mL). The range in decrease was −15 to + 3 μU/mL. DHM post-pasteurization insulin concentrations were not associated with pool milk age.p = 0.003; p = 0.034).DHM total post-pasteurization insulin concentrations did differ by pool donor number: DHM pools with one donor had lower insulin concentrations than pools with 2 donors , and caloric density .DHM insulin concentrations did not differ between term and preterm pools. The log of DHM pool insulin concentrations was positively associated with DHM fat content . This relationship may reflect the underlying relationship between maternal BMI and both human milk insulin concentrations , and milA strength of our study is usage of pre- and post-pasteurized DHM pools produced by a HMBANA milk bank, as opposed to small batch lab-generated pools. An additional strength of the study is the large sample size, which allows for an accurate estimate of the variation in pool characteristics. Our inability to link DHM pools and the total IgA and insulin concentrations to outcomes of recipient infants, or to characteristics of individual donors may be considered a weakness. Furthermore, these samples were generated from one milk bank where 9% of pools consisted of ≥3 donors over the days of pool production studied, which may not be representative of other milk banks. These findings help inform milk bank practices to optimize the benefit of DHM provided to recipient infants. Additionally, these findings help inform neonatologists about the mean and variability of bioactive components in DHM fed to premature infants.In conclusion, we have shown that DHM pools yield a wide range in concentrations of two potent bioactive components: total IgA and insulin. The number of donors included in a DHM pool affect both the final concentrations and the variability in total IgA and insulin. As such, there may be a benefit to standardizing the procedures for pool generation across independent milk banks to provide a more uniform “dose” of these bioactives to recipient premature infants. Lastly, these data support the need for both social and cultural initiatives to ensure donor milk banks have enough donation availability to meet pooling goals.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.BY, LB, RH, and NK contributed to the conception and design of the study. LB, RH, BY, and KM collected the data. BY organized the database. BY and KM performed the statistical analysis. BY and KM wrote the first draft of the manuscript. All authors contributed to manuscript revisions, read, and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Aerobic capacity is a strong predictor of cardiovascular mortality. Whether aerobic capacity influences myocardial ischemia and reperfusion (IR) injury is unknown.To investigate the impact of intrinsic differences in aerobic capacity and the cardioprotective potential on IR injury.We studied hearts from rats developed by selective breeding for high (HCR) or low (LCR) capacity for treadmill running. The rats were randomized to: (1) control, (2) local ischemic preconditioning (IPC) or (3) remote ischemic preconditioning (RIC) followed by 30 minutes of ischemia and 120 minutes of reperfusion in an isolated perfused heart model. The primary endpoint was infarct size. Secondary endpoints included uptake of labelled glucose, content of selected mitochondrial proteins in skeletal and cardiac muscle, and activation of AMP-activated kinase (AMPK).At baseline, running distance was 203±7 m in LCR vs 1905±51 m in HCR rats (p<0.01). Infarct size was significantly lower in LCR than in HCR controls . IPC reduced infarct size by 47% in LCR (p<0.01) and by 31% in HCR rats (p = 0.01). RIC did not modulate infarct size . Phosphorylaion of AMPK did not differ between LCR and HCR controls. IPC did not modulate cardiac phosphorylation of AMPK. Glucose uptake during reperfusion was similar in LCR and HCR rats. IPC increased glucose uptake during reperfusion in LCR animals (p = 0.02). Mitochondrial protein content in skeletal muscle was lower in LCR than in HCR (0.77±0.10 arbitrary units (AU) vs 1.09±0.07 AU, p = 0.02), but not in cardiac muscle.Aerobic capacity is associated with altered myocardial sensitivity to IR injury, but the cardioprotective effect of IPC is not. Glucose uptake, AMPK activation immediately prior to ischemia and basal mitochondrial protein content in the heart seem to be of minor importance as underlying mechanisms for the cardioprotective effects. Aerobic capacity is a strong predictor of cardiovascular mortality –3. RegulA common method to induce cardioprotection is ischemic conditioning, which consists of brief non-lethal episodes of ischemia and reperfusion prior to a lethal period of ischemia ,9. The cThe aim of the present study was to investigate the impact of aerobic capacity on IR injury, and to study the influence of aerobic capacity on the cardioprotective efficacy of IPC and RIC in an experimental model using hearts from rats with high or low running capacity.We studied male rats selectively bred to be either high capacity (HCR) or low capacity (LCR) runners. 48 HCR rats and 50 LCR rats were included in total. The development of the rat model of HCR and LCR rats has been comprehensively described previously ,16. The Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health . The Danish Animal Experimental Inspectorate approved the experimental work (authorization id. 2012-15-2934-00623 and 2018-15-0201-01446).All animal experiments were carried out in agreement with the Danish law for animal research  and the guidelines from in vivo procedure, followed by isolation and in vitro perfusion of the heart in the Langendorff model. Hearts were then subjected to 30 minutes stabilisation, 30 minutes global no-flow ischemia and 120 minutes reperfusion. HCR and LCR rats were randomized to either: group I. Control, group II. IPC, or group III. RIC . The hearts were rapidly excised and mounted in a Langendorff apparatus and perfused at a constant pressure of 80 mmHg. The perfusion buffer was equilibrated with 95% O2 and 5% CO2 to maintain a pH between 7.35–7.45. The temperature was kept constant at 37±0.5°C during the whole perfusion protocol. The left atrial auricle was resected and a balloon-catheter , connected to a pressure transducer, was inserted into the left ventricular cavity for continuous hemodynamic measurements. The balloon volume was adjusted to obtain a left ventricular end-diastolic pressure of 7–10 mmHg. The coronary flow was continuously measured by an in-line flow probe . All data were acquired and digitally analysed using a dedicated core software platform .Animals were anesthetized with pentobabiturate ), connected to a rodent ventilator , and ventilated with atmospheric air during the escribed ,20. The Exclusion criteria were left ventricular developed pressure (LVDP) below 110 mmHg at the end of stabilisation, coronary flow of more than 20 mL/minute or continuous ventricular fibrillation during stabilisation or reperfusion.At the end of reperfusion hearts were removed from the perfusion apparatus and immediately frozen at −80°C for a minimum of 20 minutes and subsequently cut into ≈1.5 mm slices. Slices were immersed in 1% 2,3,5-triphenyltetrazolium chloride  at 37°C and pH 7.4 for 3 minutes rendering vital tissue deep red and infarcted tissue pale. Hearts were stored in 4% formaldehyde  for 20–28 hours to enhance the contrast between vital and infarcted tissue. Each heart slice was weighed and scanned on a flatbed scanner . The area of left ventricle (LV), which corresponded to the area at risk (AAR), and area of infarction (IS) were assessed manually by observer delineation using computer assisted planimetry . Infarct size was expressed as a percentage IS of AAR. Measurements were adjusted to the wet weight of each individual slice. All analyses were performed in a blinded manner.3H2O production from d-[2-3H]-glucose added to the KH buffer ‐glucose was performed by anion exchange chromatography on an AG 1-X8 resin column . The 3H2O was dissolved in 10 mL OptiPhase scintillation solution  and the amount was quantified by β-scintillation counting on a Tricarb® 2900TR liquid scintillation analyser (Packard). The glucose concentration was calculated by dividing the disintegrations per minute by the specific activity of glucose in the perfusate. The results were plotted against time and coronary flow at sample time, and the glucose uptake rate was calculated.Myocardial glucose uptake rate was assessed from the rates of H buffer ,22. The Western blot analyses were performed in two separate series. In the first series, we used cardiac tissue to assess how IPC and RIC affected enzymes involved in substrate metabolism and related the mitochondrial proteins: AMP-activated Kinase (AMPK), Acetyl-CoA Carboxylase (ACC), Glycogen Synthase (GS), Prohibitin-1 (PHB1) and Voltage-dependent anion channel (VDAC). In the second series we used both skeletal and cardiac tissue to assess the content of 3 mitochondrial proteins: Cytochrome c oxidase IX (COX IX), β-hydroxyacyl-CoA dehydrogenase (β-HAD) and citrate synthase (CS).1 (#2426), VDAC (#4661), COX IV  and 1:3000 (cardiac muscle) 5% skim milk). The following primary antibodies were purchased from Abcam and utilized as follows: CS , β-HAD . After incubation with primary antibodies, membranes were incubated with horseradish peroxidase-conjugated secondary antibody at RT for 1 hour in a 1:5000 TBST solution with 1% BSA (for COX IV a 1:10.000 solution was used). The proteins of interest were visualized with chemiluminescent substrate . Arbitrary protein intensity were quantified with an UVP imaging system  and normalized to the total amount of protein loaded in the corresponding lane using Stain Free Technology [The Western blot analyses were performed in accordance with standard protocols, as previously described ,24. In schnology ,26.In the first series, data are presented as fold change and phosphorylated proteins are expressed relative to their non-phospohorylated from. In the second series, data are expressed as arbitrary units.Data are presented as mean ± SEM, unless otherwise indicated. Statistical analyses of infarct size, myocardial glucose uptake during stabilisation and activation of signalling pathways were performed by one-way ANOVA. Two-way ANOVA with repeated measurements was used for hemodynamic function and myocardial glucose uptake during reperfusion. Bonferroni post hoc test was perfomed for multiple comparisons. Analyses of mitochondrial protein content were performed by t-tests. All statistical calculations were performed using GraphPad Prism . p≤0.05 was considered significant. The required sample size was estimated from previously published work using the isolated heart model .At age 3–4 months, the animals underwent treadmill tests, and the longest distance of three separate runs was lower in LCR than HCR rats . At 8 months of age, LCR rats had a significantly higher body weight than HCR rats .LCR rats had lower IS than HCR rats  . In bothDuring stabilisation, LCR control rats had higher LVDP than HCR control rats  . Rate prDuring reperfusion, LVDP, HR and RPP were similar in LCR and HCR control rats . IPC incThe ratio of pAMPK/AMPK did not differ significantly between LCR and HCR control rats . IPC didThe absence of AMPK activation was supported by similar ACC and GS phosphorylation in LCR and HCR animals, and none of the regulators were affected by IPC or RIC. Similarly, the mitochondrial proteins PHB1 and VDAC were not affected by animal type or IPC and RIC.Myocardial glucose uptake was similar in all groups during stabilisation .IPC increased myocardial glucose uptake in LCR (p = 0.10) and HCR rats, but only statistically significantly in HCR rats (p<0.01). RIC did not change the glucose uptake.During reperfusion myocardial glucose uptake was higher in LCR controls than in HCR control rats (p<0.01). IPC did not change the uptake in LCR rats. IPC increased myocardial glucose uptake in HCR animals (p<0.01) .COX IV, β-HAD and CS were measured in control rats. The mitochondrial markers COX IV, β-HAD and CS in tibialis anterior muscle were lower in LCR rats than in HCR rats  . In cardThe main finding of the present study is that LCR rats are less sensitive to IR injury than HCR rats, indicating that aerobic capacity may influence endogenous sensitivity to IR injury. Despite this difference, the efficacy of IPC was preserved and equal in both phenotypes, indicating that the cardioprotective potential was independent of differences in aerobic capacity. RIC was not effective in either phenotype and may reflect a challenge in translating the effect of RIC to an older rat model .Here, we used a rat model that was derived from a genetically heterogeneous stock (N/NIH) and developed by selective breeding based on running capacity as a surrogate marker of aerobic capacity (16). Through generations of selective breeding, two distinct phenotypes have evolved . LCR ratOur results are consistent with the findings by Høydal et al, who demonstrated similar or even worse cardiac- and cardiomyocyte contractile function following myocardial infarction in HCR rats compared to LCR rats, measured by echocardiography and isolated perfused heart evaluation . Hussainin vivo and in vitro models [The cardioprotective effect of IPC is consistent and reproducible in all animal species studied until now. The efficacy is in the range of 25–45% IS reduction depending on the experimental setting, animal strain, and duration of ischemia ,42. We eo models . Most pro models –46, and LCR hearts produced enhanced cardiac work, with increased LVDP during stabilisation, while there was no difference in glucose uptake compared to HCR. This finding could indicate a more efficient handling of glucose in the LCR animals, possibly by the upregulated carbohydrate metabolism in LCR rats as described by Bye et al . IPC incIPC as well as exercise induce cardioprotection by AMPK activation –49, suchThe cardiovascular health benefits of physical activity are multifactual mediated by a favorable endocrine milieu . IL-6 isAerobic capacity is tightly linked to mitochondrial numbers and function, and in the heart IPC modifies the deleterious effects of IR injury on mitochondria. In skeletal muscle, increased content of mitochondrial oxidative enzymes are a classic marker of adaptation to increased physical activity . Our obsPatients suffering from metabolic syndrome or diabetes mellitus have aggravated outcome after acute myocardial infarction . While tin vitro rat model limits transferability to human physiology. In vitro induction of IPC may potentially lack in vivo elements of IPC including the neurohumoral response. We aimed to study the impact of exercise in the most powerful conditioning modality, which is the in vitro setting of IPC. When evaluating myocardial energy metabolism and signal transduction we are limited by glucose as the only available substate and rapid metabolic changes, such that we may have missed windows of opportunity to detect rapid temporary changes.The use of an Sensitivity to IR injury is associated with differences in intrinsic aerobic capacity but the cardioprotective efficacy of IPC is unchanged, while RIC is not effective in either HCR or LCR. The cardioprotection in LCR rats is not associated with measurable differences in myocardial glucose uptake, AMPK activation and mitochondrial protein content prior to onset of ischemia.S1 FigRatio of infarct size over area at risk. IS: infarct size, AAR: area at risk, CON: control, IPC: local ischemic preconditioning, RIC: remote ischemic preconditioning. * p<0.05, ** p<0.01. n = 4–5. Values are presented as mean ± SEM.(DOCX)Click here for additional data file.S2 FigDuring stabilisation two sample points are displayed, one before IPC (9 min) and one after induction of IPC (29 min). During reperfusion samples are displayed over time. CON: control, IPC: local ischemic preconditioning, RIC: remote ischemic preconditioning, min: minutes. * p<0.05, ** p<0.01, *** p<0.001. n = 4–5. Values are presented as mean ± SEM.(DOCX)Click here for additional data file.S1 Raw images(PDF)Click here for additional data file.


Up to 50% of men with poor prognosis, non-seminoma germ cell tumours (GCTs) die with standard BEP  chemotherapy. An intensive regimen, CBOP/BEP , met response targets in a randomised, phase II trial  61%–85%).To assess long-term outcomes and late toxicity associated with CBOP/BEP.Patients with poor prognosis extracranial GCT were randomised to 4xBEP or CBOP/BEP . Low-dose, stabilising chemotherapy before entry was permitted. Response rates (primary outcome) were reported previously. Here, we report secondary outcomes: progression-free survival (PFS), overall survival (OS) and late toxicity. Prognostic factors and the impact of marker decline are assessed in exploratory analysis.Eighty-nine patients (43 CBOP/BEP) were randomised. After median 63 months follow-up, 3-year PFS is 55.7%  for CBOP/BEP and 38.7%  for BEP , p = 0.079). Three-year OS is 65.0%  and 58.5% , respectively , p = 0.49). Twelve-month toxicity was affected by subsequent treatments, with no clear differences between arms. Stabilising chemotherapy was associated with poorer PFS , p = 0.017), whereas unfavourable marker decline, in 60 (70%) patients, was not.Although not powered for PFS, results for CBOP/BEP are promising. Impact on OS was less clear (and will be affected by subsequent therapy). Further study in an international phase III trial is warranted.ISRCTN 53643604. •Intensive induction chemotherapy met response targets in poor prognosis germ cell tumours.•Three-year progression-free survival was 56% for CBOP/BEP and 39% for BEP.•Impact on survival was less clear and will be affected by subsequent therapy.•Further study in an international phase III trial is warranted. However, there is a well-characterised poor prognosis group for whom cure rates remain less than 50% . Attemptet al. [TE23 (ISRCTN 53643604) was a randomised, phase II trial aiming to assess the efficacy and safety of a dose-intense regimen, CBOP/BEP , in these patients. The regimen, originally developed by the Royal Marsden Testicular Tumour Unit based on Wettlaufer In the primary analysis, CBOP/BEP met response targets: 74% of patients achieved a complete response or partial response marker negative  . The triHere, we report long-term data from the trial, including efficacy outcomes and late toxicity. In addition, the role of early marker decline for predicting long-term outcomes is considered.22.1Eligible patients were ≥16 years with extracranial GCT and at least one International Germ Cell Cancer Collaborative Group (IGCCCG) poor prognosis feature . Diagnoses were based on histology; or elevated AFP and/or HCG in a patient with a testicular tumour; or unequivocally elevated markers (AFP>1000 ng/ml or HCG>5000iu/l) in men aged <45 years without a testis tumour but with an otherwise appropriate clinical picture. Applicable regulatory and ethics approvals, and written informed consent, were obtained.2.22 of cisplatin 20 mg/m or carboplatin AUC3 and etoposide or vincristine for two days) before enrolment. Randomisation at the coordinating trial unit (accessible by telephone to recruiting teams) used minimisation based on preprotocol chemotherapy, primary tumour site, centre and a random element.This was an open-label, parallel, phase II, multicentre, UK trial with randomisation (1:1) to BEP or CBOP/BEP. Eligible patients not deemed fit enough to receive protocol treatment (by the treating investigator) could be stabilised with low-dose chemotherapy  were administered. At weeks 5 and 6, 2 mg of vincristine and 15000iu of bleomycin on day 1 were administered. At weeks 7–15, 3 cycles of Indiana-style BEP administered as for the control arm, except with modified bleomycin dose of 15000iu weekly. Prophylactic granulocyte-colony stimulating factor (G-CSF) was mandated (in week five of CBOP/BEP and during each BEP cycle in both arms) from January 2008 following an Independent Data Monitoring Committee recommendation.The control arm comprised 4 cycles of Indiana-style 5-day BEP [Assessments and management during treatment have been reported previously . The pro2.4The primary outcome (reported previously) was favourable response rate: the proportion of patients achieving complete response  or partial response  with negative markers.The current analysis focuses on secondary outcomes: progression-free survival ; overall survival  and toxicity (Common Terminology Criteria for Adverse Events (CTCAE) v3.0) at 12 months (+/− 2 months). Data on pulmonary and audiometry investigations performed at this time were insufficient.2.5PFS/OS analyses were based on Kaplan-Meier curves and hazard ratios (HRs) derived from Cox regression models with 95% confidence intervals (CIs). Exploratory analyses considered the impact of IGCCCG poor risk features , receiptIn multivariable models, treatment effect was adjusted for any factors with p < 0.1 in univariate models. Power was limited, particularly for OS, where number of events was small. All randomized patients were included on an intent-to-treat basis with the exception of models incorporating early marker decline, where 3 patients who stopped treatment before the relevant marker assessment were excluded. Subgroup analyses considered the impact of early marker decline in the BEP arm and in those who did not receive stabilising chemotherapy.Late toxicity data are tabulated by treatment arm for surviving patients (regardless of subsequent treatments) with no formal comparative tests.Analyses were performed using Stata (StataCorp), version 14.3Between June 2005 and December 2009, 89 patients were enrolled from 16 UK centres ; 43 of them were randomised to CBOP/BEP . The meaMedian follow-up was 63 months – similar between arms. All surviving patients had a minimum 58-month follow-up, with the exception of 6 who were lost to follow-up . Of these, 2 had had progression events . Survival status at ≥5 years was obtained through GPs for these 2 patients, and for a furtherBEP patient who was lost to follow-up.3.1Time to normalisation could not be calculated for 3 patients  who only received one cycle of treatment in the trial and so did not have the relevant marker assessment. Sixty of eighty-six (70%) of patients had unfavourable marker decline, and this was similar in the two arms . As might be expected, high baseline AFP or HCG was correlated with unfavourable marker decline. The reverse trend was seen for LDH – a small number of patients had high (>10 ULN) LDH values, and these patients were more likely to have favourable marker decline. There were no other associations between IGCCCG factors or receipt of stabilising chemotherapy and marker decline .3.2There were 48 PFS events , and median PFS was 30 months . Estimated 3-year PFS was 38.7% (95% CI: 24.7%–52.4%) in the BEP arm and 55.7% (39.7%–69.0%) for the CBOP/BEP arm; hazard ratio (HR) = 0.59 (0.33–1.06), p = 0.079 a. Rates There were 37 deaths . Median survival time was just less than 6 years in the BEP arm and had not yet been reached for the CBOP/BEP arm. Estimated 3-year OS was 58.5% (43.0%–71.2%) in the BEP arm and 65.0% (48.8%–77.2%) for the CBOP/BEP arm; HR = 0.79 (95% CI: 0.41 to 1.52), p = 0.49 b.3.3Estimated 3-year PFS was higher in the group with unfavourable marker decline compared with those with favourable decline, though this trend was non-significant . Results were similar in the subgroup who did not receive stabilising chemotherapy and in the subgroup of BEP patients.In univariate models, receipt of stabilising chemotherapy was associated with poorer PFS . No signEstimated 3-year OS was higher in the group with unfavourable marker decline compared with those with favourable decline, although this was non-significant because of the low power of the model . Results were similar in the two subgroup analyses.Results for univariate and multivariable models for OS were similar to those for PFS – the only significant associations were for receipt of stabilising chemotherapy and mediastinal primary tumours . Treatme3.4At 12 months, there were 65 surviving patients , of which 11  had active disease. Burden of toxicity was similarly low in both arms . CTCAE g3.5Thirty-five patients relapsed or their disease progressed during the trial , including all patients with a treatment failure. Thirty patients  received treatment for relapse. Of the remaining five, three died around the time of diagnosis of relapse/progression; one was felt not to be fit enough for salvage treatment and died two months later; and the remaining patient did not require further treatment and remained alive at the end of the follow-up. Of those treated, all but 5 received combination chemotherapy , most commonly paclitaxel, ifosfamide and cisplatin (TIP) (13 patients), but a range of other regimens were used. Only 1 patient (BEP arm) had a complete response and 9  had a partial response with negative markers.4Long-term data from this randomised, phase II trial support the conclusion of the primary analysis, which demonstrated that CBOP/BEP met response targets and was feasible to deliver across UK centres. Although the trial was not powered to compare efficacy outcomes, a trend suggesting a PFS benefit was observed . Survival rates in the two arms were more similar, although slightly higher with CBOP/BEP. This is to be expected given that survival rates will be influenced by subsequent treatment. Twice as many patients received salvage treatment after BEP . This phenomenon of fewer patients in the dose-intense arm was also seen in the GETUG13 trial [Our previous systematic review highlighted that – despite a number of phase II and III randomised trials of first-line treatment for intermediate and poor prognosis of GCTs conducted internationally in recent years – no single approach has emerged as clearly superior to BEP . A notabCBOP/BEP takes a different approach to other treatments by delivering intensification upfront. An advantage of this approach is that maximal therapy is delivered when the cancer is at its most bulky, enabling a more rapid control of symptoms and potentially also reducing the development of resistance. Additional benefits, from a patient perspective, are that the regimen may be modified to fit the individual's clinical situation and the intensive phase is given when patients are often in hospital with disease-related morbidity. One difficulty is that it does not fit well with the approach of picking patients for intensive treatment based on marker decline. A further dose-intense approach, demonstrating potential in early phase, single arm studies, is accelerated BEP ,13, and A factor in the lack of success of some previous trials is that recruitment potential does not match the need. It is also important to be realistic about what benefits are achievable, particularly in terms of survival, which will be influenced by subsequent treatment and events. Some previous studies have been underpowered to detect realistic effects or have failed to recruit to target. Accrual to TE23 – across 16 UK centres – took twice as long as originally anticipated (4 years rather than 2 years), hence not meeting the criteria for immediate progression to a phase III study. Clearly, major international collaborations will be required to recruit to well-powered phase III trials in a timely manner. In addition, future trials in this patient group should look to novel methodology for evaluating treatments for rarer cancers, particularly given that there are a number of treatment strategies which might benefit from simultaneous evaluation .et al. [Another factor in evaluating treatment strategies is patient selection. For many years, IGCCCG criteria have provided a simple system – based on a large, international data set – for identifying patients for whom standard BEP treatment is less likely to be successful , and triet al.  have recThe TE23 cohort included a high proportion (40%) of men diagnosed on the basis of markers/clinical picture, rather than histology, and a substantial number of patients (27%) were deemed to require stabilising chemotherapy before full-dose treatment. As such, they may represent a particularly poor risk group, and this is reflected in poorer outcomes for BEP  relative to contemporary series, including the pooled data set mentioned previously . OutcomeIn TE23, use of stabilising chemotherapy was the factor most clearly associated with poor outcomes. The most likely explanation for this is that the clinician's assessment of disease at the outset – which may not be fully captured with current risk factors – may provide the best indicator of the likely success of treatment. However, we can not exclude a detrimental impact of the use of initial low-dose therapy/delay in commencing full-dose therapy.Targeting more intensive treatment at those patients most likely to benefit is desirable. One strategy is to select patients based on marker decline after a single BEP cycle. Within the IGCCCG poor risk group, inadequate marker decline has been shown to be independently associated with worse PFS and OS  and, in A limitation of this study is the small sample size, which is insufficient for complex multivariable modelling. The lack of a clear effect of early marker decline may reflect this – although it is notable that no trend was present. A further limitation is that outcome data beyond 5 years were not collected, although it is notable that few events (either PFS or OS) occurred after 3 years. A strength of this study is the randomised nature, providing a contemporary group of patients treated with BEP, and largely complete follow-up data up to 5 years.5CBOP/BEP has shown promise for treating men with poor prognosis GCT in a randomised, phase II trial – response targets were met, and a PFS benefit is suggested. Impact on survival is less clear and will be influenced by subsequent treatment. The regimen warrants study in a phase III trial.Cancer Research UK had no role in the study design; the collection, analysis and interpretation of data; the writing of the report; nor the decision to submit for publication.R.A.H. reports activities outside the submitted work, including receiving non-financial support from Janssen, grants and personal fees from MSD, personal fees from Bristol Myers Squibb, grants from CRUK, advisory board fees and travel expenses from Nekta and personal fees and non-financial support from Roche. F.H.C., J.D.W., J.S., I.H. and S.P.S. have nothing to disclose.


Hypothyroidism (HT) and carotid artery stenosis (CAS) are complications of radiotherapy (RT) in patients with head and neck cancer (HNC). The impact of post-RT HT on CAS progression remains unclear.Between 2013 and 2014, HNC patients who had ever received RT and were under regular follow-up in our hospital were initially screened. Patients were categorized into euthyroid (EU) and HT groups. Details of RT and HNC were recorded. Total plaque scores and degrees of CAS were measured during annual extracranial duplex follow-up. Patients were monitored for CAS progression to > 50 % stenosis or ischemic stroke (IS). Cumulative time to CAS progression and IS between the 2 groups were compared. Data were further analyzed based on the use or nonuse of thyroxine of the HT group.p = 0.02). Multivariate Cox models showed comparable CAS progression (p = 0.24) and IS occurrence (p = 0.51) between the 2 groups. Moreover, no significant difference was observed in time to CAS progression (p = 0.49) or IS (p = 0.31) among patients with EU and HT using and not using thyroxine supplement.333 HNC patients with RT history were screened. Finally, 216 patients were recruited (94 and 122 patients in the EU and HT groups). Patients of the HT group received higher mean RT doses (HT vs. EU; 7021.55 ± 401.67 vs. 6869.69 ± 425.32 centi-grays, Our results did not demonstrate significant effects of HT and thyroxine supplementation on CAS progression and IS incidence in patients with HNC after RT. Radiation-induced thyroid disorders are well-known complications after radiotherapy (RT) in patients with head and neck cancer (HNC) , 2. The HT may increase atherogenic risks through increased blood pressure and low-density lipoprotein cholesterol, homocysteine, and high-sensitivity C reactive protein levels . StudiesFrom January 1, 2013 to December 31, 2014, HNC patients who had ever completed RT and received regular follow-up at radio-oncology and neurology departments of Linkou Chang Gung Memorial Hospital were initially prospectively recruited, but the data were retrospectively reviewed. In this study, we aimed to detect the impacts of hypothyroidism in the early phase of post-RT vasculopathy, patients with previous stroke or > 50 % CAS at enrolment were assumed to be vulnerable to vascular events were excluded in this study to improve the homogeneity of cerebrovascular risks of the study population. Demographic data and details of risk factors for common stroke, such as dyslipidemia, hypertension, diabetes mellitus, heart disease, and cigarette smoking, were obtained from all the recruited patients. Laboratory data at enrolment, including glycated hemoglobin and low-density lipoprotein cholesterol, were recorded. Furthermore, medication records, particularly on the use of antiplatelets, statins, or thyroxine, were collected. In addition, all patients received regular thyroid function monitoring at enrolment, including free T4 and thyroid stimulating hormone (TSH) . IMRT is inherently limited by the physical properties of the photon beam, resulting in unavoidable irradiation of normal tissues at low to moderate doses even at substantial distances from the tumor . In the We used Philips HDI 5000  or Acuson Sequoia  5–10 MHz real-time B-mode imaging system and a 3.0 MHz pulsed-wave color Doppler spectrum analyzer to follow up the carotid and vertebral arteries after RT. B-mode examinations in the sagittal  and transverse views of the extracranial carotid arteries were used to detect stenotic features. The degree of CAS was defined according to the standard ultrasound criteria . PersonnPatients were grouped based on their thyroid function at enrolment. Patients with normal TSH (≤ 5 mIU/L) and free T4 level were categorized in the euthyroid (EU) group. Patients with elevated TSH (> 5 mIU/L), including overt HT and subclinical HT, or patients who had already received thyroxine treatment for HT at enrolment were categorized into the HT group.All the patients received serial thyroid function (TSH and free-T4) follow-up every 6 months. Furthermore, serial CDU studies were performed annually to monitor CAS progression. The main outcomes of interest in this study were CAS progression and upcoming ischemic stroke (IS). We defined CAS progression as > 50 % stenosis on the B-mode or peak systolic velocities ≥ 120 cm/s based on the hemodynamic criteria at any internal carotid artery or common carotid artery in the follow-up CDU study. The presence of upcoming IS was confirmed by clinical stroke symptoms with compatible brain image findings.t test to examine differences in continuous data between the study groups. In addition, categorical variables were compared using the chi-square test or Fisher’s exact test. Both event risk and time to event (CAS progression or IS) between the study groups were compared using a multivariable Cox proportional hazards model with adjustments for the selected variables  that might confound outcomes. Furthermore, the adjusted curves of time to event for each group were depicted using the multivariable Cox model. Moreover, time to CAS progression and IS were further analyzed during the subgrouping of patients in the HT group based on the use or nonuse of a thyroxine supplement. Statistical significance was set at p < 0.05.Power analysis was performed to estimate the participant numbers in each group. Alpha error with 0.05, statistical power with 0.9, and allocation ratio with 1 were set up. The estimated proportions of stroke events of the HT and EU groups between 18 and 64 years old were set up with 3.3 % and 2.4 % according to previous study . The estp = 0.02) and was less male dominant . Additionally, the mean age of patients enrolled , mean time interval between RT and patient enrolment , and baseline TPS at enrolment  were similar between the groups  were categorized into the EU group and the remaining 122 (56 %) who had abnormal thyroid function, including those with clinical and subclinical HT, were categorized into the HT group  groups. Time to CAS progression and time to IS were compared between the study groups. The cumulative incidence plot showed similar trends of CAS progression  and The mean follow-up periods were similar between the EU (192.26 ± 33.71 weeks) and HT  between the EU and HT groups  or IS (p = 0.31) among patients of EU, HT without thyroxine supplement, or HT with thyroxine supplement  and 1.7  in the HT group patients with thyroxine supplementation. The adjusted HRs of CAS progression and IS were 0.62  and 0.26 , respectively, in the HT patients without thyroxine supplement.IS (adjusted HR = 0.58; 95 % CI = 0.11–2.94; IS p = 0.1 among pOur single-center study did not demonstrate that post-RT HT could further potentiate the risks of CAS progression and IS in patients with post-RT vasculopathy. Similar to previous study, our results also showed patients with post-RT HT received higher radiation doses Table  3]. Bot. Bot3]. Post-RT vasculopathy in HNC patients is a common and rapidly progressive complication, but at present, few effective treatments are available to alleviate post-RT vasculopathy in these patients Table  13, 27,,27, 28. Inflammation, fibrosis, and endothelial proliferation play crucial roles in post-RT vasculopathy . Anti-inOur study had several limitations. First, the number of enrolled patients was too small to demonstrate clinical significance. Real-world studies investigating medication treatments in post-RT vasculopathy usually enrolled more than 1000 patients. However, in claims database studies, HT diagnosis depends on clinician coding. Determining real thyroid function might be difficult, and subclinical HT might not be registered. In future, a prospective cohort study with more patients may help to reduce this limitation and provide clear answers. Second, the TSH and free-T4 level of the same patient may change over time. Patients categorized into the subclinical hypothyroidism initially may develop overt hypothyroidism during follow-up period. Therefore, we combined overt and subclinical hypothyroidism into the same group. This may have possibility to dilute the clinical results. Third, there was no universal protocol for thyroxine management in these recruited patients. There was no general guidance for the initiation of thyroxine supplement in clinical or subclinical HT after RT. The compliance of thyroxine supplement in each patient may confound the study results. Besides, the associated symptoms and vascular risk factors may interfere with the clinician’s decision, and this may also confound the study results. A prospective study with standardized thyroxine treatment protocol can better reveal the real benefits of thyroxine treatments. Third, evaluations of the vascular complication may not be comprehensive, and the follow-up period may not have been sufficiently long in this single-center study. Furthermore, high vascular risk patients with severe CAS were not enrolled. These could have led to an underestimation of the actual clinical events, particularly silent infarctions.Interactions between thyroid function, CAS progression, and stroke prevention in HNC patients after RT is complex. So far, no practical clinical guidance exists for this population. Our results did not demonstrate the influence of HT or the clinical benefits of thyroxine supplementation on CAS progression or IS prevention in these patients. Future studies are warranted to assess the vulnerable subgroups whose post-RT HT should be carefully monitored and treated, particularly patients who may have increased post-RT vascular complications.


ACVR1 and rs1043784 in BMP6, and primary angle-closure glaucoma (PACG) and pseudoexfoliation glaucoma (PXG).Glaucoma is a polygenic neurodegenerative disease and the second most common cause of blindness in Saudi Arabia. To test the hypothesis that genetic variants in the genes involved in the bone morphogenic protein (BMP) signaling pathway may be associated with glaucoma, we investigated the association between 3′ untranslated region variants, rs12997 in In a case-control study, TaqMan® real-time PCR-based genotyping was done in 444 subjects consisting of 250 controls, 101 PACG and 95 PXG cases, and tested for genetic association with glaucoma-types and other clinical phenotypes.ACVR1 exhibited significant 2-fold increased risk of PACG (p = 0.005) in women but not in men. Similarly, genotype analysis also showed that subjects carrying rs12997[G/G] genotype were at > 2-fold risk of PACG that remained significant after adjustment for age, sex, and Bonferroni correction in the recessive model. Furthermore, this effect was also significant in women only. In PXG, the rs12997[G/G] genotype showed a significant trend towards increased risk of the disease  but did not survive the Bonferroni correction. Regression analysis showed that rs12997[G/G] genotype was a significant predictor of PACG independent of age, sex, and rs1043784 genotypes. Likewise, age and rs12997[G/G] genotype showed significant effect on PXG outcome. The rs12997[A/G] genotype showed significant association with cup/disc ratio as compared to wild-type (p = 0.005) in PXG. Genotype and allele frequencies of rs1043784 in BMP6 did not show any significant association either with PACG or PXG.Rs12997[G] allele in ACVR1 gene involved in the BMP signaling pathway is significantly associated with PACG and PXG in a Saudi cohort. This is the first study to associate this variant/gene with PACG and PXG. However, further studies would be needed to replicate these findings in a large population-based cohort.Our results suggest that the polymorphism rs12997 in the  Obstruction in the outflow pathway of the aqueous humor due to structural changes in the trabecular meshwork (TM) and the resultant elevated intraocular pressure (IOP) are believed to be one of the major contributing factors leading to the damage of the optic nerve head (ONH), retinal ganglion cell (RGC) death and subsequent loss of vision in glaucoma . The preCDKN2B-AS1, TGFBR3-CDC7 and FNDC3B) linked to POAG which may contribute to the regulation of the TGF-β signaling [Members of the transforming growth factor-β (TGF-β) superfamily of growth factors have been implicated in glaucoma pathogenesis –6. Recenignaling .ACVR1) is a BMP type I receptor of the TGF-β receptor subfamily. Mutations in the ACVR1 gene are known to cause fibrodysplasia ossificans progressiva (FOP), a genetic disease characterized by progressive heterotopic ossification [Acvr1 has also been reported to function as a tumor suppressor gene in the mouse lens [BMPs are known to control a variety of biological functions in the cells . Like thfication , 11. Besuse lens . Bone mouse lens , 14 In-vuse lens .ACVR1, and rs1043784 in BMP6. The variants rs12997 and rs1043784 located in the 3’UTR region of the ACVR1 and BMP6 genes, respectively, and may result in disruption of microRNA (miRNA)/mRNA binding [It can thus be speculated that members of the complex TGF-β/BMP signaling pathway may have a role in maintaining TM homeostasis as well as glaucoma pathogenesis . Likewis binding , therebyA retrospective case-control study was conducted. The study was approved by the Institutional Ethical Committee at College of Medicine  and conformed to the principles of the Declaration of Helsinki. All the participants provided a written informed consent. Patients of Saudi origin with clinical diagnosis of PACG, PXG and normal controls were enrolled at King Abdulaziz University Hospital, Riyadh, Saudi Arabia.n = 101) were diagnosed based on clinical evidence of anatomically closed angle showing the occurrence of appositional or synnechial closure of the anterior chamber angle (at least 270o of the angle is occluded); raised IOP (≥21 mmHg); presence of optic disk damage with cup/disc ratio of at least 0.7 (in at least one eye); and loss of peripheral or advanced visual field [n = 95) showed the presence of flaky exfoliation material along the pupil edges or anterior lens capsule, glaucomatous optic neuropathy and associated visual field loss, and high IOP in either or both the eyes as described previously [n = 250) recruited from our ophthalmology screening clinics served as controls. The participants were: > 40 years of age, with normal IOP, and free from any form of glaucoma on clinical examination. Subjects refusing to participate were excluded.PACG patients  were used to genotype rs12997 and rs1043784, respectively on ABI 7500 Real-Time PCR System (Applied Biosystems) under conditions recommended by the manufacturer .t-test (2-groups) and one-way ANOVA (3-groups) was used to compare continuous variables. Regression analysis was performed to test the effects of multiple factors such as age, sex, and genotypes on POAG. Statistical tests were performed using SPSS version 22 . Besides, SNPStats online software (https://www.snpstats.net/start.htm) was also utilized for testing genotype associations. A p < 0.05 (2-tailed) was considered significant. Bonferroni’s correction was used to adjust for multiple testing and corrected p-values < 0.01 was considered where applicable.Hardy-Weinberg Equilibrium (HWE), allele, and genotype associations were tested using Chi-square analysis. Besides, Student’s p = 0.319) but significantly older in the PXG (p < 0.001) as compared to controls. Similarly, in terms of gender distribution, there was preponderance of men, except in the PACG group. However, the distribution was non-significant in both the PACG (p = 0.080) and PXG (p = 0.100) groups as compared to controls.The demographic data of subjects included in the study is shown in Fig. p = 0.050), wherein gender-stratification showed that women with G allele exhibited a significant ~ 2-fold increased risk of PACG (p = 0.005). Likewise, PXG group also exhibited a significant trend (p = 0.056) but did not show any significant gender distribution.The distribution of allele frequency of rs12997 according to glaucoma type and gender in cases and controls is shown in Table p = 0.475) or PXG cases (p = 0.446) as compared to controls. Furthermore, gender-stratification also yielded no significant association. Also, there was no significant deviation from HWE.The allele frequency distribution of rs1043784 according to glaucoma type and gender in cases and controls is shown in Table The genotype analyses of rs12997 according to glaucoma type and gender-stratification are shown in Tables correction = 0.05/5 = 0.01) in the recessive model. However, the significance was lost after Bonferroni correction in the co-dominant model. Furthermore, the association was significant in women but not in men, and the p-value showed significant association even after adjustment for age and multiple testing (Table As shown in Table ng Table .p = 0.049) in co-dominant model. No gender-specific association was observed with PXG .Rs1043784 variant did not show any significant association with PACG and PXG Table . BesidesTable p = 0.004). Post-hoc analysis showed that A/G genotype exhibited increased cup/disc ratio as compared to A/A (wild-type) genotype (p = 0.005). Figure Figure ACVR1 gene involved in BMP signaling and patients with PACG and PXG in a Saudi cohort.In the present study, we report a previously unreported association between variant rs12997 in the ACVR1  gene expression is still not completely understood. ACVR1 can function via the TGF-β/BMP signaling through a variety of different mechanisms. Mutations in ACVR1 have been reported to show increased responsiveness to certain BMP ligand activation in a variety of cell types, leading to over-activation of ACVR1 and dysregulated BMP signaling [ACVR1 has been reported in breast cancer [The regulation of ignaling –22. Actiignaling . The comignaling . Besidesignaling . Besidest cancer , and witt cancer .Acvr1-deficiency (loss-of-function) was found to increase osteogenesis by activating Wnt signaling and decreasing the expression levels of these Wnt inhibitors [Interestingly, ACVR1 has been reported to function as a critical regulator of the BMP/Wnt signaling pathway to promote proliferation and metastasis , 28. Genhibitors . Based oACVR1 may influence the risk of these glaucoma types. Nevertheless, rs12997 is located in the 3’UTR region of the gene, which are known to regulate mRNA stability. The functional analysis of the 3’UTR region of ACVR1 has reported the involvement of specific miRNAs in regulating its gene expression [ACVR1 may affect the binding of miR-330-3p and result in loss of ACVR1 regulation [ACVR1 with G allele may result in its over-expression as compared to the wild-type allele, thereby showing an association with PACG and PXG by mechanism(s) regulating the ACVR1/BMP/Wnt signaling pathway as described above. Besides, Wnt signaling pathway play a key role in ECM cell behaviour and elasticity [ACVR1 may result in dysregulated Wnt signaling causing ECM abnormalities in the TM and elevated IOP. Further in-vitro and molecular studies are needed to support this hypothesis.PACG is a more common form of glaucoma in Asia , 34, invpression . It has gulation . Hence, asticity , and stuasticity . It is tBMP6 and PACG and PXG, indicating BMP6 may not have a major role in glaucoma pathogenesis. However, BMP6 locus has been associated with lung function in a GWAS [BMP6 or other BMPs in glaucoma cannot be ruled out and needs further investigations [Our study showed no association between variant rs1047384 in n a GWAS . Likewisn a GWAS . Thus thigations .ACVR1 in PACG and PXG. Furthermore, linkage with other causal variant(s), gene-gene or gene-environment cannot be ruled out.The results of this study need cautious interpretation due to its certain limitations. The study examined a relatively small sample size. On the basis of MAF observed in our study population and assuming an OR of 2.0 (α = 0.05), the study exhibited a power of 0.82 and 0.80 for rs12997 association in PACG and PXG, respectively. Likewise, variant rs1043784 demonstrated powers of 0.70 and 0.68 to detect association with PACG and PXG, respectively. However, to detect an effect of 1.5 or less, as is most commonly seen in genetic association investigations, a multicenter study with larger sample-size needs to be performed to confirm these results. Besides, our results do not provide any functional/mechanistic evidence for the role of rs12997 in ACVR1 gene is associated with PACG and PXG, and suggests that ACVR1, a member of the BMP signaling pathway may play an important role in the complex pathogenesis of these diseases. However, as pointed out earlier, our results need to be replicated in other cohorts of different ethnicity and in a large population-based sample size to draw definite conclusions and evaluate the utility of this variant/gene as a potential genetic biomarker in glaucoma. Besides, based on the reports of miRNA-based regulation of ACVR1 [ACVR1 gene biology may offer potential therapeutic drug target in glaucoma.In conclusion, our study reports for the first time that genetic variant rs12997 in the of ACVR1 , 36, und


Focal segmental glomerulosclerosis (FSGS) causes renal fibrosis and may lead to kidney failure. FSGS and its common complication, proteinuria, are challenging to treat. Corticosteroids are ineffective in many patients with FSGS, and alternative treatments often yield suboptimal responses. Repository corticotropin injection , a naturally sourced complex mixture of purified adrenocorticotropic hormone analogs and other pituitary peptides, may have beneficial effects on idiopathic FSGS via melanocortin receptor activation.N = 27), and a 12-week chronic RCI dose range study (N = 34). Primary outcomes were proteinuria and renal pathology improvements for measures of renal fibrosis, tubular damage, glomerular injury, and total kidney injury score. Impact of RCI treatment was also determined by assessing urinary biomarkers for renal injury, podocyte expression of podoplanin (a biomarker for injury), podocyte effacement by electron microscopy, and histological staining for fibrosis biomarkers.Two studies in a preclinical  puromycin aminonucleoside FSGS model assessed the effect of RCI on renal function and morphology: an 8-week comparison of a single RCI dose with methylprednisolone  demonstrated statistically significant improvements with RCI 30 IU/kg in the 8-week study and RCI 60 IU/kg in the 12-week study. RCI treatment improved levels of urinary biomarkers of kidney injury (KIM-1 and OPN), expression of podoplanin, and podocyte morphology. RCI also reduced levels of desmin and fibrosis-associated collagen deposition staining. Methylprednisolone did not improve renal function or pathology in this model.These results provide evidence supporting the improvement of FSGS with RCI, which was superior to corticosteroid treatment in this experimental model. To the authors’ knowledge, this is the first evidence that a drug for the treatment of FSGS supports podocyte recovery after repeated injury. Focal segmental glomerulosclerosis (FSGS) is a disease that attacks kidney glomeruli and causes scarring, leading to kidney damage. FSGS is a renal condition frequently associated with nephrotic syndrome (NS) and is one of the most common forms of glomerular disease , 2. FSGSFirst-line treatment for idiopathic FSGS is oral corticosteroids . The mecp < 0.001), with 62% partial response and 8% complete response at 6 months with RCI in 13 FSGS patients who had failed multiple first-line therapies , RCI 30 IU/kg, and MP 2 mg/kg). The second study used a long-term 12-week PAN model  with an extended dose range of RCI for the treatment of FSGS. Disease induction with PAN occurred on day 0 (50 mg/kg), with treatment starting on day 7. Reinjury occurred with lower-dose PAN (20 mg/kg) on days 14, 21, and 28 in the 8-week model, and additional injury occurred on day 35 in the 12-week model  model, resulting in FSGS-type lesions and podocyte damage observed in NS , 28, 29.During the 8- and 12-week studies, multiple disease endpoints were analyzed to assess renal function and kidney morphology. Primary dependent measures were levels of proteinuria and scores for renal fibrosis, tubular damage, and glomerular injury. A score for interstitial inflammation was also measured, and the 4 scores were summed to yield a total kidney injury score. Scoring was performed by a certified pathologist and was graded from 0  to 5 (severe).am and 5 pm central standard time. Rats were housed 3 or 4 animals per cage in standard shoebox cages (16x8x8 inches). Aspen shavings were used for bedding in home cages. All study animals were kept on a diurnal 12-h light/dark cycle, with ample light used for routine inspection and cleaning. Ambient temperatures were maintained between 65 °F and 85 °F. Standard rat chow pellets were given freely. All animals had ad libitum access to potable water via water bottle. Rats were provided with enrichment devices such as Nylabone® products, paper twists, or other devices approved by the attending vet.All experiments were performed between 8 All in-life experiments were conducted in the site vivarium, with dosing and urine collection completed in the controlled procedure room. Animals were observed daily, including weekends and holidays, to assess general health and well-being and were weighed at least twice per week.All rat urine assays were performed with 24-h urine collections in metabolism cages with 1 rat per cage. Animals were normalized across 24 h in the metabolism cages, urine tubes were replaced, and animals remained in the metabolism cages for an additional 24 h for the duration of the urine collection. Animals had unlimited access to food and water. All histology procedures, including immunohistochemistry (IHC) staining, were completed on site. Enzyme-linked immunosorbent assay (ELISA) was conducted in the laboratory space adjoining the Meso Scale Discovery platform. All image analyses were completed in a designated imaging room on-site. All experiments were completed in the same building, on the same floor, and in the same wing of the floor.Animal sample size was determined using the resource equation method, with consideration of minimizing unnecessary animal usage, per our site guidelines . This eqDosing methodology was selected on the basis of normal treatment routes of administration. For example, in clinical settings, RCI is given subcutaneously. The most common route of administration of corticosteroids is oral dosing. The doses for RCI were determined by multiple previous in-house studies across animal models that have shown efficacy. As for MP dosing, high-dose treatment in human FSGS for prednisone is 1 mg/kg/day, with lower doses of 0.5 mg/kg/day used for maintenance . Rat dosRCI  and saline were administered subcutaneously, whereas MP was administered by oral gavage. In the 8-week model, FSGS was induced with 4 IV doses of PAN . RCI (30 IU/kg) was administered every other day beginning on day 7, whereas MP 2 mg/kg was given daily beginning on day 7. In the 12-week model, FSGS was induced with 5 IV doses of PAN , and RCI  was administered every other day beginning on day 7. Naive rats were treated with saline and no PAN to mimic handling of experimental rats , and 24-h total protein was calculated to assess proteinuria. Urine creatinine (uCr) was determined with the Jaffe method, a colorimetric plate-based assay . Two technical replicates were included for each sample, with the average of the replicates used for the final value.Urinalysis of kidney injury biomarkers osteopontin (OPN) and kidney injury molecule-1 (KIM-1) was performed via multiplex ELISA on the Meso Scale Diagnostics  Discovery system. KIM-1 and OPN were evaluated on day 28 after the third PAN booster dose (20 mg/kg), at peak proteinuria in the 8-week model, and again at day 56 after 4 weeks of treatment following the final PAN injection. An additional time point at day 42 was added for the 12-week model, corresponding to peak proteinuria in the study. Samples were tested via rat Kidney Injury Panel-1 , and values were expressed as ng/mL. The calculated values from the ELISA were normalized for each animal to the uCr concentrations in mg/mL, resulting in a ratio of ng:mg of target:creatinine concentration. Two technical replicates were included for each sample, with the average of the replicates used for the final value. Because the efficacy comparison was only in the PAN treatment (diseased) groups, naive  samples were pooled between the 8- and 12-week studies for ELISA comparisons to gain a more robust negative control baseline. Little variation in naive samples was expected based on previous experience of standard values for this age range, and animal numbers were limited in each study for ethical reasons.Animals were sacrificed, and kidneys were immediately removed. Cross sections of the central portion of the left and right kidneys were fixed in 10% neutral buffered formalin and processed to paraffin blocks for routine histology and IHC. In the 12-week PAN study, additional portions of renal cortex from both the left and right kidneys were fixed for analysis by electron microscopy (EM). All kidneys were stained with haematoxylin and eosin (H&E) and periodic acid-Schiff (PAS) for pathology scoring; Masson’s trichrome, Sirius Red/Fast-Green, and IHC staining were used to assess fibrosis and podocyte biomarkers. Slides stained with H&E, PAS, and Masson’s trichrome were sent to Seventh Wave Laboratories  for a renal pathology assessment by a board-certified veterinary pathologist who was not blinded to treatment groups, but did not have details on the specific study drug. EM samples for the 12-week study were sent to Charles River Laboratories , where they were evaluated for treatment-related changes to the glomerulus by a board-certified veterinary pathologist who was not blinded to treatment groups. All IHC staining  was performed on the Ventana  Discovery Ultra automated stainer. The antibody used for PDPN was Novus  NB110–962423.Slides were scanned on a NanoZoomer S210  at 20x or 40x magnification. Tissue images were analyzed through Visiopharm  quantitative image analysis software. For quantification of fibrosis, the kidney cortex was outlined at the region of interest (ROI), and interstitial fibrosis was quantitated per area of total tissue. Glomeruli from all areas of the kidney cortex were selected as ROIs from both the left and right kidney sections. An average of ~ 200 glomeruli per animal was selected to decrease bias due to focal pathology. Two individuals randomly identified the ROIs used in the final analyses. The software was trained to identify and quantitate staining of interest. All slides were batch processed using the same algorithm for all slides in each analysis. Naive samples were pooled for imaging analysis across studies, similar to the ELISA analysis.t tests as indicated for continuous normally distributed data. Non-normally distributed continuous and categorical data were compared with nonparametric Kruskal-Wallis (ANOVA) with post hoc tests, Mann-Whitney for 2-group comparison, or Fisher’s exact test. Graphs are presented as mean ± standard error of the mean unless otherwise noted. A significance level of p < 0.05 was established for all statistical tests.Comparisons between groups were performed through 1-way or 2-way analysis of variance (ANOVA) and to check the assumption of equal variances, with appropriate post hoc tests for multiple comparisons or In the 8-week study, one naive control died after routine bleeding, which was categorized as a laboratory handling error. In the 12-week study, 2 animals in the RCI 30 IU/kg group lost more than 20% of their body weight by day 47 or day 49 and were sacrificed according to our humane animal use protocol. Both animals had elevated proteinuria from disease induction, even prior to treatment, and maintained elevated proteinuria levels compared with all groups until their death. The lack of any animal deaths in the higher dose group of 60 IU/kg suggests the deaths were not likely treatment related but were caused by an extreme sensitivity to PAN injury. These animals were not included in any analyses. No additional controls or modifications were needed in this study, as the animal use guidelines implemented here account for changes in animal health, and the number of unexpected deaths was low .All study procedures, including for animal use welfare, were performed in accordance with the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals. The study protocol was reviewed and approved by the Institutional Animal Care and Use Committee of Mallinckrodt Pharmaceuticals.Animals were randomized by urine protein levels to ensure no significant differences were observed between groups. Baseline body weights and urine protein were measured before disease induction Table .Table 1p < 0.05), with a 38% reduction in peak mean urine protein levels at day 28 compared with saline and a 29% total reduction in area under the curve (AUC) over the study period  in the 8-week model  rats were assessed for comparison throughout our studies to confirm the disease phenotype induced by PAN. Treatment of rats with puromycin at 50 mg/kg at day 0, followed by booster doses of 20 mg/kg at days 14, 21, and 28 in the 8-week model and an additional dose at day 35 in the 12-week model, resulted in maximum urine protein levels of approximately 600–700 mg/24 h. RCI treatment of 30 IU/kg significantly reduced proteinuria (p < 0.05), and with RCI 60 IU/kg in the 12-week model versus saline, 5.1 ± 2.5 vs 8.8 ± 2.4 (p < 0.05). Treatment with MP did not reduce disease histopathology or the calculated total kidney injury score compared with saline in the 8-week model  , whereas MP treatment showed higher KIM-1 levels compared with saline on day 28 and 56 on average   were analyzed in both 8- and 12-week models Figs.  and 4. Nage Fig. a, b. Trep < 0.01)  at day 28 in the 8-week model, whereas MP significantly increased OPN levels compared with saline (p < 0.05). Treatment with RCI significantly lowered OPN concentrations at day 42 for RCI 10, 30, and 60 IU/kg (p < 0.05)   Fig. e. RCI 30p < 0.05), whereas MP treatment was unable to prevent PDPN loss, with lower mean area staining than saline   Fig. . PDPN stp < 0.05) compared to saline  in both models  and 60 IU/kg (p < 0.001) compared with saline  in the 8-week model  in the 12-week model , a cellular mechanism of fibrosis , was alsels Fig. a, b. In ine Fig. b. SiriusOverall, RCI improved glomerular function and pathology after repeated PAN injury in Sprague-Dawley rats. RCI reduced proteinuria, improved EM measures of podocyte morphology, decreased urinary biomarkers of renal damage, and increased IHC markers of podocyte function. RCI also reduced histopathology scores of fibrosis, interstitial inflammation, tubular injury, and glomerular changes after injury. By comparison, MP treatment alone was not beneficial for any of the relevant endpoints in these models. The early benefit in proteinuria reduction by MP could not be maintained throughout the study, and overall renal injury in this group was no better than in saline controls, often with higher mean severity scores than no treatment.after podocyte damage in a rodent model of FSGS. Rather than simply preventing symptoms of FSGS, RCI was shown to reverse damage to podocytes, with improved podocyte structure and function even after repeated renal injury in the 12-week model. These preclinical models demonstrate the therapeutic benefit of RCI in treatment of ongoing FSGS. The authors are not aware of another FSGS drug shown to improve damage to podocytes after repeated injury.Importantly, the improvements observed with RCI occur Results from this study are similar to published data showing statistically significant reductions in proteinuria with RCI in patients with FSGS , 35. In These results indicate that in an extended non–immune-mediated model of FSGS, steroid-mediated adverse effects from chronic treatment may outweigh early efficacy seen in FSGS models of shorter duration . AlthougThe increased PDPN expression demonstrated in this study may be a benefit of MCR-mediated RhoA activation, explored in previous studies –18. The Although a steroid treatment condition was included in the 8-week PAN group to model traditional steroid treatment, the 12-week PAN model did not include an MP group for comparison and was designed as a dose-response study for RCI. The two studies also showed slight differences in baseline age and weight. Kidney function was evaluated by proteinuria; additional measures such as blood urea nitrogen, serum creatinine, and urinary albumin were not included.In the current study, RCI treatment was effective in reducing PAN-induced renal damage and podocyte injury, which were not observed by treatment with MP. These results provide further evidence for a unique mechanism of MCR agonism in the treatment of FSGS, which was superior to steroid treatment in this experimental model.