Pleurotus ostreatus, turning them into edible biomass. The OLPR was used alone (OLPR), or in mixtures with wheat straw (WS : OLPR 1 : 3 v/v and WS : OLPR 3 : 1 v/v). Mycelial colonization was hastened by 3.7 days in WS : OLPR 1 : 3 (v/v). Yields were comparable to control (WS) in WS : OLPR 3 : 1 (v/v). Organic matter loss decreased with increasing proportions of OLPR in substrates. The nutritional value of mushrooms was improved by lower fat and sodium contents, in WS : OLPR 1 : 3 (v/v) and WS : OLPR 3 : 1 (v/v), and higher total protein, crude fiber, iron, and total carbohydrates contents in WS : OLPR 3 : 1 (v/v), compared with those of control. Polyunsaturated fatty acids, mainly linoleic acid, were the most abundant in mushrooms. Monounsaturated fatty acids increased in mushrooms of the substrates containing OLPR. A good predictive model of partial least square regression analysis showed different relationships of mushroom palmitic, oleic, linolenic, palmitoleic, and stearic acids with substrate composition. Findings suggested the use of OLPR as a supplement to commercial wheat straw and as a tool to reduce the negative impacts of their hazardous disposal on the environment.In several Mediterranean countries, olive pruning residues (OLPRs) are abandoned or burned leading to several environmental problems. Valorization of these agrowastes could be a challenge for the primary decomposer  Pleurotus, otherwise known as oyster mushrooms, rank second in the world mushroom market and are the most popular in China [Pleurotus ostreatus (Jacq.). P. Kumm. 1871 is particularly treasured compared with other species of the same genus because of its delicious taste, richness in proteins, carbohydrates, minerals  and vitamins . It is low in fat [Mushrooms of the genus in China . Pleurotw in fat  and contw in fat . The musw in fat  and calow in fat .P. ostreatus has considerable economic value because of its flexible nature allowing it to grow on a variety of agricultural wastes [Moreover, l wastes  due to il wastes , 8. It il wastes  that reql wastes .P. ostreatus [Olea europaea) is the most economically important oil-producing crop in many Mediterranean countries [P. ostreatus mushroom and therefore to test their effect on mushroom production and nutritional value.As mushrooms grow by bioconversion of agricultural wastes , their nstreatus , althougstreatus  and olivstreatus  were suistreatus , especiastreatus , and depstreatus . Nowadaystreatus . Immensestreatus , 26. Oliountries . From 75ountries . Olive tountries , generatThe experiment investigated the effect of four substrates on mushroom production and nutritional composition. In particular, the first substrate was wheat straw (WS) or control substrate, the second was olive pruning residues (OLPR), and the third and fourth substrates were a mixture of WS and OLPR, done on volume basis: WS : OLPR 1 : 3 (v/v) and WS : OLPR 3 : 1 (v/v). A complete randomized design was adopted with four treatments (substrates) and ten replicates (10 bags) per treatment.Wheat straw and olive pruning residues (OLPR) were procured from a local private enterprise  in fresh state. Olive pruning residues consisted of a mixture of thin woody sticks and leaves left after pruning, fermented outdoor for one year before use. Both tested substrates were sun-dried for two days prior to use. They were then pasteurized at 60–65°C with boiling water for eight hours and cooled down to the spawning temperature (25°C) .P. ostreatus (strain M 2175) was prepared in glass jars at the laboratory of Food Technology of the Faculty of Agricultural Engineering and Veterinary Medicine. Spawn was added with a rate of 5% w/w to the substrate and 50 g of gypsum CaCO3 (2% w/w in terms of dry weight) was added in order to adjust the substrate pH. Inoculated substrates were filled into perforated transparent polyethylene bags (60 cm length × 40 cm width). Holes were evenly made on the sides of the bags, which were placed in a cropping chamber in dark conditions at 23–25°C and 85–90% relative humidity until complete mycelial colonization. Fourteen days after incubation, the stage of pinhead formation was then stimulated by lighting, reduction of the room temperature to around 16°C, and ventilation to keep CO2 levels below 900–2300 ppm.Cereal grain spawn of Prior to filling, squares of 5 × 5 cm were drawn on each bag. The time to full mycelial colonization (100% MC) was recorded when all squares became white. The time to harvest of first flush (HF1) was determined as number of days after spawning (DAS). For every treatment, the number of mushroom flushes, biological yield (g/bag), and economic yield (g/bag) were assessed. Economic yield (g/bag) corresponded to the total weight of effective fruit bodies after removal of the base of stalks . In addiUsing ten representative samples selected per replicate (bag) relative to each treatment, physical characteristics of mushrooms were evaluated by measuring mushroom weight (g), pileus diameter (cm), stipe diameter (cm), and stipe length (cm) and by determining the ratio of pileus diameter over stipe length (PD/SL) as a qualitative indicator.Analyzed substrate properties , includeN × 6.25) [Mineral composition of substrates  was analyzed using atomic absorption spectrophotometry following AOAC  standard × 6.25) . Fat con × 6.25) . An enzy × 6.25) . Total c × 6.25) . Fatty a × 6.25) . All tes × 6.25) , 37.N × 4.38) [Mushroom composition was analyzed using fresh samples of mushroom pileus. Mushroom samples were analyzed for fat and crude fiber contents using the AOAC  procedur × 4.38) . Mineral × 4.38) ). Total Using the program Statistical Package for Social Sciences SPSS 25®, one-way ANOVA and Duncan tests were applied for data analysis. Pearson's correlations were established between substrates and mushroom composition. A simple regression was performed to test the relation between organic matter loss (dependent variable) and OLPR proportion in the substrate (predictors). Additionally, partial least squares regression (PLSR) was applied for multivariate analysis, using XLSTAT statistical and data analysis solution, Addinsoft 2019, Boston, MA, USA. The Jackknife (LOO) test was used for cross-validation of the resulting models. Confidence levels of 95% or 99% were adopted for statistical tests.p < 0.05) significant on averages of biological yield, number of flushes, and time to harvest the first flush, while it was not significant on time to complete substrate colonization, biological efficiency, and mushroom weight.Results of one-way ANOVA   showed tThe substrate OLPR was not completely colonized by the mycelium; therefore, it was not productive, and it was excluded from the results part. In comparison with control, mushroom production was hastened by 3.7 days and delayed by 11.3 days in the substrates WS : OLPR 1 : 3 (v/v) and WS : OLPR 3 : 1 (v/v), respectively. In addition, there was a significant reduction in the average number of flushes in substrates containing OLPR. Though in the substrate WS : OLPR 3 : 1 (v/v), averages of biological yield, biological efficiency, and economic yield were comparable to control, and these indicators were significantly reduced in the substrate WS : OLPR 1 : 3 (v/v).r2 = 0.85) between the proportion of OLPR and organic matter loss in the substrates, which reflects a gradual decrease in means of the latter with increasing values of the former.Moreover, results of the regression analysis  showed aThe three lignocellulosic components , cellulop=0.46), stipe diameter (p=0.48), stipe length (p=0.24), and PD/SL ratio (p=0.47). Stipe diameter ranged between 0.8 and 1.0 cm, pileus diameter between 6.4 and 7.3 cm, stipe length between 4.5 and 5.4 cm, and PD/SL ratio between 1.3 and 1.6 , crude fiber (r = –0.81), carbohydrates (r = –0.99), total soluble sugars (r = –0.97), and glucose (r = –0.91).Results in pvalue = 0.05 in terms of potassium (r = –0.73) and a strong positive interrelationship at pvalue = 0.01 in terms of sodium (r = 0.81).Analysis of mineral composition  showed tThe analysis of the fatty acid profile of substrates  showed tX-matrix) and mushroom fatty acids as dependent ones (represented by the Y-matrix). The coefficient r2 between Y and  gives an upper bound of how well the model explains the data and predicts new observations. The cross-validation q2 cum defines the stability of the model and sets the lower bound of how well the model explains the data. In addition, the root-mean-squared error (RMSE) measures the difference between observed and predicted values of a model. It was suggested that a smaller value of RMSE indicates that the model fits better the experimental data [RMSE was low for the built model  analysis was used to define the possible interrelationships between chemical composition of substrates  as independent variables  and Y variables between the inner and outer ellipses (X variable(s) to variation of relative Y variable(s).Correlations on axes ellipses  indicateellipses  were calThe predictive model  delineatOyster mushroom could help managing the disposal of agrowastes , especiaThe substrate WS : OLPR 1 : 3 has hastened the fruit formation by 3.7 d in comparison with control, compared with a hastening by 3.4 d with WS : OLPR 3 : 1 substrate as reported by Koutrotsios et al. . A similP. ostreatus in comparison with that obtained in the commercial control of the current study. Mushrooms obtained in all treatments were marketable showing a high PD/SL ratio. They had lower weight, comparable pileus diameter, and higher stipe length than those reported by Tesfay et al. [Moreover, the biological efficiency obtained in wheat straw in the current study was higher than that reported in the earlier works , 55, 56.y et al. . In addiy et al. . SmallerP. ostreatus.The incorporation of olive pruning residues in the growing substrate has ameliorated the mushroom nutritional value. The use of WS : OLPR 1 : 3 has caused an increase in protein, carbohydrates, crude fiber, and iron contents in mushrooms, while that of WS : OLPR 3 : 1 (v/v) and WS : OLPR 1 : 3 (v/v) substrates has caused a decrease in fat and sodium contents. The latter had lower moisture, carbohydrates, and crude fiber contents than those in other early studies on various substrate types , 58. TheDiets rich in fiber decrease the incidence of several diseases , and lowFurthermore, as the intake and synthesis of fatty acids from the substrate were not addressed in the literature, mushrooms may have obtained the palmitoleic acid from the wheat straw substrate; for this, fatty acid was not initially present in the substrates containing OLPR. Mushrooms may synthesize the myristic acid indirectly from substrates, as it was initially absent in all substrates types but detected in mushrooms produced in substrates containing OLPR. The strong correlation between oleic acid content in mushrooms with that of substrates suggests a direct uptake of this fatty acid, discarding the mushroom's need to synthesize it or to obtain it through conversion reactions. Moreover, the presence of arachidic acid in substrates containing OLPR and its absence in mushrooms may suggest its complete conversion into other fatty acids by the oyster mushroom.The valorization of OLPR by their bioconversion into a nutritious food has been evidenced in the present study. Farmers may benefit from their use in low proportion by producing enough yields from a lower number of harvests. The main advantage of using this underestimated agricultural by-product is in the improvement of the nutritional value of mushrooms. Results suggest the potential use of OLPR as a nutritious supplement for the commercial wheat straw substrate.


To investigate the inflammatory factors and lymphocyte subsets which play an important role in the course of severe coronavirus disease 2019 (COVID‐19). A total of 27 patients with severe COVID‐19 who were admitted to Tongji Hospital in Wuhan from 1 to 21 February 2020 were recruited to the study. The characteristics of interleukin‐1β (IL‐1β), IL‐2 receptor (IL‐2R), IL‐6, IL‐8, IL‐10, tumor necrosis factor‐α (TNF)‐α, C‐reactive protein (CRP), serum ferritin and procalcitonin (PCT), and lymphocyte subsets of these patients were retrospectively compared before and after treatment. Before treatment, there was no significant difference in most inflammatory factors  between male and female patients. Levels of IL‐2R, IL‐6, TNF‐α, and CRP decreased significantly after treatment, followed by IL‐8, IL‐10, and PCT. Serum ferritin was increased in all patients before treatment but did not decrease significantly after treatment. IL‐1β was normal in most patients before treatment. Lymphopenia was common among these patients with severe COVID‐19. Analysis of lymphocyte subsets showed that CD4+ and particularly CD8+ T lymphocytes increased significantly after treatment. However, B lymphocytes and natural killer cells showed no significant changes after treatment. A pro‐inflammatory response and decreased level of T lymphocytes were associated with severe COVID‐19. A pro‐inflammatory response, particularly the level of IL‐2R, IL‐6, TNF‐α and CRP, were associated with severe COVID‐19. The SARS‐CoV‐2 infection affect primarily T lymphocyte, particularly CD8+ T cells. In general, COVID‐19 is an acute resolved disease but it can also be deadly, with a 2.3% case fatality rate.+ and CD8+ T cells in the peripheral blood of a SARS‐CoV‐2‐infected patient is significantly reduced, whereas the status of CD4+ and CD8+ T cells are excessive activation.A cytokine storm is also associated with apoptosis of lymphocytes, leading to severe and transient lymphopenia.With increasing evidence on the key pathophysiological role of inflammatory factors in patients with COVID‐19, immunomodulatory agents including corticosteroids, tocilizumab, and lucitanib have been considered for use in clinics. However, more laboratory and clinical evidence for their use are needed.+ and particularly CD8+ T lymphocytes increased significantly after treatment. However, B lymphocytes and natural killer (NK) cells showed no significant change after treatment.In this study, the characteristics of several inflammatory factors , IL‐6, IL‐8, IL‐10, TNF‐α, CRP, serum ferritin, and procalcitonin [PCT]) and lymphocyte subsets of 27 patients with severe COVID‐19 patients were examined. We aimed to find appropriate targets for early intervention in patients with severe COVID‐19 by comparing relevant laboratory indicators before and after treatment. We found that levels of IL‐2R, IL‐6, TNF‐α, and CRP decreased significantly after corticosteroid therapy, followed by IL‐8, IL‐10, and PCT. Analysis of lymphocyte subsets showed that CD422.12 < 93% on room air, or PaO2/FiO2 ≤ 300 mm Hg, patients whose pulmonary imaging progresses greater than 50% in 24 to 48 hours were managed as severe pneumonia). Five patients had type 2 diabetes and eight had hypertension. Patients with a history of hematologic disease, autoimmune disease, or tumors were excluded from the study.We recruited 27 patients  who were confirmed to have COVID‐19 by Real‐time Fluorescent RT‐PCR Kit, and who had been admitted to Tongji Hospital from 1 to 21 February 2020. According to the Diagnosis and Treatment Protocol of New Coronavirus Pneumonia  from the National Health Commission of China,All of the patients received supplemental oxygen. All of the patients were given abidol  and five patients were given lopinavir/ritonavir  together as antiviral therapy. Moxifloxacin  was used to prevent secondary infection. Methylprednisolone  was administered to attenuate lung inflammation and was given for no more than 1 week. Blood laboratory tests and chest computed tomography were performed every 5 to 7 days. All 27 patients experienced improvements in their condition and as of 12 March 2020, eight male patients and nine female patients had been discharged from hospital. Blood samples of the patients were collected before treatment and after the condition improved significantly .2.2This study received ethical approval from the Medical Ethics Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. All participants gave written informed consent, and the study was carried out in accordance with the Declaration of Helsinki.2.3Levels of IL‐1β, IL‐2R, IL‐8, IL‐10, and TNF‐α were measured using the IMMULITE 1000 Immunoassay system . Levels of IL‐6 and PCT were determined by electrochemiluminescence immunoassay . CRP and serum ferritin were measured using latex‐enhanced immunoturbidimetry . All procedures were carried out according to the manufacturers’ instructions.2.4+CD4+CD8− T lymphocytes, CD3+CD4−CD8+ T lymphocytes, CD3−CD19+ B lymphocytes, and CD3−CD16+CD56+ lymphocytes among the total lymphocytes were obtained as follows: (a) 100 μL of whole blood was diluted with 400 μL of Iscove's modified Dulbecco's medium; (b) the cells were labeled with antibodies ; (c) the cells were fixed and permeabilized; and (d) the cells were analyzed using FACSCanto flow cytometer. The absolute numbers of different lymphocyte subsets were calculated by multiplying the percentages by the total lymphocyte count .The total number of lymphocytes in peripheral blood was counted using a hemocytometer. The percentages of CD32.5t test. A P < .05 was considered statistically significant.All statistical analyses were performed using SPSS 22.0 . Data are expressed as mean ± standard deviation and were compared using the independent‐samples 33.1The median age of male and female patients was 61 and 60 years, respectively. Before treatment, lymphocyte subsets and inflammatory factors were evaluated  of the 27 patients were compared before and after treatment  were decreased by 57.1% (12/21) of patients, and the minimum was 147/µL. CD8+ T lymphocytes  were decreased in 85.7% (18/21) of patients, and the minimum was 54/µL. NK cells  were decreased by 57.1% (12/21) of patients, and the minimum was 27/µL. Lymphopenia improved after treatment in these patients. Among the lymphocyte subsets, the CD8+ T lymphocytes showed the most significant improvement, followed by CD4+ T lymphocytes. Overall, T lymphocytes and total lymphocytes levels improved significantly. However, the changes in B lymphocytes and NK cells were not significant (Table Lymphopenia is very common in COVID‐19 and is associated with disease severity.y 57.1% 1/21 of pa4The COVID‐19 outbreak is a major challenge for clinicians. The disease pathogenesis remains to be fully characterized, and no pharmacologic therapies of proven efficacy yet exist. The immune responses plays important roles in controlling respiratory virus infections.+ T cell function,Higher levels of proinflammatory cytokines have been associated with lung damage.CRP plays an important role in innate immunity as an early defense mechanism against infections. Another inflammatory plasma marker that is extensively used in clinical practice is the ferritin. Unlike many bacterial infections, viral infections are commonly characterized by increased circulating ferritin concentrations.+ and particularly CD8+ T lymphocyte subtypes were reduced in patients with severe COVID‐19, which is consistent with the general characteristics of viral pneumonia+ T cells, provide broader and more lasting cross‐reactive cellular immunity with fewer limitations of strain‐specific restriction.+ and CD8+ T cell counts in the peripheral blood samples of patients with severe COVID‐19. In general, a significantly negative correlation has been reported between the number and function of both CD4+ and CD8+ T cells.In our study, CD4+ T cells were higher with better immune response than in male patients with COVID‐19.It had been reported that COVID‐19 mostly affected men because immune genes are more expressed on the X chromosome. Female patients had lower level of inflammatory factors, while CD4Although studies have provided evidence that cytokine storms and immunopathology can occur during pathogenic human coronaviruses infections,Some limitations of this study should also be acknowledged. This was a retrospective, single‐center, observational study, and unavoidable biases occurred when including participants. Furthermore, the sample size was very small. Despite these limitations, the study reflects the “real life” clinical situation.+ T cells. The lymphocytes function in patients with severe COVID‐19 need to be further clarified.In conclusion, a proinflammatory response, particularly the level of IL‐2R, IL‐6, TNF‐α, and CRP, were associated with severe COVID‐19. The SARS‐CoV‐2 infection affect primarily T lymphocyte, particularly CD8The authors declare that there are no conflict of interests.MN researched literature and wrote the first draft of the manuscript. F‐BT and D‐DX collected the patients’ clinical data. BY designed the investigation, and reviewed and modified the manuscript. All authors have read and approved the final version of the manuscript.This study received ethical approval from the Medical Ethics Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. Written informed consent for publication of these clinical details was obtained from each patient. Copies of the consent form are available for review by the editor of this journal.


To evaluate the influence of automated visual field (VF) testing on intraocular pressure (IOP) in patients with ocular hypertension (OHT) or glaucoma.We conducted a prospective observational study in the glaucoma department at Quinze-Vingts National Ophthalmology Hospital in Paris. Ninety-five right eyes of 95 patients followed for glaucoma or OHT were included. IOP was measured three times using a Nidek NT-510 non-contact tonometer within a maximum of 5 min before and after VF testing. Sub analyses using logistic regression analysis were performed to evaluate the impact of gender, age, central corneal thickness (CCT), mean deviation (MD) of the VF, VF test duration and filtration surgery on IOP fluctuations.P = 0.4). The average change in IOP was 0.15 ± 1.82 mmHg. Using multivariate analysis, no effect of the VF test on IOP was found , whether based on duration of the VF test (P = 0.18) or the MD (P = 0.7) after adjustment for age, gender, CCT and history of glaucoma surgery. Similarly, there was no significant difference within different types of glaucoma, including open-angle glaucoma (P = 0.36), chronic angle closure glaucoma (P = 0.85) and OHT (P = 0.42). The subgroup of patients with an IOP elevation ≥2 mmHg had a significantly higher VF test duration (P = 0.002).There was no significant change in IOP after VF testing, with IOP’s 15.14 ± 4.00 mmHg before and 14.98 ± 3.33 mmHg after the VF (VF testing does not influence IOP as measured with a non-contact tonometer. Visual field (VF) testing and intraocular pressure (IOP) measurement are essentials for the diagnosis, monitoring and treatment of glaucoma , 2. IOP , in 52 eyes of 27 patients with POAG, Martin et al. [, in 71 eyes of 45 patients, found no impact of VF testing on IOP. Most of these studies used both eyes of the same patients or were retrospective. Moreover, no clear mechanism explaining this IOP rise following VF testing has been found. Several hypotheses have been suggested, including the examination in a dark room inducing a persistent mydriasis or a psychic stress leading to a sympathetic response that transiently elevates IOP [Some studies have already investigated these possible IOP changes following VF testing, but with discordant results. Recupero et al.  concluden et al. , in 61 pn et al. , in 71 eates IOP , 16–18. We performed a prospective observational study in the glaucoma department of Quinze-Vingts National Ophthalmology Hospital in Paris. All patients were informed of the purpose of the study, and their consent was obtained in accordance with the declaration of Helsinki and approved by our CPP-Ile-de-France Ethical Committee (number 10793). Patients selected for inclusion had been followed for over 1 year for glaucoma or OHT and were over 18 years of age. They were required to have undergone at least three VF tests prior to inclusion. Exclusion criteria were difficulty in IOP measurement and any corneal pathology that might influence non-contact IOP measurement.All VF tests and IOP measurements were performed by the same examiner with the same devices. VF analysis was performed with the Humphrey visual field analyser (HFA) 24–2 or 10–2 Swedish Interactive Threshold Algorithm standard program, or the OCTOPUS 101 G2 program and C08 program, or the Frequency doubling technology (FDT) matrix 24–2 pattern on undilated eyes. VF testing always started with the right eye. All VF tests were performed in the same department and under the same condition of illumination in a dark room. During the Humphrey and Octopus visual field tests, the near prescription lens was set as needed, and the fellow eye was occluded with a black patch. For the FDT matrix, the patients were measured with their distance correction if the spherical equivalent was outside of the range from − 3.00 to + 3.00 and no correction if inside this range. We ensured that patients were seated comfortably and perfectly understood the test. Mean deviation (MD) of HFA 24–2 or Octopus G2 were used for statistical analysis when patients had HFA 24–2 + HFA 10–2, Octopus G2 + Octopus C08 or HFA 24–2 + FDT performed during the same consultation. If patient had only a HFA 10–2 performed, the MD of this VF was used. The MD of FDT matrix was not used for MD analysis. IOP was measured within a maximum of 5 min before and after VF testing by the same Nidek NT-510 non-contact tonometer  which have a test-retest variability of 6.4% .At each time point, 3 consecutive measurements were taken to obtain a mean IOP value. We selected measurements only of right eyes to minimize intraindividual variations.The change in IOP from immediately before to immediately after the visual field test was analysed with the paired t-test to account for intra-patient correlation. Multivariate linear mixed models were used to analyse IOP change adjusted for age, gender, central corneal thickness (CCT), visual field test duration, MD of visual field, and history of glaucoma surgery. Sub analyses of patients with IOP elevation or decrease ≥2 mmHg was done as it was in most studies concerning IOP variation after VF test , 13, 14.P = 0.4, paired t-test)  . Baseline characteristics are presented in Table 02 Table . Patient2 = 0.12) whether based on the duration of the VF test (P = 0.18) or the MD (P = 0.7) after adjustment for age, gender, CCT and history of glaucoma surgery  or the subgroups of patients with chronic angle closure glaucoma (P = 0.85) or OHT (P = 0.42).On multivariate analysis, no impact of the VF test on IOP was found , whether based on the duration of the VF test (P = 0.18) or the MD (P = 0.7) after adjustment for age, gender, CCT and history of glaucoma surgery, analysing all patients. Patients with a ≥ 2 mmHg IOP elevation had a mean VF test duration of 12.8 ± 2.6 min, which was a longer test duration than for other subjects  in 94 glaucomatous eyes [P < 0.001) and following visits without VF examinations . They explain the rise in IOP by a stressful perception of VF testing by the patients, leading to a sympathetic response that transiently elevates IOP [In the literature, several studies have already studied this subject and have led to conflicting results. Two studies showed an impact of VF testing on IOP. Recupero et al. reported a significant mean increase in IOP after automated VF testing (ous eyes . The meaous eyes , 20. Simous eyes  reportedates IOP . NeverthConversely, three studies showed no differences. Rebolleda et al.  studied Our study confirms that there is no effect of visual field testing on IOP. One may therefore continue to schedule visual fields just prior to the consultation in clinical practice when preferable, which facilitates the coordination of care for the doctor and patient and helps to ensure proper compliance with patient follow-up.


Tree spatial patterns in dry coniferous forests of the western United States, and analogous ecosystems globally, were historically aggregated, comprising a mixture of single trees and groups of trees. Modern forests, in contrast, are generally more homogeneous and overstocked than their historical counterparts. As these modern forests lack regular fire, pattern formation and maintenance is generally attributed to fire. Accordingly, fires in modern forests may not yield historically analogous patterns. However, direct observations on how selective tree mortality among pre‐existing forest structure shapes tree spatial patterns is limited. In this study, we (a) simulated fires in historical and contemporary counterpart plots in a Sierra Nevadan mixed‐conifer forest, (b) estimated tree mortality, and (c) examined tree spatial patterns of live trees before and after fire, and of fire‐killed trees. Tree mortality in the historical period was clustered and density‐dependent, because trees were aggregated and segregated by tree size before fire. Thus, fires maintained an aggregated distribution of tree groups. Tree mortality in the contemporary period was widespread, except for dispersed large trees, because most trees were a part of large, interconnected tree groups. Thus, postfire tree patterns were more uniform and devoid of moderately sized tree groups. Postfire tree patterns in the historical period, unlike the contemporary period, were within the historical range of variability identified for the western United States. This divergence suggests that decades of forest dynamics without significant disturbances have altered the historical means of pyric pattern formation. Our results suggest that ecological silvicultural treatments, such as forest restoration thinnings, which emulate qualities of historical forests may facilitate the reintroduction of fire as a means to reinforce forest structural heterogeneity. Fire effects on the spatial arrangement of trees have shifted dramatically in a mixed conifer forest over the course of 80 years. In 1929, fires maintained the clustered, patchy distribution of trees, lowering the average size of tree groups. In 2008, severe fires spread throughout the homogeneous forest canopy, yielding few and scattered trees. These fires mediated heterogeneous, uneven‐aged forest structures at fine scales through partial and periodic tree mortality, stimulating fire‐adapted understory plants, and creating temporally and spatially variable conditions for tree regeneration  forest plots that were stem‐mapped immediately before harvesting in 1929, approximating forests before EuroAmerican settlement characteristics, and again in 2008, representative of contemporary long‐unburned forests with a history of logging. We used a physics‐based fire model to simulate fire spread in each of the two time periods and then estimated fire‐caused mortality based on species and tree size. We hypothesized that mortality would be clustered and density‐dependent in the historical period due to pre‐existing spatial variability typical of historical forests; we further hypothesized the residual forest structure would retain a mosaic of tree groups of diverse sizes. In contrast, we expected that mortality patterns would be more random and density‐independent because the 2008 counterpart plots were comparatively homogeneous with continuous canopy as opposed to discrete tree groups  of trees ≥ 9.1 cm dbh were recorded in 1929 prior to partial harvesting. The understory fuels were mapped into broad cover type patches . Stem mapping of trees ≥ 10 cm dbh in MC9 and MC10 occurred again in 2008. In 2007, we remapped only 3.4 ha in MC11 because a road had built through it. For brevity, we refer to the 2007 and 2008 measurements as the 2008 measurement period. Additional information on the history of these plots and prior research is provided in Hasel et al.  shrubs, and shrubs of other species, represented by standard surface fuel models timber‐litter 3 (TL3), timber‐litter 1 (TL1), grass‐shrub 2 (GS2), and shrub 2 (SH2), respectively. Because understory vegetation had almost entirely disappeared by 2008 as gaps in the forest filled with trees, we simulated those surface fuels as a homogenous layer of TL3.We simulated fire behavior with the Wildland‐urban interface Fire Dynamics Simulator. Using a computational fluid dynamics approach, numerical solutions are solved in a domain composed of discretized voxels over a series of time steps  from simulation results. We used these results to estimate mortality following Parsons et al. ; we appl2.3α = 0.05. Point pattern analyses were conducted in Programita ver. 2018  here, whose test statistic was the average tree dbh located r away from another tree. By comparing the empirical statistics to a null model of random labeling where dbh is randomly shuffled among tree locations, we could assess whether trees distanced r away from another tree were smaller or larger than the mean aspatial tree dbh. In addition, we used a mark variogram, γdbh(r), to assess whether tree sizes were spatially correlated. The test function 12(dbhi‐dbhj)2 described the semivariance in tree dbh between two trees, i and j, located r distance apart. We compared the empirical statistics to a null model of random labeling to determine whether dbh between trees located r distance apart were more or less variable than expected by chance.We used mark correlation functions to describe the spatial structure of tree sizes, aiding interpretation of fire effects. Mark correlation functions yield statistics of an appropriate test function averaged over all pairs of trees at distance 2.5gall(r), and after fire, galive(r). Univariate pair correlation functions measure the average number of points, that is, trees, at distance r from a point, normalized by dividing by the expected number of points. The empirical pair correlation functions are compared to a set of functions realized from a null model. In these analyses, we used an inhomogeneous Poisson point process model. This null model randomly distributed points using an intensity field parameterized by an Epanechnikov smoothing kernel at a bandwidth of 20 m and a resolution of 1 m. We chose an inhomogeneous over a homogeneous Poisson process to account for intensity gradients in the observed data. Any values above or below expectation reflected that trees were spatially distributed as aggregated or uniform patterns, respectively.The first pair of univariate pair correlation functions described the patterns of living trees, both before fire, r from a type 1 point, normalized by the density (points per area) of type 2 points ‐ galive,dead(r), concisely referred to here as gcluster(r); gdead,dead(r) measured the relative density of fire‐killed trees near fire‐killed trees and galive,dead(r), the relative density of fire‐killed trees near surviving trees. Thus, gcluster(r) estimated whether fire‐killed trees were more, less, or equally common around surviving trees than around other fire‐killed trees. The null expectation was 0, whereas higher values indicated clustering of mortality, and lower values indicated the dispersion of mortality. Second, we measured density dependence of mortality with the difference gdead,dead+alive(r)−galive,dead+alive(r). This compared the relative density of all trees near a dead tree minus the relative density of all trees near a surviving tree. The null expectation was 0; higher and lower values indicated a bias toward mortality in areas of higher or lower density, respectively. For the null models of gcluster(r) and gdens.dep(r), we randomly labeled points’ types, alive or dead, rather than moving points because the locations of survival or mortality are conditioned on the spatial pattern of trees before a fire  in 1929 versus 2008. Tree groups were identified following the approach whereby trees within a group are within a limiting distance, here 6 m, from another member tree  to over 94% of trees  in 2008.3.2Spatial and aspatial distributions of surviving and fire‐killed trees were markedly different across time periods. Whereas tree mortality and survival were arranged in a patchy mosaic in 1929, only scattered trees were estimated to survive in 2008 Figure . In bothz scores of gall(r), averaged across r, ranged from 5.4 to 7.6 across sites  ranged from 1.8 to 2.4 ; r‐averaged z: 3.1—9.6). Further, dead trees had more neighbors than surviving trees (gdens.dep.(r); Figure r‐averaged z: 3.1—6.4).Simulated fires markedly altered the spatial patterns of trees in 1929. Trees were initially aggregated; as a measure of aggregation magnitude, s Figure . Aggrega4 Figure . Mortali4 Figure , with clgall(r)r‐averaged z: 1.8—2.2; Figure galive(r) . The spatial distributions of the fire‐killed trees were less clustered , as measured by gcluster(r). These killed trees were also in locations of higher tree density (gdens.dep.(r); r‐averaged z: 3.1—3.3). Compared to 1929, the magnitude of all measures—tree aggregation before and after a fire, and clustering and density dependence of fire‐killed trees—were all lower 2008.Trees were less aggregated in 2008 . The coefficient of variation (CV) of prefire tree group size in 1929 versus 2008 differed in MC9 , but not in MC10 , nor in MC11 .Before fire, tree groups were more numerous and larger in 2008 than in 1929 Figure . In 1929p = .01) and MC11 (p < .01) but was not significantly different in MC9 (p = .07). Further, residual tree groups were less variable in size in 2008 (CV from 0.32 to 0.46) than 1929.After fire, tree groups were fewer in number, smaller in size, and less variably sized in 2008 than 1929 Figure . Plots iThe effect of fires on tree group size distributions differed greatly between time periods. In 1929, trees within larger groups were more likely to be killed Figure . For exa4Fine‐scale heterogeneity in forest structure is increasingly recognized as a salient characteristic of forests that historically experienced frequent fire  forests in China  preferentially attack moderate to larger individual trees. Since larger trees tend to be aggregated, as in our study, tree mortality patterns resulting from mountain pine beetle may appear as less density‐dependent. Adding further complexity, the impacts of these disturbances are also conditioned on the tree patterns resulting from preceding disturbances ; Formal analysis (lead); Methodology (lead); Resources ; Software ; Visualization (lead); Writing‐original draft (lead); Writing‐review & editing (lead). Chad Hoffman: Conceptualization ; Funding acquisition ; Investigation ; Methodology ; Resources ; Software ; Supervision ; Writing‐review & editing . Brandon Collins: Conceptualization (supporting); Data curation ; Funding acquisition ; Project administration ; Resources ; Supervision ; Writing‐review & editing . Eric Knapp: Data curation ; Writing‐review & editing . William Mell: Methodology ; Software ; Validation ; Writing‐review & editing .This work was partially supported by a research partnership between the US Forest Service Pacific Southwest Research Station and UC Berkeley College of Natural Resources (project no. 16‐JV‐11272167‐063), the US Forest Service Pacific Southwest Research Station and Colorado State University (project no. 17‐JV‐11272138‐074), and the US Forest Service Pacific Northwest Research Station and Colorado State University (project no. 19‐JV‐11261987‐085).AppendixS1Click here for additional data file.


Nicotinamide riboside (NR) is a NAD+ precursor vitamin and pterostilbene (PT) is potent sirtuin activator found in blueberries. Here, we tested the effect of combined NR and PT (NRPT) on whole blood NAD+ levels and safety parameters in patients with AKI.Preclinical studies have identified both NAD+ levels were measured by liquid chromatography-mass spectrometry and safety was assessed by history, physical exam, and clinical laboratory testing.We conducted a randomized, double-blind, placebo-controlled study of escalating doses of NRPT in 24 hospitalized patients with AKI. The study was comprised of four Steps during which NRPT (5 subjects) or placebo (1 subject) was given twice a day for 2 days. NRPT dosing was increased in each Step: Step 1250/50 mg, Step 2500/100 mg, Step 3750/150 mg and Step 41,000/200 mg. Blood NAD+ levels at 48 h compared to 0 h in patients receiving placebo (p = 0.05). There was a trend for increase in NAD+ levels in all NRPT Steps individually at 48 h compared to 0 h, but only the change in Step 2 reached statistical significance , and there was considerable interindividual variability in the NAD+ response to treatment. Considering all Steps together, NRPT treatment increased NAD+ levels by 37% at 48 h compared to 0 h (p = 0.002). All safety laboratory tests were unchanged by NRPT treatment, including creatinine, estimated glomerular filtration rate (eGFR), electrolytes, liver function tests, and blood counts. Three of 20 patients receiving NRPT reported minor gastrointestinal side effects.AKI resulted in a 50% reduction in whole blood NAD+ levels in hospitalized patients with AKI. In addition, NRPT up to a dose of 1000 mg/200 mg twice a day for 2 days is safe and well tolerated in these patients. Further studies to assess the potential therapeutic benefit of NRPT in AKI are warranted.NRPT increases whole blood NADNCT03176628, date of registration June 5th, 2017. Acute kidney injury (AKI) is common, growing in incidence, and associated with significant morbidity and mortality. AKI is most commonly diagnosed in patients following major invasive surgical procedures, in the setting of sepsis, or following administration of certain drugs or contrast dyes, particularly in patients with underlying hypertension, diabetes, or chronic kidney disease . There i+) is a key cellular factor linked to metabolism and longevity [+ levels in the kidney that results from a combination of decreased NAD+ biosynthesis and increased NAD+ consumption [+ augmentation by niacinamide has been shown to prevent various etiologies of experimental AKI in mice, and an initial pilot study has demonstrated the safety of niacinamide in patients undergoing cardiac surgery [+ and SIRT1 modulation as a potential therapeutic approach in AKI [Nicotinamide adenine dinucleotide  is a combination of nicotinamide riboside (NR), a form of vitamin B3, and pterostilbene (PT), a polyphenol found in blueberries . NR is icinamide . Throughged mice . PT is aged mice , that haged mice . Therefong SIRT1 . NR suppng SIRT1 , systeming SIRT1 , exercisng SIRT1 , and musng SIRT1 . Furthery 40–90% . To date+ levels following NRPT administration, with prespecified secondary goals to determine the dose of NRPT that safely achieves at least a 50% increase, and up to 100% increase, from baseline in whole blood NAD+ levels. During each Step, NRPT (5 subjects) or placebo (1 subject) was given twice a day for 2 days , the following schedule was followed: on Day 1 blood was collected 30 min before NRPT or placebo administration and then at 2 h, 4 h, 6 h, 12 h, 24 h, and 48 h. Safety assessment and laboratory tests were performed at 24 h and 48 h after the first dose. After completion of each Step, NADSafety assessment was performed by patient interviews, vital sign measurements, physical examination, and laboratory tests, including complete blood count and comprehensive metabolic panel. Assessment of adverse events included specific questioning about nausea, abdominal pain, vomiting, diarrhea, and rash. Adverse events were characterized as probably related, probably not related, or unknown and all adverse events were reviewed by a data safety monitoring board (DSMB).Basis and was re-packaged by the Massachusetts General Hospital (MGH) pharmacy in capsules identical to placebo. The MGH pharmacy performed randomization based on the allocation schedule with a 5:1 chance of being assigned to the NRPT group. Both patients and study staff were blinded to the treatment. The study was approved by the MGH Institutional Review Board (IRB) and was registered on ClinicalTrials.gov, identifier NCT03176628. The study was initiated on November 1st, 2017 and completed on September 11th, 2018.NRPT was supplied by Elysium Inc. in the form of Study subjects were selected from hospitalized patients at the MGH.Male or female hospitalized patient, age ≥ 18 years.Developed AKI during the hospitalization, defined by an increase in serum creatinine by ≥0.3 mg/dL within 48 h; or an increase in serum creatinine to ≥1.5 times baseline. Although urine output totals per 24 h were available for all patients, they were not recorded on an hourly basis as part of routine care, precluding the use of urine output as an inclusion parameter for AKI.Hemoglobin ≥10.0 g/dL3Absolute neutrophil count ≥1500/mm3Platelet count ≥100,000/mmTotal bilirubin ≤1.5 x upper limit of normalALT and AST ≤2.5 x ULNAdequate hematological and liver function, as assessed by the following laboratory requirements:Able to provide written informed consent in compliance with the Human Investigation Review Committee (IRB).Inclusion criteria:Exposure to any investigational agent within 30 days prior to enrollment.Known allergy to any of the study drugs or their excipients.Currently pregnant (confirmed with a positive serum pregnancy test) or nursing.Unstable or clinically significant concurrent medical condition, psychiatric illness or social situation that would, in the opinion of the investigator, jeopardize the safety of a subject and/or their compliance with the protocol.2 as determined using the Modification of Diet in Renal Disease equation) prior to current hospitalization.Baseline chronic kidney disease (CKD) stage 4–5  serum creatinine criteria: stage 1, 150–200%; stage 2, 200–300%; stage 3 > 300% increase from baseline. As AKI developed during the hospitalization for all participants, baseline creatinine was defined as the last creatinine value prior to AKI, i.e. during the same hospitalization. In addition, we recorded the most recent creatinine value available in the electronic medical record prior to the current admission.+ measurement was performed from blood samples as previously described [+ was measured by liquid chromatography-mass spectrometry (LC-MS) by Keystone Bioanalytical. In brief, samples were centrifuged and 0.11 mL of supernatant was mixed with 100 μL of 0.5 M PCA in water in HPLC vial. Fifty microliters of 5 μg/mL of 13C5-nicotinamide adenine dinucleotide in 0.5 M PCA  and 0.5 mL of 0.5 M PCA in water were added. Ten microliters were injected onto the LC-MS. NAD+ was quantitated based on d5-NAD+ as an internal standard. 0.5% formic acid in water was mobile phase A and 0.5% formic acid in acetonitrile was mobile phase B. The mass spectrometer was set to monitor for the transitions of 664.4 → 524.0 (NAD+) and 669.4 → 529.3  in a positive ion mode.NADescribed . In brie+ levels) and safety data results at the completion of each Step of the protocol. The investigators assessed and recorded any adverse event in detail including the date of onset, description, severity, time course, duration and outcome, relationship of the adverse event to study drug, an event diagnosis, if known, and any action(s) taken. All adverse events were reported to the DSMB and to the manufacturer of Basis.An independent DSMB comprised of 3 physicians reviewed efficacy data . Data from different Steps were analyzed separately and collectively for Steps 1–4 vs 0 h (baseline) and vs placebo. Adverse events were analyzed descriptively and characterized as probably related, probably not related, or unknown. Data are expressed as analyte or laboratory value mean ± standard deviation in the Tables and mean ± standard error of the mean in the Figures. The study adheres to CONSORT guidelines. Statistical comparison of means is performed by two-tailed unpaired Student’s t test or ANOVA depending on the number of groups analyzed. The null hypothesis was rejected at p < 0.05.All randomized patients were included in intention-to-treat analyses. NADOf 1090 hospitalized patients that were screened, 96 (8.8%) had AKI and 33 (3%) of these patients with AKI met the study’s inclusion criteria. Sixty-three patients with AKI were excluded because of malignancy, an increase in liver enzymes or total bilirubin, or decrease in platelets. Of the 33 patients who met the inclusion criteria, 24 agreed to participate in the study across a total of four Steps . The highest mean increase in NAD+ was achieved in Step 3 , but this increase was not statistically significant. As shown, the change in whole blood NAD+ levels following NRPT administration demonstrated significant inter-subject variability. When all NPRT Steps were combined, NRPT treatment demonstrated a significant increase in whole blood NAD+ levels at 48 h compared to baseline . No significant change in whole blood NAD+ levels was noted following NRPT treatment at earlier time points. Notably, whole blood NAD+ levels in patients with AKI receiving placebo decreased by 50% at 48 h .Compared to the baseline timepoint, NRPT at all doses increased whole blood NAD8 h Fig. a, but onNRPT was safe in all Steps in patients with AKI. Compared to placebo, there was no significant difference in creatinine and eGFR in patients treated with NRPT at 48 h Fig. .Fig. 4RNRPT was safe at all doses with only minor side effects reported that resolved without intervention Table . One pat+ levels and action as a novel therapeutic approach in AKI [The incidence of AKI in hospitalized patients is about 1.6% with mortality rate of up to 35% . AKI is h in AKI , 9.+ levels in human AKI, with or without intervention. Across all treated individuals, NRPT treatment resulted in a significant increase in whole blood NAD+ at 48 h. In placebo treated individuals, AKI resulted in a 50% decrease in whole blood NAD+ levels at this time point. NRPT at all four tested doses was safe and well tolerated. These results motivate additional studies of NRPT as a potential therapeutic option in AKI.To our knowledge this is the first study describing whole blood NAD+ levels. The only dose of NRPT that resulted in a statistically significant increase in whole blood NAD+ levels at 48 h as compared to 0 h was 500 mg/100 mg in Step 2, yielding a 47% increase. The Step 3 dose resulted in a 67% increase in whole blood NAD+ levels that was not statistically significant. Thus in retrospect, the initial goal of a 50 to 100% increase in NAD+ levels was likely set too high, in large part because it was based on studies in healthy volunteers [+ levels with kidney injury (50% decrease after 2 days of AKI).Our prespecified secondary endpoint was to determine the dose of NRPT that safely achieves at least a 50% increase (and up to a 100% increase) from baseline in cellular NADlunteers  without + levels after 24 h  and no effect during earlier time points  [+ at 24 h vs 0 h was 46% for both 300 mg and 1000 mg of NR and only 12% for 100 mg of NR. NAD+ was not measured beyond 24 h in this study [+ levels , but with delayed effect to 48 h. This could represent slower incline in cellular NAD+ in patients with AKI or the effect of PT. We have previously tested the combination of NR and PT (NRPT 250 mg/50 mg and 500 mg/100 mg) in 120 elderly healthy volunteers, with the focus on NAD+ levels at later time points, namely after 30 and 60 days of treatment [+ levels in the healthy elderly population increased by 40% in NRPT 250/50 mg and 90% in NRPT 500/100 mg treatment groups and the effect was sustained until 60 days [+ levels by 23 and 47% respectively at 48 h. It is possible that the increase in NAD+ values would have been greater with more prolonged dosing and at later time points.NR alone was previously tested at similar doses  in a study of 12 healthy volunteers, resulting in a significant increase in peripheral blood mononuclear cell NADand 8 h) . The relis study . We now Reduced kidney function often leads to drug toxicity because almost half of all medications used are eliminated via the kidney . NRPT wa+ levels could be further impacted by dietary or inflammatory factors that accompany AKI in more acute settings, as with major cardiac surgery or sepsis. Additionally, because of our enrollment strategy, NRPT was administered relatively late during AKI; after being identified as having an increase in serum creatinine within 48 h, patients had an additional day prior to starting treatment to allow for the consent process, randomization and study drug blinding by pharmacy. Third, NAD+ was measured in whole blood, which may not accurately reflect kidney tissue NAD+ levels, and PT was not measured.Several limitations warrant mention. First, inherent in a pilot, dose-ranging trial, this study was of modest sample size and treatment duration. There was no control of timing of discharge from the hospital, so two out of four placebo patients were discharged prior to completion of the study, limiting the power of our comparisons of treatment versus placebo. Second, the study focused on individuals who could provide written informed consent after the onset of AKI. As a result, we enrolled subjects with mild AKI due to a range of non-life threatening etiologies. We acknowledge that the impact of NRPT on blood NAD+ levels among patients with mild AKI. However, there is considerable interindividual variability in the NAD+ response to NRPT and the increase occurs at 48 h, suggesting strategies with earlier administration should be considered in future studies. NRPT is safe and well tolerated at all doses in patients with AKI. Further phase II studies are needed to evaluate the efficacy of NRPT on renal outcomes, including individuals with more severe injury and a broader range of AKI etiologies.NRPT increases whole blood NAD


Programmed cell death ligand 1 (PD‐L1) immunohistochemistry is used to determine which patients with advanced non‐small‐cell lung cancer (NSCLC) respond best to treatment with PD‐L1 inhibitors. For each inhibitor, a unique immunohistochemical assay was developed. This systematic review gives an up‐to‐date insight into the comparability of standardised immunohistochemical assays and laboratory‐developed tests (LDTs), focusing specifically on tumour cell (TC) staining and scoring. A systematic search was performed identifying publications that assessed interassay, interobserver and/or interlaboratory concordance of PD‐L1 assays and LDTs in tissue of NSCLC patients. Of 4294 publications identified through the systematic search, 27 fulfilled the inclusion criteria and were of sufficient methodological quality. Studies assessing interassay concordance found high agreement between assays 22C3, 28‐8 and SP263 and properly validated LDTs, and lower concordance for comparisons involving SP142. A decrease in concordance, however, is seen with use of cut‐offs, which hampers interchangeability of PD‐L1 immunohistochemistry assays and LDTs. Studies assessing interobserver concordance found high agreement for all assays and LDTs, but lower agreement with use of a 1% cut‐off. This may be problematic in clinical practice, as discordance between pathologists at this cut‐off may result in some patients being denied valuable treatment options. Finally, five studies assessed interlaboratory concordance and found moderate to high agreement levels for various assays and LDTs. However, to assess the actual existence of interlaboratory variation in PD‐L1 testing and PD‐L1 positivity in clinical practice, studies using real‐world clinical pathology data are needed. Since the approval of the first immune check‐point inhibitor in 2011,For each immune check‐point inhibitor, however, a separate immunohistochemistry (IHC) PD‐L1 assay has been developed. The PD‐L1 IHC 22C3 PharmDx assay was used in clinical trials assessing efficacy of pembrolizumab, and is therefore Food and Drug Administration (FDA)‐approved and Conformité Européenne (CE)‐marked as a companion diagnostic for prescription of this drug.et al.,et al.et al.et al.,Using all these different assays to test for PD‐L1 expression in one pathology laboratory is not feasible. Not only would it be expensive and time‐consuming to run so many different tests for each patient, most laboratories will not have both staining platforms (i.e. Dako and Ventana/Roche) needed for these tests at their disposal. Furthermore, the number of tests that can be performed is restricted due to limited tissue availability.A systematic search of PubMed, Embase and Cochrane Library was performed, using the search terms ‘lung cancer’ and ‘PD‐L1’ with all relevant synonyms . Since PD‐L1 IHC was validated in histological specimens, studies examining cytological specimens only were excluded. Studies that did not perform adequate statistical analysis to compare assays  were also excluded. Only articles written in English and containing original published data were eligible for inclusion.Methodological quality of all articles remaining after full text reading were appraised by using a revised form of the Quality Assessment of Diagnostic Accuracy Studies 2 (QUADAS‐2) tool for assessing risk of bias.The following data were extracted from each study included after appraisal of methodological quality: first author’s name, year of publication, sample size, type of cancer of included patients, type of material used for PD‐L1 testing, type of standardised assay and/or LDT used for PD‐L1 testing, scoring method, cut‐off values, number of observers scoring PD‐L1, type of statistical analysis and results from comparison between assays, observers and/or laboratories. This review focuses on concordance of tumour cell (TC) staining and scoring, as treatment decisions for NSCLC patients are based on scoring of PD‐L1 expression on TCs in clinical practice. However, as scoring of PD‐L1 expression on immune cells (IC) could become relevant to clinical practice in the future, we also extracted data on concordance of IC staining and scoring and included this as Data The search in PubMed, Embase and Cochrane Library yielded 4294 unique hits after removal of duplicates , and classifying the cases by the dichotomous cut‐off criteria included in the scoring system resulted in higher concordance levels for all antibodies (Light’s κ range = 0.59–0.80). Other studies also assessed interobserver concordance for multiple cut‐offs, and many found concordance levels to be lower for the 1% cut‐off compared to the 50% cut‐offet al.Sixteen of the 27 included studies examined interobserver concordance  has only approved durvalumab as consolidation treatment in stage III NSCLC patients whose tumours show PD‐L1 expression of ≥1%.Finally, we assessed concordance of PD‐L1 IHC assays and LDTs between laboratories. Only a limited number of studies assessed this type of concordance, especially compared to the large number of studies assessing interassay and/or interobserver concordance. Most of the studies assessing interlaboratory concordance found high agreement for all standardised assays, while one study found lower agreement for LDTs.This systematic review has some limitations. Most importantly, there is significant heterogeneity between the studies included, especially in the choice of antibodies tested and the statistical methods used to analyse concordance. This prohibits pooling of data and complicates proper comparison of results between studies. Most studies, however, used similar samples for PD‐L1 testing, i.e. formalin‐fixed paraffin‐embedded material from tumour resections or biopsies from NSCLC patients. This supports comparability between studies. Conversely, this also provides a disadvantage: it only allows for comparison of PD‐L1 IHC assays and LDTs in histology, while in clinical practice PD‐L1 immunostaining is frequently performed on cytological specimens. Comparison of PD‐L1 IHC assays and LDTs in cytological NSCLC specimens falls beyond the scope of this review, but would be worth evaluation in a separate study. Finally, many of the included studies were not of high methodological quality, with only five studies being judged as having low risk of bias. Excluding the studies with the highest level of risk of bias, however, has improved the overall quality of this review.To conclude, this systematic review has shown that interassay concordance of TC staining is generally high between the standardised assays 22C3, 28‐8 and SP263 and properly developed and validated LDTs. Nevertheless, the use of clinically relevant cut‐offs may lead to lower levels of interassay concordance, indicating that these assays and LDTs cannot simply be interchanged. Interobserver agreement, moreover, is generally high for all assays and LDTs, but decreases with use of the 1% cut‐off. Lastly, interlaboratory concordance seems to be high for standardised assays and moderate for LDTs, but has not been studied sufficiently to draw definitive conclusions. Studies using real‐world clinical pathology data are necessary to assess whether use of different PD‐L1 IHC assays and LDTs, scoring by different pathologists and use of different pre‐analytical variables actually lead to differences in PD‐L1 positivity between laboratories in clinical practice.B. K. and S. W. received research grants from AstraZeneca, MSD and Roche Diagnostics. S. W. also received research grants from BMS, NextCure and Pfizer. M. H. received research grants from AstraZeneca, BMS, MSD, Pfizer, Roche and Roche Diagnostics. S. B. declares no conflicts of interest. None of the grant suppliers were involved in the study design, collection, analysis and interpretation of data, writing of the manuscript or the decision to submit the manuscript for publication.Table S1. Search syntax in PubMed, Embase and Cochrane Library.Table S2. Inclusion and exclusion criteria.Table S3. Quality assessment of included studies.Table S4. Study characteristics of all studies included for data extraction and analysis.Table S5. Results from studies assessing inter‐assay concordance of TC staining.Table S6. Results from studies assessing inter‐observer concordance of TC scoring.Table S7. Results from studies assessing inter‐laboratory concordance of TC staining.Table S8. Results from studies assessing inter‐assay and/or inter‐observer concordance of IC staining/scoring.Data S1. Supplementary results: concordance of IC staining and scoring.Click here for additional data file.


The uric acid metabolism pathway is more similar in primates and humans than in rodents. However, there are no reports of using primates to establish animal models of hyperuricaemia (HUA).To establish an animal model highly related to HUA in humans.n = 5/group). Blood samples were collected over 8 h, and serum uric acid (SUA) level was determined using commercial assay kits. XO and PNP expression in the liver and URAT1, OAT4 and ABCG2 expression in the kidneys were examined by qPCR and Western blotting to assess the effects of inosine on purine and uric acid metabolism. The validity of the acute HUA model was assessed using ulodesine, allopurinol and febuxostat.Inosine  was intraperitoneally administered to adult male rhesus monkeys (Inosine (200 mg/kg) effectively increased the SUA level in rhesus monkeys from 51.77 ± 14.48 at 0 h to 178.32 ± 14.47 μmol/L within 30 min and to peak levels (201.41 ± 42.73 μmol/L) at 1 h. PNP mRNA level was increased, whereas XO mRNA and protein levels in the liver were decreased by the inosine group compared with those in the control group. No changes in mRNA and protein levels of the renal uric acid transporter were observed. Ulodesine, allopurinol and febuxostat eliminated the inosine-induced elevation in SUA in tested monkeys.in vivo and explore the disease pathogenesis.An acute HUA animal model with high reproducibility was induced; it can be applied to evaluate new anti-HUA drugs  Hyperuricaemia (HUA) occurs because of purine metabolism aberrations and is typically characterized by increased uric acid formation or reduced uric acid excretion. HUA is defined as a serum urate concentration that allows urate saturation in bodily fluids (>7.0 mg/dL) (Roddy and Choi Uric acid production and metabolism are complex processes involving various factors that regulate uric acid production in the liver and reabsorption or excretion from the kidneys and gut (Mori and Percudani SLC22A12) is an important organic anion transporter that maintains uric acid homeostasis. It is located in the brush border of proximal tubular epithelial cells and mainly mediates the reabsorption of uric acid. Intracellular urate is released through glucose transporter 9 . A multiple specific anion transporter, organic anion transporter 4 (encoded by SLC22A11), is located in the apical membrane of epithelial cells and has been demonstrated to promote urate reabsorption , GLUT9 (SLC2A9), ABCG2 and OAT4 (SLC22A13).In this study, we induced HUA in the rhesus monkey, an Old-World non-human primate, via intraperitoneal injection of inosine, a key precursor in purine metabolism. The effects of three highly specific inhibitors of uric acid synthesis, ulodesine, allopurinol and febuxostat, were evaluated in this model, along with the levels of serum urate; hepatic Inosine was purchased from Sigma-Aldrich . Allopurinol was purchased from Nanjing Dolai Biological . Ulodesine (lot no. 20150727) and febuxostat (lot no. 2014-FB-0902) were procured from WuXi AppTec, WuXi, Shanghai, China). The uric acid assay kit was purchased from Nanjing Jiancheng Biological Engineering Institute , and the transcriptor first-strand cDNA synthesis kit  was obtained from Roche . Premix Taq™ (code no. RR003A) was purchased from Takara Biotechnology , and TriPure isolation reagent (lot # 94013920) was purchased from Roche . Ketamine hydrochloride (lot no. 161201) was purchased from Jiangsu Zhongmu Beikang Pharmaceutical . Antibodies against XO (ab109235-10000), PNP (ab109559), URAT1 , GLUT9 (Ab223470) and ABCG2 (Ab207732) were purchased from Abcam . The antibody against OAT4  was purchased from ABclonal Biotechnology . The RIPA lysis buffer and BCA protein concentration assay kit were purchased from Beyotime . The polyvinylidene fluoride membrane was purchased from Millipore , and all other chemicals were from Sigma-Aldrich .Animal welfare and experimental procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals . Washington (DC), National Academies Press (US). And all efforts were made to minimize animal suffering. In this study, to determine the changes in liver XO, PNP, and renal transporter mRNA and protein expression following inosine administration, liver and kidney tissue samples had to obtain. To minizine pain and distress, type-B ultrasound was used for obtaining the fresh tissue samples. After puncture, the needle, which was accurately positioned and not near any major blood vessels such as the portal vein or inferior vena cava, was rapidly withdrawn; hence, the animals recovered quickly with little to no pain and no postoperative bleeding or infection. During the study, no death had occurred, and all animals were fully recovered by the end of this experiment.g for 15 min at 10 °C; serum SUA level was determined using commercial assay kit.Monkeys were used as experimental animals to approach the optimal dose of inosine to induce highly repeatable acute HUA statuses in the test animals. The monkeys were randomly assigned to four groups of five animals each. The control group was intraperitoneally injected with 0.9% saline, whereas the other groups were intraperitoneally injected with inosine in 0.9% saline at doses of 75, 100 and 200 mg/kg. Blood samples were collected at 0, 0.5, 1, 2, 4 and 8 h after administration. Serum was separated by centrifuging the blood samples at 5000×g for 15 min at 10 °C, and then stored at −80 °C until use. Serum SUA concentration was determined using commercial kit, according to the manufacturer’s instruction.Monkeys were randomly assigned to four groups of five animals each. The control animals were intraperitoneally injected with 0.9% saline. Experimental groups were intraperitoneally injected with the following: 200 mg/kg inosine in 0.9% saline, 200 mg/kg inosine followed by 2 mg/kg ulodesine and 200 mg/kg inosine followed by 2 mg/kg febuxostat. Blood samples were collected 0, 0.5, 1, 2, 4 and 8 h after administration. Serum was separated by centrifuging the blood samples at 5000×Monkeys were randomly assigned to three groups of five animals each. The control animals were injected intraperitoneally with 0.9% saline. The experimental groups were intraperitoneally injected with the following: 200 mg/kg inosine in 0.9% saline and 200 mg/kg inosine followed by 2.5 mg/kg allopurinol. Blood samples were collected 0, 0.5, 1, 4 and 8 h after administration; the SUA level was determined as described above.Monkeys were randomly assigned to three groups of five animals each and exposed to inosine and allopurinol as described in the previous section. Approximately, 20 mg of the liver and kidney tissues (based on volume estimates) was obtained under B-mode ultrasound guidance at 1 h after inosine injection. Briefly, the animals were anaesthetized by intramuscular injection of 5–10 mg/kg ketamine hydrochloride. Their abdomens were then shaved and disinfected with iodophor. The liver and kidneys were located by scanning the intercostal area of the right upper quadrant or lateral waist subcostal abdomen using Biosound Esaote MyLab 30CV  fitted with a 3.5-Hz transducer (Esaote) and fixed to a guide frame. Biopsies were obtained using a biopsy gun with the range set to 15 mm and positioned perpendicularly to the puncture target and away from large vessels such as the portal vein and inferior vena cava. After puncture, the needle was withdrawn quickly. The tissues were stored in liquid nitrogen until the extraction of total RNA and proteins.XO (GenBank: NM_000379.3), PNP (GenBank: NM_001193551), URAT1 (GenBank: AB738914.1), GLUT9 (GenBank: NM_020041.2), OAT4 (GenBank: NM_018484) and ABCG2 (GenBank: NM_001032919.1) were amplified by quantitative real-time PCR (qRT-PCR) on a CFX384 Touch™ Real-Time PCR Detection System . The polymerase chain reaction (PCR) conditions were as follows: initial denaturation for 5 min at 95 °C, and 40 cycles of 5 s at 95 °C and 30 s at 60 °C. Gene expression was quantified by the ΔΔCt method with CXF386 software , and normalized to GAPDH (GenBank: NM_001195426.1) as an internal control. Primers were designed using Primer 5.0 and synthesized by Takara Biotechnology   using 1 mL of TriPure . RNA integrity was evaluated by 1% agarose electrophoresis, and the samples were stored at −80 °C. Subsequently, 2 μg of RNA was reverse-transcribed using Transcriptor for Strand cDNA Synthesis , according to the manufacturer’s protocol. The obtained cDNA was stored at −20 °C. , Japan) .Protein samples were extracted from the renal cortex tissues and quantified using a BCA protein concentration assay kit. Protein samples were separated on a 10% SDS-PAGE separation gel and transferred onto a 0.45 μm polyvinylidene fluoride membrane. The membranes were blocked with 5% skim milk in TBST buffer  for 30 min and subsequently incubated with primary and secondary antibodies. Primary antibodies were as follows: rabbit monoclonal antibodies against XO , rabbit polyclonal antibodies against PNP , URAT1 , GLUT9  and OAT4 ; and mouse monoclonal antibodies against ABCG2  and β-actin . Reactivity was detected using an anti-rabbit IgG (whole molecule)-peroxidase antibody  or anti-mouse IgG (whole molecule)-peroxidase antibody . Immunoreactive bands were detected with ECL reagents, and the densitometry analysis of the immunoblot results was conducted using ImageJ software .p < 0.05.Data are reported as mean ± standard error of mean (SEM), and the groups were compared using GraphPad Prism 8 software  using the one-way analysis of variance followed by Tukey’s post hoc test for multiple comparisons. Differences were considered significant at p< 0.01). However, in the two lower inosine dose groups (75 and 100 mg/kg), stable elevation in the SUA level was not observed at the corresponding time points. Indeed, the injection of 100 and 75 mg/kg inosine did not significantly increase the SUA level at 0.5, 1, 2, 4 and 8 h after administration compared with the injection of saline (The SUA level increased rapidly within 30 min after injecting a high dose (200 mg/kg) of inosine, which peaked at 1 h, and then decreased gradually, returning to the baseline level within 4 h of inosine administration. The SUA level increased from 51.77 ± 14.48 μmol/L at 0 h to 178.32 ± 14.47, 201.41 ± 42.73 and 134.89 ± 30.1 μmol/L at 0.5, 1 and 2 h, respectively, after inosine administration, compared with that in the control monkeys  to a nearly undetectable level (p< 0.05) of animals in the treatment group compared with that in the control group    and 7.As a key precursor in purine metabolism, inosine is metabolized by PNP to hypoxanthine, which is subsequently metabolized by XO to xanthine, generating uric acid. Potassium oxonate, a selective competitive inhibitor of uricase, has been used previously to induce HUA in rodents for drug screening  intake of inosine in 30 male patients caused an increase in the SUA level, resulting in asymptomatic HUA. However, the SUA level returned to the normal level after the withdrawal of inosine Sun . Based oEpidemiological studies have shown that the level of blood uric acid in a population is related to the sex and age of individuals. The incidence of HUA in men is higher than that in women (Du et al. PNP mRNA level was increased by inosine compared with saline. The protein level of PNP was not significantly increased in monkeys after treatment. Furthermore, we observed that the XO mRNA and protein levels in the liver were decreased by inosine compared with saline in monkeys after treatment. It is known that XO is the target of allopurinol and febuxostat in lowering the SUA level. In this study, inosine significantly downregulated the expression of XO mRNA and protein. The mechanism underlying these effects of inosine requires further analysis. A possible underlying mechanism is the initially increased SUA inhibits the expression of XO, the key enzyme involved in UA formation to avoid excessive accumulation of SUA, the final product of purine metabolism. Uric acid production and metabolism are complex processes involving various factors that regulate uric acid production in the liver and reabsorption or excretion from the kidneys and gut.We demonstrated that inosine-treated rhesus monkeys exhibit symptoms of HUA. PNP and XO are key enzymes involved in the purine metabolism pathway. Our results showed that the Organisms can positively regulate their enzyme activities in response to the changes in the internal and external environments despite wide fluctuations in the external environment by changing the rate of biochemical reactions depending on their physiological needs (Rodwell and Kennelly However, HUA animal models without organ damage may prevent studies of chronic diseases. There are advantages and disadvantages to different animal models. Acute HUA is easily restored to baseline level after the withdrawal of stimulators, whereas persistent HUA has serious negative effects on several organs including the kidney, heart and vessels. When developing rational models, researchers should consider the experimental purposes and conditions. HUA animal models will be valuable for drug testing, as shown by the results showing that they respond to XO inhibitors and other drugs that the lower uric acid level or inhibit purine nucleoside phosphatases.in vivo of novel ani-HUA drugs targeting both PNP and XO prior moving to clinic trial, as well as a useful system for HUA pathophysiology studies.Our study suggested that the intraperitoneal injection of inosine would induce a reliable acute HUA symptom in rhesus monkeys, and the model described here appeared to be a rational platform to evaluate therapeutic activities


Interhemispheric interactions in stroke patients are frequently characterized by abnormalities, in terms of balance and inhibition. Previous results showed an impressive variability, mostly given to the instability of motor‐evoked potentials when evoked from the affected hemisphere. We aim to find reliable interhemispheric measures in stroke patients with a not‐evocable motor‐evoked potential from the affected hemisphere, by combining transcranial magnetic stimulation (TMS) and electroencephalography. Ninteen stroke patients  were studied for 6 months after a first‐ever stroke in the middle cerebral artery territory. Patients underwent four evaluations: clinical, cortical, corticospinal, and structural. To test the reliability of our measures, the evaluations were repeated after 3 weeks. To test the sensitivity, 14 age‐matched healthy controls were compared to stroke patients. In stroke patients, stimulation of the affected hemisphere did not result in any inhibition onto the unaffected. The stimulation of the unaffected hemisphere revealed a preservation of the inhibition mechanism onto the affected. This resulted in a remarkable interhemispheric imbalance, whereas this mechanism was steadily symmetric in healthy controls. This result was stable when cortical evaluation was repeated after 3 weeks. Importantly, patients with a better recovery of the affected hand strength were the ones with a more stable interhemispheric balance. Finally, we found an association between microstructural integrity of callosal fibers, suppression of interhemispheric TMS‐evoked activity and interhemispheric connectivity. We provide direct and sensitive cortical measures of interhemispheric imbalance in stroke patients. These measures offer a reliable means of distinguishing healthy and pathological interhemispheric dynamics. We used transcranial magnetic stimulation–electroencephalography (TMS–EEG) to characterize interhemispheric dynamics in stroke patients. We provide a direct evidence of interhemispheric imbalance in stroke patients. TMS–EEG offers a reliable assessment of cerebral state during evolution of stroke. AHaffected hemisphereCCcorpus callosumCSTcorticospinal tractEMGelectromyographyFAfractional anisotropyGPFTgrip pinch force testHChealthy controlsICAindependent component analysisICCintraclass correlation coefficient.IHBinterhemispheric balanceIHCohinterhemispheric coherenceIHIinterhemispheric inhibitionISPinterhemispheric signal propagationLHleft hemisphereM1primary motor cortexMDmean diffusivityMEPmotor‐evoked potentialMNIMontreal Neurological InstituteMRImagnetic resonance imagingNIHSSnational institute health stroke scaleRHright hemisphereRMTresting motor thresholdTMStranscranial magnetic stimulationUHunaffected hemisphere1Poststroke alterations in cerebral excitability affect the balance between the two hemispheres, the interactions of which are crucial in the execution of unilateral and bimanual movements , which takes advantage of the strengths of the two techniques. TMS–EEG allows to focally stimulate a precise cortical area and, at the same time, to monitor neural activity both in the stimulated area and in the interconnected networks with an excellent temporal resolution and a good spatial resolution  were enrolled in the study. Inclusion criteria were (a) first‐ever chronic ischemic stroke, that is, at least 6 months after the stroke event; (b) presence of hemiparesis due to an ischemic left or right subcortical or cortical lesion in the territory of the middle cerebral artery. Exclusion criteria were (a) history of seizures; (b) severe general impairment or concomitant disease; (c) patients older than 80 years; and (d) treatment with benzodiazepines, baclofen, and antidepressants. Table 2.2Clinical evaluation was made using the National Institutes of Health Stroke Scale (NIHSS) and the GPFT. The latter was performed with a digital hand‐held dynamometer . The patient was placed seated with the elbows resting on the table top, with a 90° flexion of elbow and neutral forearm , which produces a monophasic pulse of ∼80 μs length. The position of the coil on the scalp was defined as the M1 site in which TMS evoked the largest MEPs in the relaxed first dorsal interosseous (FDI) muscle of the hand contralateral to the stimulation. The coil was oriented tangentially to the scalp at about 45° angle away from the midline, thus inducing a posterior–anterior current in the brain. The intensity of stimulation for single‐pulse TMS was adjusted to evoke a 1 Mv MEP. To this aim, we first tested the resting motor threshold (RMT), defined as the lowest intensity that produced MEPs >50 μV in at least 5 out of 10 trials in the relaxed FDI of the right hand .2.42 stimulator with a 50 mm figure‐of‐eight coil (Magstim Company Limited), which produces a biphasic waveform with a pulse width of ∼0.1 ms. Coil positioning was the same used for corticospinal evaluation. Intensity of stimulation was set at 90% of the RMT. When RMT was not recordable in the AH due to lack of MEP response, TMS was set at the same value of the UH. Each session consisted of 80 TMS pulses applied at a random ISI of 2–4 s over M1 of both the hemispheres separately, that is, the UH and the AH. The order of stimulation of the two hemispheres was counterbalanced across patients. During the entire session, patients were seated on a dedicated, comfortable armchair in a soundproofed room. A TMS‐compatible DC amplifier  was used to record EEG activity from the scalp. The EEG was continuously recorded from 29 scalp sites positioned according to the 10–20 International System, using TMS‐compatible Ag/AgCl pellet electrodes mounted on an elastic cap. Additional electrodes were used as ground and reference. The ground electrode was positioned in AFz, while the reference was positioned on the tip of the nose. EEG signals were digitized at a sampling rate of 5 kHz. Skin/electrode impedance was maintained below 5 kΩ. Horizontal and vertical eye movements were detected by recording the electrooculogram to offline reject the trials with ocular artifacts.Analysis of cortical activity was performed with TMS–EEG. TMS was carried out using a Magstim RTMS–EEG data were analyzed offline with Brain Vision Analyzer (Brain Products GmbH) and EEGLAB toolbox running in a MATLAB environment . As a first step, data were segmented into epochs starting 1 s before the TMS pulse and ending 1 s after it. We first removed and then replaced data, using a cubic interpolation, from 1 ms before to 10 ms after the TMS pulse from each trial. Afterward, data were downsampled to 1,000 Hz and band‐pass filtered between 1 and 80 Hz (Butterworth zero phase filters). A 50 Hz notch filter was applied to reduce noise from electrical sources. Then, all the epochs were visually inspected and those with excessively noisy EEG were excluded from the analysis. Independent component analysis (INFOMAX‐ICA) was applied to the EEG signal to identify and remove components reflecting muscle activity, eye movements, blink‐related activity, and residual TMS‐related artifacts basing on previously established criteria  is computed as normalization of cross‐spectrum by the two auto‐spectra. For each frequency f, the coherence value is a real number between 0 and 1. Coherence values were then obtained by averaging the values over all the epochs for the alpha band (8–13 Hz), which was found as the natural frequency of M1, as revealed by a time/frequency decomposition based on a complex Morlet wavelet transform (cycles = 3.5). Finally, event‐related coherence With AH) and for the UH (IHCohUH).Therefore, a coherence increase in the frequency band during 2.53); (d) diffusion weighted spin‐echo echo planar imaging (twice‐refocused SE EPI) , collecting seven images with no diffusion weighting (b0) and 61 images with diffusion gradients applied in 61 non collinear directions (scan time: 11 min).Structural brain MRI was obtained in a single session using a head‐only 3.0 T scanner , equipped with a circularly polarized transmit‐receive coil. The acquisition protocol included the following sequences: (a) dual‐echo turbo spin echo ; (b) FLAIR ; (b) morphological 3D‐T1‐weighted magnetization‐prepared rapid acquisition gradient echo (MPRAGE)  and from CAMINO (www.camino.org.uk). After correction for eddy current induced distortions and involuntary motion , the diffusion tensor was estimated voxel‐wise . A lesion mask was created for each patient by assigning a value of 1 to every voxel corresponding to a lesion and a value of 0 elsewhere. T1‐weighted images were warped into the Montreal Neurological Institute space. The same transformation was applied to the corresponding lesion mask. A probabilistic lesion map, indicating the percentage of patients with a lesion in a given area, in the LH, was obtained by combining every patient's lesion masks.For each patient, lesions were outlined in the MPRAGE images, using a semi‐automated local threshold contouring software  the Huynh–Feldt ε correction was used. Pairwise comparisons were corrected by the Bonferroni method.All data were analyzed using SPSS version 22 . Prior to undergoing analysis of variance (ANOVA) procedures, normal distribution of clinical and neurophysiological data was assessed by means of Shapiro–Wilks' test. Level of significance was set at eas) and small detectable change (SDC)  for an n sample size as:SEMt‐test. To assess the sensitivity of ISP, IHB and IHCoh in distinguishing the group of healthy volunteers from stroke patients, we used intraclass correlation coefficient (ICC), which indicates how strongly units in the same group resemble each other. For the ICC calculation, we considered all the between‐subjects sources of variance and we computed it as:To assess whether test–retest reliability of these measures was different in stroke patients as compared with healthy volunteers, we compared SDC of the sample of patients with a sample of 20 healthy volunteers tested in a previous study of our group  and of the two‐hemisphere activity (IHB). Moreover, we explore linear relationships between ISP and IHCoh from the AH and UH stimulation with (a) FA of the CST afferent from the AH and UH and (b) FA of the splenium (Region V of the CC) given its specific role of this structure in the interhemispheric connection of motor .All the 19 patients were successfully screened with NIHSS (mean score 6.63 ± 0.80). Six patients showed a score between 1 and 4 (mean score 3.5 ± 0.34), indicating a “minor stroke” severity; the other 13 patients showed a score between 5 and 11 (8.07 ± 0.45) indicating a “moderate stroke” severity. Eight patients were excluded from the evaluation with the GPFT due to excessive spasticity of the hand (MAS > 2). Analysis of the GPFT did not reveal any significant difference between the two experimental sessions  nor for the UH/RH . Analysis of the UH‐SICI showed a significant effect of ISI  = 14.355; p < .001; ε = .489). Post hoc analysis showed a lower corticospinal excitability at an ISI of 1 and 2 ms compared to ISIs at 5, 7, 10, and 15 ms ; a significant difference was observed also between the ISI between 3 and 7 ms (p = .003). Analysis of the UH‐LICI did not show any significant main effect nor interactions.Twelve patients did not have a sufficiently stable 1 mV MEP to test IHI, ICF/SICI, and LICI in the AH. Of these, nine patients did not show any evocable MEP; the other three patients showed sufficiently stable MEPs of about 50 μV thus we could assess RMT, although at a very high intensity value, respectively, 96, 89, and 87% of MSO  = 5.855; p = .022; ε = .159). Post hoc analysis revealed no differences between the activation of the two hemispheres in stroke patients (p = .073) nor in HC (p = .131). The second analysis aimed at testing differences in the interhemispheric dynamics revealed a group × stimulation × hemisphere interaction  = 14.009; p = .001; ε = .318). No main effect was observable of the MEP factor (p = .947) nor in interaction with other factors . Post hoc analysis comparing the two groups revealed a lower excitability in stroke patients, compared to HC, both when stimulating the AH  and when stimulating the UH . Post hoc analysis comparing the two hemispheres reactivity in HC, revealed a higher excitability over the stimulated hemisphere, compared to the contralateral one, as expected . In stroke patients, this difference was significant only when stimulating the UH  but not when stimulating the AH . When we considered only the patients with a clearly MEP evocable from the AH, the analysis confirmed the significant group × stimulation × hemisphere interaction  = 14.525; p = .001; ε = .398). Post hoc analysis confirmed that TMS‐evoked cortical activity was significantly inhibited stimulating both the hemispheres of HC  and when stimulating the UH of stroke patients with AH‐RMT (p = .027) but not when stimulating the AH (p = .945).Figure F = 8.242; p = .007; ε = .210). Post hoc analysis comparing the two groups, revealed a higher ISP  when stimulating the AH in stroke patients compared to HC ; while no difference was observable when stimulating the UH . When comparing the two stimulated hemispheres, stroke patients showed a higher ISP  when stimulating the AH compared to the UH (p = .001), whereas, as expected, HC did not show any significant difference between the two stimulations (p = .6).Figure F = 5.12; p = .015; ε = .142). Post hoc analysis revealed that IHB was significantly higher in stroke patients compared to HC (2.53 ± 2.45 vs. 1.18 ± 0.87). Analysis on IHCoh did not reveal any main effect of group or stimulus factors, nor any interaction. Analysis of test–retest reliability for stroke patients revealed the following SEMeas for ISPAH (0.249), ISPUH (0.187), IHB (0.87), IHCohAH (0.065), and IHCohUH (0.099) with the following SDCindiv necessary to exceed measurement noise for ISPAH (0.691), ISPUH (0.521), IHB (2.41), IHCohAH (0.181), and IHCohUH (0.274). Analysis of test–retest reliability for healthy volunteers revealed the following SDCindiv necessary to exceed measurement noise for ISPAH (0.288), ISPUH (0.287), and IHB (0.343) . Figure group for both groups. To reduce SDCgroup below 0.1 in stroke patients the following sample sizes would be needed: 50 for ISPAH, 35 for ISPUH and more than 60 for IHB. The same analysis conducted on healthy volunteers revealed that 10 subjects would be needed for ISP regardless the hemisphere of stimulation and 15 for IHB. Analysis of SDC comparison showed that a higher reliability of these measures in healthy volunteers compared to stroke patients . Analysis of ICC revealed a high reliability in distinguish between the two groups for IHB , ISPAH  and ISPUH , but not for IHCohAH  nor for IHCohUH .Figure 3.4Nine patients were able to complete the entire structural evaluation with MRI. Seven patients were excluded for the presence of cardiac pacemaker (three patients) or metal implants (four patients) the remaining three patients were not able to complete the entire scanning. Probabilistic lesion maps indicate the percentage of patients with lesion in a given brain area overlaid onto a T1‐weighted image in Montreal Neurological Institute space Figure . For a d3.5R = −.564; p = .035; second: R = −.782; p = .002) . Correlations conducted on structural data were significant only for FA of the CC in the posterior midbody section (pCC). Figure r = −.733; p = .012) but not after AH stimulation   but not after AH stimulation . No significant correlations were observed with FA of the CST  but not with the M1‐M1 spectral coherence .Correlation conducted on clinical data were significant only for the GPFT scores, which were linearly related to IHB in both the experimental sessions (first: ) Figure . No sign) Figure . A signiT Figure  nor with4Here, we provide the first detailed characterization of interhemispheric dynamics in unilateral stroke patients, basing on neurophysiological, structural, and clinical measures. We found that the stimulation of the AH did not result in any interhemispheric suppression of TMS‐evoked cortical activity onto the UH, as normally observable in the healthy brain. Surprisingly, the stimulation of the UH revealed, on the contrary, a preservation of the interhemispheric suppression mechanism, similarly to what we observed in the healthy brain. Then, we computed an index, that is, IHB, reflecting the balance between the two hemispheres, which we recently validated in a large sample of 50 old and young healthy volunteers  compared to stimulation of UH in patients (SD = 0.22) and in HC . We also observed a larger distribution of the ISPAH values ranging from 0.27 to 3.71, compared to ISPUH in patients (0.27–1.35) and ISP in HC . These results demonstrate that ISP is able to discriminate the different pattern of interhemispheric transmission from the AH and UH, regardless the presence of MEP. From a physiological point of view, the inconsistent ISP pattern when tested from the AH, is likely due to an abnormal inhibitory/excitatory activity originating from the lesioned hemisphere  are needed to observe an SDC of 0.1. This might be due to the higher variability observed in the cortical reactivity to TMS, especially when testing the lesioned hemisphere. Importantly, correlation analyses between cortical, and clinical and structural data were highly reproducible, a result that supports their use in clinical evaluation. Finally, analysis of ICC showed that subjects in the same group resemble each other in terms of ISP and IHB. This result is important because it shows that ISP and IHB are reliable measures in distinguish healthy and pathological interhemispheric dynamics. Differently, our results showed a low ICC reliability of IHCoh, a result that can explain the absence of correlation with structural data after 3 weeks.The physiological nature of the novel interhemispheric TMS–EEG indexes presented here is not completely understood. However, we recently found a strict correlation between our TMS–EEG indexes and the traditional TMS–EMG of IHI, that is, IHI, tested with two coils over the M1 of the two hemispheres  and right‐sided (13 patients) lesions that lead us to collapse all the ipsilesional the AHs in one side, with a reduction of the spatial accuracy. Another limitation of the study lies in the impossibility to accurately estimate the intensity of stimulation for the AH in some patients. However, we demonstrated that this factor did not affect our results. Indeed, our results were also confirmed in a subgroup of stroke patients with an evocable MEP from the AH. Moreover, only a low number of patients completed the clinical and the structural evaluation, thus our conclusions regarding the correlation analysis should be considered as exploratory. Finally, it is conceivable that the suppression of TMS‐evoked activity is produced, at least to some extent, by a degradation of the activation spreading through biological tissue (Määttä et al., In conclusion, the main contribution of this study lies in the proposal of new TMS–EEG indexes of interhemispheric dynamics in stroke patients. These measures show high reliability in distinguishing healthy and pathological interhemispheric dynamics and, more importantly, can be evoked even in absence of a stable MEP, which is often unreliable in stroke patients. These findings could be of high relevance from a clinical point of view. Indeed, a part from the intrinsic limitations of the technique, for example, the time of acquisition and analysis of the signal, TMS–EEG provide noninvasive and in vivo measures of the cortical state during the evolution of the cerebral reorganization right after a stroke event, as we recently demonstrated in acute stroke patients (Pellicciari et al., The authors declare no conflicts of interest.Elias Paolo Casula: Conception and design of the work, data analysis, manuscript writing, revision of the work. Maria Concetta Pellicciari: TMS–EEG data analysis. Sonia Bonnì: Data collection, patient's recruitment. Barbara Spanò: MRI data analysis. Viviana Ponzo: TMS–EEG and TMS–EMG data collection. Ilenia Salsano: MRI data collection and analysis. Giovanni Giulietti: MRI data analysis. Alex Martino Cinnera: Functional and behavioral data collection. Michele Maiella: TMS–EEG data collection and analysis. Ilaria Borghi: TMS–EEG data collection and analysis. Lorenzo Rocchi: Manuscript writing, revision of the work. Marco Bozzali: MRI data analysis, revision of the work. Fabrizio Sallustio: Patient's recruitment, revision of the work. Carlo Caltagirone: Revision of the work. Giacomo Koch: Conception and design of the work, revision of the work.