The built HSVR model consistently met the most stringent criteria when subjected to various statistical assessments. A mock test also assured the predictivity of HSVR. Consequently, this HSVR model can be adopted to facilitate drug discovery and development.The vast majority of marketed drugs are orally administrated. As such, drug absorption is one of the important drug metabolism and pharmacokinetics parameters that should be assessed in the process of drug discovery and development. A nonlinear quantitative structure–activity relationship (QSAR) model was constructed in this investigation using the novel machine learning-based hierarchical support vector regression (HSVR) scheme to render the extremely complicated relationships between descriptors and intestinal permeability that can take place through various passive diffusion and carrier-mediated active transport routes. The predictions by HSVR were found to be in good agreement with the observed values for the molecules in the training set ( Curcuma longa), has a great beneficial potential in treating cancer, diabetes, osteoarthritis, antianxiety, and even novel coronavirus disease 2019 (COVID-19) [Oral administration is the predominant route for medication that can be manifested by the fact that ca. 56% of unique drugs approved by FDA in 2018 were orally administrated [OVID-19) ,4 and yeOVID-19) .Drug absorption mainly relies on solubility and intestinal permeability , which iABCB1), breast cancer resistance protein , MRP2 (ABCC2), MRP3 (ABCC3), MRP4 (ABCC4), MRP5 (ABCC5), MRP6 (ABCC6), MRP7 (ABCC10), MRP8 (ABCC11), and MRP9 (ABCC12) [SLC15A1), concentrative nucleoside transporter 1 , concentrative nucleoside transporter 2 , equilibrative nucleoside transporter , organic cation transporters 1 , organic cation/carnitine transporter 1 , organic cation/carnitine transporter 2 , monocarboxylate Transporter 1 , organic anion transporting polypeptide 2B1 , serotonin transporter , and apical sodium-dependent bile acid transporter; (ASBT. SLC10A2) [In addition, the ABC transporters including P-glycoprotein  , and theSLC10A2)  can be fMCT1, SLCA1, organPeff) of the gastrointestinal (GI) tract segments, namely duodenum, jejunum, ileum, and colon, in human, rat, and mouse [Peff, which is expressed as cm/s, can be calculated byQ is the perfusion buffer flow rate; A represents the surface area within the intestinal segment that can be computed by the radius of the intestinal segment (R) and the length of the perfusion intestinal segment (L) [Of various in vitro assay systems to measure intestinal permeability, human colorectal adenocarcinoma cells (Caco-2), Madin−Darby canine kidney cells (MDCK), and parallel artificial membrane permeability assay (PAMPA) are commonly used , and thend mouse . The parment (L) ,(2)A=2πWhen compared with in vitro assays, in vivo tests provide a closer to real-life environment, but they are costly, time consuming, and sometimes inhumane, and are subjected to discrepancies by a number of factors such as individual differences in intestinal cell surface and epithelial cell integrity , especiaIn silico technologies have been seamlessly integrated into the drug discovery and development and they especially provide valuable advantages in ADME/Tox profiling due to their extremely fast throughput and low cost . As suchAccordingly, it is extraordinarily difficult, if not entirely infeasible, to develop a robust in silico model to predict the intestinal permeability with the consideration of those critical factors governing the perplexing efflux and influx active transport and passive diffusion mentioned above. Such challenge, nevertheless, can be solved by the novel ML-based hierarchical support vector regression (HSVR) scheme devised by Leong et al.  since HSPeff, molecular weight (MW), log D, log P, hydrogen-bond acceptor (HBA), hydrogen-bond donor (HBD), and polar surface area (PSA) in density form for the training and test molecules. Thus, the unbiased partition of data samples can be ascertained [Of all molecules selected in this study, 53 and 13 molecules were randomly assigned to the training set and test set, respectively, with a ca. 4:1 ratio. ertained .2, whereas surface areas of the others are less than 600 Å2.It is not trivial to establish the AD of predictive models prior to model development to identify the outliers and exclude them from data collection . There aVarious SVR models were built based on various descriptor combinations and runtime parameters, and three SVR models, denoted as SVR A, SVR B, and SVR C, were selected to construct the SVR ensemble, which, in turn, was subjected to regression by another SVR to generate the HSVR model. The optimal runtime parameters of SVR A, SVR B, SVR C, and HSVR, are listed in Peff values. These three SVR models, which unanimously selected four descriptors with different combinations , were adPeff values by SVR A, SVR B, SVR C, and HSVR for the molecules in the training set. The predictions by SVR A, SVR B, and SVR C are generally in good agreement with the observed values for most of the molecules in the training set, as depicted by their small MAE and s values (Max (0.91), MAE (0.25), s (0.15), and RMSE (0.29) and the largest r2 parameter (0.83), suggesting that SVR B executed better than SVR A and SVR C for the molecules in the training set. Nevertheless, SVR B produced not only the lowest r2 and Y-scrambling, and their almost zero values of <s values . Furtherlidation , signify of 0.02  when subR models .Peff values by SVR A, SVR B, SVR C, and HSVR for the molecules in the test set. Nevertheless, the mean absolute errors calculated by SVR A, SVR B, and SVR C increase from 0.33, 0.25, and 0.15 in the training set to 0.39, 0.26, and 0.24 in the test set, respectively , in which SVR A, SVR B, SVR C, and HSVR yielded absolute residuals of 0.25, 0.20, 0.06, and 0.03, respectively. As such, HSVR statistically functioned better than SVR A, SVR B, and SVR C, as manifested by all parameters listed in r2 value (0.93) as compared with its counterparts in the SVRE. In addition, there is a little chance that HSVR was produced by chance correlation, as manifested by its nearly zero value of <The HSVR model was yielded by the regression of the SVR ensemble based on the predictions of all samples and statistical evaluations in the training set  and its > (0.02) .Max declined from 0.50 in the training set to 0.47 in the test set. s values of 0.29, 0.24, 0.20, and 0.14, respectively. Similar observation that HSVR gave rise to smaller absolute residuals than its counterparts in the SVRE can also be noted in the test set. The absolute prediction deviation of compound 59, for instance, was 0.04 yielded by HSVR, whereas SVR A, SVR B, and SVR C gave rise to the absolute residuals of 0.35, 0.38, and 0.24, respectively. HSVR normally generated consistent and small errors in both training and test sets, as depicted by those parameters listed in q2 (0.81) in the test set and the smallest difference between r2 and r2 and q2 or between r2 and When applied to the molecules in the test set, slight performance decreases can be observed for HSVR. For instance, RMSE increased from 0.17 in the training set to 0.26 in the test set . Neverthq2 values and smallest deviation-related parameters. The superiority of HSVR in the outlier set can be plausibly due to the broad applicability domain encompassed by HSVR as compared with its SVR counterparts in the ensemble and, more importantly, the more robust nature of HSVR makes it more practically useful in real applications [When applied to the outliers, HSVR even showed more pounced predominance, as indicated by those statistical parameters listed in ications .Peff values predicted by HSVR for the molecules in the training set, test set, and outlier set. It can be conceived that the residuals are approximately evenly allocated on both sides of x-axis along the range of predicted values in all datasets, suggesting that there is no significant systematic error associated with HSVR. The unbiased predictions can be further rendered by its almost negligible average residuals that were 0.00, −0.10, and 0.09 in the training set, test set, and outlier set, respectively  are not applicable to these drugs. The subsequent relationship between both measured systems was initially instituted and checked based on those common seven drugs and the resulted scattered plot is displayed in r value of 0.80, suggesting that it is plausible to validate the derived HSVR model by those novel molecules assayed by Lennernäs, which is consistent with the fact that the rat SPIP Peff values can be useful to predict human intestinal permeability [To imitate real world challenges, the derived HSVR model was checked by all marketed drugs assayed by Lennernäs , of whiceability .r value between experimental human log Peff and predicted rat log Peff was 0.79. The negligible difference between both numbers (0.80 vs. 0.79) suggests that HSVR can almost reproduce the experimental observations. Accordingly, this mock challenge by 11 marketed drugs apparently assured the predictivity of HSVR and it is plausible to adopt this HSVR model as a surrogate for preliminary estimation of human intestine permeability in the process of drug discovery and development.P in intestinal permeability [nrot), charge, and fraction ionized at pH 7.4 (cFI7.4) to intestinal permeability [Numerous in silico models have been reported to predict intestinal permeability ,53,54,55eability , whereaseability .n-octanol–water partition coefficient (log P) and the n-octanol–water partition coefficient at pH 6.5 (log D). Moreover, it was proposed by Balimane et al. that log P and log D should be adopted to predict the intestinal permeability since log P alone is not sufficient enough to accurately render this complicated process [P and log D were adopted by this study , plausibly due to the descriptor–descriptor interaction [nN+O. The negative correlation between nN+O and log Peff (−0.29) is also consistent with the fact that permeability can decrease with MW [It has been observed that PSA is profoundly implicated in membrane permeability in passive diffusion , which i with MW .Peff is seemingly obscure, as illustrated by Peff for each histogram bin of HBD for all molecules included in this investigation. This peculiar relationship can be plausibly attributed to the fact that the substrates of PepT1 and P-gp, which are the most abundant SLC and ABC transporters, respectively, in jejunum [It has been postulated that hydrogen bond, which can be characterized by HBA and HBD, plays a critical role in intestinal P-gp-mediated transport  and HBD  jejunum , can int jejunum ,26. The  jejunum ,63. As sν is a size-related descriptor which measures the ratio of largest to smallest dimension. It can be observed in Peff for each histogram bin of shadow-ν, that Peff generally increased with shadow-ν for all molecules selected in this investigation, suggesting that molecules with larger shadow-ν have higher permeability than their smaller counterparts.Shadow-Peff for all molecules enrolled in this study, with an insignificant r value of −0.12 , which is possibly associated with molecular size, polarity, and/or polarizability , is closof −0.12 . This inμ in intestinal permeability has been recognized [μ can describe the solute‒solute and solute–solvent dipole interactions [μ and permeability. In addition, it has been observed that ligands can interact with the efflux transporter P-gp and the influx transporter PepT1 through dipole interactions [μ in intestinal permeability.The significance of the descriptor cognized  since μ ractions , as demoractions , leadingractions ,70,71, gν, and nN+O, which was selected in place of MW in this study (vide supra), are closely linked to molecular size, and the molecular size is a determining barrier factor in intestinal permeability as postulated [It is of interest that most of descriptors selected in this study are associated with passive diffusion, which is consistent with the fact that passive diffusion is the major route for intestinal permeability for many administrated drugs . Additiostulated ,73,74.Peff for each histogram bin of CSA. It can be observed that Peff did not show substantial variations with CSA initially, yet the Peff value greatly dropped once CSA was larger than 75, which is very similar to the trend observed for MR  models and ADME/Tox properties that should be taken into account ,76. ActuPeff for those molecules selected in this study and the largest absolute maximum r was merely 0.29 between nN+O and log Peff , but no acceptable models were yielded (data not shown) . This chlog Peff , depictiPeff values for a given compound within close range. Chemical structures without defined stereochemistry  were discarded from the collection. All molecules included in this study, IUPAC names, SMILES strings, CAS registry numbers, logarithm of observed Peff values, and references to the literature are listed in A comprehensive literature search was carried out to retrieve in vivo permeability data from a variety of sources to construct quality data for this investigation. Of various assay systems, 74 compounds, which were measured by SPIP in rat jejunum with pH 6.5 phosphate buffered saline (PBS), were adopted from various sources ,96,97,98d,p) by the Gaussian package  since it has been demonstrated elsewhere that predictive models with the selection of DFT descriptors can perform better [EHOMO), lowest unoccupied molecular orbital energy (ELUMO), free energy (ΔG), molecular dipole (μ), and its maximum absolute components (|μ|Max) of each molecule were also retrieved from the optimization calculations.All molecules selected in this study were subjected to full geometry optimization using the density functional theory (DFT) B3LYP method with the basis set 6-31G and E-Dragon (available at the website: http://www.vcclab.org/lab/edragon/). Additionally, the cross-sectional area (CSA) was also computed using the method modified by Muehlbacher et al. since it was implicated in membrane permeability [Ka values. Specifically, molecules with only one pKa value are defined as neutrals, whereas those with more than one pKa value are designated as zwitterions, acids, and bases when their largest pKa values are larger than 7 and the smallest pKa values are smaller than 7; the largest and smallest pKa values are smaller than 7; and the largest and smallest pKa values are larger than 7, respectively.More than 200 one-, two-, and three-dimensional molecular descriptors, which can be categorized as electronic descriptors, spatial descriptors, structural descriptors, thermodynamic descriptors, topological descriptors, and eability . Moleculr2 > 0.80 [r2 ≧ 0.64 was adopted. Descriptor normalization, which was designated to reduce the possibility of descriptors with broader ranges outweighing those with narrower ranges, was implemented byjth original and normalized descriptors of the ith sample, respectively; jth descriptor; and n is the number of samples.Descriptor filtration was initiated by discarding those descriptors missing for at least one sample or barely displaying discrimination against most of samples. It was suggested by Topliss and Edwards that the probability of spurious correlations can be reduced by removing those descriptors with intercorrelation values of 2 > 0.80 . AccordiDiscovery Studio due to its effectiveness and efficiency [Descriptors are one of the critical factors influencing the performance of predictive models . The inificiency . Furtherficiency .Discovery Studio and the outliers were determined. The remaining compounds were randomly divided into the training set and test set with a ca. 4:1 ratio as proposed to build and validate the models, respectively [Discovery Studio. In addition, it was suggested by Golbraikh et al. that only high levels of chemical and biological similarity between the training samples and test samples can develop a sound model [It is of necessity to identify the outliers prior to the model development, which can be done by examining the molecular distribution in the chemical space . As suchectively , using tnd model . As suchHierarchical support vector regression (HSVR), which was originally proposed by Leong et al. , was evosvm-train module in LIBSVM (software available at: http://www.csie.ntu.edu.tw/~cjlin/libsvm) using those samples in the training set with various descriptor combinations and SVR run parameters. The derived models were validated by svm-predict using those samples in the test set. The regression functions, namely ε-SVR and γ-SVR, were tried, and radial basis function (RBF) was selected as the kernel function due to its simplicity and greater performance [ε-SVR and ν-SVR, the corresponding ε and ν, cost C, and the kernel width γ were automatically run by the systemic grid search algorithm in a parallel fashion.The principle and implementation of HSVR has been detailed elsewhere and the architecture of HSVR schemeis depicted in Figure 1 of Leong et al. . Brieflyformance . The runThe principle of Occam’s razor was applied to the construction of SVR ensemble (SVRE), which suggests that the fewer the ensemble members, the better . More spThe difference between the observed value (n samples in the dataset were computedThe root mean square error (RMSE) and the mean absolute error (MAE) for r2 and q2 in the training set and external set, respectively, by the following equationThe developed models were further assessed by the correlation coefficients r2 to gauge the model performancek were derived from the regression line  without the intercept, whereas Additionally, Ojha et al.  also proY-scrambling or Y-randomization was applied by randomly permuting the log Peff values, viz. Y values, to refit the previously derived models while the descriptors remained unchanged. This procedure was repeated 25 times, as suggested in [The developed model was further subjected to internal validation using the leave-one-out cross-validation scheme, producing the correlation coefficient ested in , to giveCCC) using QSARINS [nTR and nEXT are the numbers of samples in the training set and external set, respectively; Additionally, the predictivity in the external dataset was evaluated by the correlation coefficients  QSARINS (12)qF12r in Equations (18)–(21) denotes r and q in the training set and external set, respectively. Finally, the predictivity of developed models were further assessed by combining the most stringent criteria collectively suggested by Golbraikh et al. , Ojha etIntestinal permeability plays a pivotal role in systemic drug absorption that, in turn, can be of critical importance to drug efficacy. As such, intestinal permeability is one of the important drug metabolism and pharmacokinetics parameters that should be assessed in the process of drug discovery and development. An in silico model to predict the intestinal permeability can be of great value to drug discovery and development. Nevertheless, intestinal permeability is an extremely complex process that can take place through various routes, namely passive diffusion and carrier-mediated active transport. thus distinct descriptor combinations as well as various relationships are needed to depict these variations in different mechanisms. The novel machine learning-based HSVR scheme, which concurrently possesses the advantageous characteristics of a local model and a global model, namely larger coverage of applicability domain and higher degree of predictivity, respectively, was adopted in this investigation to construct a QSAR model to predict the intestinal permeability. The constructed HSVR showed great prediction accuracy for the molecules in the training set and test set, respectively, with superior predictivity and statistical significance. When subjected to a mock test by a group of molecules to mimic real challenges, the built HSVR model also performed equivalently well. In addition, the selected descriptors can render those interactions associated with passive diffusion and active transport. Accordingly, it can be asserted that this HSVR model can be utilized as an accurate and dependable predictive tool, even in the high throughput environment, to facilitate drug discovery and development by predicting the intestinal permeability of hit and lead compounds even when they are virtual. Additionally, the development of HSVR also paves the way to create more in silico models to predict oral absorption, drug stability in stomach, and bioavailability in the future.


Even patients with lymphoproliferative diseases may develop a persistent chronic pain not responsive to usual treatments due to changes in antibody production and to some treatments like radiotherapy, chemotherapy, and the administration of monoclonal antibodies, which further impair the immune defense and induce chronic inflammatory phenomena acting as a substrate for a persistent chronic pain. Five patients with indolent lymphoproliferative diseases were treated for severe pain nonresponsive to other pain reliever treatments with SCS applied with an All-in-One Shot (OS) procedure. For all patients, the estimated survival time was of 5 years or more. All patients showed a significant reduction of the intensity of pain: the mean Numerical Rating Scale was 7.4 before treatment and 2.2 after. No patient developed adverse events. Supported by the data of this study, we believe that the habit to deprive patients with an indolent form of lymphoproliferative diseases of the possibility to reduce the intensity of chronic pain by SCS treatment is extremely reductive and frustrating. The pain is an epiphenomenon of several pathological processes. A constant stimulation of the nociceptive pathways produces changes in the ion channels, receptors, and nerve synapses, which, together with an increased diffusion of neurotransmitters and neuromodulators, facilitates the depolarization of peripheral and central neurons –5. Theredura mater with loss of cephalorachid fluid, infections and paresis due to possible compression of the spinal cord, extravasation of serum at the pulse generator plant site, feeling of numbness or persistent pain localized to the area below the implant or at the electrode implant site, allergic reactions, and attempts of rejection for implanted materials. Over time, a stimulation change may occur due to cellular changes in tissues surrounding the electrodes caused by the displacement of the electrodes, loosening of the electrical connections, defects in the lead, or alterations in the charge of the batteries. Spinal cord stimulation is mainly applied in the failed back surgery syndrome (FBSS), neuropathic pain, peripheral ischemic pain, amputation pain and visceral pain  is among the main innovative techniques to control the otherwise intractable chronic pain. SCS uses low voltage current that reaches the spinal cord through the application of epidural leads that stimulate nerve fibers blocking or weakening the transmission of pain –21. The ral pain , 29, andLymphoproliferative diseases can be classified as aggressive or indolent. The aggressive forms need immediate intensive treatment to improve the expectancy of life. Instead, Indolent lymphoproliferative diseases have a slow growth and are usually treated only in case of systemic symptoms, symptomatic splenomegaly, bulky mass over 7 cm, and progressive leukemic serum effusions. The indolent forms require a 3- to 6-month watch-and-wait period to verify whether the disease may turn aggressive; this strategy is linked to three main reasons: the good long-term prognosis measured in years, the absence of benefits with chemotherapy that however may induce severe side effects, and the possibility of these indolent forms going through multiple reactivations over time requiring different chemotherapy treatment , 31. ChrIn the last 2 years, five patients with B-cell lymphoproliferative diseases with a life expectancy of more than 5 years complaining for an intense persistent pain resistant to numerous pain-relieving treatments were treated with SCS at the Department of Pain Therapy Monaldi Hospital—AORN Ospedali dei Colli, Naples, Italy. Before implanting the SCS apparatus, individual prognostic scores were examined.The International Prognostic Score (CLL-IPI) was calculated for two CLL patients, the FLIPI was calculated for two follicular lymphoma (FL) patients, and the International Prognostic Scoring System (ISSWM) was calculated for one patient with WM. The SCS stimulation was considered effective when a pain reduction >50% was obtained. The intensity of pain was assessed according to the McGill Pain Questionnaire (MPQ), recording the Numerical Rating Scale (NRS) of the pain average: minimum pain, maximum pain, and pain during exertion . At the baseline visit, each patient signed an informed consent for the anonymous use in clinical investigation and scientific publication, according to Italian laws on privacy and with the rules of our ethics committees. All patients were evaluated with ECOG Performance Status (PS). Patients, according to the CARE guidelines, underwent physical examination, routine blood chemistry tests, and serum markers of HBV, HCV, and HIV infections  –42. To rp < 0.05). We assessed individual NRS changes in pain before and after SCS with the Wilcoxon signed-rank test using the Reliable Change Index, which indicates whether a change in the NRS score is significantly greater than expected based on the reliability test–retest. In the reliable change, index X1 represents a subject's pre-test score, X2 represents the post-test score in the same subject, and Sdiff is the standard error of the difference between the two test scores. Sdiff can be computed directly from the standard error of measurement (SE) according to the following: RC: X1 – X2/SDIFF, where SDIFF = √2(SE) 2, and SE is computed as follows: SE = s√1 – rxx. We used a 95% confidence interval, meaning that we considered RCI values of 1.96 or higher statistically significantly.The Wilcoxon signed-rank test was used to evaluate changes in NRS before and after SCS in each patient, a non-parametric test useful for assessing whether two repeated measurements in a single patient differ significantly  and a slight decrease in gamma globulin serum levels. He had an IGHV mutated status, no cytogenetic abnormalities, serum β2 macroglobulin = 2 mg/L, and PS = 2. His CLL-IPI showed an intermediate risk with 79.3% 5-years survival possibility. He was negative for serum markers of HBV, HCV, and HIV infections. In February 2019, he was treated with OS dorsal SCS, implanting two leads with tip in T7; the chosen waveform was BURST DR with the following parameters: frequency 40 Hz, intraburst 500 Hz, pulse length 1,000 ms, Max Amplitude 2 mA  pulsed radiofrequency treatment (PRF). She was affected by CLL stage II according to Rai staging. A lymphocytosis  and a slight decrease in gamma globulin serum levels were the only abnormalities in the blood tests. She had an IGHV mutated status, no cytogenetic abnormalities, serum β2 microglobulin = 3 mg/L, and PS = 2. Her CLL-IPI showed a low risk with a 93.2% possibility of 5-year survival. He was negative for serum markers of HBV, HCV, and HIV infections. The NRS was 8 in April 2019 when she was treated with an OS dorsal SCS implanting two leads with a non-aligned tip in T6 (left) and T7 (right); the chosen waveform was BURST DR with the following parameters: frequency 40 Hz, intraburst 500 Hz, pulse length 1,000 ms, Max Amplitude 2 mA  and serum B2-microglobulin = 2.2 (mg/L). Her ISSWM was 0 with a median survival of 142 months. Her PS was 2. All serum markers of HBV, HCV, and HIV infections were negative. She was treated with an OS SCS implanting one lead with tip in C2–C3 in July 2019; the chosen waveform was high frequency at 10 kHz; the sweet spot of the electric field was set in the e1–e2 dipole  for pulmonary FL. No other side of the disease was demonstrated by a [18f] fluoro-D-deoxyglucose positron emission tomography/computed tomography (FDG-PET/CT) performed in February 2019. At that time, the LDH serum value was 200 IU/L, and all other blood tests were normal. In March 2019, he performed a RT (24Gy) and in May 2019, a FDG-PET/CT showed a complete remission of pulmonary lesion.No pain relief was observed after DRG pulsed radio frequency (PRF) thermal ablation of the transverse process of D4 performed in May 2019. The patient was in stage IE according to Ann Arbor staging, his PS was two, and his FLIPI was one, a value indicating a low-risk category with a 10-year overall survival of about 70%. Serum markers of HBV, HCV, and HIV did not show an ongoing infection, while an anti-HBc positivity was a clue of a past HBV infection. In September 2019, still suffering for an intractable persistent intercostal pain with NRS of 6, he was treated with right D3 and D5 DRG stimulation. The implant was done using an OS procedure with the following parameters: continuous stimulation frequency, 30 Hz; pulse width, 300 ms; max amplitude range, 0.350–0.450 mA . The NRSz = −2.12; p = 0.034). The reliable change index for NRS score at the two different time points showed a RCI of 2.07 and an effective size of 3.11. These data confirm that HFSCS has produced a significant reduction of pain in our five patients.Five patients with lymphoproliferative diseases were treated for severe pain nonresponsive to other pain reliever treatments with SCS, two had CLL, one had WM, and two had FL; two were females and three were males; the median age of the patients was 69 years (range 53–70). The PS was two in all cases. The SCSs implanted were lumbar in one patient, cervical in two, and ganglionic in two. For all patients, the estimated survival time was of 5 years or more. The mean NRS before treatment was 7.4, with highest peak at 10 and the lowest at six, whereas the mean NRS after treatment was 2.2, with highest peak at five and lowest at one . The WilWe conclude that the high-frequency SCS was useful for all patients with lymphoproliferative diseases included in the present study.No patient had ongoing HIV, HCV, and HBV infections. None of these five patients experienced adverse events or infections signs.The most common indications for SCS are FBSS, complex regional pain syndrome, brachial plexopathy, diabetic neuropathy, post-herpetic neuralgia not responding to other treatments, and pain associated with vascular pathology or secondary to damage of peripheral nerves. All these indications do not foresee their use in the onco-hematological patients. In these patients, the greater risk of both systemic and local infections could jeopardize the success of the implant. The reduced prognosis of these patients, the greatest risk of infections and the high cost of the procedure are the reasons for the scarce use of this technique in this patient setting. Unfortunately, changes of the immune system occurring in patients with lymphoproliferative diseases may favor the onset of intractable persistent chronic pain. Furthermore, some treatments like radiotherapy, chemotherapy, and the administration of monoclonal antibodies may further impair the immune defense of these patients and generate chronic inflammatory phenomena acting as a substrate for the development of persistent chronic pain. Some surgical diagnostic techniques like VATS in our patient 1 may contribute to generate a chronic persistent pain syndrome. Therefore, in patients with lymphoproliferative diseases, there are reasons inducing a persistent chronic pain not responsive to usual treatments for whom the SCS should be taken into consideration.All five patients in this study were in good general condition, with a PS of 2, and had an indolent lymphoproliferative disease, and for all of them, a survival of 5 years or more was expected. The implant and use of the SCS in a one-shot procedure were safe in all five treated patients who did not develop adverse events and infections. Of note, this procedure is reversible in case of ineffectiveness .The habit to deprive patients with lymphoproliferative diseases of the possibility of being treated with SCS just because for them a reduced survival duration is expected is extremely reductive and frustrating. Even if in an evaluation of the cost-effectiveness ratio, the expected duration of life has a relevant role, it is inhumane not to reserve also for patients with indolent lymphoproliferative diseases the possibility to take advantage of the benefits SCS may offer. All this considering, there is no reason to deny these patients the possibility of accessing SCS treatment. The data presented in this study might confirm this opinion, but further studies are needed to conclude on this topic.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Written, informed consent was obtained for the publication of these case reports.AS and AP: conceptualization. AS, CS, MD, PB, AMS, ESag, ESar, and DT: data curation and investigation. AS and BC: methodology. AS: project administration, writing—original draft, and writing—review and editing. All authors have read and agreed to the published version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


After adjustment for various demographic parameters by multivariate analyses, male sex (standard β = 0.23), EG (0.19), and diabetes (0.09) were associated with higher AGE levels; PG (−0.18) and smoking (−0.19) were associated with lower AGE levels. Age, visual acuity, intraocular pressure, glaucoma medications, lens status, and systemic hypertension were not associated with AGEs. The high AGE level in EG suggested that specific oxidation and glycation mechanisms underlie the glaucoma pathogenesis associated with pseudoexfoliation syndrome.Advanced glycation end products (AGEs), which are the products of a non-enzymatic reaction between reducing sugars and other macromolecules, are critical in aging, as well as metabolic and degenerative diseases. To assess the involvement of AGEs in glaucoma, skin autofluorescence (sAF) level, which is a measurement of AGEs’ accumulation, was compared among Japanese patients with glaucoma (316 with primary open-angle glaucoma (PG) and 127 exfoliation syndrome and glaucoma (EG)) and controls (133 nonglaucomatous controls) . The sAF values were estimated from the middle fingertip using a 365 nm light-emitting diode for excitation and detection at 440 nm emission light. The estimated AGE values (arbitrary unit) were 0.56 ± 0.15, 0.56 ± 0.11, and 0.61 ± 0.11 in the control, PG, and EG groups, respectively ( Progressive optic neuropathy and visual field loss characterize glaucoma, a leading cause of irreversible blindness worldwide  includinWe previously identified a significantly lower antioxidant capacity level and higher hydroxylinoleates, oxidation products of the linoleates, in blood samples from subjects with PG and EX as compared with those in control samples ,10,11. IAdvanced glycation end products (AGEs) are various lipids, proteins, or nucleic acids that undergo glycation as a result of chronic exposure to glucose or other reducing sugars. AGEs accumulate in long-lived proteins such as collagen and have been indicated as components of oxidative stress generation in tissues ,17,18. FAGE formation and accumulation are associated with metabolic diseases such as diabetes mellitus ,24,25 ann = 316), exfoliation glaucoma (EG) (n = 127), and non-glaucomatous controls (n = 133). The participants underwent ophthalmologic examinations including measurement of the best-corrected visual acuity (BCVA) and IOP by Goldmann applanation tonometry, slit-lamp biomicroscopy, and funduscopy. One author (M.T.) measured the IOPs on the day of sample collection. In addition, the highest IOPs recorded in the previous medical records were investigated. The lens status (phakia/pseudophakia) and the number of glaucoma medications used by the patients were obtained from medical records. A fixed combination eyedrop was counted as two medications. The subjects were interviewed about current smoking habits and history of diabetes, and systemic hypertension. PG was diagnosed based on open iridocorneal angles bilaterally, the characteristic appearance of glaucomatous optic neuropathy such as enlargement of the optic disc cup or focal thinning of the neuroretinal rim, corresponding visual field defects in at least one eye, and no evidence of secondary glaucoma bilaterally. EG was diagnosed based on an open iridocorneal angle and characteristic pseudoexfoliation material deposits on the anterior capsule or pupillary margin in at least one eye. If both eyes were eligible, the eye with the worse mean deviation was included in the PG or EG group. Visual field defects were identified using the Humphrey Visual Field Analyzer . Control subjects were consecutively recruited from the patients who visited the ophthalmology outpatient clinics at the Matsue Red Cross Hospital, Matsue, Japan and the Iinan Hospital, Iinan, Japan. The controls, aged 20 years and older, had no clinical signs of glaucoma and did not use glaucoma medications, and the highest IOP of the control subjects was lower than 20 mmHg. The lens status was not considered. The eye of a study participant with the better BCVA was included in the analyses. If both eyes had the same BCVA, the right eye was included. For the PG, EG, and control groups, eyes with ocular diseases other than glaucoma and cataract and decimal BCVA worse than 0.01 were excluded. Since the association between diabetes and AGEs has been well documented [The current study adhered to the tenets of the Declaration of Helsinki. The institutional review board of the Matsue Red Cross Hospital approved the research conducted at the Matsue Red Cross Hospital  and at the Iinan Hospital . All participants provided written informed consent for inclusion in the study. Subjects were recruited consecutively at the Division of Ophthalmology, Matsue Red Cross Hospital, and the outpatient clinic at the Iinan Hospital. A total of 576 eyes of 576 Japanese subjects  71.6 ± 12.8 years) comprised the study population, i.e., subjects with PG . The sAF values obtained with the excitation and emission wavelengths of 365 nm and 440 nm, respectively, were used to estimate the levels of AGE accumulation. The obtained sAF levels are correlated positively with the level of the hyperglycemia-associated AGE, Nδ-(5-hydro-5-methyl-4-imidazolone-2-yl)-ornithine (MG-H1) . The sAFThe finger clip feature of the device enables measurement of the intensity of the fluorescence from the middle finger of the non-dominant hand where the least skin melanin is present. Although the intensity of autofluorescence from veins is approximately 1.5-fold higher than that of skin, the influence of the veins is negligible in the measurement of the sAF using the AGEs Sensor, since the fingertips have only capillaries and no veins. Therefore, the fingertip is suitable to avoid non-specific skin fluorescence . TrainedThe measured AGEs were expressed in arbitrary units. According to our pilot study, the coefficient of variation and intraclass correlation coefficient (Cronbach’s α) of three repeated AGE measurements were 6.7 ± 7.3% and 0.938, respectively.t-tests, and the differences in categorical data, i.e., sex, lens status, smoking, diabetes, and hypertension were calculated using the G-test followed by the post-hoc Fisher’s exact probability test. To correct for multigroup comparisons, p values of 0.0167 and 0.0033 for the unpaired t tests or Fisher’s exact probability test were considered to be significant at 5% and 1%, respectively, based on Bonferroni’s method. Possible correlations among AGEs and other parameters were assessed by linear regression analyses with Pearson’s correlation coefficient for continuous data and the unpaired t-test for categorical data. To adjust for differences in parameters among groups, possible associations among AGEs and various parameters were tested further using multiple regression analyses. All statistical analyses were calculated using JMP Pro statistical software version 14.2 .The data are expressed as the means ± SD for continuous variables and in numbers and percentage for categorical variables. For the statistical analyses, the decimal BCVA was converted into the logarithm of the minimum angle of resolution. For comparisons among the three study groups, the differences in continuous data, age, BCVA, IOP, highest IOP, and number of glaucoma medications were calculated using one-way analysis of variance followed by post-hoc unpaired p = 0.0007) and those of the PG  group, whereas the levels were equivalent in the control and PG groups (p = 0.5120).The subject demographic data are shown in r = 0.11, p = 0.0063), while the BCVA, IOP on the day of the AGE measurement, highest IOP recorded, and number of glaucoma medications used were not correlated with the AGEs  and systemic hypertension , and no previous history of smoking could have weakened the detection power for involvement of those factors. The absence of parameters of glaucoma severity, such as visual field sensitivity and retinal nerve fiber thickness in the statistical analyses, was also was a study limitation.In conclusion, the levels of AGEs in subjects with EG were higher than those with PG and non-glaucomatous control subjects. This result suggested that specific oxidation and glycation mechanisms underlie the pathogenesis of glaucoma associated with EX.


Mycotoxins are secondary metabolites produced by some filamentous fungi, which can cause toxicity in animal species, including humans. Because of their high toxicological impacts, mycotoxins have received significant consideration, leading to the definition of strict legislative thresholds and limits in many areas of the world. Mycotoxins can reduce farm profits not only through reduced crop quality and product refusal, but also through a reduction in animal productivity and health. This paper briefly addresses the impacts of mycotoxin contamination of feed and food on animal and human health, and describes the main pre- and post-harvest systems to control their levels, including genetic, agronomic, biological, chemical, and physical methods. It so highlights (i) the lack of effective and straightforward solutions to control mycotoxin contamination in the field, at pre-harvest, as well as later post-harvest; and (ii) the increasing demand for novel methods to control mycotoxin infections, intoxications, and diseases, without leaving toxic chemical residues in the food and feed chain. Thus, the broad objective of the present study was to review the literature on the use of ozone for mycotoxin decontamination, proposing this gaseous air pollutant as a powerful tool to detoxify mycotoxins from feed and food. Climate change is expected to negatively affect all dimensions of food and nutrition security and safety—including availability, access, utilization, and quality of principal food commodities—which are key aspects of food systems that are also challenged by the predicted increase in the global population ,2. AgricAspergillus spp., ochratoxin A (OTA) from Aspergillus spp. and Penicillium spp., fumonisins, type A trichothecenes (HT-2 toxin (HT-2) and T-2 toxin (T-2)) and type B trichothecenes from Fusarium spp., and patulin from P. expansum [1) [1 and moniliformin toxins [Mycotoxins are chemical compounds synthesized as secondary metabolites by some filamentous fungi. The mycotoxins of most agricultural relevance are aflatoxins from expansum . They vaexpansum ,7,8, in nsum [1) , in sowsnsum [1) ,11; in hn toxins ; and in n toxins ,14,15.1 and aflatoxin B2 (AFB2), the strongest hepatocarcinogenic molecule known, also showing genotoxic properties, as evaluated by the World Health Organization (WHO)-International Agency for Research on Cancer in 1993. Moreover, the hydroxylation of the AFB1 and AFB2 involves the production of aflatoxin M1 and M2. Other major human mycotoxicoses have resulted from exposure to ergot, tricothecenes, ochratoxins, 3-ninotropropionic acid, ZEA, and fumonisins [Some mycotoxins have an elevated carry over rate from feed to milk, likely contributing to mycotoxin intake in human populations, which are also threatened . The heamonisins . In addimonisins . Althougmonisins . Mycotoxmonisins ,22. The −1, depending on the mycotoxin, the food/feed commodity, and the country taken into account  and WHO, leading to the definition of strict legislative thresholds and limits in many areas of the world. Differently, in several African countries, the consumption of mycotoxin-contaminated foods is still a significant risk, especially for children, immunocompromised people, and rural populations . Howevert e.g., ,24). The. The−1, t  use not only as a food-sanitizing agent in coping with food decay during storage, but primarily for its potential in the inactivation of toxic fungi and in the detoxification of mycotoxins from feed and food.Overall, the control of mycotoxin contamination follows two strategies: prevention of their production  and detoxification  . The preThe strategies developed to limit the accumulation of mycotoxins in feed and food are numerous and can be classified according to the intervention moment . TheThe pre-harvest control of mycotoxins is based on the control of contamination levels in crops to be used as food and feed components. In general, these systems are based on preventive strategies, which aim to avoid the development of contamination, operating on the predisposing factors that facilitate the production of mycotoxins.Agronomic practices such as planting/harvesting timing, crop rotation, tillage, and management of irrigation, highly affect the mycotoxin contamination of crops in the field . Early hFusarium colonization of kernels was higher for the organic system than the conventional one. Studies conducted in Northern and Central Italy (in 2010 and 2011) on maize and wheat have not shown differences between organic and conventional farming systems in terms of the presence of mycotoxin-producing fungi. The incidence of Fusarium spp. was similar in conventional and organic wheat, but was significantly different in maize. Aspergillus and Penicillium incidence was linked to weather more than farming system; 2010 was the most favorable for Fusarium species . Cereal systems . These r systems ,46. Simi systems  in Brazi systems  evaluateThe use of modern technologies within a precision farming system could effectively help to prevent chemical contaminants like mycotoxins. Nazarizadeh et al.  describeA. flavus and aflatoxin in pre-harvest; for example, Dorner and Cole [A. flavus and A. parasiticus significantly reduced aflatoxin contamination. In addition, Cleveland et al. [F. verticillioides had the beneficial carry-over effect of excluding fumonisin-producing strains and preventing them from producing fumonisins. Luongo et al. [F. verticillioides and F. proliferatum in maize residues by non-pathogenic Fusarium species. The ability of fungal antagonists to cope with toxic fungi is, however, related to macro- and micro-climatic conditions in the antagonist–pathogen interaction. The efficiency of mycotoxin bio-control agents depends on their ability to colonize the target substrate and to be active under different environments in the field or during storage without inducing effects that compromise the end use quality of the commodity [Bacillus thuringiensis, which encodes for production of a crystal protein that is toxic to common lepidopteran corn pests) has been shown to significantly lower the levels of common mycotoxins compared to non-Bt isolines [Biological systems are based on strategies that can compete in field environments with toxic fungi. Different microorganisms have been reported as bio-control agents of and Cole  showed td et al.  reportedo et al.  also desommodity . Insect ommodity . In seveisolines ,57,58,59Insect damage is well recognized as a collateral factor in mycotoxin development. Fungal colonization or insect infestation of crops must be controlled by appropriate use of registered insecticides, fungicides, and other proper practices within an integrated pest management control . In partAlthough pre-harvest approaches should be preferred, in the perspective of preventing mycotoxin contamination, the development of toxic fungi is inevitable under certain environmental conditions. Therefore, appropriate storage practices and other post-harvest control systems are necessary to minimize the final mycotoxin content of foods and feeds. Several strategies are available for the fungal inactivation and mycotoxin degradation. These can be classified as (micro)biological, physical and chemical approaches.2) and high CO2 concentration contributes to the avoidance of fungal development and mycotoxin production [2 concentration (from 5 to 1%) can decrease Aspergillus growth and aflatoxin production [2) concentration is a valid method to control grain quality post-harvest. Recently, Moncini et al. [F. graminearum, F. langsethiae, A. flavus and F. verticillioides, and aflatoxins production by Aspergillus were significantly decreased when artificially contaminated corn and wheat grains were exposed to a highly purified N2 controlled atmosphere (98.5% ± 0.5). These results indicate that a N2-controlled atmosphere acts as an eco-friendly tool that could be transferred to a large-scale system for grain storage in order to avoid or reduce chemical treatments. Many of the above strategies are applicable to prevent toxic fungal development and mycotoxin formation. However, once mycotoxin contamination has occurred, feed and food associated with various mycotoxins must be managed through post-harvest detoxifying procedures.Appropriate storage methods may prevent mold growth, and optimal temperature and humidity of store houses can also reduce mold growth and prevent mycotoxin production . A relaoduction . Decreasoduction . Since gi et al.  documentSaccharomyces cerevisiae and Pichia carribica showed high potentiality to degrade patulin as a less toxic compound during fermentation in juices, as well as to inhibit patulin production by Penicillium expansum during post-harvest [Trichosporon mycotoxinivorans can deactivate OTA into the nontoxic form after incubation for 2.5 h. Molnar et al. [S. cerevisiae has been reported as the most efficient microorganism for AFB1 adsorption, since yeast cell walls allows the cells to adsorb a range of compounds from the environment [S. cerevisiae cell wall was shown to bind T-2 [Aspergillus, Penicillium, and Rhizopus are also able to detoxify mycotoxins; Clonostachys rosea can be used as a biocontrol agent in cereals, where it can produce zearalenone lactonohydrolase, thus avoiding other fungal growth on zearalenone-contaminated media [Aureobasidium pullulans can inhibit OTA accumulation and decrease aspergillosis occurrence and was therefore used as a biocontrol agent in wine grapes [A. flavus and A. parasiticus and a horseradish peroxidase from Raphanus sativus showed AFB1 degradation activity. A purified extracellular enzyme, myxobacteria aflatoxin degradation enzyme, from the bacterium Myxococcus fulvus ANSM068 showed much degradation ability toward AFG1 and AFM1 . Brevibacterium species are capable of degrading OTA, due to the release of highly active proteolytic carboxypeptidase enzymes (EC 3.4). A recent report revealed that OTA was significantly (75–85%) reduced by a carboxypeptidase and peptides present in liquid cultures of Bacillus subtilis CW14 [Biological approaches for mycotoxin decontamination by using microorganisms  have been used efficaciously for the management of mycotoxins in feed and food. Mycotoxin-degrading bacteria have been isolated from various sources like rumen and intestinal flora, soil, and even water. Improved feed utilization due to interactions between lactic acid bacteria (LAB) strains and mycotoxins  have been reported ,66. In p-harvest ,69. Simir et al.  developeironment . In addibind T-2 . Bioconted media ; and Aure grapes . In addie grapes . A peroxlis CW14 .A. parasiticus) of healthy kernels. During storage, transportation and marketing, low moisture levels should be maintained by avoiding leaking roofs and condensation arising from inadequate ventilation. A study conducted by Fandohan et al. [Physical methods of mycotoxin decontamination include various procedures such as drying, cleaning, mechanical sorting and separation washing, thermal inactivation, and irradiation. Rapid drying of agricultural products reduces the moisture level, creating less favorable conditions for fungal growth and proliferation, and insect infestation . Hamitonn et al.  demonstrn et al.  reportedn et al. . Thermaln et al. ,82, or tn et al. . Ultravin et al. . Irradian et al. ). The grn et al. .3), organic acids (formic and propionic acid) and other agents. Ammonization of grains not only reduces aflatoxins, fumonisins, and OTA to undetectable levels, but also inhibits toxic fungal growth. However, this method is not permitted in the European Community for human foods [3O4 nanoparticles), essential oils and their main bioactive compounds . These novel techniques are apparently promising, effective, and low-cost strategies that can offer eco-friendly solutions for the control of toxic fungi and mycotoxins in agricultural and food industries [Numerous chemical agents are suitable for mycotoxin control involving bases (such as ammonia and hydrated oxide), oxidizing agents . But human beings make extra O3 every day (“foe O3”). This ground-level O3 is formed when exhaust gas emissions from vehicles (mainly represented by nitrogen oxides and volatile organic compounds) interact with sunlight, especially in the summer; the entire complex of reaction is known as “photochemical smog”. When O3 is above threshold levels , it can3 is a powerful oxidizing unit and therefore a potent disinfecting agent, and its use may have many advantages in the food industry, such as in food surface hygiene, sanitation of food plant equipment, reuse of waste water, disinfection of packaging materials, and control of noxious organisms in stored products. Microbicidal action of O3 is gaining attention due to the fact of zero residues on the product and no need of aeration to remove the gas [Being made up of three fairly stable oxygen atoms, O the gas .3 generation at a commercial level [3. Ozonisers adopting this method expose O2 molecules to a high voltage of electrical discharge, so initiating the formation of free radical O2 and thereby generating O3 [Ozone can be generated starting from dry oxygen through machineries, and the electric corona discharge or UV radiation are the mostly used methods for Oal level . Howeverating O3 . Breakagating O3 . 3 was formally approved as an antimicrobial agent for the sanitization of foods in order to tackle the environmental and occupational contamination related to the use of chlorine [3 merely dissociates into O2 and related radicals. For this reason, the application of O3 technology in food chains has been considered safe and effective by the WHO, and is now recognized as a “green technology” [3 must be considered one of the most powerful oxidant disinfectants, being 1.5- and 1.3-fold stronger than chlorine and hydrogen peroxide against bacteria, virus, algae, and fungi [3 is able to disable noxious organisms such as pathogenic microbes and storage pests, by oxidizing their vital cellular components. In particular, O3 acts against unsaturated lipids in the microbial cell membranes causing a leakage of their contents, and, eventually, microbial lysis [3 causes widespread oxidation of internal cellular proteins reducing their growth and causing rapid cell death  to be Generally Recognized as Safe (GRAS) for bottled water disinfection early in 1982 . In 2001chlorine . Indeed,hnology” . With a nd fungi . Ozonatind fungi . The mecal lysis . In addienhanced . Howevervitamins . For thivitamins ,108,109.3 is a few hours in the presence of food/feed. When bubbled into water, O3 dissolves, partially forming hydroxyl radicals that can oxidize the microorganisms/contaminants more efficiently than molecular O3 itself [3 [3 gaseous form is considered the more useful application in decontaminating mycotoxins. Both methods have been successfully used to reduce postharvest diseases, viability of toxic fungi and mycotoxin accumulation in food/feed products. The last review on the application of O3 to prevent and degrade mycotoxins dates back to 2010. Here, an updated knowledge of the topic acquired in the last ten years is reported.Ozone can be directly applied to food/feed in its gaseous form or dissolved in aqueous solutions and applied as ozonized water. In the gaseous form, the half-life of O3 itself . Howevertself [3 . Ozonizetself [3 , but thehttps://www.sciencedirect.com/ and https://www.scopus.com/), and searching for the terms “ozone” or “oxidant agent”, and “mycotoxins”, “aflatoxins” or “trichothecenes”, and “food” or “feed”, is shown in 3 as a strategy to prevent/degrade mycotoxins have been already published [3 as a decontamination tool during the processing and storage of many food products, including cereal grains [3 technology seems to be increasing, as demonstrated by the big rise in studies published in the last few years  documented the higher effectiveness and the practical advantages of a gaseous application of O3 than aqueous solutions. This method is a more useful type of application and it has been successfully used in reducing mycotoxin accumulation in many kinds of food/feed products . The re cereals ,129,130.As reported above, ozonation is a chemical mean for food/feed processing, that has been used on many kinds of products or intermediates. Most of the reviewed experiments (81%) were carried out on cereals, in particular wheat and corn, and their derivatives such as flour ,131,132,3 was able to degrade aflatoxins in many food/feed portions and in several operation conditions  requires longer O3 exposure until the lactone ring is opened by the O3 treatment . Conseqcinogens  reported3 concentration and/or longer treatment times [3 to 9,10 double bond with the net addition of two atoms of O2, with the remainder of the molecule left unaltered  was conducted on trichothecenes type A such as HT-2 and T-2 toxins, and type B such as DON ,135 in mnt times ,137,138,nt times , initialratoxins . The remum genus ) and cit species ). It is bination ,143,144.bination ,146.The present review paper on mycotoxin contamination of feed and food highlights (i) the lack of effective and straightforward solutions to control mycotoxin contamination in the field at pre-harvest, as well as later at post-harvest, and (ii) the increasing demand for novel methods to control mycotoxin infections, intoxications, and diseases, without leaving toxic chemical residues in the food and feed chain.3 is able to inhibit fungal growth, sporulation and germination, by offering negligible loss of nutrients or sensory qualities in food/feed. However, its antimicrobial activity is very dependent on vegetable/fungus species, growth stage, concentration, and exposure time. In addition, degradation products, formed after O3 treatment of these residues, have not exactly been determined, and this seems to be the most crucial obstacle on this subject. In vivo and in vitro toxicological tests should be conducted to screen the effects of degradation products on human and animal health. Through emerging new techniques (reactive oxygen/nitrogen species), as well as improvements and innovations in O3 generation and application systems, certainly the subject will be evaluated more effectively in the future, facilitating enhanced control of both quality and safety parameters of ozonized foods/feeds. For effective and safe use in processing, optimum O3 concentration, contact time, and other treatment conditions should be defined for foods and feeds. A pilot test must be conducted for each case before starting a commercial application, since every O3 application is unique. Industrial facility of O3 technology remains to be developed for large scale treatment of food/feed products, requiring input from different disciplines.Ozone application has given promising results for important problems in the food industry, such as mycotoxin and pesticide residues. In particular, O


Multiple public health problems have been caused by various coronavirus strains over the last few years, such as the middle eastern respiratory syndrome (MERS), severe acute respiratory syndrome (SARS), and COVID-19. COVID-19, which is also known as coronavirus disease 2019, was first detected in Wuhan, China, and has significantly impacted people's health and lives. Additionally, it has led to a pandemic, and the virus has spread to over 121 countries worldwide. There is numerous information available regarding this virus. A detailed and extensive study of the morphological and histopathological findings will help understand and diagnose the disease. As it is a new disease, it is challenging to understand the mechanism of the action and disease pathology due to the limited availability of data from autopsies or biopsies. However, as the detailed mechanism of injury remains unclear, this paper aims to review the postmortem gross and histopathological findings of various organs that have been affected with coronavirus, focusing on the pulmonary, cardiac, and hematologic findings. This paper emphasizes the postmortem findings of the effect of the coronavirus disease on multiple organ systems.Advance search of the keywords on PubMed was used, limiting the search to the last five years. The eligible article is narrowed based on relevance containing postmortem findings of the novel virus; COVID-19. A total of 25 full-text articles were selected and used in the review of this paper. COVID-19 is a pandemic that has affected a large percentage of the world’s population. The total number of COVID-19 cases worldwide as of July 17, 2020, is over 13.9 million, which has led to over 593,000 deaths [COVID-19 is a novel coronavirus that has ribonucleic acid (RNA) as nuclear material. It was declared a pandemic by the World Health Organization on March 11, 2020 . The fouThere is a wide range of clinical spectrums of COVID-19, which ranges from the asymptomatic or paucisymptomatic forms to the conditions that are characterized by respiratory failure, which requires mechanical ventilation and admission to the intensive care unit. Very severe cases can lead to multi-organ dysfunction syndrome (MODS), which can result in sepsis or shock. The clinical manifestations have been divided into the following three categories based on the severity:Mild diseaseThis is an uncomplicated or mild illness. Individuals show mild symptoms, such as a mild fever, dry cough, sore throat, nasal congestion, malaise, headache, or myalgias. This comprises 81% of the total cases, while some patients with moderate disease present with respiratory symptoms such as cough, shortness of breath, and tachypnea. No signs and symptoms of severe disease are present .Severe diseaseThis occurs in 14% of cases. The symptoms in these patients range from dyspnea, a respiratory rate of >30, and a blood oxygen saturation of less than 93% . They prCriticalThese patients present with respiratory failure, cardiac injury, septic shock, and/or MODS, or multi-organ failure (MOF). This comprises 5% of the total cases [The individuals who develop symptoms and/or are exposed to the virus are tested for COVID-19. A nasal swab is collected as a sample. During the analytic stage, real-time reverse transcription polymerase chain reaction (RT-PCR) is the molecular test of choice for the diagnosis of SARS-Cov-2 infections, whereas antibody-based tests were introduced as a supplemental tool. Post-analytically, the results should be interpreted carefully with the help of both the molecular and serological findings .People across the world are practicing social and physical distancing to control the spread of this fatal virus. Those who have been exposed to someone who is infected with the disease must be quarantined, and those who test positive and develop symptoms must isolate for 14 days. According to a study conducted in China, the median incubation period for COVID-19 was estimated to be 5.1 days with a 95% confidence interval with a range of 4.5 to 5.8 days. Approximately 97.5% of those who will develop symptoms will do so within 11.5 days with 95% CI, with a range of 8.2 to 15.6 days [The causes of death from COVID-19 range from acute respiratory distress syndrome , type I respiratory failure , sepsis , acute cardiac injury 72/94; 77%), heart failure , alkalosis , hyperkalemia , acute kidney injury , and hypoxic encephalopathy  [2/94; 77%Several studies have demonstrated how the COVID-19 hyperinflammatory response is one of the primary causes of death that affects the heart vessels, liver, kidney, and other organs. Macrophages are the major cells that are activated in response to an injury by supporting and activating the stem/progenitor cells, clearing the damaged tissue, remodeling the extracellular matrix to prepare the scaffolding for regeneration, and promoting angiogenesis -10.Postmortem pulmonary findings in COVID-19 patientsThe bronchoalveolar fluid in patients with severe COVID-19 has shown a large quantity of chemokine from the macrophages; this was also evident in the immunostaining of postmortem tissue from a COVID-19 patient. The CD169+ lymph node, subcapsular, and splenic marginal zone macrophages expressed the SARS-CoV-2 entry receptor ACE2. They showed that these macrophages contained the SARS-CoV-2 nucleoprotein (NP), although the small conditional RNA (scRNA)-seq analysis of human tissue failed to identify an ACE2 expression on most tissue-resident macrophages . AccordiMinimally invasive autopsies of three COVID-19 patients in Chongqing, China revealed damage to the alveolar structure with minor serous and fibrin exudation and hyaline membrane formation . The celPostmortem cardiovascular findings in COVID-19 patientsLike other viruses, coronavirus is associated with cardiomyopathy and myocarditis. Infected patients may develop lymphocytic, eosinophilic, or giant cell/granulomatous myocardial inflammation, which results in infectious dilated cardiomyopathy . The myoHematology, renal and other findings in COVID-19 patientsA study that was performed in Brazil showed fibrin thrombi in the alveolar arterioles of 80% of patients with COVID-19. This is evidence of the hypercoagulable state in severely ill patients, which leads to a ventilation-perfusion mismatch in the lungs and a peripheral ischemic event . The hypAutopsy reports of 10 patients from Hospital Graz II, Austria, which is the second-largest public and academic teaching hospital in Austria, showed thrombosis of the small-sized and mid-sized pulmonary artery at different stages in all patients, bronchopneumonia in six patients, and hepatic congestion in eight patients. The other findings in the liver included hepatic steatosis, portal fibrosis, lymphocytic infiltrates and ductular proliferation, lobular cholestasis, and acute liver cell necrosis, along with central vein thrombosis. Other common findings included renal proximal tubular injury, focal pancreatitis, adrenocortical hyperplasia, and lymphocyte depletion of the spleen and lymph nodes . AlthougThe hematologic dysfunction can be explained by the virus-induced procoagulant and coagulopathy, virus invasion, and damage of the lymphocytes. This resulted in lymphopenia, which is the depletion of white pulp that leads to a predisposition to deep vein thrombosis and pulmonary thromboembolism .Extrapulmonary findings from certain comorbidities, such as diabetes and hypertension, are related to septic shock, and other changes, including superficial perivascular dermatitis, orchitis, myositis, myocarditis, alteration in the renal glomeruli, and endothelium of small vessels and cerebral cortex, are related to the COVID-19 virus .The most common postmortem findings were the pulmonary findings, which included diffuse alveolar damage in almost all cases. Other pulmonary findings, including acute respiratory distress syndrome and tissue from the lungs, were usually positive for the 2019-nCoV antigen. The other significant extrapulmonary findings require further research, as understanding the pathological findings will be helpful for the treatment plans, including the use of anticoagulants in patients in hypercoagulable states. There is a strong association with the hyperinflammatory state, which can be explained by most of the signs and symptoms that are exhibited by COVID-19 patients, including most of the pathological findings.


More than 30 years ago, the Nordic Gene Bank established a long-term experiment on seeds stored under permafrost conditions in an abandoned mine corridor in Svalbard, as a tool to monitor storage life under these conditions. The study included seeds from 16 Nordic agricultural and horticultural crops, each represented by two or three cultivars . All seeds were ultra-dried to 3–5% moisture before being sealed in glass tubes. Germination tests were performed in accordance with the International Seed Testing Association (ISTA) protocols. At the initiation of the experiment, the samples showed good germination with the median value at 92%. The overall picture remained stable over the first twenty to twenty-five years. However, the variation became larger over time and at 30 years, the median value had dropped to 80%. At the lower end, with a high drop in germination, we found rye, wheat, and English ryegrass. At the upper end, we found Kentucky bluegrass and cucumber. The lowest germination was found in samples with the highest initial seed moisture levels. Pre-storage conditions are likely to be of major importance for longevity.  The rock temperature was stable, which meant that it was independent of an external energy supply. As a tool to monitor storage life under these permafrost conditions, a long-term seed storage experiment was initiated. The experiment started in 1986 and included samples for germination monitoring until 2086, thus it was termed “the 100 year experiment”. Important crops for Nordic agriculture and horticulture were included. The investigation is still ongoing, and in this paper, we summarize the results after the first 30 years.Most food plants produce seeds that can be stored under low temperature and moisture conditions. Much of our knowledge on seed longevity is based on artificial ageing experiments, where seeds are exposed to suboptimal conditions of elevated temperature and moisture for some weeks, and storage life is predicted based on the seed moisture content, storage temperature, and seed lot characters ,2. Such The overall picture of seed germination development over storage time across all accessions (species and cultivars) is illustrated by boxplots . A lowerCucumis sativus L.) lots and the two rye  lots. This also shows that interspecies differences are not of importance for explaining seed longevity. The seeds of all the species remained viable after 30 years in permafrost under the given conditions . Crops, but also cultivars, within the same crops showed different results. Similar patterns have also been observed in other long-term experiments, both under ambient ,17 or −1Solanum lycopersicum L. var. lycopersicum) [For instance, the potential seed longevity of barley was found to be best if it was harvested a week or so after grain filling was completed . The samersicum) . Furtherersicum) ,27. In oLolium perenne L.) lots , two out of four wheat (Triticum aestivum L.) lots , one of the three timothy (Phleum pratense L.) lots (Timothy 1), one of the three barley (Hordeum vulgare L.) lots (Barley 1), and in the one seed lot of meadow fescue (Schedonorus pratensis (Huds.) P. Beauv.). The literature shows that especially rye, but also some forage grasses, can be relatively short-lived [Lactuca sativa L.) lots , two of the three carrot (Daucus carota subsp. sativus (Hoffm.) Schübl & G. Martens) lots , one of the four wheat lots (i1-Wheat), one of the three barley lots (Barley 2), one of the three timothy lots (Timothy 2), one of the two cauliflower (Brassica oleracea L. var. botrytis) lots (Cauliflower 2), one of the two oilseed rape (Brassica napus L.) lots (Rape 1), and one out of the three red clover (Trifolium pratense L.) lots (Redclover 1). The most long-lived, with a loss in germination less than 5% after 30 years of storage, were found in all of the three beet (Beta vulgaris L.) lots, all of the three onion (Allium cepa L.) lots, both of the cucumber lots, both of the Kentucky bluegrass (Poa pratensis L.) seed lots, the only cabbage (Brassica oleracea L.) seed lot, and in the last of the red clover, cauliflower, barley, wheat, oilseed rape, carrot, and lettuce seed lots. Other studies have also shown that beet seeds as well as cucumber seeds have retained a high germination level over time, but, in contrast to our study, onions are generally found to be short-lived [Our results show good performance for many of the vegetables, while the picture is more varied for cereals and forage species . Table 1rt-lived ,29,30,31rt-lived ,29,30,31For all samples, the variation between the highest and lowest moisture content over the ten years was between 0.3% and 0.8% . The datThe study has so far revealed valuable results concerning the longevity of seeds after 30 years in permafrost. Nine out of the 38 seed lots showed a germination loss exceeding 15%, which is the level recommended by the FAO for carrying out regeneration. Rye and ryegrass in particular showed a rapid decline, while many of the vegetables showed a low decline in germination. The results are relevant for the seeds in the first Nordic back-up collection stored in the abandoned coalmine. A given storage condition is an essential characteristic for comparing results with other experiments. Here, seeds were stored in permafrost with a stable sub-zero temperature. We had no reference material at −18 °C conditions. Despite the limited number of samples and the lack of a −18 °C control, the observations add knowledge about the longevity of seeds.In total, 38 seed lots covering 16 crops were included in the study . Each spThe lots were from different cultivars and origins. No cultivation details are available except that the seed lots should be of good quality. Initial germination tests were conducted (y0). Before storage, all the seeds were dried to 3–5% internal moisture content using a Munters  dehumidifier adjusted to 10% RH in a room at 25 °C. After that, they were placed in a freezer overnight before they were sealed in glass ampoules. Each seed lot was divided into sub-samples of 2 × 500 seeds for each withdrawal. The sub-samples were boxed and labelled with the date for withdrawal, transported to Longyearbyen, Svalbard, and placed in the abandoned coalmine corridor. The temperature inside the coalmine was measured as −3.5 °C (± 0.2 °C).The first set of samples was tested in December 1986 (year 0), with a plan to have the last tests in 2086 (year 100). Since the start of the experiment, 11 boxes have been retrieved. The moisture content of the seed was monitored from year 0 to year 10 . The gerFor cereals, in-between paper methods (BP) were used; the seeds were placed on one moist paper with a second paper on top, and thereafter rolled and placed vertically in plastic. Inside the roll, the seeds germinated, and the seedlings developed. For grasses, red clover, and vegetables (except for beet), a Jacobsen apparatus was used , which iR software was used for statistical examination. One value was missing for year 5 for red clover (cultivar Jokioinen). It was replaced with the average of the four nearest neighbor values: 76.0%. The short descriptor names of the eleven test results are year 0, year 2.5, year 5, year 7.5, year 10, year 12.5, year 15, year 17.5, year 20, year 25, and year 30, where year 0 represents the results of the trial that started in 1986. Summary statistics and boxplots were used to overview the data. The R function ‘time series’ was used to illustrate the fluctuation of the results over the 11 test occasions over the first 30 years. We calculated the loss in germination (Δ germ) by averaging the germination percentage of the three first test occasions  minus the test occasion at y30. We then categorized the samples into three: the most short-lived group , the intermediate group (Δ germ 5% to 15%), and the most long-lived .


This approach eliminates the generation of toxic gases, simplifies the washing steps, and reduces the time required to prepare rGO. To validate the proposed method, we present spectroscopical and morphological studies, using energy-dispersive X-ray spectroscopy (EDS), UV-visible spectroscopy, infrared spectroscopy, Raman spectroscopy, scanning electron microscopy (SEM), and transmission electron microscopy (TEM). Thermal gravimetric analysis (TGA) is used to analyze the thermal properties of GO and rGO. This eco-friendly method proposes a complete guideline protocol toward large-scale production of oxidized graphene, with potential applications in supercapacitors, fuel cells, composites, batteries, and biosensors.The oxidative exfoliation of graphite is a promising approach to the large-scale production of graphene. Conventional oxidation of graphite essentially facilitates the exfoliation process; however, the oxidation procedure releases toxic gases and requires extensive, time-consuming steps of washing and reduction to convert exfoliated graphene oxide (GO) into reduced graphene oxide (rGO). Although toxic gases can be controlled by modifying chemical reactions, filtration, dialysis, and extensive sonication are unfavorable for large-scale production. Here, we report a complete, scalable, and green synthesis of GO, without NaNO Althougon atoms . Keepingon atoms ,10 or oxon atoms ,12. The on atoms , whereason atoms ,15,16. Son atoms ,18,19 toon atoms ,21.2−sp3 hybridization as a direct consequence of the degree of oxidation acquired during the oxidation process [4 and NaNO3 in concentrated H2SO4. This procedure involves the generation of toxic gases, such as NO2 and N2O4, and the residual Na+ and NO3− ions are difficult to remove during the processes of synthesizing and purifying GO [Typically, the oxidation–reduction of graphite uses strong oxidizing agents to yield graphene oxide (GO), an insulating carbon material . To date process  . Hummerfying GO ,30,31. W3. The authors successfully demonstrated that this modification does not decrease the yield of the product, but reduces toxic gas evolution and makes the disposal of wastewater more straightforward due to the absence of Na+ and NO3− ions [To circumvent these environmental and technical issues, Chen et al.  have impO3− ions . NeverthO3− ions . Hence, To support this direction, Abdolhosseinzadeh et al.  reported3 [To the best of our knowledge, a reliable method with the capability to supply the large demand for pristine GO and (undoped) rGO by using a green approach, a short-sonication time, and viable washing steps is still lacking. In this communication, such a method is presented, considering the environmental problem and technical limitations. We successfully demonstrate, through several spectroscopy and morphological studies, an eco-friendly and complete guideline protocol for large-scale production of GO by excluding NaNO3  and addi3 .2SO4, ACS reagent, 95.0–98.0%), potassium permanganate , hydrochloric acid , and citric acid  were obtained from Sigma Aldrich. All chemicals were used as received, without further purification. Graphite powder , sulfuric acid  was added, and the solution was stirred for 20 min at 2O2, 30%, Merk) and stirred up to turn the color of the solution from dark brown to yellowish. The resulting graphite oxide suspension was washed with 1:10 HCl solution and distilled water several times, each for 10 min by means of centrifugation at 3000 rpm until the pH was at ~6. The precipitated material was dried at 80 °C for 24 h.It is important to stress once more that the synthesis of GO is based on the approach of Ref. . HoweverInitially, 50 mg of resulting solid was dispersed in 500 mL of distilled water by sonication for 0.5 h, employing an ultrasonic bath  in continuous operation. Bath sonication is preferred to tip sonication because the direct positioning of the tip in the medium results in strong material fragmentation . The obtThe characteristic absorption spectra of GO and rGO were recorded using UV-vis spectroscopy . Infrared spectra were collected using a Fourier transform infrared spectrometer Jasco FT/IR-4000 . The thermal stability of GO and rGO was investigated using thermogravimetric analysis . The structure and surface morphology of the obtained materials were taken out on a transmission electron microscope  operating at 80 kV, and a scanning electron microscope  equipped with a JEOL-made dispersive X-ray spectrometer (EDS) with the accelerating voltage of 15 kV. Raman spectra were obtained using a Jasco NRS-500  spectrometer with a 532 nm laser wavelength . SEM samples were prepared by drop casting on aluminum substrates and dried at 80 °C for 2 h. Similarly, TEM and Raman samples were prepared by drop casting onto formvar-coated copper grids and glass substrates, respectively.The main intent of this work is to propose a general eco-friendly approach to large-scale production of oxidized graphene. In this sense, we have selected CA because it is a green and low-cost alternative for conventional reducing agents of GO  , C−1), C−1), and C−1). The broad peak observed at 3426 cm−1 is due to the hydroxyl groups  and more stable oxygenated functional groups, respectively [ectively . On the ectively . TGA cur−1 and the G peak at −1. Other less intense peaks also have been detected: the 2D peak at −1 and the D+G peak at −1. The D and D+G (the combination of D and G bands) peaks are ascribed to structural imperfections of GO and rGO  wD/IGI) is a common index for the density of defects on GO and rGO. As shown in earlier studies [The peak ratio between the intensity of D and G peaks  of rGO is shifted with respect to the 2D peak (−1) of GO. The latter was impossible to be fitted, and the maximum intensity value was removed from the data (−1. Furthermore, the 2D peak became more symmetrical in rGO, indicating the presence of few-layer-reduced graphene oxide.Unlike the 2D peak of monolayer graphene, which has high intensity, the 2D peak of GO and rGO is complex in shape and evolves with the number of layers . The prethe data c. The reSEM and TEM micrographs of GO and rGO are shown in 3 [.This asseveration is supported by TEM analysis. 3 ,30.Figu3 in the oxidation and adding CA in the reduction, which demonstrated to be an effective and inexpensive reducing agent for GO. EDS measurements showed a reduction effectivity from 0.5 to 1.5 h, while GO is in contact with CA under vigorous agitation. UV-visible spectra confirmed the reduction of GO into rGO. FTIR analysis demonstrated the presence of oxygen functional groups, which decreased in intensity after reduction. The thermal stability of rGO was found to be higher compared to GO. Raman spectra showed a high intensity of the D peak in GO, indicating the presence of basal/edge defects; however, the characteristic 2D peak of graphene arose in rGO. SEM measurements showed that the semitransparent layers were relatively free of impurities. TEM analysis presented similar features. However, rGO exhibited a more regular, transparent, and thin nanosheet. The proposed method prevents toxic gas evolution, prolonged sonication, and complicated washing steps, making it effective at preparing oxidized graphene on a large scale.In summary, we have established a complete and green protocol for the preparation of GO and rGO by excluding NaNO