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+{"text": "Although overall incidence is rare, leukemia is the most common type of childhood cancer. It accounts for 30% of all cancers diagnosed in children younger than 15 years. Within this population, acute lymphocytic leukemia (ALL) occurs approximately five times more frequently than acute myelogenous leukemia (AML) and accounts for approximately 78% of all childhood leukemia diagnoses. Epidemiologic studies of acute leukemias in children have examined possible risk factors, including genetic, infectious, and environmental, in an attempt to determine etiology. Only one environmental risk factor (ionizing radiation) has been significantly linked to ALL or AML. Most environmental risk factors have been found to be weakly and inconsistently associated with either form of acute childhood leukemia. Our review focuses on the demographics of childhood leukemia and the risk factors that have been associated with the development of childhood ALL or AML. The environmental risk factors discussed include ionizing radiation, non-ionizing radiation, hydrocarbons, pesticides, alcohol use, cigarette smoking, and illicit drug use. Knowledge of these particular risk factors can be used to support measures to reduce potentially harmful exposures and decrease the risk of disease. We also review genetic and infectious risk factors and other variables, including maternal reproductive history and birth characteristics. Although overall incidence is rare, leukemia is the most common type of childhood cancer, accounting for 30% of all cancers diagnosed in children younger than 15 years . Within In the United States, the incidence of childhood ALL increased between 1975 and 2002; however, this increase was not statistically significant. The observed increase may be due in part to changes in diagnostic practice and accuracy and improved reporting. The incidence rate of childhood ALL is now stable at three to four new cases per year per 100,000 children who are younger than 15 years, with a peak incidence at approximately 2\u20135 years of age . For chiThe incidence of ALL, particularly T-cell ALL, is slightly higher among boys than girls. . Girls, Throughout childhood, the incidence rate of ALL among African American children is approximately half the rate among Caucasian children. During the first few years of life, the incidence rate of AML among African American children is approximately one-third the rate among Caucasian children; however, African American children \u2265 3 years of age have higher rates than Caucasians .Until the early 1980s, leukemia was the most common cause of death due to cancer among children in the United States . The morThe overall cure rate for childhood ALL is now approximately 75\u201380%; however, for AML the cure rate is between 40 and 45% . In earlEpidemiologic studies of acute leukemias in children have examined a number of possible risk factors  in an effort to determine the etiology of the disease. Only one environmental risk factor (ionizing radiation) has been significantly linked with either ALL or AML; most environmental risk factors  have been weakly or inconsistently associated with either form of childhood leukemia.Because childhood leukemia is a rare occurrence, prospective studies are difficult to conduct, and therefore studies most frequently use a retrospective case\u2013control design. Although easier to conduct, these studies have several inherent limitations . For exaThe environmental risk factors we discuss in this article include some of the more researched and controversial topics including ionizing and nonionizing radiation, chemicals such as hydrocarbons and pesticides, alcohol, tobacco use, and illicit drug use. Knowledge of these particular risk factors can be used to support measures to alleviate potentially harmful exposures and reduce the risk of disease. We also review genetic and infectious risk factors and other variables, including maternal reproductive history and birth characteristics.Ionizing radiation is one of the few exposures for which the causal relationship with childhood leukemia, particularly AML, has been established . The magNumerous epidemiologic studies have investigated the potential association between childhood leukemia and paternal ionizing radiation exposure in the workplace before conception or preconception, also referred to as \u201cpreconception paternal irradiation\u201d (PPI). Some studies have found an association , whereasScotland from 1958 to 1990 , includiOntario, Canada, near five nuclear installations Southern England, near two sites , and in With regard to diagnostic X rays, Mechanisms of potentiation by PPI are discussed in detail elsewhere . Maternain utero radiation exposure and the development of leukemia\u2014particularly related to the atomic bomb exposures in Japan at the end of World War II, the 1986 Chernobyl (Russia) accident, or exposure to diagnostic X rays. An increased risk of childhood leukemia in the offspring of atomic bomb survivors has yet to be shown  and developed leukemia in less time when compared with adults . SeveralAlthough inconclusive, to date, radiation exposure secondary to the Chernobyl accident has not been shown to increase the risk of leukemia in children who were exposed after birth . Recent Numerous epidemiologic studies have been conducted to determine whether an association exists between exposure to nonionizing EMFs and childhood leukemia; some have found a small association  while othttp://www.ehponline.org/docs/2006/9023/abstract.html).The chemical classes most commonly associated with childhood leukemia are hydrocarbons and pesticides. Studies have examined the relationship between childhood leukemia and direct exposure to these chemicals   as well Hydrocarbons are organic compounds made up primarily of carbon and hydrogen atoms. Examples of hydrocarbon compounds include gasoline and trichloroethylene (spot remover). Hydrocarbons are found in many household and industrial products including paint removers and thinners, and solvents, which are used to dissolve other chemical substances.http://www.ehponline.org/docs/2006/9023/abstract.html).The most widely recognized hydrocarbon is benzene, a ubiquitous chemical used in the manufacture of paints and plastics and as a constituent in motor fuels and hobby glues. It is also formed during incomplete combustion of fossil fuels . Benzene is a known human carcinogen. It has a strong positive exposure\u2013response relationship with leukemia, particularly AML, at a range of exposures not much higher than the current legal standard for workers as recommended by the National Institute for Occupational Safety and Health . A recenIn a review by In a separate large case\u2013control study, only paternal long-term exposure to plastics during the preconception period showed an increased risk of childhood leukemia, though not statistically significant  .There is much speculation about the biological mechanism by which paternal occupational exposures may affect the offspring\u2019s risk for leukemia. Potential mechanisms include genetic alteration of the father\u2019s sperm, which may transmit cancer susceptibility to the child, transplacental fetal exposure after the father brings a toxic exposure into the home, and direct postnatal exposure to chemicals brought home on the clothing   or indirSpecific occupations found more frequently among mothers of children with leukemia include metal manufacturing or processing, textiles, and pharmacist . IncreasA recent study in California suggested that children living in areas with high levels of point source carcinogenic hazardous air pollutants (HAPs) are at an increased risk of developing leukemia  . Selectea]pyrene   Table 2Table 2. In a recent case\u2013control study conducted in France, for the first time insecticidal shampoo treatment of pediculosis was found to be associated with childhood ALL and AML  . VariousStudies have shown an increased risk for AML, particularly among very young children, associated with maternal alcohol consumption during pregnancy . Shu et It is unclear whether maternal or paternal cigarette smoking before or during pregnancy is a risk factor for developing childhood leukemia . Two stuMaternal marijuana use before and during pregnancy has been associated with childhood AML and ALL. Despite a small sample size, findings from a Children\u2019s Cancer Group study showed a 10-fold risk of childhood AML associated with maternal use of marijuana just before or during pregnancy . The autin utero genetic events has been suspected for many years based on concordance studies on twins with leukemia  from newborns who later developed ALL (This concept that some cases of childhood leukemia originate oped ALL . Infant oped ALL .in utero (CYP1A1) and NAD(P)H quinine oxi-doreductase 1 (NQO1) variants has been found to be worse than that of patients who lack these variants /E2A-PBX1] support the theory that not all cases of childhood ALL develop in utero . The assin utero . Furthervariants .Although genetic syndromes account for only a small proportion of childhood leukemias, certain inherited diseases are associated with a higher risk of developing leukemia. Examples of these diseases include Fanconi anemia , Bloom sSiblings of children with leukemia are at greater risk of developing leukemia than children whose siblings do not have the disease . Also, ahttp://www.ehponline.org/docs/2006/9023/abstract.html).Several observations contribute to the theory that a transmissible agent is potentially involved in the oncogenic process of childhood leukemia. First, the peak incidence of childhood leukemia and that of common childhood infections both occur among children 2\u20135 years of age, the age group least likely to possess sophisticated immune systems . Second,An alternative hypothesis to Kinlen\u2019s suggests that the mechanism of \u201cdelayed infection\u201d is responsible, and that an abnormal response to the infection promotes a second event critical for the development of ALL . SupportConflicting results have been reported from studies of the relationship between maternal history of fetal loss and risk for ALL or AML . A histop < 0.001) and father\u2019s  ages  ages . For chi02) ages .Several studies have described an association between increased birth weight and childhood ALL, particularly ALL developing early in life . Increashttp://www.ehponline.org/docs/2006/9023/abstract.html).For information on medications/therapeutic agents, military history and nutrition in relation to childhood leukemia, see Supplemental Material  is an important step in the reduction of the overall burden of the disease. In general, benzene and ionizing radiation are two environmental exposures strongly associated with the development of childhood AML or ALL. However, identifying other environmental risk factors associated with leukemia is difficult for several reasons:inability to confirm and quantify exposures;lack of a prospective cohort;presence of different variants of leukemia; presence of confounders; andinadequate understanding of the pathophysiology of leukemia .Future attempts to overcome these obstacles  could possibly uncover other environmental risk factors associated with childhood leukemia and mitigate potentially harmful exposures to reduce the risk for disease. Future studies, when appropriate, should attempt to use common questionnaires, address timing and route of exposure, document evidence that exposure has actually been transferred from the workplace to the child, and store biological samples when possible.In the Abstract and in the section \u201cRisk Factors,\u201d the sentences \u201cOnly two environmental risk factors (benzene and ionizing radiation) have been significantly linked to ALL or AML\u201d in the original manuscript published online have been changed here to \u201cOnly one environmental risk factor (ionizing radiation) has been significantly linked to ALL or AML.\u201d"}
+{"text": "However, few data exist associating parental exposures and ras mutations in their children. Now a team including NIEHS grantee Leslie L. Robison of the University of Minnesota report that parents\u2019 chemical exposures may be associated with distinct ras mutations in their children with acute lymphoblastic leukemia (ALL).A variety of carcinogens have been shown to induce ras mutations. A total of 127 out of 837 ALL cases exhibited ras mutations in the K- or N-ras genes. Earlier studies have reported a 5\u201320% frequency of ras mutations among patients with ALL.This study used data from a large case\u2013control study of childhood ALL conducted by the Children\u2019s Oncology Group in Southern California. DNA samples from the study children were examined for ras mutation in the children. Use of drugs such as marijuana, LSD, and cocaine was associated with increased risk of N-ras mutation . Paternal use of amphetamines or diet pills was associated with a four-fold increase in N-ras mutation. Maternal exposure to solvents and plastics during pregnancy raised the risk of K-ras mutation about three-fold and seven-fold, respectively, and maternal exposure to plastics after pregnancy was associated with an eight-fold higher risk. Maternal and paternal exposure to oil and coal products and other hydrocarbons before and during pregnancy was associated with about a two-fold greater risk of N-ras mutation.A number of parental chemical exposures were associated with significantly increased risks for ras mutations. The authors conclude that parental exposures to hydrocarbons and mind-altering drugs, chemicals that have been previously suggested to increase the risk of childhood leukemia, are related to specific ras mutations in childhood ALL.In previous studies, parental occupational exposure to hydrocarbons  has been linked to elevated childhood leukemia risk. The present study has extended these findings to include drugs of abuse and additional chemical exposures, and to link them to"}
+{"text": "I read with interest the recent review by Although atomic bomb survivors offer the clearest evidence of leukemia risk after childhood exposures to ionizing radiation, studies of children exposed to fallout in other contexts should not be downplayed. In utero exposure to ionizing radiation has been a known causal factor for childhood cancer for > 50 years. Although The association between preconception paternal irradiation (PPI) and childhood leukemia has always been controversial. Two of the major objections to the \u201cGardner hypothesis,\u201d as When interpreting the evidence for a PPI effect in atomic bomb survivors, it is important to consider what is known about potential mechanisms. As reviewed by To summarize, it is not unreasonable to observe that the weight of evidence generated to date supports the idea that preconception, prenatal, and postnatal exposures to ionizing radiation are all risk factors for childhood leukemia."}
+{"text": "Environ Health Perspect 115:138\u2013145 (2007)], benzene was incorrectly noted to be associated with the development of childhood acute lymphocytic leukemia (ALL) and acute myelogenous leukemia (AML). The authors note that although benzene is a known carcinogen associated with adult leukemia, in general, it is not associated with the development of childhood AML or ALL. Ionizing radiation is the only environmental exposure strongly associated with the development of childhood leukemia.In the conclusion of the article \u201cRisk Factors for Acute Leukemia in Children: A Review\u201d by Belson et al. ["}
+{"text": "Non-small-cell lung cancer (NSCLC) is the leading cause of cancer death. Early detection of NSCLC will improve its outcome. The current techniques for NSCLC early detection are either invasive or have low accuracy. Molecular analyses of clinical specimens present promising diagnostic approaches. Non-coding RNAs (ncRNAs) play an important role in tumorigenesis and could be developed as biomarkers for cancer. Here we aimed to develop small nucleolar RNAs (snoRNAs), a common class of ncRNAs, as biomarkers for NSCLC early detection. The study comprised three phases: (1) profiling snoRNA signatures in 22 NSCLC tissues and matched noncancerous lung tissues by GeneChip Array, (2) validating expressions of the signatures by RT-qPCR in the tissues, and (3) evaluating plasma expressions of the snoRNAs in 37 NSCLC patients, 26 patients with chronic obstructive pulmonary disease (COPD), and 22 healthy subjects.In the surgical tissues, six snoRNAs were identified, which were overexpressed in all tumour tissues compared with their normal counterparts. The overexpressions of the genes in tumors were confirmed by RT-qPCR. The snoRNAs were stably present and reliably detectable in plasma. Of the six genes, three  displayed higher plasma expressions in NSCLC patients compared with the cancer-free individuals (All < 0.01). The use of the three genes produced 81.1% sensitivity and 95.8% specificity in distinguishing NSCLC patients from both normal and COPD subjects. The plasma snoRNA expressions were not associated with stages and histological types of NSCLC (All > 0.05).The identified snoRNAs provide potential markers for NSCLC early detection. Non-small-cell lung cancer (NSCLC) is the number one cancer killer in the USA and worldwide . The oveNon-coding RNAs (ncRNAs) are functional transcripts that do not code for proteins, however, play a major role in regulating almost every level of gene expression . In addiO-ribose methylation of rRNAs or snRNAs, whereas SNORAs are guides for the isomerization of uridine residues into pseudouridine [SnoRNAs represent one of the largest groups of functionally diverse trans-acting ncRNAs currently known in mammalian cells . NcRNAs ouridine ,21. Accuouridine . Furtherouridine ,23. AlthTo define and validate snoRNA signatures whose altered expressions were associated with early stage NSCLC, we obtained surgical specimens from 22 stage I NSCLC patients who had either a lobectomy or a pneumonectomy. The cases consisted of 11 patients with squamous cell carcinoma (SCC) and 11 patients with adenocarcinoma (AC) . Pearson test showed that each gene's the relative expression level by RT-qPCR and log 2-transformed signal intensity value by microarray significantly correlated . The observations suggested that the identified snoRNAs could be confirmed by a different technique, and thus the differential expressions of the identified genes in the surgical specimens were based on accurate quantification. Furthermore, no significant expression difference of the six snoRNAs by RT-qPCR (P > 0.05) was observed for the stage I tumor samples with different histological types. The observation provides further evidence that the elevated snoRNA expressions in cancer tissues are not histologically specific changes.Although previous reports have documented presence of detectable quantifies of miRNA in plasma, whether snoRNAs stably exist in the cell free body fluid remain unknown. To determine if the snoRNAs were present in plasma, we first prepared two RNA pools containing equal amounts of RNA from plasma of five cancer patients and five healthy individuals, respectively. Expression of each snoRNA was then measured by RT-qPCR in the pooled RNA samples. All tested snoRNAs had \u2264 32 Ct values in both pools, indicating that the snoRNAs existed in plasma. To determine if the snoRNAs were stably and reliably detected in plasma, we first assessed the stability of endogenous ncRNAs in plasma, because it contains high levels of RNase activity. Plasma obtained from three healthy subjects was split into two parts, respectively. One part of each sample was treated with Ribonuclease A at final concentration of 10 \u03bcg/mL, whereas the second part was not added with Ribonuclease A. Expressions of the six snoRNAs and a miRNA, miR-21, were measured by using RT-qPCR in parallel. The abundance of all snoRNAs and the miR-21 in the plasma samples with the different treatments was fairly equable (P > 0.05) , demonstrating that the plasma snoRNAs were resistant to RNase digestion. To further verify the stability of the plasma snoRNAs, plasma was collected from another three healthy individuals. Each sample was divided into 4 parts. The first aliquot from each specimen was processed immediately for isolating RNA, while others were stored in -80\u00b0C and processed for RNA isolation on day 1, 7 and 30. Expressions of the snoRNAs were measured at the same time in these specimens that were processed from the different time points. Expression level of miR-21 was also simultaneously assayed on the specimens. Each of the snoRNAs and miR-21 displayed equal expression levels between the samples, respectively . Therefore, like miRNA, snoRNAs are present in a stable form and consistently measurable in archived plasma samples.To determine specificity of snoRNA quantification by RT-qPCR assay in plasma, SNORD76 and SNORD78 that are located in the same chromosomal region (1q25.1), were synthetically generated . Each one subjected to two independent RT-qPCR reactions, where in each reaction there were present PCR primers specific to only one of the two genes. Amplification only of the appropriate gene matching the specific primer was observed, indicating that RT-qPCR assay could detect snoRNA with high specificity. Furthermore, given that the average size of snoRNAs (70 to 90 nt) was longer than that of miRNAs (22 nt), we evaluated whether the RT-qPCR assay would only detect snoRNA but not DNA sequences. RNA preparation extracted from two plasma samples was divided into two identical portions, which were then treated with or without DNase I Reaction Buffer, respectively. The expression level of the snoRNAs in each of the two groups was quantified by RT-qPCR. There was no difference in the gene expression levels between RNA extracted from the samples treated with DNase I and RNA from the samples without treatment (P < 0.05). The result suggests that the snoRNAs could be specifically detected without contaminating genomic DNA in RNA preparations.To determine the sensitivity of detecting snoRNA by RT-qPCR assay in plasma, the total RNA was isolated from plasma of three healthy subjects and then diluted in diethyldicarbonate water by ten orders of magnitude, respectively. The serially diluted RNAs served as experimental samples for measuring expression of the snoRNAs. The results showed excellent linearity between the RNA input and the Ct values for RT-qPCR. Furthermore, the assay had a dynamic range of at least six orders of magnitude (R2 = 0.997), and was capable of detecting al least 100 copies of the target snoRNA genes. Altogether, the snoRNAs could be accurately and reliably measured in blood plasma by the RT-qPCR platform.RT-qPCR assay was successfully performed in all plasma samples of 37 NSCLC patients and cancer-free individuals including 26 patients with chronic obstructive pulmonary disease (COPD) and 22 healthy subjects (Table Receiver-operator characteristic (ROC) analyses were applied to evaluate the capability of using the three snoRNAs to discriminate NSCLC patients from healthy individuals and COPD patients. As shown in Table We then evaluated the combination of the three snoRNAs for identification of NSCLC. The three snoRNAs in combination produced 0.8935 AUC, being considerably higher than those of each individual gene (All P < 0.05) in identification of NSCLC group from healthy group Table . Accordi2 < 0.50, P > 0.05). The data suggested that expressions of the snoRNAs were complementary to each other, and further supported that the combined analyses of the genes outperformed a single one used alone. Therefore, the panel of the snoRNAs provides a reasonable power for the early detection of NSCLC in plasma.Spearman rank correlation analysis indicated that the estimated correlations among expression levels of the three snoRNAs in plasma were low  . The finding that altered expressions of the snoRNAs are found not only in advanced stage, but also in early stage NSCLC might be an important characteristic if they are to be employed for early detection. Additionally, there were no significant differences of sensitivity and specificity for the panel of snoRNAs in discriminating lung SCC and AC patients from healthy controls (P > 0.05) . Therefore, the combined analysis of the three genes in plasma had equal diagnostic efficiency for the two major histological types of NSCLC. There was no association of the changes of the three genes with the age, gender, ethnic, and smoking packer-year of the participants (All p > 0.05) .In the present study, we profiled snoRNA expression signatures of early stage NSCLC by performing microarray analysis on surgical tissues. Aberrant expressions of the identified signatures were well confirmed by RT-qPCR assay. To the best of our knowledge, this is the first study to globally analyze snoRNA expression patterns in human tumor tissues. Furthermore, we demonstrated that like miRNAs, snoRNAs remained intact and were readily detectable in plasma by using RT-qPCR. More importantly, based on the discoveries, we developed a panel of plasma-based snoRNAs as potential biomarkers for early stage NSCLC. Our data might provide compelling evidence that dysregulations of the snoRNAs could play an important role in lung tumorigenesis, and measuring plasma snoRNAs might serves as a potential noninvasive approach to improve diagnosis of NSCLC.Although rarely being reported, malfunction of some snoRNAs have recently been considered to contribute to carcinogenesis. For instance, adeno-associated viruses integrated their genome into mouse genome, causing liver cancer . InteresMost of the previously identified lung cancer associated molecular genetic changes were related to the smoking status. Furthermore, some of the changes were associated with lung inflammatory diseases, especially COPD . The useAlthough the results look promising, the sensitivity (81.1%) and specificity 95.5%) of the snoRNAs are still not yet efficient for routine clinical application. To surmount the problem, we need to identify additional cancer-associated ncRNAs that can be added to the current ones so that the diagnostic efficacy of the plasma-based approach could be improved. The fundamental mechanism supports this premise is that although only about 352 snoRNAs were analyzed, more than 500 snoRNAs might exist in the human genomic sequences . Further.5% of thWe have defined and developed a panel of snoRNAs, whose altered expressions were associated with early stage NSCLC. We demonstrated that the snoRNAs existed in a stable form and were reliably measurable in plasma. Detection of the class of ncRNAs in plasma could potentially be used as a noninvasive diagnostic tool for early NSCLC. Nonetheless, a large multi-center clinical project to further validate the full utility is required before it could be adopted in routine clinical setting.To define and validate snoRNA signatures whose altered expressions were associated with early stage NSCLC, surgical specimens were obtained from 22 lung cancer patients who had either a lobectomy or a pneumonectomy between March 6, 2000 and June 23, 2003 at the University of Maryland Medical Center. All cases were diagnosed with histologically confirmed stage I NSCLC, comprising 11 patients with SCC and 11 patients with AC  as previously described ,16. The http://www.affymetrix.com). The normalized microarray data underwent further analysis as described in statistical section.SnoRNA profiling was performed by using GeneChipR Array . The array comprised 7,815 probe sets that were designed to analyze small non-coding RNAs. Microarray experiments were done with all 22 matched malignant and noncancerous sample pairs according to the manufacturer's instructions as described in our previous report . Briefly\u03bcmol/l of each primer and 2\u00d7 SYBR Green PCR Master mix (Qiagen). At the end of the PCR cycles, melting curve analyses were performed. All assays were performed in triplicates, and one no-template control and two interplate controls were carried along in each experiment. Expression levels of the snoRNAs were calculated using comparative cycle threshold (Ct) method as previously described [Expressions of the identified snoRNAs were first validated in the surgically resected tissues and then tested in plasma by using real-time SYBR green RT-qPCR assay. Briefly, 10 ng of plasma RNA was polyadenylated by poly(A) polymerase and reverse transcribed to cDNA using miScript Reverse Transcription kit  according to the manufacturer's instructions. RT-qPCR was performed using miScript SYBR Green PCR kit (Qiagen) with the manufacturer provided miScript Universal primer and the snoRNA-specific forward primers in ABI PRISM 7900 Real-time PCR system . The primers were designed based on the snoRNA sequences obtained from the gene database of The National Center for Biotechnology Information. The primer sequences for the snoRNAs and their amplification profile are available upon request. Each PCR reaction was carried out in a volume of 25 \u03bcl containing 2 \u03bcl of the cDNA, 0.1 escribed ,16. Ct vescribed ,16,45,46http://www.broad.mit.edu), BRB-ArrayTools version 3.6 (http://linus.nci.nih.gov/BRB-ArrayTools.html), and microarray software suite 4 (TM4) (http://www.tm4.org). We then performed tree visualization by using Java Treeview 1.0 . Pearson's correlations were used for the comparison of RT-qPCR and microarray data. ROC curve analysis was done using plasma expression level of each gene for the NSCLC patients, COPD patients, and normal controls by Analyse-it software . Using this approach, AUC identified optimal sensitivity and specificity levels at which to distinguish NSCLC patients from healthy individuals or COPD patients, and corresponding thresholds were calculated for each snoRNA. In addition, Spearman rank correlation was carried out to analyze the correlation between the expressions of the identified snoRNAs. Moreover, the associations between the expression levels of the snoRNAs and both clinicopathologic and demographic characteristics of the cases and controls were evaluated by using univariate and multivariate logistic regression models. All P values shown were two sided, and a P value of < 0.05 was considered statistically significant.To find snoRNAs that were differentially expressed between paired NSCLC specimens and corresponding noncancerous tissues, we first analyzed the normalized microarray data by using GenePattern (The authors declare that they have no competing interests.FJ designed the study, performed research, analyzed data and wrote the paper. JL, YM, JS, and LY performed microarray and RT-qPCR analyses. RL performed analyzed data and wrote the paper. ZL performed data mining analyses. MG recruited patients, obtained written consents, and collected clinical data. All authors have read and approved the final manuscript.SnoRNAs that show changes in clinical specimens of lung cancer patients. SnoRNAs differentially expressed in non-small cell lung cancer tissues versus normal lung tissues and plasma of cancer patients and control subjects.Click here for file"}
+{"text": "Daphnia pulicaria. We selected on size in each of four populations that differ in the frequency of sex, and evaluated correlated responses in a life table. Size at advanced adulthood, reproductive output, and adult growth rate clearly showed greater responses in high-sex populations, with a similar pattern in neonate size and r. This pattern is expected only when trait correlations are favored by selection and the frequency of sex favors the creation and demographic expansion of highly fit clones. Juvenile growth and age at maturity did not diverge consistently. The inter-clutch interval appeared to respond more strongly in low-sex populations, but this was not statistically significant. Our data support the hypothesis that correlated selection is the strongest driver of genetic correlations, and suggest that in organisms with both sexual and asexual reproduction, adaptation can be enhanced by recombination.Genetic correlations among traits alter evolutionary trajectories due to indirect selection. Pleiotropy, chance linkage, and selection can all lead to genetic correlations, but have different consequences for phenotypic evolution. We sought to assess the mechanisms contributing to correlations with size at maturity in the cyclic parthenogen  Genetic correlations among traits can alter the evolutionary trajectory of a trait across the adaptive landscape because traits are indirectly subjected to the selection pressures aimed at correlated traits  in one population, and by allelic variation at different pleiotropic genes in another population, the slopes could differ. Of course, if genes with pleiotropic effects on two traits were fixed within a population, there would be no correlated response to selection.A and B, are governed by variation at two linked loci, a and b. In one form of allelic variation, alternate alleles at loci a and b lead to different slopes of association between traits A and B. If allelic variation takes this form, we expect there to be no net trait correlation within a population, and thus our clonal selection on body size would yield no response in a second trait.If genetic correlations are driven by linkage, we expect that populations with different frequencies of sex will differ in their correlated responses. However, specific predictions depend on the underlying distribution of allelic variants. We can consider a simplification of the problem where two traits, a and b may lead to trait variation that slides along a common slope. Such variation would lead to the observation of genetic correlations among traits within a population. In this case, correlations that arise out of chance linkage are expected to be quickly eroded away by recombination in populations with relatively high frequencies of recombination. Therefore, correlated responses to selection would be stronger in populations with lower frequencies of sex. As these associations arise out of chance events, there is no force driving correlations to be similar in different populations, and therefore we expect slopes of association to differ among populations.Alternatively, alternate alleles at loci Daphnia, these fit clones can spread through demographic expansion, shielding highly favored combinations from sexual erosion.The situation under correlations which arise from linkage due to selection is more complicated. As with chance linkage, any correlations which do arise are going to be eroded by recombination, and we might expect this to result in populations with high frequencies of recombination to have smaller genetic correlations between traits than in populations with lower recombination. However, this expectation ignores the role of recombination in novel allele combinations with high fitness, and it is possible that the creative potential of sex outweighs the erosive effects of sex. If the net effect of sex is beneficial, and a particular correlation holds a selective advantage, sex would be creating high fitness clones that adhere closely to the optimal trait correlation. In taxa with the potential for asexual reproduction, such as If the erosion of favorable combinations outweighs their origin, the net effect of sex is detrimental. This may occur, for example, if the capacity for asexual demographic expansion is limited or if few of the potential combinations of alleles are in fact advantageous. In this case, the predicted differences between populations with high and low frequencies of sex are qualitatively similar to the situation under chance linkage: high-sex populations should have smaller correlated responses than low-sex populations. However, the magnitude of the difference would be smaller than in the chance linkage situation, because selection is still countering the erosion via sex to some degree.D. pulicaria from the field, established them as clonal lineages in the lab, and selected clones with extreme body sizes. In the second phase, we assayed life history characteristics of the selected clones in a common garden life table experiment.We divided our project into two phases. In the first phase, we isolated individual D. pulicaria  near the W. K. Kellogg Biological Station in southwest Michigan,  lakewater. These were initially kept at 10\u00b0C, at a 12:12 L:D photoperiod and fed a standard lab preparation of the green alga Scenedesmus auratus. Animals were allowed to reproduce asexually and thus form clonal lineages. In three populations, \u223c90% of the animals isolated resulted in lineages that were available at the start of the selection procedure. However, only about 50% of the animals isolated from Warner Lake yielded lineages. This resulted from a combination of relatively poor survivorship of the originally isolated individuals and a relatively high tendency for those who did survive to produce solely male offspring. In previous work on the Warner population  from our base population samples showed that observed heterozygosities in Bristol and Little Long matched expected heterozygosities, but that there was heterozygote excess in Pine and Warner. Inbreeding coefficient (FIS) estimates were 0.02 in both Bristol and Little Long, but significantly negative in Warner and Pine . The ratio of observed to expected multilocus genotypic diversity was significantly lower than expected in Warner (ratio = 0.4), Pine (0.3), and Bristol (0.5), but not Little Long (1.0). These results are consistent with greater effects of clonal selection in populations with lower frequencies of sex.A recent population genetic analysis . Prior to the initiation of selection, clones were acclimated to the eventual life history assay conditions: 20\u00b0C, 12:12 L:D, and 3 \u00d7 10We selected on body size in two episodes. In the first, we identified approximately 30 large and small clones in each population. In the second, these were further reduced to the 10 largest and smallest. As it was impossible to perform the initial body size assay on all clones concurrently, we divided the first episode of selection into two blocks, quantifying body size on half of the clones from each population during each block.To assay body size, we first divided each clonal lineage into two sub-lines in order to allow for any remaining maternal effects variation to be incorporated in within-clone variation  was chosen for the life table. These individuals were raised under the conditions described above, and observed daily. For each of the 10 replicate individuals per clonal lineage, we recorded length at birth, at maturity, and when the 4th clutch was released. We also recorded clutch size, offspring sex ratio, and date for the 1st through 4th clutches produced. From these data, we calculated composite traits including juvenile and adult specific growth rates , inter-clutch intervals and S) by subtracting the overall mean body size of a group in a particular body size assay from the mean body size of the clones selected at the completion of that same assay. The response to selection (R) was calculated by subtracting the overall mean body size of a group from the mean of the selected clones as measured in the subsequent assay. Broad-sense heritability (H2) was then calculated as the ratio R/S , frequency of sex in the field (high or low), population nested within sex frequency (treated as a random effects variable), and an interaction between sex frequency and selection. The interaction provides the critical test of our core predictions.We tested for correlated responses to selection on size at maturity in each population separately with clone as our unit of replication. We used univariate two-tailed We were interested in whether the slope of the association between size at maturity and other traits was similar across populations. We estimated regression slopes, and then tested whether they were significantly different among populations in separate linear models for each trait. In these models, the correlated trait was the dependent variable, with size at maturity, population, and a population-by-size at maturity interaction as independent variables. To determine if slopes are different across populations, we tested the significance of the interaction term.All statistical analyses were conducted in Systat 12.0 . Archived phenotypic data can be accessed from Dryad under doi: 10.5061/dryad.fv3cb.Body size of clones was distributed approximately normally in each base population . As variP < 0.0001 in all cases except Warner, where P = 0.0229). Size differences of the final set of clones selected from the second body size assay ranged from 0.45 mm (Warner) to 0.68 mm (Little Long). This meant that the average size of large clones was 30% larger than small clones in the Warner population, 39% larger in Little Long, 51% larger in Pine, and 53% larger in Bristol  and sex frequency or mean body size for the four populations. However, in three of the four populations, H2 was higher when selecting for small body size  than those with low-sex investment in the field . Linear models showed a significant sex frequency-by-selection interaction in L4, reproductive output, and adult specific growth rate, and marginally significant interactions for L0 and r  and again when the fourth clutch (L4) was released  and 7.2% (Little Long) (We measured the correlated response to selection in two size traits: body length at birth (Lreleased . Unsurprulations . L0 is nle Long) ; Table 4Correlated responses of the number of offspring produced were stronger in high-sex populations than in low-sex populations , but wer4 because it is scaled to body size. In contrast, juvenile specific growth rate showed no correlated response to selection on size at maturity in three of the four populations . ICI is not strictly a reproductive feature in nificant C.r. Age at maturity, adult growth, and inter-clutch interval each had a significant association in one population . Linear models testing whether slopes differed across populations  found no support for differences except in size at birth . Detailed results from the regression analysis are given in Supplementary Regression analysis unsurprisingly paralleled correlated divergence, with significant regression slopes generally for size, reproduction, and Understanding phenotypic evolution for a trait requires an understanding of the indirect selection pressures it experiences via genetic correlations. Those indirect pressures are a consequence of both the selection exerted on correlated traits, and the mechanisms of correlation by which phenotypic traits can be connected. We have experimentally evaluated the correlated responses to clonal selection on body size in populations that differ in their history of sexual recombination with the aim of understanding the mechanisms of connection. These differences allow us to potentially distinguish among pleiotropy, chance linkage, and selective advantage as mechanisms underlying genetic correlations. Furthermore, sexual recombination itself remains a significant puzzle in evolutionary biology because it simultaneously has the capacity to demolish and assemble genomes with high fitness . Of the potential mechanisms contributing to trait associations, this was predicted only when there was both selection for a particular correlation and the net effect of the higher frequency of sex was to promote adaptation through the creation of high fitness clones. If this is true, we would predict that the strengths of the correlations are tighter in the high-sex populations than in the low-sex populations. However, we cannot evaluate this prediction effectively because we did not conduct the final life table assay on the full set of clones initially isolated. Although our clonal selection procedure allows us to estimate the slope of the relationship efficiently, the absence of the middle-value clones from the life table means that we do not know how tight that relationship is.For three traits , the magnitude of divergence was clearly greater in populations with a high historical frequency of sex, and a similar trend was evident in two other traits  suggested greater divergence in low-sex populations. This could be consistent with a role for chance linkage or selection, if the net effect of sex was deleterious. There was no statistical support for differences among populations in the slope of the relationship , which iWe found little support for the notion that the association between body size and other life history traits differed among populations, except for size at birth. In principle, this can arise under any of the mechanisms contributing to genetic correlations. There was no significant difference between high and low-sex populations, but the trend was for high-sex populations to have a larger difference than the low-sex populations. This is opposite of what is expected under chance linkage. Under the mechanism of pleiotropies arising from different genes in different populations, there should be no effect of sex. However, even though the trend is insignificant, it is worth considering the possibility that selection is contributing to the correlation between size at maturity and size at birth, but that the optimal correlation differs among populations (discussed below).If genetic correlations among life history traits were largely driven by pleiotropies, recombination frequency should have no effect on the divergence of correlated traits when selection is applied directly to a key trait. In this case, we would expect to see the same degree of correlated divergence in all populations if each population harbored the same distribution of allelic variation, but this occurred for none of the traits examined. The magnitude of correlated divergence in age at maturity was idiosyncratic with respect to sex, with no evidence that the association slope differed among populations. This is consistent with pleiotropies that emerge from the same genes in each population, if allelic variation differs among populations.Daphnia is constrained by brood chamber size) would be moderate, and of timing traits would be small. Instead, traits from all three classes showed a strong effect of historical frequency of sex. This should not be interpreted to mean that pleiotropy is absent from these correlations, as it is possible for there to be a shared pleiotropic basis for a correlation that is common to all populations onto which selection builds the correlation further.Given that the traits we measured differ in their similarity to size at maturity, the directly selected trait, we expected some traits, particularly body size at later adulthood, to be driven by pleiotropy. For example, it seems reasonable that the shared genetic basis of size at other ages would be strong, of reproductive output  as a primary competitor. In addition, all four lakes have similar productivity, near the upper part of what is considered oligotrophic, as estimated from Spring measures of total phosphorus (C\u00e1ceres and Tessier We have argued that our four populations are ecologically similar, and in the broader context of potential habitats, they are. From the perspective of zooplankton like Nonetheless, the lakes are not identical, and this creates the potential for selection for differences in the precise correlations among traits. The predictions we described with regard to similarity or dissimilarity of correlations across populations are based on the assumption that the same correlations are selected for in each population. For the traits where we have inferred an important role for selection due to greater correlated differences in the higher sex populations, it is hard to see what could lead to similar correlations across populations if there were in fact selection for different correlations. Even if there were real differences in slope that we did not have the power to detect, that would still implicate selection as the driving mechanisms behind the correlations. In fact, the slope of the association with size at birth was different across populations, and this may be due to different selection pressures.r, this is not indicative of fitness of Daphnia in lakes, where seasonal succession and other environmental variation may have strong impacts on what traits and trait combinations are advantageous at different times. Long-term fitness in Daphnia in the field will depend on the contribution of an individual to future generations via both asexual and sexual reproduction, and genotypes may differ in the reproductive mode in which they have an advantage. It would be very interesting to compare the field performance of clones that differ in body size and degree of deviation from the correlation in their population.We have detected a quantitative genetic signature of selection on some correlations, but we do not have data that test the fitness consequences of the correlations in the wild. Although we estimated Daphnia, capitalizing upon the power of sex to create advantageous combinations of alleles probably depends on the opportunity for fit clones to expand through asexual reproduction. This raises the question of what constraints operate to determine an optimal frequency of sex in cyclic parthenogens, as it is easy to imagine that as the frequency of sex increases, the expansion of fit clones would decrease.Although genetic correlations of life history traits and the evolutionary consequences of recombination have been studied extensively, few attempts have been made to juxtapose them in a single study. This is somewhat surprising, because an essential element of debate about the evolutionary role of recombination is the direction and extent to which it influences favorable combinations of traits. Relatively few study systems offer an opportunity to consider populations that vary in recombination frequency, and most considerations of the effect recombination has on correlations have focused on examining genetic correlations before and after a limited number of rounds of recombination in exclusively sexual species. In our system, the only situation that is expected to yield stronger correlated responses to selection in high-sex populations compared with low sex is when genetic correlations result from selection for the correlation and the creative power of sex outweighs its destructive power. For Daphnia genome, and the long history of demographic and trait-based ecological work, means that Daphnia provide the opportunity to combine approaches with studies like ours to gain a more complete picture of complex trait evolution.It is apparent that no single approach will provide a general understanding of the evolution of complex traits. Currently, there is a creative tension between molecular methods that seek to reduce complex traits to variation at the nucleotide level, and quantitative genetic methods that seek to integrate the multifaceted influences on complex traits. Both are important avenues of research, as we have yet to reach a point where general, predictive principles can accurately define future evolutionary trajectories of interlinked, complex traits. The recent sequence of the"}
+{"text": "Purpose. The histological diagnosis of Mycobacterium tuberculosis (MTB) remains a diagnostic challenge despite different methods. Immunohistochemistry (IHC) not only could confirm granulomatous tissue involvement but also can demonstrate MTB antigen immunolocalization. This study tries to clarify the details of immunohistochemical staining for MTB with pAbBCG. Materials/Methods. Twenty-three confirmed TB granulomatous tissue samples were studied by Ziehl-Neelsen and immunohistochemistry (IHC) staining with pAbBCG. Samples were selected from the archive of the Department of Pathology, National Research Institute of Tuberculosis and Lung Disease, Tehran, Iran. Results. IHC staining was positive in all samples, whereas Ziehl-Neelsen was positive in 9 cases out of 23 (39.1%). Tissue types used were pleural tissue, lymph nodes, and lung tissue. IHC showed positive coarse granular cytoplasmic and round, fragmented bacillary staining. In this study, epithelioid cells clearly showed more positive staining at the periphery of the granuloma rather than the center of granuloma. There is also positive staining in endothelial cells, fibroblasts, plasma cells, lymphocytes, and macrophages outside the granuloma. Conclusion. Considering the criteria of positive immunohistochemical staining of TB granulomatous reactions, this stain not only highlights the presence of mycobacterial antigens for tissue diagnosis, but also could morphologically localize its distribution in different cells. Histological diagnosis of tuberculosis (TB) has long been an important issue in anatomical pathology. On the other hand, extrapulmonary TB comprises 10\u201315% of infections with Mtb. Following HIV prevalence increase, extrapulmonary TB is rising as well .Considering the limitations in sensitivity and specificity of Ziehl-Neelsen staining , mycobacGranulomatous reactions, and in some cases, nongranulomatous reactions such as the presence of foamy macrophages  or mycob4 bacilli per each slide is required to reach diagnosis.Detection of beaded bacilli (which are more commonly observed in the necrotic zone) by Ziehl-Neelsen staining of tissue specimens  indicateConsidering the mechanism of Ziehl-Neelsen staining and its relatively low sensitivity and specificity, this staining technique has limited diagnostic value and inadequate ability for pathophysiological assessment of Mtb antigens in the tissue. Searching for mycobacterial antigens using IHC is a well-recognized technique with special application in research projects. This technique is based on the production of a variety of polyclonal and monoclonal antibodies in tissue reactions. Several studies have reported the sensitivity of this technique can be 64 to 100% for the detection of mycobacterial antigen. Limited numbers of studies have discussed in depth positive tissue staining method for Mtb . HoweverRecent studies on the variable morphology of TB bacilli emphasize the importance of IHC in searching for different morphological variations of TB antigens in the tissue.The present study compares the sensitivity and specificity of the two techniques of Ziehl-Neelsen and IHC staining for field diagnosis of TB. It also evaluates the value of accurate description of IHC staining with pAbBCG in tissues with granulomatous reaction due to TB with regard to diagnosis, immunolocalization, and morphology of TB bacilli in this technique.A total of 50 patients undergoing simultaneous biopsy and tissue culture with positive tissue culture for Mtb during 2005\u20132009 were selected from the MRC Department Masih Daneshvari Hospital. Using the archives of the Pathology Department of this hospital, which is a referral center for pathological lung lesions, H&E slides of the selected patients were evaluated. Cases with small number of granulomatous lesions or tissue volume were excluded from the study. Eventually, 23 tissue samples of 23 TB patients with adequate tissue and number of granuloma were chosen for different staining techniques. Characteristics and type of samples were retrieved from the pathology reports. Paraffin-embedded blocks were stained using Ziehl-Neelsen and IHC staining. Seven control tissue specimens were also selected from the archives of the Pathology Department. These specimens had granulomatous reactions due to foreign body, fungi, or hydatid cyst. These specimens were also stained similar to the abovementioned samples. Mycobacteriology Research Center of Masih Daneshvari Hospital is the national reference center and educational collaborating center of the WHO office in Eastern Mediterranean Region. The study conducted after Masih Daneshvari Hospital ethic committee.2O2 were used for 10 minutes.For immunohistochemical staining, 3-4 micron sections were cut from the tissue block and incubated overnight at room temperature. After deparaffinization and fixation with 99\u201370% alcohol, specimens were rinsed with distilled water and PBS. In order to remove endogenous peroxidase, methanol and 3% HAfter retrieval and cooling down the tissues, they were rinsed with distilled water and PBS and specimens were blocked with antiserum. Antibody was prepared with Tween 20 + Tris (Merck 1.088387) at a 1\u2009:\u20091000 dilution. In the first phase, primary antibody  (pAbBCG) was reacted in a wet environment at room temperature for 30\u2009min and after rinsing the specimens with PBS, and secondary antibody (Envision K5007) was added. After staining with chromogen, hematoxylin was used as the contrast dye. For each staining, one negative and one positive control were also considered. Staining was done as fine granular cytoplasmic, coarse granular cytoplasmic and bacillus staining. Different areas and cells in tissue specimens were evaluated; type of granuloma (with or without necrosis), and presence of multi-nuclear giant cells, epithelioid cells, necrotic zones, lymphocytes, plasma cells, peri-granuloma macrophages and fibroblasts and perigranuloma endothelial cells were assessed by a junior and a senior pathologist. Positive staining was defined as staining of 10% of epithelioid cells in the granuloma. For other types of cells, any form of staining was considered as positive.Ziehl-Neelsen staining was performed according to the standard protocol. In summary, tissue specimens were deparaffinized and rinsed with consecutive dilutions of alcohol (96% to 70% ethanol). After heat fixation, specimens were washed with carbol fuchsin for 4 minutes and incubated with HCL. Counterstaining was done using Brilliant Green for 20\u2009s. After rinsing, samples were allowed to dry at room temperature.A total of 23 cases were evaluated, out of which 17 (73.9%) were males. Type of tissue in understudy cases was pleura , lymph node  , and lung tissue . Granuloma, in the form of necrotizing poorly organized granuloma, was 9 cases (39.1%), necrotizing well-organized granuloma in 5 cases (21.7%), necrotizing nonpoorly organized granuloma in 4 cases (17.3%), and non-necrotizing well-organized granuloma observed in 3 cases (13%). In all biopsy samples, IHC staining yielded positive results for both fine and coarse granular cytoplasmic and round bacillary staining in epithelioid cells , 19 (82.6%), 3 (13%), 5 (21.7%), 1 (4.3%), 8 (34.8%), and 1 (4.3%), respectively, . NecrotiZiehl-Neelsen stain method for the diagnosis of TB is positive in one-third of cases with confirmed TB infection. Detection of tuberculosis in tissue slides is still based on the histological pattern of the granuloma that has several differential diagnoses with different treatments .Presence of mycobacterial antigens and tissue morphology can be evaluated with IHC technique. Eliminating background with proper technique could provide 100% specificity if coarse granular cytoplasmic and bacillus staining are considered. These obtained results are comparable with the findings of a study by Goel and Budhwar, reporting 64\u2013100% sensitivity for IHC and zero to 44% sensitivity for Ziehl-Neelsen staining .Moreover, the results of monoclonal antibody testing  also shoIn order to achieve appropriate staining by this technique, great attention must be paid to dilution and technique of IHC with polyclonal antibodies. If adequate attention is not paid, fine granular staining in the background can cause false positive reactions provided that the pathologist is not familiar with this technique. This is among the main limitations of working with this type of antibody. However, in case of positivity of this staining as coarse granular cytoplasmic staining, stained fragmented bacillia or stained bacilli form, percentage of specificity for this staining technique is highly increased. It should be emphasized that coarse granular cytoplasmic staining or positively stained fragmented bacilli of any degree were not observed in control samples. These findings are in accordance with those of Madhu Goel and Budhwar, describing small bacillary fragments and antigenic dusts .On the other hand, new findings have demonstrated that morphological modifications of tubercle bacilli in circular or intermediate shapes even in the tissue can be live TB bacilli. This issue has been well described by Velayati et al. . In the Another finding is the presence of fragments of TB bacilli in macrophages, fibroblasts, plasma cells, lymphocytes and even endothelial cells outside the granuloma indicating that the mentioned cells play an active role in histopathogenesis of TB.A similar study  mentioneThus, aside from the positivity of lymphocytes for the presence of TB bacilli antigens in IHC staining with pAbBCG, our study is uniquely important in revealing the presence of these antigens in plasma cells, endothelial cells, and fibroblasts. This issue needs to be further investigated in terms of TB pathogenesis.Ziehl-Neelsen staining has low sensitivity and requires the presence of intact bacilli. Logani et al.  demonstrOur study showed that the IHC technique has high sensitivity and specificity. It also indicates localization of cells infected with mycobacterium or cells containing TB bacilli antigens. It somehow reveals the dissemination of TB bacilli and its antigens in tissues as well. In this study, epithelioid cells in the periphery of granuloma were stained more positively than the epithelioid cells at the center of granuloma or adjacent to the necrotic zone. Using anti-MPT64 in tissues affected with TB granuloma, Purohit et al.  found thThere are some limitations to this study. First, we have selected 23 cases from 50 specimens that were culture positive for MTB; thus, the study has bias to show sensitivity and specificity exactly. Secondly, pAbBCG is polyclonal antibody for Mtb complex. Although we have compared the sensitivity and specificity of this method with Ziehl-Neelsen staining, the aim of study is illustration of immunolocalization of Mtb Ag for use in diagnostic or research field, not comparing sensitivity with specificity. Further studies should be carried out on suspected tissues involvement with Mtb bacilli through IHC and use of monoclonal antibodies.Furthermore, pathologists must be acquainted with adequate staining pattern, elimination of background staining, and type of selected antibody. This method is especially important for use in countries with high prevalence of TB as a technique with early diagnostic value in tissue specimen. In early diagnosis, this technique can reduce related morbidity and mortality and decrease the rate of complications due to misdiagnosis and mistreatment of TB."}
+{"text": "Increased droughts due to regional shifts in temperature and rainfall regimes are likely to affect forests in temperate regions in the coming decades. To assess their consequences for forest dynamics, we need predictive tools that couple hydrologic processes, soil moisture dynamics and plant productivity. Here, we developed and tested a dynamic forest model that predicts the hydrologic balance of North Patagonian rainforests on Chilo\u00e9 Island, in temperate South America (42\u00b0S). The model incorporates the dynamic linkages between changing rainfall regimes, soil moisture and individual tree growth. Declining rainfall, as predicted for the study area, should mean up to 50% less summer rain by year 2100. We analysed forest responses to increased drought using the model proposed focusing on changes in evapotranspiration, soil moisture and forest structure . We compared the responses of a young stand  and an old-growth forest  in the same area. Based on detailed field measurements of water fluxes, the model provides a reliable account of the hydrologic balance of these evergreen, broad-leaved rainforests. We found higher evapotranspiration in OG than YS under current climate. Increasing drought predicted for this century can reduce evapotranspiration by 15% in the OG compared to current values. Drier climate will alter forest structure, leading to decreases in above ground biomass by 27% of the current value in OG. The model presented here can be used to assess the potential impacts of climate change on forest hydrology and other threats of global change on future forests such as fragmentation, introduction of exotic tree species, and changes in fire regimes. Our study expands the applicability of forest dynamics models in remote and hitherto overlooked regions of the world, such as southern temperate rainforests. Climate and forests are dynamically linked through the spatial and temporal variability of soil moisture Forest gap models use a variety of approaches to model forest hydrology. While some gap models use a simple bucket water balance model Forest gap models have successfully simulated the dynamics of a variety of forest types including temperate rainforests of the southern hemisphere This study introduces a forest gap model specifically designed for assessing the responses of temperate rainforests in southern South America to increased drought. The model provides accurate estimates of forest water fluxes and incorporates dynamical linkages among rainfall regimes, soil moisture, and individual tree growth. We assessed model performance by comparing the results with detailed field measurements of water cycling in a stand located on northern Chilo\u00e9 Island, Chile (41\u00b050\u2032S). We also conducted a sensitivity analysis of the response of current forests to drought, i.e. when rainfall is decreased. Model predictions of forest hydrology (evapotranspiration and soil moisture) and structure  under increased drought predicted for 2100 in the study area were compared for a young-secondary (YS) and an old-growth (OG) forest stand to analyze differences in responses to expected changes in rainfall.Estaci\u00f3n Biol\u00f3gica Senda Darwin (EBSD), with permission granted by the owner. Fragments of secondary and primary forests occur over rolling hills of low altitude (50\u2013100 m) dispersed in a matrix of bogs, shrublands and grazing pastures. The present landscape has been shaped by a history of widespread use of fire to clear land for pastures since the late 1800s, followed by selective logging of remaining forest patches The study was conducted on northern Chilo\u00e9 Island, Chile , Drimys winteri (Winteraceae) and Nothofagus nitida (Nothofagaceae), with the common presence of Tepualia stipularis (Myrtaceae) and several Myrtaceae tree species in the understory. Ferns  and angiosperms (e.g. Gesneriaceae and Bromeliaceae) growing epiphytically are frequent. Detailed descriptions of structure and dynamics of this forest type have been previously published Floristically, forests of the study area belong to the North Patagonian temperate rainforest type FORMIND-CL v.1.0) that includes calculations of hydrologic balance. The model is based on FORMIND, a forest model comprehensively tested to simulate the dynamics of temperate rainforests in SSA FORMIND is a generalized forest growth model that simulates the spatial and temporal dynamics of uneven-aged, mixed species forest stands t) as a mosaic of interacting forest patches of 20\u00d720 m, which is the approximate crown size of a large mature tree in the forest. Within these patches, stand dynamics is driven by competition for light and space following the gap model approach dbh). Tree mortality can occur either through self-thinning in densely populated stands, tree senescence, gap formation by large falling trees, slow tree grow, or external disturbances (e.g. windthrow). Gap formation links neighboring forest areas. Tree regeneration rates are formulated as maximum rates of recruitment of small trees at dbh threshold of 1 cm, with seed loss through predation and seedling mortality being incorporated implicitly Here, we introduce an individual-oriented dynamic forest model , vertically averaged over the soil depth z (mm), was considered as central state variable d is the Julian day of the year, n is the porosity ; dPnet is the net precipitation falling to the soil surface (mm day\u22121); dTr is the transpiration rate (mm day\u22121); and Q is the soil drainage (mm day\u22121). Both n and z are assumed to be time-invariant parameters \u03b8, m3 water/m3 soil, i.e. dimensionless) can be calculated as follows Soil moisture n\u2022z. Both the local vertical and horizontal variability of soil moisture are considered negligible at the daily timescale, assuming an equal propagation of the wetting front and equal soil moisture redistribution over the rooting zone dPnet) is described by,dEc is the canopy interception (mm day\u22121), defined here as the total daily rainfall  that is retained by the canopy and is evaporated so that it does not reach the ground. Following dEc asymptotically approaches the canopy retention capacity and can be modeled at daily intervals as:tS is the canopy water retention capacity of the stand at year t and h\u03b1 is a parameter describing the slope of the saturation curve. The parameter h\u03b1 represents, in a simplified terms, the complex process of water partitioning into throughfall and stem flow tS depends on leaf area index of the forest patch at simulated year t (tLAI) and is calculated by the expression maxLAI is the maximum leaf area index of the forest and hf is a shape parameter. We avoided unrealistic canopy interception values in the model by setting dEc\u200a=\u200adP when dEc>dP.Daily net precipitation falling to the soil surface ) was modeled according to s\u200a=\u200a1), soil water is permitted to percolate at a rate equivalent to the saturated hydraulic conductivity of the soil  from:PB is the gross biomass production of the tree (\u00b5mol carbon dioxide m\u22122 s\u22121), and WUE is a parameter denoting water-use efficiency at stand level. PB is obtained from the rate of single-leaf photosynthesis following PB  of trees is obtained from Tr by the length of the active photosynthetic period per year.Water-use efficiency describes the proportion of water used for the assimilation of a unit of carbon in the photosynthesis dPET, mm day\u22121) describes a physical limit for the amount of water that can be held and transported away from the canopy under given climatic conditions. Evaporation is neglected in the model; therefore, it is assumed that maximum water losses by vegetation are limited by the difference between dPET and the canopy interception of the day (dEc), as follows:dPET is calculated using a modified Penman-Monteith expression in case of aerodynamic conductance dRn, J m\u22122 day\u22121):\u03b3 is the psychometric constant , L is the latent heat of vaporization of water (ca. 2.56\u00d7106 J kg\u22121 slightly depends on temperature). The rate of change of saturated vapor pressure with temperature  is calculated as The daily potential evapotranspiration  following \u03c9(s), 0\u2264 \u03c9(s) \u22641 \u03c9(s) is implemented as a daily reduction factor due to water scarcity by,wp\u03b8 is the wilting point, and msw\u03b8 represents a threshold when enough soil moisture is available for potential tree biomass production. We calculated msw\u03b8 from:fc\u03b8 is the soil field capacity. wp\u03b8, msw\u03b8, and msw\u03b8 are expressed as normalized soil moisture. In the model, the wilting point (wp\u03b8) determines the minimum soil moisture content necessary for tree biomass production. Thus, we assumed a linear reduction of biomass production when soil water content was between msw\u03b8 and wp\u03b8. The water required for biomass production of trees is completely removed from the soil compartment when soil moisture reaches msw\u03b8 \u200a=\u200a1), after the calculation of maximum possible transpiration of trees. Both biomass production and water supply are reduced until the water needed for biomass production corresponds with wp\u03b8. The calculated rate of biomass production influences tree respiration rate through maintenance and growth respiration, which are calculated subsequently in the model (see The dependence of water uptake for tree biomass production on soil moisture is described by a function representing a reduction factor due to water scarcity (\u03c4 (day) can be expressed as an exponential distribution given by \u03bb is the mean time interval between rainfall events (days). Total daily rainfall (dP) depends on the amount of rain of each event , which is also assumed to be an independent random variable, expressed by an exponential probability density function \u03b7 is the mean depth of rainfall events (mm day\u22121). Both 1/\u03bb and \u03b7 parameters are calculated for each season of the year. We obtained daily global radiation (dRg) from EBSD instrumental records (period from May 1998 to February 2009). dRg varied among seasons in relation to daily rainfall . Therefore, in the model, dRg was distributed as a Gaussian variable whose mean and standard deviation depended on dP . Values of \u00b5Rg and \u03c3Rg were obtained from instrumental records and varied depending on a threshold value of 1 mm of dP and the season of the year. Daily temperature  was simulated by a Gaussian random variable with parameters  that varied according to season of the year.Rainfall time series, representing the frequency and depth of rainfall events, were constructed as series of random numbers generated by probability distributions. The interval between rainfall events, 2 ha\u22121) and size (dbh) distribution in a young secondary and an old-growth North Patagonian forest stand (hereafter YS and OG respectively) found in a flat forested area at EBSD , in each plot. We eliminated two trees from our stemflow estimates that died during the study period. We converted the volume of collected water to millimeters of rain assuming that the surface of the collectors equals the projected tree crown area. Crown area was approximated by the area of an ellipse. Throughfall collectors, 0.12\u00d72 m long  gutters were held, with a slight inclination, 0.5 m above the ground at three different locations within each plot. Collectors were connected with a funnel to a 25 l polythene container. Soil matric potential was measured every 30 minutes with four sensors per plot (WatchDog Data Loggers 450 and 800) placed approximately in every quarter of each plot, beneath the canopy and at ca. 15 cm soil depth. Continuous soil moisture measurements were obtained for the period January 2007 to March 2009.We estimated net precipitation using throughfall measurements taken in two YS forest plots , adding stemflow (water running down the stems). We conducted these measurements of volume accumulated during rainfall events occurred between June 2007 and December 2010. During this time period, we also analyzed hourly records of rainfall from the meteorological station at EBSD to obtain daily incident rainfall above the canopy. Rainfall events considered in the analysis occurred with a separation of at least two hours without rain to allow for full drip from the forest canopy. Stemflow collectors consisted of a 2 mm thick smooth polycarbonate sheet molded around the stem to form a funnel. A hose led from the lowest point of the funnel to a 25 l polythene container, where the stemflow volume was collected after each rain event. Stemflow collectors were placed in 10 randomly selected trees of the two main canopy species, FORMIND-CL v.1.0 and their values are shown in We used a previous model parameterization for North Patagonian forests including all main canopy tree species (11 tree species) occurring in the studied forests. The calibration, validation and robustness of this parameter set to reproduce forest stand structure is discussed in detail by hf describing the relationship between leaf area index and canopy water storage capacity was calibrated following \u22121 at a leaf area index of 5.0 as measured by maxLAI was set to 5.5 following the maximum value observed in other Chilean temperate rainforests h)\u03b1 was set according to common values for broad-leaved temperate trees WUE using transpiration estimates of D\u00edaz et al.Tr\u200a=\u200a296 mm year\u22121 and PB\u200a=\u200a32.9 tC ha\u22121. The selected WUE was then confirmed by comparison with reported values from other temperate rainforests wp\u03b8, fc\u03b8 and soilk) to average values dP >0). These calculations were done only for seasons with >85 daily records. We avoided the potential overestimation of annual rainfall by normalizing predicted seasonal rainfall sum by prescribed seasonal rainfall averages . We also compared model results with field measurements of hydrologic balance at yearly temporal scales in temperate rainforests elsewhere in Chile (i.e. independent studies).We compared field measurements of net precipitation with model predictions at daily temporal scale. For this analysis we selected 50 rain events, representing field measurement intervals <20 days long, for which accumulated rainfall during the event  was correctly measured by EBSD weather station, and for which <50% of containers were filled completely during the rain event. Hydrologic parameters used for model estimation of net precipitation are indicated in dbh distributions predicted by the model with measured structure of both YS and OG North Patagonian stands. Model comparison for the YS was performed after 60 years of succession, with succession initiated from a treeless state. To compare OG forest structure, we initiated simulations with stand inventory data and run the model for 1000 years to allow the simulated stand to reach dynamic equilibrium. We compared data at the end of the simulations with the known OG structure We tested model performance to reproduce forest structure under current climate. For this analysis, we compared tree basal areas and stand PRECIS-DGF model, PRECIS-DGF suggests a 50% decrease in rainfall during the growing season by year 2100. We used the daily scale output of the PRECIS-DGF model for year 2100 to calculate seasonal climatic parameters for year 2100. We linearly interpolated this seasonal rate of change between 1998\u20132009 and 2100. We used this scenario to set the limit of change in rainfall by year 2100 and developed climatic scenarios covering the change from current climate to the business-as-usual scenario. Climate scenarios were developed by gradually changing current climate parameters 1/\u03bb and \u03b7 until they reached the estimated value for year 2100, i.e. 50% of the current value. First, we reduced \u03b7  multiplying current values by 0.9 to 0.5 in steps of 0.1. Then, we increased the parameter 1/\u03bb  multiplying current values by 1.1 to 1.5 in steps of 0.1. These two parameters were first varied separately (keeping the second parameter constant) and then both together. Parameter variations produced a total of 36 climate scenarios, i.e. six levels of each of the two rainfall parameters, including the current climate scenario  from 0 to 100% of the current value  predicted for this century in the study area. To this end, we used a regional climate model downscaled for Chilean landscapes . Soil moisture increased during austral fall and winter . Modeled soil moisture was lower in OG than in YS  was negligible when \u03b7 was kept constant at its current value  and decreases in AGB by 27% of the current value (\u22121). Main changes in basal area and AGB of OG stand were predicted when current \u03b7 and 1/\u03bb parameters were multiplied by 0.6 and 1.3, respectively  in YS attributable to changes in rainfall parameters . In contectively .We developed and evaluated the performance of an individual- and process-based dynamic forest model that incorporates detailed calculations of water cycling for temperate rainforests of southern South America. The model allows for the investigation of dynamic linkages between rainfall trends and forest processes at stand scale. Parameters selected for this study were taken from the literature or calibrated using our own field data , thus th2). Further, model predictions of daily net precipitation were based on average estimated LAI values for YS because of the lack of field measurements. LAI is a relevant variable to understanding biogeochemical cycles The accuracy of model predictions regarding forest composition and structure were comparable to previous results , which wsoilk, which was calibrated specifically for soils from northern Chilo\u00e9 Island. Applying this model to forests developing on other soil types in SSA (e.g. volcanic originated soils) will require a site-specific calibration of soil parameters , and consequently a higher water demand for biomass production. Under increased drought, water demand for biomass production in OG forest is not fully covered by soil moisture supply, which causes the predicted decline of AGB and basal area .Here, we focused on developing an accurate model for assessing the influence of hydrologic processes involved in forest dynamics. Using the model, we quantitatively demonstrated the relative importance of soil moisture on forest structure . We exclThe rise of atmospheric carbon dioxide concentration and ensuing climate change are influencing water-use efficiency of forests Here, we deliberately excluded the influence of expected regional changes in temperature on forest processes (e.g. tree growth) to rather emphasize direct impacts of drought on model results. However, the model suggests that changes in PET due to expected warming trends in the study region are negligible compared to the strong impacts of increased drought . Our simNothofagus forest of SSA To the best of our knowledge, this is the first application of a forest gap model in temperate rainforests of SSA that integrates dynamic calculations of forest hydrology. The present work uses the best information available to ensure that climate patterns were directly comparable to hydrologic field measurements used for model calibration. However, our results on climate change impacts should be interpreted with caution because our baseline climate is constrained to a short period (1998\u20132009) within a long-term trend of rainfall (time period 1901\u20132005). Long-term monitoring of forest hydrology and dynamics can corroborate our results. To date, long-term monitoring (>10 years) of forest hydrology is lacking in forests of SSA. As more empirical data becomes available, the model can be revised and updated. The model developed here allows for the analysis of multiple environmental factors driving forest dynamics. For example, our model can help us understand the long-term responses of regional forest to drought events induced by El Ni\u00f1o Southern Oscillation that amplify background tree mortality rates in We developed and applied a forest dynamic model to analyze the impact of climate-driven increased drought on ecological and hydrological processes. The developed model was accurate for depicting forest hydrology at stand scales (i.e. <100 ha) and allowed the analysis of the dynamical linkages among rainfall regimes, soil moisture variation, and individual tree growth. Using the model we demonstrated that evergreen, broad-leaved temperate rainforests in southern South America are expected to be highly sensitive to future climate change, particularly increases in drought during parts of the year. Increased summer drought predicted for this century will likely impair biomass carbon accumulation, and amplify background tree mortality rates in this region. The developed model expands the range of applicability of gap models to assess climate change impacts in remote and understudied regions of the world, such as temperate forests of the southern hemisphere. It also represents an advance in the development of simple, general models to account for complex and dynamical processes operating at multiple spatial scales in forests.Figure S1FORMIND-CL v.1.0.A diagram of the hydrologic submodel of  Interaction between processes and variables in the hydrologic submodel, and their respective time scales of calculations. Arrows indicate whether the results of a model calculation influence the calculations of another submodel. Blue boxes represent analyzed variables of this study. All calculations are done in yearly time steps in the model, excepting the ones indicated in the dashed box. Variable notations follow the text. AGB: Above-ground biomass, LAI: leaf area index.(PDF)Click here for additional data file.Figure S2Weather generator results. Density functions of daily rainfall and daily mean temperature predicted by the weather generator compared to observed weather records from EBSD weather station.(PDF)Click here for additional data file.Figure S3Weather generator results. Comparison between simulated and observed climatic patterns during the year. Simulations were run for 100 years using parameters in (PDF)Click here for additional data file.Figure S4Forest composition predictions. Model results for forest composition using different model versions compared to field data. Simulations run under the same conditions detailed in Methods section.(PDF)Click here for additional data file.Figure S5Drought induced simulations with warming included. Changes in hydrologic components and forest structure when warming and increased drought was considered. PET: Potential evapotranspiration (mm year\u22121), T: transpiration (mm year\u22121), Ec: canopy interception (mm year\u22121), ET: evapotranspiration (mm year\u22121), BAT: total basal area (m2 ha\u22121), BT: Total biomass (tC ha\u22121). Result of a two-sample Wilcoxon test is shown on the upper right of each panel. Pink lines, drought induced simulations with warming included, blue lines drought induced simulations without warming, circles represent the values of simulation results. Note different scales for the axes.(PDF)Click here for additional data file.Figure S6Sensitivity of evaporatranspiration. Changes in evapotranspiration (ET) of the old-growth stand under current climate when using different water-use efficiency values (WUE). Simulations run under the same conditions detailed in Methods section.(PDF)Click here for additional data file.Appendix S1FORMIND-CL v.1.0.Calculation of canopy photosynthetic rate in (PDF)Click here for additional data file."}
+{"text": "Modeling above- and belowground carbon stocks, we analyzed the carbon balance of the investigated tropical forest. The simulated carbon balance of this old-growth forest is zero on average. This study provides an example of how forest models can be used in combination with forest inventory data to investigate forest structure and local carbon balances.Tropical forests are carbon-dense and highly productive ecosystems. Consequently, they play an important role in the global carbon cycle. In the present study we used an individual-based forest model (FORMIND) to analyze the carbon balances of a tropical forest. The main processes of this model are tree growth, mortality, regeneration, and competition. Model parameters were calibrated using forest inventory data from a tropical forest at Mt. Kilimanjaro. The simulation results showed that the model successfully reproduces important characteristics of tropical forests . The estimated aboveground biomass (385 t/ha) is comparable to biomass values in the Amazon and other tropical forests in Africa. The simulated forest reveals a gross primary production of 24 t Tropical forests have long been recognized as highly productive ecosystems . As suchMt. Kilimanjaro supports high biodiversity due to its unique range of vegetation zones and climatic conditions. Savannas and different forest types can be found along the elevation gradient . Before The forest model FORMIND was applied here to reproduce the patterns of an almost undisturbed tropical forest at Mt. Kilimanjaro. Specifically, we assessed (1) the patterns in number of trees, basal area, aboveground biomass, stem size distribution, and leaf area index. (2) We calculated a local carbon balance of the investigated forest. (3) We asked if this lower montane forest is a carbon sink or source.2 [www.kilimanjaro.biozentrum.uni-wuerzburg.de) in cooperation with the Kilimanjaro National Park (http://www.tanzaniaparks.com/kili.html). Andreas Hemp, Markus Fischer and Ingolf Steffan-Dewenter are Spokesperson of the DFG project and are responsible for the permits for the investigated forest at Mt. Kilimanjaro. This study was not carried out on private land and we confirm that the field studies did not involve endangered or protected species.At Mt. Kilimanjaro the submontane and lower montane forest belt between 1,000 m a.s.l. and 1,800 m a.s.l. has to a large extent been converted to small-scale coffee-banana plantations, the so-called \u201cChagga home gardens\u201d, a special type of agroforestry. The maximum population density in the area is 1,000 persons per km2 . The troHeinsenia diervilleoides  and Strombosia scheffleri (24%), Entandophrangma excelsum (6%) and Garcinia tansaniensis (5%). In total, we discovered 93 trees and 16 tree species at the test study site. The observations of the study site were linear scaled up to one hectare to be comparable to our simulation results of the forest model (1 ha forest simulations).Concerning this forest inventory, for each tree with DBH > 10 cm the specific position, stem diameter, tree height, crown expansion, and species identity were determined. The forest inventory of all five research sites were used to parameterize the model. These forest research sites distinguish from each other by forest structure and species composition mainly due to the spatial location and disturbance history. For testing the simulation outcome we used a nearly undisturbed research site  was calculated as the balance of carbon sequestration and carbon emissions , mortality, recruitment, and competition for light and space. The simulated forest area was divided into patches according to the typical size of tree fall gaps (20 m x 20 m). The vertical leaf distribution and light availability was calculated for each patch. The biomass growth of each tree was determined on the basis of a carbon balance including photosynthesis, respiration, biomass allocation, and litter fall. Photosynthetic production in the canopy depends on local light availability. Forests of up to 1,000 hectares can be simulated over a time period of several centuries. In this study the FORMIND model was used to investigate the biomass production and mortality of the forest ecosystem. In addition, above- and belowground carbon stocks and the resulting net ecosystem exchange  is a well-established classification method in vegetation ecology ,46. For We analyzed forest succession on 1 hectare over 1000 years, starting with bare ground conditions and without climate limitations. To assess the variability in the forest model we had 10 repeated simulation runs. Two types of analysis are presented: First, for a comparison of the model output with field data, we calculated the mean of simulated forest attributes over the last 300\u20131,000 years, based on the assumption that the forest is in the equilibrium state for the entire period. In particular, we analyzed aboveground biomass, basal area, stem numbers (dbh>10 cm), stem size distribution, and leaf area index (LAI). Second, we analyzed forest attributes which are difficult and costly to measure in a real forest, namely gross primary production (GPP), aboveground respiration of the vegetation, the resulting net primary production (NPP), and mortality. Additionally, we calculated above- and belowground carbon stocks and net ecosystem exchange (NEE). Details of the forest model and all parameter values are given in For this study, most of the FORMIND model parameter values were determined from the local forest inventory , while some parameter values were taken from the literature see . ParametTo test the developed parameterization for the forest at Mt. Kilimanjaro we compared simulated aboveground biomass, basal area and stem numbers with the field data for the test study site . The comparison is made on the level of plant functional types (PFT). We found that shade-tolerant (PFT1&2) and intermediate shade-tolerant (PFT3) tree species are most dominant in the investigated forest. Especially tall-growing tree species corresponding to PFT1 dominate the study site. The simulated forest reproduced this high PFT1 fraction see  for all odm/ha) was 2% higher than observed in the field data  was close to the observed field data .The total basal area of the simulated data  than in the field inventory  was repeatedly measured at different heights  and ranged from 6 at ground level to 4 at 2.0 m above ground. The LAI in the simulated forest at 2.0 m height was similar to the observed values see . Howevercha-1yr-1  was approximately 4.7 tcha-1yr-1. The total loss of aboveground biomass due to mortality events was in the same magnitude as NPP (4.7 tcha-1yr-1), which is typical for forests in the late successional phase [In the simulated mature forest, gross primary production (GPP) reached 23.5 tyr-1 see . Respiraal phase ,51.c/ha in the living vegetation, 32 tc/ha in the deadwood and 28 tc/ha in the soil . Uncertain parameters were calibrated using forest inventory data. However, parameters related to growth processes are difficult to estimate and repeated forest inventories would be necessary . As no sTo conclude, this study shows that process-based forest models can be used to investigate the carbon balance of tropical forests on a local scale. Aboveground biomass and net ecosystem exchange are especially important for the analysis under different conditions. It would be interesting to investigate forest dynamics in the context of climate change  and land use  in future studies. Using FORMIND, disturbances can be simulated in a straightforward manner because this model is individual-based . ThiS1 Dataset(XLSX)Click here for additional data file.S1 Text(PDF)Click here for additional data file.S2 Text(DOCX)Click here for additional data file."}
+{"text": "Bone marrow-derived stem cells (BMDSCs) play an essential role in organ repair and regeneration. The molecular mechanisms by which hormones control BMDSCs proliferation and differentiation are unclear. Our aim in this study was to investigate how a lack of ovarian or/and thyroid hormones affects stem cell number in bone marrow lineage. To examine the effect of thyroid or/and ovarian hormones on the proliferative activity of BMDSCs, we removed the thyroid or/and the ovaries of adult female rats. An absence of ovarian and thyroid hormones was confirmed by Pap staining and Thyroid Stimulating Hormone (TSH) measurement, respectively. To obtain the stem cells from the bone marrow, we punctured the iliac crest, and aspirated and isolated cells by using a density gradient. Specific markers were used by cytometry to identify the different BMDSCs types: endothelial progenitor cells (EPCs), precursor B cells/pro-B cells, and mesenchymal stem cells (MSCs). Interestingly, our results showed that hypothyroidism caused a significant increase in the percentage of EPCs, whereas a lack of ovarian hormones significantly increased the precursor B cells/pro-B cells. Moreover, the removal of both glands led to increased MSCs. In conclusion, both ovarian and thyroid hormones appear to have key and diverse roles in regulating the proliferation of cells populations of the bone marrow. The transplantation of stem cells has been proposed as a novel approach for the treatment of patients with several diseases, including ischemic heart injury . VariousThyroid and ovarian hormones regulate cellular homeostasis, proliferation, apoptosis, and differentiation . InvestiThe formation of new blood vessels is essential for successful bone marrow stem cell transplantation and tissue regeneration . On the Ovarian estrogens regulate the proliferation and growth of sex organs and other tissues related to reproduction, but their role is not limited to reproductive functions. Estrogens also play an important role in the balance between bone formation and resorption, and their loss causes critical physiological and pathological problems .Given the above-mentioned facts, there is a growing interest to study hormonal influence on the proliferation of stem cells, especially the role of the thyroid and ovarian hormones in females. Many patients are already being subjected to bone marrow samples, with the purpose of isolating hematopoietic stem cells from bone marrow, without prior knowledge of the proliferative state of the cells to be transplanted. Increased or decreased stem cell proliferation maybe a significant factor in determining an efficient therapeutic result. To ensure greater success in transplantation, the integration of transplanted stem cells in vivo or ex vivo is favored by the increased proliferative capacity of the stem cells. In this study, we analyzed proliferative activity by detecting bromodeoxyuridine (BRDU), which is incorporated by cells during the S-phase of mitosis. This marker does not require immunohistochemistry and can be quantified by flow cytometry .In the present study, we used bone marrow-derived mesenchymal stem cells, which have been reported to be very efficient in regenerative medicine due to their multi-lineage differentiation character and high potential to repair tissues. We compared the proliferative activity of these cells with other bone marrow-derived stem cells (BMDSCs) of rats subjected to the same conditions (ovariectomy and/or thyroidectomy). This enabled us to analyze the role of hormonal interactions (in vivo) in regulating BMDSCs expansion. We used specific markers and flow cytometry to assess the response of affected cell populations. Our results revealed diverse effects of thyroid and ovarian hormonal loss on BMDSCs proliferation.The characterization of the estrus stage of the animals was successfully determined by Pap smear; we determined the time of sexual inactivity between recurrent periods of estrus (the diestrus stage) as well as characterized the period of preparation for estrus (the proestrus stage) for all of the groups. Only the ovariectomized groups were permanently in the diestrus stage, indicating a successful ovariectomy .p value of <0.05 was considered statistically significant. The TSH levels of the thyroidectomized (T) group and the ovariectomized and thyroidectomized (O + T) group were significantly higher after the surgeries compared to the TSH levels before the surgeries. The TSH of the ovariectomized (O) and the control (C) groups did not change after the surgery compared to samples taken before the surgery  number was affected by the surgeries. MSCs were identified by the presence of the CD44H+, CD54+, CD73+, CD106+, CD34\u2013, and CD45\u2212 phenotypes A,C. The Our analysis of the total mononuclear cell proliferation did not reveal any significant difference between the studied groups A. IntereThe use of BMDSCs has made a number of contributions to the study of regenerative medicine; however, the hormonal regulation of the biological activities of these cells is not fully understood. Experimental thyroidectomy and ovariectomy in animal models have led to many advances in the fields of cell biology, endocrinology, and metabolism, particularly with regard to studying the roles of thyroid and ovarian hormones in regulating mechanisms of cell survival, proliferation, differentiation, and programmed cell death. In the present study, we investigated endocrine-dependent events of bone marrow stem cell survival and proliferation upon the removal of the thyroid gland or/and ovaries. Some previous studies have studied the hormonal influence on cell biology by using thyroid inhibitors at the receptor and histone acetylation levels. Other studies have focused on disturbing mechanisms involved in thyroid hormone biosynthesis, cellular uptake, cellular transport, or nuclear action. These strategies are known to cause many problems, such as a disruption of oxygen consumption and peripheral metabolic abnormalities via unknown mechanisms ,35,36. IThe endothelial progenitor cells were identified via detecting for positivity of the CD31+, CD45\u2212, and CD34+ phenotypes. We found an increased endothelial progenitor cell number in the thyroidectomized group. However, our analysis did not detect any change in the endothelial progenitor cell proliferation of the ovariectomized or the control groups. Because decreased estrogen affects total bone marrow mass, we asked if a decrease in ovarian hormones could block the stimulatory effect of thyroid removal on endothelial progenitor cells proliferation. Interestingly, our results indicate that the absence of ovarian hormone does not change the increased endothelial progenitor cell number caused by thyroid hormone removal .Bone marrow EPCs are derived from CD34+ stem cells and undergo continuous differentiation into erythrocytes, thrombocytes, leukocytes, and mature endothelial cells ,37; howeOur results indicate significant thyroid hormonal action on EPCs number; however, other bone marrow signals that stimulate the proliferation of this lineage cannot be excluded. Further studies should investigate molecular pathways that govern the effect of thyroid hormones on endothelial progenitor cell proliferation. Thyroid hormone stimulates expression of its target genes through the thyroid receptor\u2019s activation . TranscrPrevious studies have proposed various mechanisms that affect precursor B cells/Pro-B cells in aged animals. These include survival factors, altered differentiation, and impaired production of precursor B cells/Pro-B cells. However, our understanding of how cell proliferation and its mechanisms are regulated by hormones remains fragmentary. One of the major goals of this study was to understand the regulation of precursor B cells/Pro-B cells number in aged female Wistar rats. Earlier works have shown that the precursor B cells/Pro-B cells pool is decreased by ~fourfold in aged adults ; howeverPrevious results have shown an association between certain regulatory genes and the developmental sequence of precursor B cells/Pro-B cells ,44. For The majority of immature bone marrow precursor B cells/Pro-B cells undergo significant proliferation to reach the optimum number ,54,55. IBone marrow-derived mesenchymal stem cells have been reported to be very efficient in regenerative medicine due to their multi-lineage differentiation character ,61 and hCell viability and proliferation are major concerns in the field of stem cell biology as well as in therapeutic applications. It is critical to understand the in vivoresponse of BMDSCs, as successful models widely used in translational research, to physiologic and pathologic endocrine alterations. Future studies could investigate if monitoring the conditions of patients receiving stem cell therapy is related to thyroid and ovarian hormones homeostasis. Overall, the strength of our findings is that they were performed in an in vivo model. In summary, the present results reveal that hypothyroidism causes a significant increase in endothelial progenitor cells, whereas a loss of ovarian hormones significantly increased the numbers of precursor B cells/Pro-B cells and decreased the bone marrow mesenchymal cell number. The removal of both ovaries and thyroid glands caused a significant increase in mesenchymal stem cells number . HoweverA total of forty female Wistar rats, aged approximately three months, with weights ranging from 250 to 350 g were used. They were separated into four distinct groups, namely (C) control, animals which were not subjected to surgery, (O) ovariectomized, (T) thyroidectomized, and (O + T). The rats were housed in ventilated cages under standard laboratory conditions as well as standard temperature (20 \u00b1 2 \u00b0C), operating on a 12-h dark/light condition with a relative humidity of 30\u201370%. Three to four animals were placed in a single cage. The cages were properly separated, identified with badges for each individual animal, and containedthe rat group number and the date of surgery. The rats had free access to water and standard animal food. The thyroidectomized rats received adequate calcium treatment. All rats that underwent surgery were anesthetized by administering intramuscular ketamine (50 mg/kg) and xylazine (10 mg/kg), then an ovariectomy or/and a thyroidectomy was/were performed. The protocol of these studies was approved by an institutional committee for laboratory animal control , and it was in agreement with the guidelines for the care and use of laboratory animals published by the ARRIVE guidelines .To confirm the loss of thyroid hormone in the thyroidectomized animals, we obtained TSH levels of various blood samples. Thirty days after the surgical procedures on the thyroid gland and ovaries, a cardiac puncture needle 25 \u00d7 07 mm was used under an anesthetic condition (ketamine 50 mg/kg) to withdraw 3 mL of blood for measurement of TSH . Then, three milliliters of 0.9% saline solution were administered into the peritoneal cavity of the animals for volume reposition to avoid dehydration.To confirm the diestrus stage, smears from all rats of all groups were obtained, examined by Papanicolaou staining, and analyzed as described by Long Evans (1922) and Mandl (1951). The protocol used for staining was modified from Hubscher et al. (2005) .After puncturing the iliac crest, the collected bone marrow blood was transferred to a 15 mL flask, and mixed with sterile phosphate buffered saline (PBS) [This procedure confirmed that the bone marrow cells had maintained their phenotypic characteristics, as well as enabled cell quantification after the surgeries. In brief, 100 mL of isolated cells were put in 12 \u00d7 75 mm falcon tubes for immunophenotyping and cytometry. The cells were maintained in PBS/5% albumin, (nine tubes per rat). Antibody labeling was used to identify the various cell types. After homogenization, the cells were incubated for 15 min with the primary antibodies. They were then washed with 400 \u03bcL PBS/5% albumin and centrifuged (1400 rpm (350 G), 5 min). The supernatant was discarded, and the cells were suspended in 100 mL PBS/5% albumin and incubated with the FITC-conjugated antibody (Rat Anti-Mouse IgG1-3 \u03bcL) for 15 min in the dark. After incubation, the cells were washed with 400 \u03bcL PBS/albumin 5% and centrifuged (1400 rpm (350 G), 5 min), then the supernatant was discarded and the cells were suspended in 100 mL PBS/5% albumin. Monoclonal antibodies were used following the incubations and washing procedure mentioned above. Finally, the cells were suspended in 250 \u03bcL PBS/5% albumin and analyzed by flow cytometer  .v/v) (in PBS buffer at 37 \u00b0C), the cells were stained with anti-BRDU-FITC-conjugated antibody. The fluorescent DNA-binding stain 7-aminoactinomycin (7-AAD) was added to the samples right before the flow cytometry procedure.BRDU-incorporated cells (representing the S-phase of the cell cycle) were analyzed with the BrdU Staining Kit Protocol Flow , according to the manufacturer\u2019s recommended instructions. In brief, cells were surface stained then fixed and permeabilized by BD Cytofix/Cytoperm Buffer. After one hour of incubation with 30% of Dnase ("}
+{"text": "Maternal smoking has been linked to lower birth weight and higher risk of childhood obesity. However, it is unknown whether grand-maternal smoking during pregnancy is associated with grandchildren birth weight and body mass index (BMI) trajectories.We investigated associations of smoking during pregnancy with birth weight, risks of overweight and BMI trajectories among 46,858 mother-child dyads and 6,583 grandmother-mother-child triads of three cohort studies of related individuals. Smoking during pregnancy was reported by mothers, and anthropometric data were provided by participants in each cohort.2  higher through adolescence and young adulthood than that of grandchildren of non-smoking mothers.Compared to grandchildren of non-smoking women, grandchildren of women who smoked more than 14 cigarettes per day throughout pregnancy were 70 g  heavier at birth, and 18%  more likely to become overweight. The mean BMI of grandchildren of women who smoked during pregnancy was 0.45 kg/mGrandmothers\u2019 smoking during pregnancy was associated with higher birth weight, higher risk of overweight, and higher BMI through adolescence and young adulthood. Poor fetal development has been increasingly recognized as a contributor to the incidence of non-communicable diseases, such as hypertension, type 2 diabetes, and cardiovascular disease ,2. WomenSeen as a fashionable behavior, the prevalence of smoking among women peaked in 1960s in U.S. ,5. StudiThe goal of this study was to examine the associations of grand-maternal smoking during pregnancy with birth weight and risk of obesity in their grandchildren. To achieve this goal, we constructed a three-generation cohort by linking data from three cohorts of related individuals: the Nurses\u2019 Mothers Cohort Study (NMCS), the ongoing Nurses\u2019 Health Study II (NHS II) and the ongoing Growing Up Today Study 2 (GUTS2). The NMCS is comprised of mothers of women participating in NHSII and GUTS2 is comprised of offspring of women in NHS II, thus allowing us to study smoking effects across three generations.NHS-II was established in 1989 when 116,430 female nurses (aged 25\u201342 years) completed a questionnaire about their lifestyle and medical history. Follow-up questionnaires are sent biennially to collect updated information. In 2001, 39,904 mothers of NHS-II participants completed a questionnaire regarding their pregnancy with their NHS-II participant daughter and additional information regarding their daughter\u2019s infancy (Nurses\u2019 Mothers Cohort Study). The Growing Up Today Study 2 (GUTS2) began in 2004 when 10,907 children  age 9\u201314 years of women participating in NHS-II completed a baseline questionnaire regarding health and growth indicators. Follow-up questionnaires have been sent biennially to update information. Additional information on GUTS2 participants\u2019 perinatal and early life exposures were collected in 2009 from 9,096 NHS-II participants with children enrolled in GUTS2. After excluding 1,652 individuals with missing information on maternal smoking status during pregnancy, we had data from 38,252 mother-offspring dyads between NMCS (F1) and the NHS-II (F2) cohorts, 8,606 mother-offspring dyads between the NHS-II (F2) and the GUTS2 (F3) cohorts, and 6,583 grandmother-mother-offspring triads . The study was approved by the Institutional Review Boards of the Harvard T.H. Chan School of Public Health and Brigham and Women\u2019s Hospital.F1 (grandmother): NMCS (F1) participants reported on whether they had smoked during pregnancy, whether they had stopped smoking during pregnancy, and in which trimester they had stopped. NMCS (F1) participants provided further information on the number of cigarettes smoked per day. Smoking during pregnancy was categorized into \u2018never smoked during pregnancy\u2019, \u2018only smoked during first two trimesters\u2019, \u2018smoked during all three trimesters, 1\u201314 cigarettes per day\u2019, and \u2018smoked during all three trimesters, \u2265 14 cigarettes per day\u2019.F2 (mother): NHS-II (F2) participants reported on whether they had smoked during pregnancy, whether they had stopped smoking during pregnancy, and in which trimester they had stopped. Smoking during pregnancy was categorized into \u2018never smoked during pregnancy\u2019, \u2018only smoked during the first two trimesters\u2019, and \u2018smoked during three trimesters\u2019.A validation study conducted among 146 NMCS participants who had also participated in the National Collaborative Perinatal Project showed that the validity of recalled maternal smoking during pregnancy was high  .F2 (mother): F2 participants reported their own height and weight at baseline, and current weight was updated on biennial questionnaires.F3 (offspring): F2 participants reported the birth weight of F3 participants in 2009. The birth weight was recorded in 6 categories ranging from \u22645, 5.0\u20135.4, 5.5\u20136.9, 7\u20138.4, 8.5\u20139.9, and \u226510 lbs. Recalled offspring birth weight is shown to be of high validity and reproducibility from thirty or more years ago . We crea2. Self-reported height and weight by adolescents and adults are known to be sufficiently valid for use in epidemiologic research [Body mass index (BMI) was calculated as the ratio of weight (kg) to height (m) squared. In adolescents < 18 y), overweight/obesity was defined as a BMI at or above the age- and sex-specific cutoffs proposed by the International Obesity Task Force (IOTF) . In adulresearch ,18. Once y, overwWe mailed NMCS (F1) participants questionnaires to report maternal age at the time of their daughters\u2019 birth, gestational age, level of education, consumption of alcohol, vegetable, fruit, and meat, physical activity, and weight gain during pregnancy. All of the questions were asked using multiple choice response options, and we converted the categorical variables to continuous variables .In F2 participants, social-economic status was measured by a question \u201chow do you feel about your standing in US society using a ladder\u201d in 2001. The ladder includes 10 levels from bottom to top, with a higher level indicating higher social-economic status. Dietary intakes were measured by 131-item food frequency questionnaire (FFQ), and we derived the AHEI-2010 score based on intake levels of 11 components, which were chosen on the basis of their association with chronic disease and mortality risk in observational and interventional studies F1 smoking with F3 outcomes: Generalized estimating equation (GEE) regression models were used to evaluate the associations of F1 smoking with F3 birth weight and BMI trajectories from childhood through young adulthood accounting for both within-person and within-sibling correlation in outcomes. Log-binomial GEE regression models were used to examine the association of F1 smoking with F3 risk of overweight or obesity accounting for the non-independence of sibling clusters and confounding variables. We adjusted for gestational age, age at birth, level of education, consumption of alcohol, vegetable, fruit, and meat, physical activity, and weight gain during pregnancy. We additionally adjusted for F2 mothers\u2019 pre-pregnancy BMI, smoking during pregnancy, social-economic status, and AHEI diet score and physical activity during pregnancy. We further plotted the F3 BMI trajectories according to F1 smoking status using a locally weighted scatterplot smoothing regression estimator with the LOESS function in R.2 groups. We collapsed grand maternal smoking into \u2018never smoked during pregnancy\u2019, \u2018smoked only during the first two trimesters or throughout pregnancy with 1\u201314 cigarettes per day\u2019 and \u2018smoked through three trimesters during pregnancy, \u2265 14 cigarettes per day\u2019, and matched by a ratio of 7:1.5:1, which made the maximum use of the F1 participants.We conducted sensitivity analysis using time-varying overweight/obesity status assessed every other year as main outcome, and applied log-binomial GEE to examine the association between F1 smoking and F3 risk of overweight/obesity. To examine whether the association between grand maternal (F1-F3) smoking during pregnancy with offspring risk of overweight or obesity was mediated by F2 maternal smoking status, we restricted our analysis to F2 mothers who never smoked during pregnancy. To further evaluate whether the association between grand maternal (F1-F3) smoking during pregnancy with offspring risk of overweight or obesity was mediated by F2 mother pre-pregnancy BMI, we matched the F1 participants by F2 pre-pregnancy BMI, which was divided into < 20, 20, 21, 22, 23, 24\u201326, 26\u201328, \u2265 28 kg/mAll statistical analyses were performed using SAS Version 9.2  and R 3.1.0.The baseline characteristics according to grand-maternal smoking status during pregnancy are shown in As expected, maternal smoking during pregnancy was associated with lower offspring birth weight (P for trend < 0.001) . However2; 95% CI: 0.14, 0.75; P = 0.004)  . The dif= 0.004) , Table 3= 0.004) .We further examined the association of grand-maternal smoking with anthropometric outcomes in an analysis restricted to F2 mothers who never smoked during pregnancy. The positive associations with birth weight, risk of overweight, and BMI during follow-up persisted in this analysis . MoreoveUsing an intergeneration study design, we found that grandmothers\u2019 smoking during pregnancy was associated with higher birth weight, higher BMI, and higher risk of overweight in youth. These findings are significant in at least two aspects. First, they suggest that the high frequency of smoking in the 1960s may have contributed to fueling today\u2019s high prevalence of childhood and adolescent obesity. If this relation holds to replication, it has important implications for the prevention of obesity and associated conditions over the following decades in developing regions of the world where smoking continues to increase, particularly among women of reproductive age . Second,The literature on the relation of grandmaternal smoking during pregnancy and grandchild birth weight and BMI trajectories is scarce. Two studies have examined the association of grandmother smoking during pregnancy with birth weight of the offspring, and positive associations were only found when restricting to certain subpopulations ,24. A poIn our study, we found that grandmother smoking was associated with higher birth weight and higher BMI in the offspring, and the associations appeared to be independent of smoking and BMI during the second-generation pregnancy. Although the mechanism is not clear, the results of our study suggest that smoking may exert trans-generational effects on offspring birth weight and risk of overweight in the third generation. In fact, a transgenerational effect has been reported by other studies, due to adverse intrauterine environments. Specifically, in humans, low birth weight mothers were more likely to have low birth weight infants and men who were born after a pregnancy complicated by pre-eclampsia were more likely to father a first pregnancy affected by preeclampsia ,26. AnimThe mechanism of smoking with obesity of the offspring has been investigated. One prospective study showed that infants exposed in utero to smoke had higher arterial cortisol and adrenocorticotropin hormone levels at birth . EpigenoThe possibility that the associations are the result of unmeasured confounding cannot be excluded. First, grandmothers who were smokers during pregnancy might be from lower social-economic status (SES), and their offspring might be likely to be obese . HoweverIn conclusion, our study showed that grandmothers\u2019 smoking during pregnancy was associated with higher birth weight and higher risk of overweight from childhood through young adulthood. These findings point to a potential contributor of the current childhood obesity epidemic in the US. Furthermore, they may be informative of the effects on future generations in regions of the world where smoking among women of reproductive age remains high.S1 Table2, 21\u201322.9 kg/m2, 23\u201324.9 kg/m2, 25\u201329.9 kg/m2, 30\u201334.9 kg/m2, \u2265 35 kg/m2).Model 1 adjusted for gestational age (quartiles), age at birth (quartiles), level of education , as well as consumptions of alcohol (continuous), vegetable (continuous), fruit (continuous), meat (continuous), physical activity , and weight gain (quartiles) during pregnancy. Model 2 adjusted for F1 smoking status , gestational age , age at birth (quartiles), as well as alcohol consumption (continuous), aHEI (quartiles), physical activity (quartiles), and BMI before pregnancy (< 20.9 kg/m(DOCX)Click here for additional data file.S2 Table2). Model 2 additionally adjusted for F2 mother smoking during pregnancy . Model 3 additionally adjusted for F2 mother social-economic status , F2 mother diet score (tertiles), and F2 mother physical activity (tertiles).Multivariate-adjusted model adjusted for gestational age (quartiles), age at birth (quartiles), level of education , as well as consumptions of alcohol (continuous), vegetable (continuous), fruit (continuous), meat (continuous), physical activity , and weight gain (quartiles) during pregnancy. Model 1 additionally adjusted for F2 mother pre-pregnancy BMI Click here for additional data file.S3 Table& Results for overweight/obesity were relative risk. Multivariate model adjusted for gestational age (quartiles), age at birth (quartiles), level of education , as well as consumptions of alcohol (continuous), vegetable (continuous), fruit (continuous), meat (continuous), physical activity , and weight gain (quartiles) during pregnancy.*Results for birth weight and BMI were regression coefficients. (DOCX)Click here for additional data file.S4 Table&Results for overweight/obesity were relative risk. Multivariate model adjusted for gestational age (quartiles), age at birth (quartiles), level of education , as well as consumptions of alcohol (continuous), vegetable (continuous), fruit (continuous), meat (continuous), physical activity , and weight gain (quartiles) during pregnancy.*Results for birth weight and BMI were regression coefficients. (DOCX)Click here for additional data file."}
+{"text": "AbstractThe diversity of organisms is being commonly accessed using metabarcoding of environmental samples. Reliable identification of barcodes is one of the critical steps in the process and several taxonomy assignment methods were proposed to accomplish this task, including alignment-based approach that uses Basic Local Alignment Search Tool (BLAST) algorithm. This publication evaluates the variability of 5' end of 18S rRNA barcoding region as expressed by similarity scores  produced by BLAST, and its impact on barcode identification to family-level taxonomic categories.In alignment-based taxonomy assignment approach, reliable identification of anonymous OTUs to supraspecific taxa depends on the correct application of similarity thresholds. Since various taxa show different level of genetic variation, practical application of alignment-based approach requires the determination and use of taxon-specific similarity thresholds. Identification of anonymous barcodes clustered in Operational Taxonomic Units (OTUs) is one of the critical steps in metabarcoding studies of living organisms. It can be accomplished via several taxonomy-assignment tools belonging to four different categories: alignment-based, probabilistic, tree-based and phylogeny-based . Alignment-based approach uses Basic Local Alignment Search Tool BLAST,  algorithRecent publication describing Classification Resources for Environmental Sequence Tags CREST,  uses folCephalobidae and Panagrolaimidae below). Published similarity measures , and non-nematode sequences placed within Nematoda (see Desmodoridae (represented by 21 taxa), Chromadoridae (30 taxa), Comesomatidae (12 taxa), Monhysteridae (21 taxa), Xyalidae (14 taxa)  and Panagrolaimidae (18 taxa), were also included for comparison  of the lgorithm . Two sepIdentity score received by the nearest ingroup taxon (sequence from the same family), i.e., the closest scoring match from the same family that is not the same sequence;Identity score received by the furthest ingroup taxon before the first outgroup taxon (sequence from the different family), i.e., the furthest scoring match from the same family immediately preceding the closest scoring match that belongs to a different family;Identity score received by the nearest outgroup taxon (sequence from the different family), i.e., the closest scoring match from a different family.GQ503078Monhystera sp. and KJ636248Mononchusaquaticus).Standard statistical measures  were calculated for alignment score, identity score and coverage when appropriate . This alone shows that 95% similarity threshold used to define families in LCAClassifier of CREST  and 95-98% (highest). Identity scores for the nearest outgroup taxon were also variable, with the highest 93-96% in Desmodoridae and the lowest 81-91% in Chromadoridae. For comparison, in the family Cephalobidae identity scores for the furthest ingroup taxon range within 96-98% and for the nearest outgroup taxon \u2013 within 95-99%. Same values in the family Panagrolaimidae are 79-99% (furthest ingroup taxon) and 73-95% (nearest outgroup taxon). What is more important is that ranges of identity scores for furthest ingroup taxon and nearest outgroup taxon showed considerable overlap for all compared families  except for the family Comesomatidae  than the furthest ingroup taxon with 100% coverage (96-97% identity). The family Panagrolaimidae presented a different challenge \u2013 all compared barcodes of the genus Halicephalobus received very low sequence coverage with the furthest ingroup (32%) and nearest outgroup (17-27%) taxa, and found no outgroup sequences with 100% coverage, even though many sequences in the reference database have full overlap with them. This can indicate that BLAST algorithm has difficulties aligning highly modified sequences of Halicephalobus.These two terrestrial families purposely chosen for comparison also present two specific challenges that were not seen in marine families. For example, in the family Comesomatidae to 86% in the family Chromadoridae, while average values were more consistent across families (97.0-99.2%) and very similar to the results described in the previous section (Results 1. Variable coverage).The results of BLAST searches are summarized in Suppl. material Desmodoridae showed relatively narrow range, 93-96% identity score to query sequence. For the remaining five families the same scores varied between 80-93% (lowest) and 92-100% (highest). 100% identity scores for the furthest ingroup taxon were noted in several cases and were caused by limited number of reference taxa that had 100% coverage with query sequence. Identity scores for the nearest outgroup taxon were also variable, with the highest 92-96% in Desmodoridae and the lowest 80-91% in Chromadoridae. For comparison, in the family Cephalobidae identity scores for the furthest ingroup taxon range within 96-98% and for the nearest outgroup taxon \u2013 within 95-97%. Same values in the family Panagrolaimidae are 79-99% (furthest ingroup taxon) and 78-95% (nearest outgroup taxon). Similarly to the preceding comparison (Results 1. Variable coverage) the ranges of identity scores for furthest ingroup taxon and nearest outgroup taxon showed considerable overlap for all families  except for the family Comesomatidae . In the family Cephalobidae the nearest outgroup taxon no longer have higher identity score than the furthest ingroup taxon (for same query sequence), making identifiication more reliable. In the case of the family Panagrolaimidae, limiting searches to sequences with 100% overlap produced no nearest outgroup hits for the genus Halicephalobus.Limiting searches to sequences with 100% overlap affected two specific issues with the families Only in one out of five analyzed families of marine nematodes, there was no overlap in ranges of identity scores between furthest ingroup taxon and nearest outgroup taxon. Remaining four marine and two terrestrial families showed considerable overlap between both values (identity score of the furthest ingroup taxon and identity score of the nearest outgroup taxon). Moreover, both values showed substantially different variability ranges and average values depending on the taxon in-question. It suggests that universal similarity thresholds applied to nematodes need to be used with great caution.Chromadoridae were as low as 86%, thus using 95% or even 90% similarity threshold to assign anonymous OTUs to families will treat such cases unidentifiable. This problem can not be solved by broadening similarity cutoffs, as it will increase incorrect taxon assignment for all families, but only by filling in the gaps in the reference databases by specifically targeting those species and genera for which no sequence data is available.Even considering only highest scoring hits of the BLAST searches for alignment-based identification of OTUs should be done with great care. Due to scarcity of nematode reference dataset, many highest scoring hits have very low identity scores, especially in case when only 100% overlapping sequences are considered. In this analysis, nearest ingroup scores for some sequences from the family Level of overlap between query and reference sequence has certain impact on identity scores in particular and on the identification process in general. While performing BLAST searches, I noticed numerous cases when outgorup taxa with lower coverage received higher identity scores than ingroup taxa with more complete coverage. On the other hand, limiting BLAST searches to sequences with only 100% coverage effectively limits the range of reference taxa to compare with \u2013 as already described in GQ503078Monhystera sp. groups within the family Xyalidae instead of the family Monhysteridae, while KJ636248Mononchusaquaticus groups within the family Monhysteridae instead of the family Mononchidae.Presence of erroneous sequences in reference databases and its impact on identification of anonymous OTUs had been extensively discussed and illustrated . In addiThe diversity of nematodes is seriously underrepresented in reference databases used for identification of anonymous barcodes (OTUs). When using alignment-based taxonomy assignment tools to identify nematode OTUs, it is important to know both (1) the lowest similarity thresholds that can be confidently applied to assign OTUs to supraspecific taxa, in order to maximize the efficiency of identification; and (2) the highest similarity thresholds that can ensure minimum number of mis-assigned OTUs.Targeted sequencing of reference taxa from underrepresented nematode families is expected to improve the efficiency of alignment-based taxonomy assignment approach. Two groups of taxa should be specifically considered: (1) those species that are completely missing from the reference databases, and (2) those species, which sequences  do not have full coverage with the barcoding region used in metabarcoding studies.It is also important to understand that universal similarity thresholds can only be applied with great caution, that taxon-specific similarity thresholds may be more effective to use, and that other taxonomy assignment methods may be more reliable for a particular dataset .Supplementary material 1Table S1. GenBank accession numbers and classification of sequences used in present analysis.Data type: listFile: oo_110493.pdfHolovachovSupplementary material 2Desmodoridae, Chromadoridae, Comesomatidae, Monhysteridae and Xyalidae) and two terrestrial  families of nematodes. Number of analyzed sequences for each family is given in parenthesis.Table S2. Variability of maximum alignment score and identity score for the nearest ingroup, furthest ingroup and nearest outgroup taxa in the BLASTN searches with variable coverage for each of five marine  and two terrestrial  families of nematodes. Number of analyzed sequences for each family is given in parenthesis.Table S3. Variability of maximum alignment score and identity score for the nearest ingroup, furthest ingroup and nearest outgroup taxa in the BLASTN searches with 100% coverage for each of five marine (Data type: listFile: oo_110495.pdfHolovachov"}
+{"text": "This study investigates a possible relationship between perceived and self-ascribed gender identity and the respective acoustic correlates in a group of young heterosexual adult speakers. For the production study, a sample of 37 German speaking subjects  filled out a questionnaire to assess their self-ascribed masculinity/femininity on two scales. A range of acoustic parameters  were measured in speech collected from a picture describing task. Results show that male speakers judging themselves to be less masculine exhibited larger vowel spaces and higher average fundamental frequency.For the perception experiment, a group of 21 listeners  judged masculinity of single word male stimuli drawn from the collected speech sample. A significant correlation between speakers\u2019 self-ascribed and listeners\u2019 attributed gender identity was found with a stronger relationship for female listeners. Acoustic parameters used by listeners to attribute gender identity include those used by speakers to index masculinity/femininity.The investigation demonstrates the importance of including self-ascribed gender identity as a potential source of inter-speaker variation in speech production and perception even in a sample of heterosexual adult speakers. The traditional gender dichotomy is being increasingly called into question, both in popular discourse as well as in the scientific domain. A popular scientific magazine  dedicatespeech? To answer this, we first have to look at which differences have been found between the sexes in terms of acoustic-phonetic speech patterns. Many studies have investigated the potential biological reasons for variation in male and female speech . We . We speech  and could show that even Queen Elizabeth II, one of the subjects in the study, has participated in this process of sound change.People learn by observing and mimicking , childrWhen it comes to gender-specific speech patterns the problem is that most linguistic studies still assume that women and men are homogeneous groups. One exception is the research area focusing on differences within women and men based on the sexual orientation of the speaker   ex ex46] exheterosexual men were the ones with the strongest wish to disclose their sexual orientation. The authors suggest this might be due to the fact that this group has \u201cthe most status to defend by signaling their heterosexuality clearly to others\u201d (p.62) . The speech material consists of utterances of words extracted from unscripted speech and the parameters investigated comprise mean fundamental frequency, variation in fundamental frequency, acoustic vowel space size and the acoustic contrast between the sibilants /s/ and /\u222b/.This investigation consists of a production study and a perception study examining the potential acoustic-phonetic cues associated with self-rated and perceived gender identity. The perception study concentrates (due to the results of the production study) on male speakers. The speech material used consists of a short bisyllabic word only and its aim is twofold: first, to assess the relationship between self-ascribed and perceived gender identity; second, to examine the acoustic cues potentially responsible for the perceived gender identity ratings. These cues are linked to the production study and comprise mean fundamental frequency, fundamental frequency pattern, first and second formant of /a/, spectral characteristics of /s/ and duration.The Written consent from all subjects taking part in the production and perception study was obtained prior to the recording of the stimuli and the perception test. The data collected and experiments conducted were part of a larger project which was reviewed and approved by the Ethics Committee of the University of Jena.The data used in this study is drawn from a larger project investigating the speech of mothers and fathers to their child and other adults over the timespan of their child\u2019s first year ,55. Due Participants were asked to fill in a questionnaire regarding their demographics and factors related to the project . The gender/sex options participants were given included \u201cfemale\u201d, \u201cmale\u201d and \u201cothers\u201d. No participant chose the last option. No explicit data regarding sexual orientation were obtained from the subjects. Data from 37 German subjects ) were collected. All speakers live in a small city (Jena) in East Central Germany, exhibiting mild dialectal influence.Participants were recorded at home by the same female experimenter using a headset-microphone (Sennheiser ew 100 G3 \u2013SK100) and a ZOOM\u2013H6 Handy Recorder. A picture describing task was used to elicit unscripted speech using 15 pictures with various objects and animals see . The expTasse (cup) and Fluss (river), the sibilant /\u0283/ in the words Tasche (bag), Fisch (fish) and Kirsche (cherry), and the vowels /i: \u025b a u:/ in the carrier words Tiger (tiger), Biene (bee), \u00c4pfel (apples), Katze (cat), Wasser (water), Kuh (cow), and Buch (book). The pictures were created to elicit each target word at least three times and comprised the sibilant /s/ in the words Self-reported gender identity ratings were obtained using two different questionnaires. The F+ scale of the German version  of the gender-role adoption , gender-role preference (desired degree of masculinity-femininity), and gender-role identity . Participants have to index for 7 items on a scale from 1 (very masculine) to 7 (very feminine), e.g. how their interests/behaviour/appearance would be evaluated tradionally, how they consider themselves, and how they would ideally like to be. In other words, the higher the scores, the higher the speaker\u2019s self-ascribed femininity.The second questionnaire used is the Traditional Masculinity-Femininity scale (TMF), an instrument recently developed for measuring gender-role self-concept . The TMFpsych package in R. For both scales reliability was high, with higher values for TMF than for GEPAQ-F+ (Cronbach\u2019s \u03b1 = .94 and = .80).Reliability of the two scales was measured by calculating Cronbach\u2019s \u03b1 using the Both questionnaires were filled in by the participants after the recordings. For each scale (GEPAQ and TMF) a mean score was calculated for each speaker and used for the correlations with the listeners\u2019 ratings and the acoustic measures.Acoustic segmentation and analysis was done using PRAAT . Start The first three formants of /i: \u025b a u:/ were measured in the middle of the vowels using PRAATs LPC formant measurement algorithm  The maximum formant value was set to 5000 Hz for the male speakers and to 5500 Hz for the female speakers. Formants were manually corrected if necessary. Mean F1 and F2 values of each vowel category for each speaker were used to calculate the size of a speaker\u2019s acoustic vowel space. This vowel space was estimated by calculating the area of the polygon enclosed by the mean formant values of the vowels in the F2xF1 space. We used Bark, a psycho-acoustic measure better reflecting the non-linearity of the perceptual system .Discrete Cosine Transformation  was useFurthermore, mean fundamental frequency (f0) and standard deviation of f0 were measured over the whole utterance of the speaker .Welch Two Sample t-tests were run to investigate gender differences in the self-rated GEPAQ and TMF scores of the speakers. To analyze the relationship between the two scores and between acoustic cues and self-rated gender identity scores, Pearson Product Moment correlations (one-sided) were run. Since correlation analyses were run for several acoustic parameters and both genders separately, p-values were corrected for running multiple tests.within the genders , thus providing a better starting point for correlations with the acoustic parameters. For this reason, unless specified otherwise, subsequent analyses will be carried out using the GEPAQ scores as a measure of gender identity.Although TMF does a better job in distinguishing the genders, GEPAQ scores are more widely distributed The investigated acoustic cues comprise the vowel space size, the mean fundamental frequency and the variation in fundamental frequency, and the sibilant contrast. Pearson's product-moment correlations were run for each acoustic parameter and both genders and Similar results were found with respect to TMF. Here too, significant correlations were found for vowel space and mean f0 in males only. For vowel space size, height and significance level are comparable to those reported for GEPAQ , while for mean f0 the correlation is weaker .Due to the results of the production study only male speakers were included in the perception experiment. The speakers consisted of a subset of the male speakers described above  and an additional data set of male speakers recorded only for the purpose of the perception test . The additional speakers also signed an informed consent form. It should be said, however, that we did not inquire further into the status of the relationships of the additional speakers, since this was not deemed relevant to the investigation at hand (i.e. attribution of masculinity due to acoustic cues).The reason for not taking all male subjects of the production study were conditions placed on the comparability of the stimuli to be used (see below).Tasse (cup), carrying an open vowel and a sibilant, taken from the speakers\u2019 recording of the picture describing task (see above). From each speaker one Tasse token was chosen. Stimuli were normalized by setting the ampliude to the same level using PRAAT. However, no modification of other prosodic parameters  was undertaken. The selected tokens were uttered in the middle of an utterance and followed by a break. In this way, the occurrence of creaky voice and coarticulatory influences were reduced to a minimum. These conditions on comparability meant that 4 of the original male speakers were excluded from the perceptual study.In contrast to the production experiment, where the speech material investigated consisted of a stretch of running speech, here only one particular word was used. This was done since we did not want other acoustic cues than the ones we are investigating  to be responsible for the ratings. Therefore stimuli were restricted to the bi-syllabic word Tasse was set to the release of the closure of the stop. Each stimulus consisted of two Tasse tokens of different speakers with a pause of 0.4s between them. After each stimulus a silence of 0.9s was added followed by a beep (with a duration of 0.5s and a frequency of 880Hz) to signal the start of the next stimulus pair to be rated.The start of The methodology of the perception test was oriented on an earlier study dealing with perceived tempo . The \u2018ExTasse tokens were chosen. After a block of 30 pairs listeners could take a break as long as they wanted. Altogether the experiment lasted approximately 30 minutes. After the perception test, listeners were asked to fill in the TMF and GEPAQ questionnaires to examine a possible relationship between listeners\u2019 gender identity and their ratings. Again, reliability of the two scales was measured by calculating Cronbach\u2019s \u03b1 using the psych package in R. Reliability scores were similar to the ones measured for the speakers\u2019 ratings (Cronbach\u2019s \u03b1 for TMF = .89 and for GEPAQ = .79).Prior to the experiment a test round was carried out with five stimulus pairs to familiarize the listeners with the design. The speakers used for the test round were also part of the main experiment but different Tasse stimuli regarding fundamental frequency, formants and spectral characteristics of /s/. In particular, the fundamental frequency in /a/, the difference in fundamental frequency between /a/ and /\u0259/ (as a coarse measure of the f0 contour of the utterance), the first and second formant of /a/, DCT 1\u20133 of /s/, and the duration of the word and of /s/ (in ms and in % of the word) were measured.Parallel to the production experiment, acoustic parameters were analyzed in the lme4 package [Linear Mixed Models as implemented in the  package  in R (ve package ) were ru2(3) = 13.82, p < .01). Thus, a relationship between self-rated and perceived masculinity exists and this relationship is affected by the gender of the listener. The data were therefore separated by listeners\u2019 gender and mean scores for each stimulus pair were calculated for the genders, respectively. A linear mixed model (LMM) with the rated masculinity scores as dependent variable, the GEPAQ scores and the listener\u2019s gender as fixed factors and the listener and speaker 1 and speaker 2 of the stimulus pairs as random effects revealed a significant interaction of the listeners\u2019 gender and the GEPAQ scores of the speaker  and lower femininity scores . However, these groups did not differ in the size of the correlations between perceived and self-rated gender identity of the speakers .We also checked for an effect of the gender identity score (GEPAQ) of the In addition, we performed a LMM including the raters\u2019 gender identity instead of their gender. Neither a main effect nor an interaction with the speakers GEPAQ score reached significance.Thus, in the case of male and female listeners rating the masculinity of male speakers, we assume the listeners\u2019 sex and not gender identity to be the contributing factor to the differences in perceptual ratings.pairs of speakers were rated, each investigated acoustic parameter is actually the difference in this parameter between the two speakers. Significant main effects were found for fundamental frequency of /a/ (\u03c72 (1) = 36.55, p < .001) and f0 contour (\u03c72 (1) = 24.2, p < .001). In addition, a significant interaction between F1 and listener gender was also found (\u03c72(2) = 15.25, p < .001). Durational patterns, F2 and spectral characteristics of /s/ did not show a significant effect. Several acoustic parameters (and their interaction with listener gender) were entered as fixed factors to the LMM (in a stepwise fashion) to look for a potential impact of this acoustic cue on the perceived masculinity of the speaker. These parameters were the fundamental frequency in /a/, the difference in fundamental frequency between /a/ and /\u0259/ as a simple quantification of f0 movement over the two syllables, the first and second formant of /a/, DCT 1\u20133 of /s/, and the duration of the word and of /s/ (in ms and in % of the word duration). Since in the perception test This study highlights three aspects of gender and speech. First, self-ascribed gender identity is not a bimodal factor but a continuous variable which exhibits considerable inter-gender variation. This was most apparent in men\u2019s responses to the GEPAQ questionnaire on positive feminine attributes. Second\u2013and confirming, in part, hypothesis 1 \u2013this idiosyncratic gender identity is indexed in speech through fine phonetic detail: men who rate themselves as more feminine have larger acoustic vowel spaces and higher mean fundamental frequencies . Third\u2013in support of hypothesis 2 \u2013the concept of masculinity/femininity is attributed by listeners using similar acoustic parameters as those used by speakers , but they also attend to additional parameters , probably based on stereotypical assumptions about gendered speech, with female speech being associated with rising f0 contours and male speech with falling contours . Indeed,The association of certain spectral cues in speech and perceived masculinity has been found before \u201343,73\u201375Our study highlights an additional gender-specific aspect regarding the transportation of gender identity in speech (in conjunction with hypothesis 3). We found an interaction of formant frequencies and the listener\u2019s gender. Particularly, the acoustic cue of a lower F1 in /a/  was used by listeners to attribute masculinity to a greater extent when listeners were female. Together with the finding that females were better raters in ascribing masculinity to male speakers this adds to research dealing with voice characteristics indicating underlying mate quality in humans , 76. It perception of masculinity/femininity in speech has a long tradition in linguistic studies, investigating the manifestation of self-ascribed gender identity in speech, and here, particularly in the speech of heterosexual adults, is rather new. Questions of sex and gender have concentrated primarily on the distinction between male and female speakers or have dealt with the sexual orientation of the speakers .er, e.g. ,38. The We are aware that the production study and the perception study are measuring different facets of the complex construct of gender identity. Self-rated and perceived gender identity were measured differently. Speakers were asked to rate their gender identity on a number of scaled items, whereas listeners were required to compare the masculinity of two stimuli. Even the results from the GEPAQ-F+ and TMF questionnaires illustrate clearly that strongly correlated yet different socio-psychological aspects of gender identity are being assessed . The diMoreover, differences in the correlation between GEPAQ and TMF also exhibited gender-specific patterns with a weaker correlation for men than women. A man scoring low on TMF, and thereby rating himself as very masculine, could still have a high self-rating on the GEPAQ items, such as emotional, helpful, kind and aware of another person\u2019s feelings. This emphasizes the difference between these scales regarding what aspects of gender identity they capture, and this is especially interesting in the light of changing gender roles with men participating more in family life and being more involved in child care than they were a few decades ago. Gender specificity with respect to the importance of gender identity being transported in speech is also reflected in the different correlations between acoustic cues and self-rated gender identity, which were only found to be significant in males.Both differences in the correlation between GEPAQ and TMF and in the correlations between self-rated gender identity and acoustic cues can be taken as an indication of the importance for males to index their sexuality, gender typicality, masculinity/femininity \u201352, 77.The perception test was only carried out using male stimuli. This was due to the finding in the production study that only male speakers were overtly indexing their self-ascribed gender identity in their speech. However, future experiments need to test female and male listeners\u2019 judgments of masculinity/femininity in female voices.Factors such as gender, ethnicity, age, and group membership have played a pivotal role in explaining inter-speaker variability in speech and idiosyncratic fine phonetic detail since the beginnings of sociolinguistics . This stS1 Data(CSV)Click here for additional data file.S2 Data(CSV)Click here for additional data file."}
+{"text": "E. coli O157 are prevalent in animal reservoirs and in the food chain, but the incidence of human infection due to E. coli O157 is rare. One of the reasons could be inability of the organism from animal origin to produce sufficient amount of Shiga toxin (Stx), which is the main virulence factor associated with the severe sequelae of infection. This study aimed to fill out this knowledge gap by investigating the toxigenic properties and characteristics of stx phage of E. coli O157 isolated from animal sources in Bangladesh.In many Asian countries including Bangladesh stx2 positive E. coli O157 of food/animal origin for stx2 gene variants, Shiga toxin production, presence of other virulence genes, stx phage insertion sites, presence of genes associated with functionality of stx phages (Q933 and Q21) and stx2 upstream region. Of the 47 isolates, 46 were positive for both stx2a and stx2d while the remaining isolate was positive for stx2d only. Reverse Passive Latex Agglutination assay (RPLA) showed that 42/47 isolates produced little or no toxin, while 5 isolates produced a high titre of toxin (64 to 128). 39/47 isolates were positive for the Toxin Non-Producing (TNP) specific regions in the stx2 promoter. Additionally, all isolates were negative for antiterminator Q933while a majority of isolates were positive for Q21 gene suggesting the presence of defective stx phage. Of the yehV and wrbA phage insertion sites, yehV was found occupied in 11 isolates while wrbA site was intact in all the isolates. None of the isolates was positive for the virulence gene, cdt but all were positive for hlyA, katP, etpD and eae genes. Isolates that produced high titre Stx (n\u2009=\u20095) produced complete phage particles capable of infecting multiple bacterial hosts. One of these phages was shown to produce stable lysogens in host strains rendering the Stx2 producing ability.We analysed 47 E. coli O157 isolates in Bangladesh carry inducible stx phages and have the capacity to produce Stx2, indicating a potential risk of E. coli O157 infection in humans.Despite low frequency in the tested isolates,  Escherichia coli O157 in humans can lead to haemorrhagic colitis and often to a more serious complication, known as haemolytic\u2013uremic syndrome. Cattle are asymptomatic reservoirs for E. coli O157 . The suspension was treated with DNase 1 (10\u00a0mg/ml) and RNase (10\u00a0mg/ml) followed by proteinase K (20\u00a0mg/ml). DNA was extracted using equal volume of Phenol: Chloroform: Isoamyl alcohol (25:24:1) followed by precipitation with double volume of absolute alcohol and 3\u00a0M sodium acetate (pH\u00a04.6). Phage DNA was dissolved in TE buffer and stored at -20\u00a0\u00b0C until further use. stx2 gene was detected in phage DNA by PCR as described previously   with and without MMC induction were analysed by FIGE to determine if there is any association between toxin production and inducibility of phages. Considerable increase in intensity of stx2 phage DNA (and thus replication) was observed after MMC induction of isolates that produced relatively high titre of toxin , low \u22643 or no ti), low \u22643 or no ti), low \u22643 or no ti), low \u22643 or no tistx2-phages to infect different hosts was analysed by plaque-forming assay and by hybridization of the plaques with a 372-bp stx2 gene probe  infected clinical isolate of S. sonnei but the phages induced from isolates originated from goat  could not infect S. sonnei. E. coli MC1061, DH5\u03b1 and S. dysenteriae 4 were infected by three phages each (Table\u00a0E. coli O157:H7 NCTC 12900 (Stx negative), while phage induced from the control E. coli O157:H7 NCTC 12079 isolate infected E. coli MC1061 and S. sonnei. Although clear infection of S. dysenteriae type 2 (data not shown) and type 4 was observed with phages from M163 and G71 isolates, respectively but these plaques were negative in plaque blot hybridization assay with stx2 probe suggesting that isolates carrying inducible phages other than stx2 phage.The ability of induced stx2 phages to infect and lysogenize different susceptible bacterial strains was assessed. Phage induced from isolates G51 successfully lysogenized E. coli MC1061, which was stable after repeated sub-cultivation of the transduced strain. The lysogenized strain produced high titre of Stx2 toxin in VTEC-RPLA assay (toxin titre 64). None of the phages from remaining isolates was found to lysogenize bacterial hosts. PFGE banding pattern of phage integrated host strain was almost identical with that of phage non-integrated strain except for disappearance of an 80\u00a0kb band in the lysogenized strain along with the intensification of a 140\u00a0kb band, which is indicative of the integration of phage DNA into bacterial host chromosome , extracted DNA from the gel and did PCR for stx2 gene. We also excised the 80\u00a0kb band from the host strain and did PCR for stx2 gene using template DNA extracted from gel. Only the intensified 140\u00a0kb band from lysogen was found positive for stx2 gene while none of the 140\u00a0kb and 80\u00a0kb bands in host strain (E. coli MC1061) was positive demonstrating the integration of stx2 gene in the bacterial chromosome.The ability of ome Fig.\u00a0. For furE. coli O157 isolates of cattle origin, particularly from Asian countries, contain defective stx phages and, therefore, cannot produce sufficient amounts of toxin required to cause severe human infection [E. coli O157 isolates from food or food-producing animal (cattle) origin have little or no ability to produce Shiga toxin in spite of being positive for the stx gene. This could be one of the reasons for a low prevalence of E. coli O157 infection in humans in Bangladesh [E. coli O157 is Stx2 and adhesin intimin; the role of other virulence factor such as enterohaemolysin, a serine protease (EspP) and catalase or peroxidase (KatP) in causing infection in human may be low [E. coli O157 infection in humans have also been reported from other Asian countries including Thailand and Malaysia [n\u2009=\u20095; 11%) can produce high titre of Stx2 using the VTEC-RPLA assay. We analysed these isolates for a set of four PCR-amplifiable genetic loci as markers that were previously reported to be linked with toxin non-producing (TNP) characteristics of E. coli O157. We found that most of the isolates were positive for these four genetic markers whereas a few isolates showed negative results for one or two of these markers. Among the 5 isolates that were capable of producing high titre of toxins, four were positive for TNP PCR, which is not in agreement with the previous study [Q933and Q21 could be a better marker for the detection of TNP strains from both human and animal origins. Previous studies demonstrated that E. coli O157:H7 strains harboring the antiterminator gene variant Q933 produced significantly higher levels of Stx2 than strains with the Q21 variant [Q933, although 5 isolates produced moderate to high level of Stx, in contrast with the previous study [Q21, which is also not in agreement with previous reports [Q21. In sum, none of the PCR methods developed to date is 100% specific for detecting toxin non-producing isolates and thus toxin assay is still the most reliable approach.Previous studies have demonstrated that nfection . In thisngladesh . As the y be low . Lower pMalaysia , 19. Howus study . Althougus study , a smallus study . Therefous study  recommen variant . None ofus study . All of  reports , 35, altstx2a and stx2d subtypes. STEC strains carrying more than one stx2 subtypes have been reported in previous studies [Typing of Stx2 provides important epidemiological characteristics of STEC isolates. In this study, we found that all isolates, except for one, carried a combination of  studies , 36. Acc studies . However studies , 39.E. coli O157 showed that only a few isolates have Stx2 phage integration sites at yehV and all isolates have intact wrbA site, which is inconsistent with previous study where wrbA was found to be the most commonly occupied site by Stx2 encoding bacteriophages [yehV is commonly occupied by bacteriophages in E. coli O157 isolated from cattle [stx2 phages at yehV site were amplification-positive for the left but not the right yehV junction, a characteristic more commonly seen in cattle isolates compared to the clinical isolates [yehV and wrbA sites in a significant number of isolates, accentuating the need for further exploration of isolates for the presence of other insertion sites.Analysis of phage integration sites in iophages . Neverthm cattle . It has isolates ; corroboE. coli O157 isolates, we examined the inducibility of stx phages present in these isolates. Apart from being the reservoir of main virulence factors of STEC, stx phages are involved in the regulation of pathogenicity factors and the development of genomic plasticity of host bacteria [E. coli O157 strains, high titre of toxin production is coupled with the production of infectious phage particles [pR\u2019 promoter [E. coli O157:H7 12,079 as a control which carries MMC inducible stx phage. Complete cessation of bacterial growth was observed in this isolate within 10\u00a0h of MMC treatment. Comparing the degree of induction of phages in E. coli O157:H7 12,079 with phages in cattle isolates, we found that phages in M163 had the same degree of induction with E. coli O157:H7 12,079 strain while phages in M133, M18, G51 and G71 were inducible at a relatively slower rate (Fig.\u00a0In order to get more insight into characteristics of TNP bacteria . Among varticles . TNP isopromoter . Howeverstx2 gene in induced phage DNA extracted from all five isolates. EcoR1 digestion of phage DNA revealed a high degree of heterogeneity among DNA bands in FIGE indicating a considerable diversity of phage genomes. However, the heterogeneity was not seen in hybridization of stx2 gene as a single band of similar size was found to be hybridized with most of the phage DNA samples. The heterogeneity in EcoR1 digested phage DNA might be due to the presence of non-stx phages present in the isolates.PCR confirmed the presence of E. coli DH5\u03b1, E. coli MC1061, clinical isolates of Shigella sonnei and S. dysenteriae type 4. More precisely, 3 of the 5 stx2 phages could infect a clinical isolate of S. sonnei. Stx gene containing bacteriophage in S. sonnei was also reported previously and studies suggested that Shigella might play a role in spreading of stx genes [S. sonnei has been increasing in patients with diarrhoea in the country [S. sonnei has been recently emerged, which carries all essential virulence genes needed for enteroinvasive pathogenesis along with an inducible Stx1 encoding prophage providing additional virulence potential related to Shiga toxin [Horizontal gene transfer plays a major role in virus evolution by creating new combinations of genetic material through pairing and shuffling of related DNA sequences \u201344. We htx genes \u201348. This country . A new vga toxin .stx2 phage induced from isolate G51 in E. coli MC1061. The lysogenized strain produced a high titre of toxin similar to parent strain without MMC induction. In PFGE analysis, XbaI digestion of chromosomal DNA of lysogenic and non-lysogenic strains of E. coli MC1061 confirmed the integration of phage DNA to MC1061 DNA. We found that a single band of approximately 80\u00a0kb disappeared from lysogenic strains while another band of approximately 140\u00a0kb in size substantially intensified, suggesting that an additional band of similar size is present in the same position (Fig.\u00a0stx2 phage DNA has genome sizes in the range from 49 to 66\u00a0kb [E. coli MC1061 (Fig.\u00a0stx lysogens occur at a low frequency and all stx phages are not capable of generating lysogens at the same frequency [stx phages induced from lysogenic strains generate lysogen rarely even under the best conditions [Among six bacteriophages used in testing lysogenic conversion of recipient bacterial strains, we identified lysogens of ion Fig.\u00a0. The stx061 Fig.\u00a0. In generequency  or lysogrequency . stx phanditions .E. coli O157 isolates producing no or low toxin, particularly in Asian countries where the prevalence of these organisms in the animal reservoir is high. Besides, detection of active and infectious phages with lysogenic properties in the current and previous [Nearly all bacterial genomes comprise of multiple active or defective phages . Prophagprevious , 55 studE. coli O157 from food and food-producing animals tested in this study are not capable of producing Shiga toxin, the main virulence factor for the organism. This might be one of the reasons that E. coli O157 infection is not prevalent in humans in Bangladesh. The non-toxigenic characteristic of E. coli O157 strains is attributed to the presence of genetically defective stx phages in isolates. Unlike non-toxigenic isolates, the study also detected a few toxin-producing isolates carrying active stx phage. Stx phages from these isolates are capable of infecting multiple bacterial hosts and producing stable lysogen in E. coli and thus converting it into a toxigenic strain. This implies that the evolution of new STEC can readily occur in the environment, particularly in areas polluted with cattle feces.The majority of"}
+{"text": "Lung adenocarcinoma (LUAD) is the most common histological type of all lung cancers and is associated with genetic and epigenetic aberrations. The tumor, node, and metastasis (TNM) stage is the most authoritative indicator of the clinical outcome in LUAD patients in current clinical practice. In this study, we attempted to identify novel genetic and epigenetic modifications and integrate them as a predictor of the prognosis for LUAD, to supplement the TNM stage with additional information.A dataset of 445 patients with LUAD was obtained from The Cancer Genome Atlas database. Both genetic and epigenetic aberrations were screened for their prognostic impact on overall survival (OS). A prognostic score (PS) integrating all the candidate prognostic factors was then developed and its prognostic value validated.p < 0.001) and recurrence-free survival . Patients in the early stages (stages I/II) and advanced stages (stages III/IV) of LUAD could be further subdivided by PS into four subgroups. PS remained efficient in stratifying patients into different OS (p < 0.001) and RFS (p = 0.005) when the low- and high-risk subgroups were in the early stages of the disease. However, there was only a significant difference in OS (p = 0.04) but not RFS (p = 0.2), between the low-risk and high-risk subgroups when both were in advanced stages.A total of two micro-RNAs, two mRNAs and two DNA methylation sites were identified as prognostic factors associated with OS. The low- and high-risk patient groups, divided by their PS level, showed significantly different OS (PS, in combination with the TNM stage, provides additional precision in stratifying patients with significantly different OS and RFS prognoses. Further studies are warranted to assess the efficiency of PS and to explain the effects of the genetic and epigenetic aberrations observed in LUAD. Lung cancer is the leading cause of global cancer-related mortality, and ranks second in the estimated new cases of cancer in both sexes in the United States . Lung adAberrant genetic and epigenetic modifications of oncogenes and tumor suppressors contribute to the tumorigenesis and progression of LUAD . GeneticIn recent years, a class of small non-coding RNA molecules, called microRNA (miRNA), has been increasingly investigated . miRNAs So far, many studies have established panels of prognostic factors that predict the outcomes of patients with LUAD, based on multiple lines of evidence. However, most studies were conducted without integration of the network constituted by dysregulations at different levels. Because LUAD represents a set of heterogeneous diseases in which aberrations can exist at genome and epigenome levels, we performed a genome-wide analysis, which should provide more comprehensive insight into survival prediction. Using the data of 445 patients from The Cancer Genome Atlas (TCGA) database, we identified prognostic value of two miRNAs, two mRNAs and two methylation sites. A prognostic score (PS) was developed by integrating these factors to stratify LUAD patients with different lengths of survival into subgroups. From our data, combining PS and the TNM stage achieved greater accuracy in predicting the prognoses of patients with LUAD, indicating that PS is a promising system for personalized and precise medicine.1, including the expression levels of 20530 mRNAs, 2228 miRNAs and 485577 DNA methylation sites, together with the outcomes of 630 patients. The exclusion criteria were listed as follows: (1) Patients whose genomic or epigenomic information was absent; (2) Genes lacking information on either their transcript (mRNA or miRNA) or DNA methylation levels in more than half the LUAD samples; (3) Patients whose survival information was unavailable. Ultimately, a total of 445 LUAD patients were included in the study, together with 16928 mRNAs, 453 miRNAs and 395963 DNA methylation sites.The genome-wide data for 706 LUAD patients was downloaded from TCGA databasep \u2264 0.1 for further analysis.A Cox regression model was used to evaluate the association of gene transcripts (mRNA or miRNA) and DNA methylation sites with lengths of overall survival (OS). A univariate Cox regression analysis was initially used, followed by the screening of included potential factors with a Afterward, considering the remaining large numbers of gene transcripts and methylated sites, we performed a Lasso-Cox analysis to screen and shrink the data. We then used multivariate Cox regression to further analyze the association between the gene transcripts or DNA methylation sites with OS, while adjusting for other clinicopathological factors.p \u2264 0.05).We retrieved the potential target genes of miRNAs that had already been shown to be significantly associated with OS from miRTarBase  . Lasso-CThe direction of association among the transcripts and methylation sites were calculated with Spearman\u2019s correlation in the 445 LUAD tissues. If an mRNA tended to increase when miRNA or methylation increased, the Spearman\u2019s correlation coefficient was positive. If an mRNA tended to decrease when miRNA or methylation increased, the Spearman\u2019s correlation coefficient was negative. We set a threshold of 0 with which to assess the candidate miRNAs, mRNAs and methylation sites. Any pair with a correlation coefficient value < 0 was considered to be negatively correlated, whereas any pair with a correlation coefficient value > 0 as positively correlated.2. The impact of the candidate methylation sites on survival was confirmed in MethSurv3  and advanced-stage (stages III\u2013IV) subgroups based on the pathological stage. The Kaplan\u2013Meier method and log-rank tests were used to assess the differences in OS and RFS between in the subgroups.p < 0.05 was considered statistically significant.All statistical analyses were performed with R version 3.4.4 . A two-tailed The clinicopathological characteristics of the LUAD patients in our study are shown in p value was only marginally significant (As shown in nificant . In the nificant .p \u2264 0.05) targets of the 26 survival-associated miRNAs. A Lasso-Cox analysis was used to select the mRNAs of the 21 genes that interacted with the corresponding survival-related miRNAs. After a multivariate Cox regression analysis of the 73 potential prognostic factors, the overexpression of two miRNAs (MIMAT0002890 and MIMAT0000426) and the hypermethylation of two sites (cg12141052 and cg16404170) were confirmed as significant predictors of worse prognosis  and RFS . As shown in p < 0.001] and RFS  between two groups.To further assess the predictive capacity of all the candidate prognostic factors, PS was established as an integrated prognostic predictor. To verify the efficiency of PS, the 445 LUAD patients were divided into two groups stratified by the median PS. The high-risk group (PS > 1.88) included 223 patients and the low-risk group (PS < 1.88) included 222 patients. As shown in p < 0.001) and RFS  when the low-risk and high-risk subgroups were in the early stages of the disease (p = 0.04) but not RFS  between the two subgroups when both were in the advanced stages of the disease  but not RFS  in the low-risk and high-risk groups. Thus, PS was proved to be a useful prognostic indicator that can supplement additional information to the TNM stage, especially for LUAD patients in the early stages of the disease. Our study suggests that the combination of the TNM stage and PS increases the accuracy in predicting the outcomes of patients with LUAD.Significantly different OS and RFS were also observed among the subgroups in further analyses , 4B. On  disease , 4C. How disease , 4D. On The past decade has witnessed rapid progress in next-generation sequencing and its increasing application in preclinical practice. In recent years, several studies have attempted to associate the transcriptome or epigenome with the clinical outcomes of patients with LUAD . Zhang eTo the best of our knowledge, this is the first study to integrate genetic and epigenetic modifications for survival prediction in LUAD patients using TCGA samples. With a comprehensive analysis and screening of mRNA expression, miRNAs and DNA methylation sites based on samples from 445 patients, we identified a set of prognostic factors from both the transcriptome and epigenome. Notably, we included the histologic subtypes and the TNM stages in our initial survival analysis. In this way, we developed a novel subgrouping system that integrates PS and the TNM stage to predict the survival of patients with LUAD.We started by identifying the clinicopathological characteristics associated with the OS of patients with LUAD. Both Cox regression and Kaplan-Meier survival analyses confirmed the significant prognostic impact of the TNM stage . Further4 was preferentially considered for the validation of our candidate miRNAs, however, the small number of miRNA targets shared between miRTarBase and Targetscan limited its use . Significantly different OS was also observed among subgroups stratified by PS plus the TNM stage (n = 65). On the contrary, PS remained consistently efficient in stratifying OS in the smokers (n = 359)  .p = 0.2) should be possibly attributed to the limited number of LUAD patients with advanced-stage disease. Last but not least, the clinical utility of PS identified here may be limited in patients with small-sized lesions because of the difficulty in extracting sufficient RNA and protein. More studies are warranted to assess the roles of these candidate prognostic factors in LUAD.There were several limitations to our study. For instance, risk factors such as packages of cigarettes and adjuvant therapy were not included in our analysis because of their interpatient heterogeneity. Moreover, the histologic subtypes was unsatisfactory in distinguishing prognoses in the multivariate analysis which could be explained by the missing histologic information for almost half the patients. It is noteworthy that the failure of PS to distinguish RFS in the advanced-stage subgroups  for LUAD from the genome and epigenome, and developed PS from them. Combining the TNM stage and PS provided additional precision in stratifying patients into significantly different OS and RFS subgroups. Further studies are warranted to assess the efficiency of PS and to explain the effects of these observed genetic and epigenetic aberrations in LUAD.DC, YS, SL, CC, and YC designed the study. DC, YS, FZ, and EZ performed the data analysis. DC, YS, FZ, XW, and XZ wrote the manuscript. GJ, SL, CC, and YC reviewed and edited the manuscript. YC and FZ afforded to conduct our study.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."}
+{"text": "The aim was to obtain such nanostructured coatings for titanium-based biomedical surfaces, which would induce multi-functional properties to implantable devices, such as the controlled release of the therapeutically active substance under the action of a magnetic and/or electric field. Thus, the unaltered laser transfer of the initial biomaterials was reported, and the deposited thin coatings exhibited an appropriate nanostructured surface, suitable for bone-related applications. The laser processing of PANI-LIG materials had a meaningful impact on the composites\u2019 wettability, since the contact angle values corresponding to the composite laser processed materials decreased in comparison with pristine conductive polymer coatings, indicating more hydrophilic surfaces. The corrosion resistant structures exhibited significant antimicrobial activity against Escherichia coli, Staphylococcus aureus, and Candida albicans strains. In vitro cytotoxicity studies demonstrated that the PANI-LIG-modified titanium substrates can allow growth of bone-like cells. These results encourage further assessment of this type of biomaterial for their application in controlled drug release at implantation sites by external activation.Composite thin coatings of conductive polymer  embedded with aminoglycoside Gentamicin sulfate (GS) or magnetite nanoparticles loaded with GS (Fe The laser processing of antimicrobial composite materials with both electrical and magnetic properties, in the form of thin coatings, remains a highly topical and challenging subject of modern healthcare-related applications. Being considered a multivalent class of biomaterials, this type of smart surface has lately received prodigious attention from the international scientific community because of their great implication in both fundamental studies and practical biomedical applications ,2.3O4) and loaded with aminoglycoside antibiotic molecules , were deposited by the matrix assisted pulsed laser evaporation (MAPLE) technique, to examine their potential as electrically and magnetically controlled drug delivery platforms. The effectiveness of the selected laser processing method is dictated by the patterning facility for depositing on different geometric shapes of substrates in a highly uniform manner 3O4@GS) wThe metallic precursors (heptahydrate ferrous sulfate and anhydrous ferric chloride) were dissolved in ultrapure water. The resulted solution was dropwise added into an ammonia aqueous solution containing Gentamicin sulfate  and main3O4@GS powders suspended in DMSO as a 3% (w/v) solutions, respectively) were poured into a pre-cooled MAPLE target holder and subsequently immersed in liquid nitrogen for 15 min. The resulted frozen targets were kept at a temperature of ~173 K by introducing active liquid nitrogen cooling during the deposition.To obtain the solid MAPLE targets, magnetite-free (with or without antibiotic addition) and antibiotic-functionalized magnetite embedded suspensions  model COMPexPro 205  that operated at a repetition rate of 20 Hz. A laser beam homogenizer was used to improve the energy distribution of the laser spot. Within our experiments, the deposition parameters were maintained constant, namely the target-substrate distance (5 cm), subsequent number of laser pulses , laser fluence (100 mJ/cm2), and laser spot area (33 mm2). During the deposition process, the frozen targets were rotated at a rate of 50 Hz, to avoid the target heating and drilling due to the multiple laser irradiations. In addition, to improve the uniformity of deposition, the substrates were continuously rotated at a rate of 50 Hz.The MAPLE experiments were carried out in a stainless steel reaction chamber at 5 Pa pressure, using a KrF* laser source (\u03bb = 248 nm and \u03c42), depending on physico\u2013chemical characterization or biological assays, respectively. All depositions were conducted at room temperature, and before the deposition experiments, all the substrates were subjected to a successive ultrasonic cleaning treatments with acetone, ethyl alcohol and deionized water for 15 min and further dried in a jet of high purity nitrogen. The as-deposited samples were denoted as PANI-LIG (pristine polymer samples), PANI-LIG:GS (composite antibiotic-loaded polymer samples). and PANI-LIG:Fe3O4@GS . For data comparison, a control set of coatings were prepared by dropcast on double side polished Si (100) and glass substrates, to evaluate the potential chemical changes occurred during the laser transfer.The simple and composite coatings were deposited onto grade 4 commercial titanium disks (12 mm in diameter and 0.2 mm thickness), transparent infrared (IR) (100) silicon slides and glass slides (10 \u00d7 10 mm\u03b1 radiation from a Cu X-ray tube (run at 15 mA and 30 kV) was used. The samples were scanned in the Bragg angle 2\u03b8 range of 20 to 70\u00b0, with a step size of 0.04\u00b0, and 6 s per step.(i) X-ray diffraction (XRD) analysis was performed to investigate the crystalline nature of the deposited coatings. A Bruker D8 Advance diffractometer , equipped with a Cu target X-ray tube, in the parallel beam setting was used. In all cases, K\u22121, with a resolution of 4 cm\u22121 was used, and for each sample, a set of 50 individual scans was acquired.(ii) Fourier transform infrared spectroscopy (FTIR) was performed for stoichiometry and chemical functions integrity investigation of the as-deposited coatings. A Shimadzu FTIR 8400 s spectrophotometer , operating in transmission mode in the range of 5000 to 500 cm(iii) Scanning electron microscopy (SEM) analysis revealed the morphological features of the deposited materials and it was performed on an ApreoS scanning electron microscope , using secondary electron beams with energies of 10 kV, on samples capped with a thin gold layer to diminish the accumulation of electric charges on the specimen surface.2, in tapping mode.(iv) Atomic force microscopy (AFM) was performed for topographical evaluation of the deposited coatings by using a MultiView 4000 Nanonics System  over a scanned area of (40 \u00d7 40) \u03bcm\u00ae software , using water as a standard solution, at room temperature, by the goniometric method. In this context, 2 \u03bcL of water was dropped in a controlled manner on samples\u2019 surface, maintaining constant the droplet volume and the dropping distance. Thus, the droplet image was recorded under a 2\u00b0 angle with respect to the plan of the examined sample. To obtain the CA values, the acquired data were fitted using the second-degree polynomial equation, and the slope values corresponding to the tangent drawn by the drop were extracted 40]. Samp2) until they reached confluence. Afterwards, they were detached using 1% Trypsin, seeded at a density of 10,000 cells/sample onto the PANI-LIG substrates. These were further incubated in standard conditions, during 48 h, to examine the morphological behavior of the osteoblast-like MG-63 cells 2) until Micrographs were acquired using an FEI Inspect S microscope  operating at 20 kV acceleration voltage.Staphylococcus aureus ATCC 25923) and Gram-negative (Escherichia coli ATCC 15224) bacteria, and also against fungal strain . Glycerol stocks of these microbial strains were streaked on nutritive agar plates and colonies were allowed to grow at different for 24 h at 37 \u00b0C.The anti-biofilm assays of the deposited thin coatings were performed according to ASTM E2180-07 (2012) standard by adjusting the protocol described in ,45. The Before testing, all deposited coatings were sterilized by UV radiation exposure for 30 min. For assessing monospecific biofilms formation, 2 mL of liquid nutrient medium (nutrient broth) was added to the sterilized sample independently placed in 6-well plates containing either test (deposited samples) or control (uncoated) substrates. The microbial inoculum consisted of a volume of 20 \u03bcL of the microbial suspension prepared by fresh colonies  prepared in phosphate buffered saline (PBS) .Biofilm formation on the materials was assessed after incubation for distinctive periods, by the viable cell count (VCC) method. Concretely, after the incubation period, the samples containing attached pathogenic organisms were gently washed with sterile physiological PBS and transferred into a new sterile tube, containing 1 mL of fresh and sterile nutrient broth and vigorously vortexed for 30 s to detach the biofilm-embedded cells. The obtained cellular suspensions  were then serially diluted, and each dilution was spot-inoculated onto nutritive agar, to achieve microbial growth and to estimate the number of colony forming units (CFU)/mL . ExperimGiven the fact that some diffraction peaks corresponding to the pristine polymer were overshadowed by other maxima corresponding to the obtained composites, we considered providing a thorough overview with respect to the nature of the deposited coatings by including the collected XRD data within two distinctive plots. Therefore, the diffractograms presented in The diffraction peaks observed in The principal diffraction peaks corresponding to the conductive polymer can be observed in all deposited samples, either pristine coating or coatings embedded with sole antibiotic or antibiotic-loaded magnetic nanoparticles. The identified diffraction maxima correspond to approximate 2\u03b8 diffraction angles of 8.98\u00b0, 10.15\u00b0, 13.83\u00b0, 15\u00b0, 17.94\u00b0, 20.36\u00b0, 25.45\u00b0, and 27\u00b0, respectively. The broad peak centered at 2\u03b8 \u2248 15\u00b0 corresponds to the (011) diffraction plane assigned to basic polymer. The peaks located at ~21\u00b0, corresponding to (020) crystal plane of orthorhombic polymer in its emeraldine salt form, could be related to the chain repeated units and parallel periodicity . The pea3O4 nanoparticles exhibits, in addition to sole polymer peaks, diffraction peaks that correspond to crystalline magnetite (marked in orange). The identified additional maxima present the following diffraction angle\u2013crystal plane pairs: 30.36\u00b0 \u2013 (220), 35.75\u00b0 \u2013 (311), 43.52\u00b0 \u2013 (400) and 53.95\u00b0 \u2013 (422), which are attributed to diffraction planes of face-centered cubic Fe3O4 in its pure spinel form [The XRD pattern corresponding to the PANI-LIG materials embedded with drug-loaded Fenel form . Changesnel form .The compositional investigation of the MAPLE coatings, performed by the identification of the main infrared maxima corresponding to the materials considered within our study, indicates a successful and quasi-stoichiometric laser transfer.For comparison reasons, we separately represented the infrared spectra of each MAPLE coating and compared it with the corresponding dropcast (DC) results . As a geIn the case of pristine PANI grafted lignin coatings (\u22121 may result from the superposition of e C=O and C=N (assigned to the quinoneimine group) stretching vibrations of PANI polymer. The adjacent low-intensity absorption bands  have specifically resulted from the stretching vibrations of carbonyl group within lignin. The characteristic vibrations identified at ~1590 cm\u22121 and ~1504 cm\u22121\u2014resulted by the stretching modes of C=C functional group within quinoid (N=Q=N) and benzenoid (N=B=N) rings within PANI, respectively, and represent a clear signature of the polymer backbone [The band identified at 1705 cmbackbone . The C=C\u22121, is particularly assigned to C=C vibration of the aromatic lignin ring. Another absorbance maxima characteristic for lignin is identified at ~1460 cm\u22121 wavenumber and is the consequence of methoxy infrared vibration.The low-intensity absorbance maximum observed within the previous values, concretely at ~1514 cm\u22121 is due to the C\u2013N strong stretching vibrations within aromatic amines in PANI [The peak at ~1300 cm in PANI .\u22121, of low intensity, may be specifically assigned to the C\u2013H in-plane bending vibrations . This particular band is also mentioned in the literature as the \u201celectronic-like band signature\u201d of polyaniline. The following infrared peaks (identified at ~1090 cm\u22121) may result from both organic compounds, as a consequence of superposed C\u2013H bending (within PANI) and \u03b2-O-4 vibration (ether bond band of lignin).The absorption maximum identified at ~1147 cm\u22121 (indicating the head-to-tail coupling within polymer structure) is related to the out-of-plane bending mode of aromatic C\u2013H groups attributed to 1,4-disubstituted aniline units [The infrared band around ~818 cmne units .\u22121, which is particularly attributed to the stretching of SO2 band within Gentamicin, can be observed. In addition, the absorbance band identified at ~1097 cm\u22121 may result from the vibrations of HSO4\u2212 group of the antibiotic. As well, we may consider that the infrared region in the range of 1110 to 1180 cm\u22121 contains absorbance maxima corresponding to both organic PANI and LIG (as discussed above) but also to Gentamicin sulfate (due to constituent C\u2013H vibrations). The 1367 cm\u22121 absorbance peak can also correspond to both PANI and GS, following the overlapping of C\u2013N modes from both compounds and C\u2013O vibrations within the aminoglycoside. The overall shifting tendency of the infrared data corresponding to PANI-LIG:GS coatings may be explained by the weak electrostatic interaction between the secondary amines within polymer structure and the antibiotic hydroxyls.In the case of antibiotic-loaded MAPLE samples  might have resulted from the weak interactions established between antibiotic and hydroxylated magnetite. The presence of Fe\u2013O bond is clearly evidenced at ~647 cm\u22121 wavenumber. On the other hand, the possible distinctive interactions between the amino functions of GS and the metallic ions within Fe3O4 might have determined the splitting of the Fe\u2013O absorbance band  [As for the infrared data corresponding to PANI-LIG coatings embedded with antibiotic-functionalized magnetite nanoparticles (ocesses) .At this point, one can state that the MAPLE technique proved an effective strategy for the synthesis of PANI-LIG-based coatings, enabling the preservation of chemical functions for all considered materials and their quasi-stoichiometric transfer.The SEM images of all MAPLE samples  reveals 3O4@GS composites. It was observed that Gentamicin sulfate and magnetite compounds are uniformly spread into the PANI-LIG matrix, but after the surface functionalization reaction, both the size and shape grains altered. Because the formation of magnetite aggregates is directly related to their nanosized dimensions [3O4@SG samples, a protective antibiotic layer was formed on the metallic nanoparticles surface (Fe3O4), almost entirely dispersed on the surface of the coating. Thus, fewer surface-active sites for particle-particle interactions [It can be observed that all PANI-LIG-based coatings formed compact and uniform layers on the substrates, and it also covered them entirely. Instead, even though all granular and preferentially spherical shaped structures are nanosized, their dimensional range is related to the composition of the coatings, as follows: 100 to 300 nm for simple PANI-LIG samples, and 40 to 100 nm for both PANI-LIG:GS and PANI-LIG:Femensions , magnetimensions ,63. By uractions  remained3O4@GS) exhibit better cohesion and a higher aggregation of grains (resulting in a more amorphous structure of coatings) than the much-ordered alignment arrangement in the case of PANI-LIG. In addition, in good agreement with literature data, that antibiotic and magnetite presence may influence the basic polymer morphology, hydrophilicity and solubility [3O4@GS coatings.In addition to that, the crystalline nature of samples is corroborated by the SEM micrographs. The composite samples  parameter indicates a predominant compact flat surface with peak-like topography exhibiting positive values lower than 1.6.Quantitative measurements of surface roughness are revealed in Rsk parameters is attributable to the reduced presence of peaks on the surface and leads to a homogenous feature of height distribution [In the case of samples with antibiotic, the substantial contrast between the RMS and ribution ,65 confiRku) parameters  supports the skewness observations indicating, once again, that the MAPLE processing method leads to predicted smooth topographies, as are desired for medical applications.As a further matter, the kurtosis (The surface wettability investigation involves the contact established between a liquid (water) and a solid (either uncoated or MAPLE deposited coatings). The contact angle (CA) is a reliable parameter in biomaterials characterization since modifying and/or controlling the wettability behavior is of great importance for cell adhesion and proliferation.CA average values are found in As a general remark, it can be observed that simple conductive polymer coatings exhibit an intrinsically hydrophilic behavior with a contact angle value of 64 \u00b1 3 (it resists water molecules). The tendency is to leave the aqueous solution or to associate its several molecules by hydrophobic interactions so that its contact with water molecules is minimal (by the formation of micelles aggregates). Moreover, it is known that the variation of the pristine PANI-LIG hydrophobicity affects both cells attachment and spread .Nevertheless, laser processing as thin coatings has a significant impact on composites\u2019 wettability, and one observes that CA values decrease in comparison with a pristine conductive polymer, indicating more hydrophilic surfaces, which results in an increase of water permeability of the composite coatings. In addition, all wettability measurements revealed a slight decreasing tendency of CA values, when compared to bare Ti substrate (69.5 \u00b1 4.5).A lower contact angle was evidenced for antibiotic-embedded polymer coatings (49.5 \u00b1 0.9), suggesting that water droplets placed on the composite coatings surface immediately penetrate into the grains. This behavior can be explained by the PANI-LIG porous nature, possessing high surface energy that causes the water to seep into the voids of the structures . In geneOne observes that earlier surface topographic results (relevant for human osteoblast differentiation and proliferation ) are ent3O4@GS 35.9 \u00b1 2.0\u00b0, the results being in good agreement with AFM observations, which obtained lower roughness values. The approximately flat surface of the sample permitted the water drop dripping, thus, justifying the small CA values obtained. Moreover, from long-term studies concerning the drop evolution in time  has significantly improved the composite coating\u2019s wettability behavior, indicating the possibility to adjust the contact angle values of chosen biomaterials to biological requirements.Ecorr, Rp , Icorr (determined by the intersection of the linear portions of the anodic and cathode curves), and CR, listed in ECorr and a lower Icorr indicate a better/good corrosion protection.The antioxidant capacity of polymeric coatings  may offer protection against various diseases, such as cardiovascular diseases, osteomyelitis or cancer , in partPolarization resistance values obtained from electrochemical impedance spectroscopy data and from Tafel curves are congruent series, supporting a decrease of corrosion current in PAN-LIG samples.2) because the electrolyte diffuses through the deposited polymer layer. However, when compared to reference Ti substrate, the enhanced corrosion behavior of PANI-LIG coatings may be assigned to limited diffusion of electrochemically-active species to the substrate. As well, compounds with lower formal potentials are considered stronger reducing agents, and in this observation, are more powerful antioxidants [As a general remark, corrosion potential differences are small because the material exposed to corrosion remains the substrate (titanium), and also the measured corrosion current refers mainly to the support material  is interpreted as a corrosion protection effect, obtained by limiting the diffusion of the support material.Furthermore, it is known that polyaniline is usually electroactive only under acidic conditions (pH less than 4 ), while Rp values were reported for the PANI LIG:GS coatings led us to assume that the initial electrochemical reactions between the PANI-LIG:GS coatings and the electrolyte happened. This may cause the partial release of the incorporated compounds resulting in limited areas exposure of the substrate to new interactions with the electrolyte. However, such potential mechanism did not severely affect the titanium, since it is naturally protected by the native oxide layer [In the case of PANI-LIG:GS coatings, we observed quick corrosion in the early stage. We presume, since the coatings are thick and stable and act as a barrier, that the compound molecules must travel a long distance from the coating to the medium. Consequently, the OCP is much slower (56 mV). Undoubtedly, as the antibiotic was added, the coating protection ability was enhanced. Comparable de layer .3O4 and Gentamicin sulfate) in the coating which results in the increasing tortuosity of the oxygen and water vapor diffusion pathways. It is known that there is a need for sufficient H2O and O2 for the rust formation and dissolution of titanium for causing corrosion. If any of these processes are prevented, the corrosion is inhibited, and deposited coatings become efficient for corrosion prevention. Therefore, it is reasonable to believe that increasing the tortuosity of the diffusion pathways could prevent H2O and O2 from accessing the substrate surface, thus, leading to good anticorrosion properties [The mechanism of the enhanced corrosion protection effect of the composite coatings, both the simple antibiotic functionalized, and magnetic nanoparticles embedded drug might be a result of the well-dispersed compounds .Similar results of electrochemical behavior are found in literature and could be related to the reduction reaction of the polymeric matrix, which contributes in increasing the cathodic currents with corrosion rates of between 0.004 and 15 mm/year ,77.Morphological investigations were performed for osteoblast-like cells cultured during 48 h in contact with PANI-LIG- based thin coatings, to reveal information on the ability of these substrates for cell attachment and sustained growth. In these regards, fluorescence microscopy imaging was performed to evaluate the cytoskeleton appearance by specific staining of the actin filaments with Texas-Red\u2013phalloidin.Considered a marker of the cell\u2019s well being, the cytoskeleton has many functions besides offering the shape of the cell . For allFrom the morphological point of view, the osteoblast-like phenotype with preferential flattened morphology was maintained for every deposited sample. Extensions of the actin filaments, the phylopodias, are strongly emphasized and would favor and strengthen the cells\u2019 anchorage onto the coating surfaces. Moreover, specific longitudinal disposal of actin microfilaments and their multiple extensions, with eccentric prominent nuclei  was obseIn addition, the normal morphology of Hoechst- stained nuclei (blue), indicated that the compounds did not interfere with the normal growth of the existing cells . One canScanning electron microscopy investigations confirmed the information obtained from immunofluorescence evaluation of the cytoskeleton and nuclei morphology. Such behavior could be related to favorable interaction of the cells at the interface with the substrates. This is supported by the abundance of branched actin filaments protuberances extending to form connections from one cell to other and with the coating substrates a,b.However, in compliance with immunofluorescence and MTS viability measurements , the morA change in cells\u2019 behavior might be induced not by the deposited composite coatings, but the interaction of the osteoblast-like cells with the inorganic nanoparticles ,82. MoreEscherichia coli, Staphylococcus aureus or Pseudomonas aeruginosa were found in the literature [The antimicrobial properties of conductive functionalized polyanilines have been investigated by exploring their interaction with microbial cells. It seems that acidic dopants on the polymer molecular chains react (by electrostatic carry charges of different polarity) with the microbial surface structures and induce membrane and cellular wall disruption resulting in cellular death in a time-exposure manner . Data reterature .S. aureus and E. coli)  . The inh coating .Additionally, the simple antibiotic coatings proved to be a sustained antimicrobial activity, with CFU/mL values decreasing continuously on increasing the incubation time.E. coli, commencing at the 48 h incubation time interval.However, the antibiotic-containing coating begins to lose its efficiency in biofilm inhibition, especially in Overall, the biological assays demonstrated that the composite coatings synthesized by MAPLE can provide an efficient protection against microbial biofilms development, without inducing any cytotoxicity towards tested MG-63 cells. A more significant reduction in the number of viable microbial cells (compared to control) was observed for simple antibiotic coatings and a compromise must be made in choosing the optimum material which fulfills the vital concerns regarding the desired application .By gathering the FTIR data , the morphological topographical and wettability results  one may conclude that all PANI-LIG based coatings were successfully MAPLE transferred by laser processing.All coatings present the specific bands of studied materials, results also validated by XRD, which exhibit the poor crystalline nature of the coatings, with low-intensity maxima emerging corresponding to the base compounds. The effective functionalization of PANI-LIG coatings was proved, being confirmed by good dispersion of the drug and magnetite nanoparticles within the polymer matrix.The antioxidant capacity of PANI-LIG based coatings has significant implications for their inclusion as biomaterials in biological media, with the PANI-LIG:GS coating proving to be the most efficient against corrosion, its Icorr being, in this case, 3.7 times smaller than for bare Ti, while its Ecorr increased 2.31 times. Our studies of cytotoxicity showed that PANI-LIG coatings might support osteoblast-like cell growth.S. aureus, E. coli, and C. albicans model strains, studied at three incubation time intervals, was assigned to PANI-LIG:GS samples. Corroborating the cytotoxicity and the antimicrobial activity measurements, along with the physico\u2013chemical characterizations, we may conclude that the proposed conductive polymer-based coatings, either simple antibiotic or magnetite\u2013antibiotic functionalized ones, have the potential for innovative systems for controlled drug release in an external-activated electric or magnetic field. The choose between them depends on the desired application. Nevertheless, further studies may, therefore, be envisioned to specifically evaluate the antibiotic release in electric and magnetic fields.The most efficient antimicrobial activity, considered as long-term protection against adhesion and biofilms developed by"}
+{"text": "Fasciolosis and dicrocoeliosis are important parasitic diseases worldwide, causing significant financial losses due to decrease in production and viscera condemnation in animals. We performed the current research to assess the epidemiology of these infections and determine their significance from an economic perspective in Arak, Iran.In total, we evaluated 118,463 sheep, 207,652 goats, and 43,675 cattle through necropsic analysis at the slaughterhouses. The average weight of sheep, goat, and cattle liver was 1000, 900, and 5000 g, respectively. The average price of liver in the market was 8 USD/kg. Moreover, the elimination of fundamental nutrients and vitamins was evaluated in infected livers. The prevalence of fasciolosis and dicrocoeliosis was determined. Analysis of variance test was applied for the statistical analysis, and the significance level was <0.05.Fasciola hepatica and Dicrocoelium dendriticum were detected in 0.56%  and 0.77%  of the animals, respectively (p=0.1). The annual economic loss attributed to fasciolosis and dicrocoeliosis was 26698.4 and 30479.2 USD, respectively. The total economic loss was 10,880, 9079.2, and 10,520 dollars in sheep, cattle, and goats, respectively. On the other hand, financial loss resulting from fasciolosis was 7160, 6098.4, and 13,440 dollars in sheep, goats, and cattle, respectively. In addition, economic loss due to dicroceliasis was 10,880, 9079.2, and 10,520 dollars, respectively.In total, Overall, fasciolosis and dicrocoeliosis in Iran always remain common in sheep, goats, and cattle that afford major economic loss of all the country also exist in Arak province. The present study could provide basic information for further examination of liver fluke infections in Iran. Fasciola species and Dicrocoelium dendriticum are recognized as the most common liver flukes through many regions of Asia including Iran [Fasciola hepatica has a broad geographical distribution as a plant-borne zoonotic disease and occurs in >51 countries, especially where sheep and cattle are reared [Farm animals play a significant role in human nutrition and socioeconomic evolution and development. Animal products such as milk and meat are a significant source of micronutrients, energy, and protein supply provided approximately 15% of energy and 30% of protein worldwide. In developing countries, with the increase in the number of residents and income growth, the use of animal protein per capita, with the global consumption of meat, is projected to increase by nearly 73% by 2050 . Trematoing Iran -10. Goating Iran . Fasciole reared ,13. Treme reared ,10,14-17Moreover, the infection has been reported in over 300 million cattle, as well as 250 million sheep around the world . In factF. hepatica infection can reach up to 77%, and losses due to fasciolosis in some areas are >70.67 \u00admillion pounds a year [F. \u00adhepatica and Fasciola gigantica species are endemic to northern and western regions of Iran [Fasciola spp. [On the other hand, indirect losses include reduced product quality, decreased rate of production, slow development, fertility impairment, and increased costs of stock replacement, and anthelmintic treatments -21. In is a year ,22. Thers a year . Consides a year ,23. Acces a year . Prevales a year . Among As a year . In fact of Iran \u201326. Repoola spp. -29.Dicrocoelium infection in slaughterhouses showed a prevalence of at least 0.1% up to 29.32% in Iran. The highest prevalence belongs to East Azerbaijan Province in northwestern, and the lowest belongs to Fars Province in the south of Iran [The financial losses due to the condemnation of livers in Asian countries were calculated to be until 17.02 million USD annually . Dicroco of Iran ,31,32.Overall, scant research has focused on the economic loss and its public health significance of fasciolosis and dicrocoeliosis at national and regional levels; in fact, there is no adequate information on this region. So, we aimed to, determine the prevalence of fasciolosis and dicrocoeliosis in ruminant and, evaluated economic loss caused by the condemnation of livers at abattoirs of Arak in the center of Iran.The project was found to be in accordance with the ethical principles and standards for conducting medical research. This project was approved by Research Ethics Committee, Faculty of Medicine of Kashan University of Medical Sciences, Iran.Arak is a city of Markazi Province, Iran . It is lIn this cross-sectional study, data were collected during 2013-2016 to evaluate the prevalence of infection in the abattoirs of Arak and determine direct economic losses, resulting from liver condemnation through postmortem analysis. Through visualization and palpation, the liver and bile duct were examined.Fasciola and Dicrocoelium parasites were evaluated in the removed liver and gallbladder. The species of parasites were identified, using the morphological characterization of F. hepatica, F. gigantica, and D. dendriticum.Overall, 369,790 slaughtered sheep, goats, and cattle were recorded during necropsic analysis. To determine the annual financial loss, the average number of slaughtered animals (per year in the abattoirs) was multiplied by the prevalence of infection in the current study and the mean price of liver in the region. We summarized the results for each year to determine the number of infected livers in animals. Using the available information, economic loss was estimated during 2013-2016. The total quantities were converted to US dollar. Moreover, direct economic loss imposed by fasciolosis and dicrocoeliosis was measured as follows:In this equation, DFL denotes direct financial loss, N represents the number of condemned livers, P is the average price of liver ($/kg), and W denotes the average weight of liver (kg). The average weight of the liver was measured by weighing 118,463 sheep, 207,652 goats, and 43,675 cattle livers. Furthermore, the average weight was measured to be 1000, 900, and 5000 g in sheep, goats, and cattle, respectively. According to interviews with local butchers, the average price of liver was 250,000 Rials/kg (8 dollars).Minerals and vitamins were measured in each 100 g of liver, according to the protocol proposed by the United States Department of Agriculture .Fasciola and Dicrocoelium species was calculated . The number of infected cases was divided by the total number of slaughtered animals and multiplied by 100. Furthermore, for the comparison of fasciolosis and dicrocoeliosis in terms of prevalence, Analysis of variance test was carried out used to compare the prevalence of fasciolosis and dicrocoeliosis in different animals. Chi-squared test was used to analyze the effect of the season on the prevalence of the disease. The significance level was considered when the probability value p<0.05.The data was processed by Microsoft Excel 2017 and IBM SPSS Statistics  for determination of significant difference between variables. To evaluate the prevalence of fasciolosis and dicrocoeliosis, the number of animals infected with Fasciola and Dicrocoelium species at Arak abattoirs, of which 2105 (0.56%) and 2884 (0.77%) were infected, respectively. The prevalence of Fasciola infection was 0.75%, 0.42%, and 0.76% in sheep, goats, and cattle, while the prevalence of Dicrocoelium infection was 1.14%, 0.60%, and 0.60%, respectively. However, the difference in prevalence rates of fasciolosis (p=0.21) and dicrocoeliosis (p=0.16) was not significant in various hosts  were slaughtered and evaluated for us hosts .Fasciola infection in all hosts was the highest during winter, and the difference was significant (p=0.04). However, no seasonal trend was found in the prevalence of dicrocoeliosis, and differences were not of significance (p>0.1).Furthermore, the prevalence of fasciolosis and dicrocoeliosis was evaluated during 4 seasons, as shown in Fasciola infection in the area study shows alarming values: 895 kg of sheep livers, 762 kg of goat livers, and 1680 kg of cattle livers were condemned. In addition, 1360 kg of sheep livers, 1135 kg of goat livers, and 1315 kg of cattle livers infected with dicrocoeliosis. Based on the analysis of market prices in Arak, the average price of 1 kg of liver was reported to be 8 dollars. The average annual loss of meat and offals, resulting from Fasciola and Dicrocoelium infections, was estimated at 26698.4 and 30479.2 USD, respectively. Overall, 7160, 6098.4, and 13,440 USD were lost due to fasciolosis in sheep, goats, and cattle, respectively. In addition, economic loss due to Dicrocoelium infection was 10,880, 9079.2, and 10,520 USD in sheep, goats, and cattle, respectively , manifestations include fever, vomiting, diarrhea, abdominal pain, hepatomegaly, urticaria and eosinophilia, and can last for months. In the chronic phase (caused by the adult fluke within the bile ducts), the symptoms are more discrete and reflect intermittent biliary obstruction and inflammation. Occasionally, ectopic locations of infection  can occur [D. dendriticum infection in humans are light and asymptomatic. In heavier infections, symptoms may include abdominal pain, biliary colic, digestive disturbances, liver abscesses hepatomegaly, cirrhosis, cholecystitis, and seldom skin rash urticaria [Recently, human fasciolosis and dicrocoeliosis occurred occasionally but are now increasingly reported from entire the world. No continent is free from these diseases, and it is likely that where animal cases are reported, human cases also exist. The number of human cases affected by an occur . Differean occur . Most inrticaria .Fasciola and Dicrocoelium infections was 0.56% and 0.77%, respectively, which was relatively insignificant. Moreover, the prevalence of fasciolosis was 0.75% in sheep, 0.42% in goats, and 0.76% in cattle. Furthermore, the prevalence of dicrocoeliosis was 0.60% in goats, 1.14% in sheep, and 0.60% in cattle. The prevalence of liver fluke infections in ruminants depends on some factors such as the environment, the climate, and the choice of diagnostic methods. Changes in climatic conditions are closely correlated with changes in the prevalence of animal fasciolosis [Based on the findings, fasciolosis and dicrocoeliosis are serious problems in the slaughterhouses of Arak, Iran, and impose great financial losses due to liver condemnation and weight loss. The prevalence of ciolosis .et al. [et al. [Obviously, husbandry conditions of the sheep  majorly contribute to the high incidence of diseases in sheep. Nevertheless, cattle and goats might show hereditary resistance. In the current research, fasciolosis (0.56%) was less prevalent in comparison with similar results which have been reported by Khoramian et al.  with 3.2et al.  with 8.4 [et al.  with 0.9Moreover, in the current research, the prevalence was lower than that found in Zimbabwe with 37% , in Kenyet al. [Fasciola infections was estimated at 3.28%, 2.76%, and 3.68% in sheep, goats, and cattle, respectively Khanjari et al. [Fasciola infections in sheep and goats in the abattoirs of Amol in the north of Iran. Moreover, Khosravi and Babbahamdy [Fasciola and Dicrocoelium diseases; therefore, today, healthier animals enter the market. Two great features differentiate the epidemiology of D. dendriticum from Fasciola species. First, moist environment is not necessary for the intermediate hosts of Dicrocoelium species, which are widely present in meadows. Second, fluke eggs are able to survive on pastures for a long time [Fasciola infections among all evaluated animals was reported during winter. This finding may be associated with meteorological evidence, which predicts many rainfalls during autumn and winter; furthermore, infection is quite prevalent in snails during rainy seasons. In general, it takes 12-16 weeks for a parasite to mature after infection; therefore, patent infection is predicted to be prevalent during winter [In some regions of Iran, the prevalence of fasciolosis is low. Similar results have been reported in slaughterhouses in some regions of Iran. In this regard, Khoramian et al.  have repbbahamdy  describebbahamdy . Compariong time . The higThe reduced rate of production, decreased growth rate of animals, and production of low-quality livestock products for humans, increased susceptibility to secondary infections, and cost of disease control measures are among the consequences of fluke infestation ,50.Furthermore, severe infestation might directly or indirectly lead to mortality through triggering or intensifying diseases. Today, scarce data are available regarding the presence of minerals and vitamins in the livers of infected animals; furthermore, the literature on this subject has not been reviewed so far.The connection between economic loss and diseases, such as fasciolosis and dicrocoeliosis, is of great significance, especially regarding the nutritional elements removed from the humans\u2019 nutrient cycle due to parasitic diseases. No records are available on the financial burden of fascioliasis and dicrocoeliosis in this region of Iran. Based on the findings, the prevalence and economic significance of fasciolosis and dicrocoeliosis were considerable in livestock slaughtered at Arak abattoirs.However, in the present study, the annual \u00adeconomic loss was still moderately high. The high prevalence of infection is indicative of appropriate conditions for the growth and survival of parasites. The present findings highlight the importance of these diseases in farm animals and indicate the need for the development of control measures by studying the economic burdens and evaluating the local epidemiology of diseases. In the present study, the total annual economic loss was higher than that reported in Asella, Ethiopia . DiffereThe current findings showed that fasciolosis and dicrocoeliosis are still major risk factors for the health of livestock products in Arak, Iran. The financial loss resulting from liver condemnation and indirect weight loss was substantial in this study; \u00adtherefore, adoption of control and prevention strategies is necessary.Fasciolosis and dicrocoeliosis were common and economically important infections in cattle, sheep, and goats at Arak slaughterhouses. Furthermore, the annual direct economic loss due to the condemnation of livers infested is relatively high. By studying the economic significance of these diseases and evaluating the local epidemiology, more practical and effective control strategies can be developed to prevent and limit the infection and reduce economic significant.MA designed, overseeing the research project, interpretation of data, and manuscript writing; EN collected the data, HH and MD helped in writing and review of the manuscript. All authors read and approved the final manuscript."}
+{"text": "Spillovers from the Affordable Care Act Medicaid expansion to other social-sector outcomes have received little attention. One that may be especially salient for public policy is the impact of expanded eligibility on jail-related outcomes. This study compares recidivism outcomes in three non-expansion counties to nearby expansion counties before and after Medicaid expansion. Using forty-eight months of arrest data from six urban county jails, we conduct comparative interrupted time series analyses to describe changes in the probability of rearrest and the number of arrests before and after Medicaid expansion. Consistent with previous literature, we find mixed results. In two case studies, Medicaid expansion is associated with decreased rates of recidivism. In the other, we find differential increases in jail-based recidivism after Medicaid expansion. We use contextual information from site visits and stakeholder interviews to understand the factors that may mediate and moderate the relationship between Medicaid expansion and return to jail. Prior to the authorization of state options for Medicaid expansion, low-income adults without dependent children were ineligible for Medicaid in most states. This population over-lapped with the jail-involved population significantly. Both groups were predominantly young, low-income, minority, and male. In 2014, men made up 85.3 percent  and younAlthough federal law permits Medicaid coverage to continue during an individual\u2019s incarceration, forty-six states do not continue coverage . The vasJail-involved individuals also have higher rates of chronic physical health conditions (such as asthma and diabetes), communicable diseases (such as HIV and Hepatitis C), mental illnesses, and substance use disorders than the general population . EstimatThese high risks stem in large part from the conditions under which justice-involved people live and from their limited ability to obtain appropriate care for their health needs. For instance, one-third of inmates taking prescription drugs do not have access to necessary medication while in jail, and more than half (60 percent) of those who require routine blood testing had no testing while in jail . AdditioProviding timely access to health-care services, particularly treatment for mental illnesses and substance use disorders, may be one way to reduce rates of reoffense. Indeed, recent evidence indicates that improved access to evidence-based treatment for mental and addictive illnesses can improve reentry outcomes . Other sEarly work found that recently released people with an SMI in King County, Washington, and Pinellas County, Florida, who obtained Medicaid coverage on release from prison were 16 percent less likely to be rearrested in the following year and spent more time in the community before rearrest (102 versus ninety-three days) than similar people who did not get Medicaid coverage . In contIn a more recent study using these same data in Washington, The U.S. Government Accountability Office (GAO) studied reentry programs in Florida and Michigan. In Florida, it found that access to community services, including through Medicaid, on release from prison was not associated with any change in the likelihood in rearrest, overall. Some groups  did experience a slight decrease in the likelihood of rearrest . During The ACA\u2019s coverage provisions (including Medicaid expansion) not only increased eligibility for previously ineligible populations, but also changed the type of behavioral health-care services that low-income adults have access to. Prior to the ACA, the public behavioral health system was funded predominantly by categorical Medicaid programs  and state and federal budgeted funding mechanisms . These funding mechanisms often did not require evidence-based treatment in specialty substance use disorder treatment programs, nor did they encourage adequate treatment capacity.Since ACA enactment, treatment providers in expansion states are generally required to meet conditions of participation in Medicaid, which require greater capacity and provision of evidence-based behavioral health care. Additionally, federal legislation and regulations attempt to standardize Medicaid benefits for behavioral health-care services. Together, the Mental Health Parity and Addictions Equity Act of 2008 and the ACA require all Medicaid managed care plans to cover treatment services for mental illness and substance use disorder as essential health benefits, and at parity with medical and surgical benefits. The ACA also requires that all enrollees from the expansion population have coverage for mental health and substance use disorder care that is at parity with medical and surgical coverage.Despite federal legislation and regulation to standardize these benefits, the fee-for-service behavioral health services provided by Medicaid vary by state, given that fee-for-service Medicaid programs are not subject to the provisions of the Mental Health Parity and Addiction Equity Act or the ACA (with the exception of expansion adults). For instance, forty-three of the fifty-one jurisdictions surveyed cover inpatient psychiatric hospitals stays, four states require a copayment for these services, and seventeen states limit these services . CoveragIn addition to changes in Medicaid benefit design and covered services, the target populations of the ACA\u2019s Medicaid expansion differ from those included in previous studies examining the relationship between access to health-care services and recidivism. Taken together, these imply that previous findings about Medicaid and recidivism may not generalize to the expansion population. In this study, we extend previous analyses to the Medicaid expansion population and provide one of the first quasi-experimental analyses of the relationship between gaining health insurance coverage and criminal justice outcomes. To do so, we compare recidivist outcomes among jail-involved individuals in three non-expansion counties with nearby expansion counties before and after Medicaid expansion.NFIB v. Sebelius, which gave states the option to expand their Medicaid program.1 We used forty-eight continuous months of individual-level booking and release dates from six urban county jails (three in expansion states and three in nearby non-expansion states) and comparative interrupted time series regression analysis to describe the level and trends of the rate of rearrest and the number of arrests before and after Medicaid expansion in expansion and non-expansion counties. We also estimate the impact of expanded Medicaid eligibility on rates of recidivism for the whole sample and for the largest racial-ethnic groups. Finally, in a qualitative analysis, we identify county-level reentry or diversion programs/policies that may explain the differential relationship by county pair (if any) between expanded Medicaid coverage and recidivism.We examine the relationship between expanded Medicaid eligibility and recidivism with an intent-to-treat analysis that takes advantage of the plausibly exogenous variation provided by Supreme Court ruling in We initially selected four county pairs\u2014Hennepin County, Minnesota (expansion) and Dane County, Wisconsin (Midwest region); Pima County, Arizona (expansion) and El Paso County, Texas (Southwest region); East Baton Rouge Parish, Louisiana, (expansion) and Hinds County, Mississippi (Southeast region); and St. Louis, Missouri, and East St. Louis, Illinois. Three of these four pairs agreed to participate and provide data for our study; the St. Louis locales declined to participate see . The iniIn the Midwest, both study counties had similar proportions of the population younger than eighteen\u201420 percent for Dane County and 22 percent for Hennepin County see . White, 2). Both counties have implemented programs to link justice-involved individuals with behavioral health-care services. In Dane County, Wisconsin, the jail has an Americorp volunteer on site three days a week to provide the jail\u2019s inmates with enrollment assistance into BadgerCare (Wisconsin\u2019s Medicaid program) prior to their release. Additionally, Dane County has a number of jail diversion initiatives, including electronic monitoring and reduced sentences for community program participation, that allows a person to remain in the community and receive community-based behavioral health services after adjudication.In addition to similar demographic characteristics, both Wisconsin and Minnesota provide similar levels and types of Medicaid coverage for behavioral health conditions see 2. Both cSimilarly, Hennepin County, Minnesota, has a countywide Integrated Access Team that identifies, screens, and refers the justice-involved for treatment and assures continuity of care whether the person is in or out of jail. Additionally, reentry staff work with the community-based case managers to connect inmates to medication assistance in the community. In addition to reentry programs, Hennepin County has diversion programs for people with behavioral health needs. In 2018 (which is after the study period in the Midwest county pair), Hennepin County opened a comprehensive social services facility that provides detoxification and mental health crisis services, employment counseling, and Medicaid eligibility assistance. All providers housed at the drop-in center accept Medicaid reimbursement for their services.However, Dane County is not a pure non-expansion county. Wisconsin expanded its Medicaid program\u2019s eligibility to 100 percent of the federal poverty level for nondisabled adults without dependents at the roughly the same time as the ACA Medicaid expansion. Thus Wisconsin had likely provided financial access to behavioral health services to the population at highest risk for criminal justice involvement. This may attenuate differences in outcomes between the Midwest pair.In the Southwest, we deliberately chose counties on the U.S.-Mexico border with large Hispanic-Latino populations\u201483 percent in El Paso and 36 percent in Pima . An estiThe two counties also have similar levels of coverage for behavioral health conditions in the Medicaid program see . HoweverIn terms of diversion and integration programs, El Paso passed the Criminal Justice Mental Health Jail Diversion Collaboration Resolution in 2011. The purpose of this resolution was to engage community stakeholders in efforts to divert both prearrest and postarrest individuals with behavioral health conditions, an estimated 30 to 35 percent of the jailed population, from the justice system to appropriate treatment.The sheriff in Pima County also implemented initiatives to reduce criminal justice involvement among individuals with behavioral health conditions in 2011. At this time, Pima County built a Crisis Response Center and Behavioral Health Pavilion to provide integrated care to those experiencing behavioral health crises and help them avoid unnecessary incarceration. Additionally, the Pima County Sheriff\u2019s Department Mental Health Investigative Support Team coordinates responses with Pima County Behavioral Health and other law enforcement agencies when individuals with behavioral health conditions are involved in criminal justice events.In the Southeast, Hinds County and East Baton Rouge had large African American populations\u201447 percent in East Baton Rouge and 72 percent in Hinds County before expansion . More thMississippi and Louisiana provide similar levels of coverage for behavioral health services, and neither state covers residential psychiatric treatment see . AdditioLouisiana has expanded its Medicaid program under the ACA, but little progress has been made in engaging local sheriffs to enroll eligible jail inmates in the state\u2019s expanded Medicaid program, even as part of reentry planning. In addition to these challenges, efforts at implementing a program to facilitate Medicaid enrollment upon discharge have been stymied by the East Baton Rouge\u2019s data system, which maintains information not by Social Security number but instead by an inmate ID unique to the jail.Similar obstacles exist in Hinds County, Mississippi. The county is under a U.S. Department of Justice consent decree for failing to provide adequate health-care services in its jail system. In addition, little capacity exists in the community behavioral health-care system to treat people leaving jail, and reentry planning is severely limited. In fact, in 2015, a newly elected sheriff terminated a mental health diversion program, which demonstrated savings to the jail system in its first year of implementation.These statistics and descriptions suggest a number of similarities between the counties in each county pair in terms of the nature of their local populations, the economic circumstances in each county, the likely pressures on the law enforcement system and efforts (or lack of them) to integrate the criminal justice and behavioral health-care systems. The qualitative data support our inferences within each county pair. However, we have also identified circumstances where changes to the state\u2019s Medicaid program  or criminal justice system practices  may drive our results toward finding no changes.Data were obtained from each county jail and include person-level booking and release dates. Arrestees in each county are given unique identification numbers upon first arrest in the county and thus could be followed over the study period. The data spanned four years in each county\u2014two before and two after Medicaid expansion. Additionally, each county pair provided individual-level characteristics of arrestees, but these characteristics differed across counties. Each county pair provided at least the gender of the arrestee. In the Southwest and Southeast counties, the county provided the race-ethnicity of the arrestee. In the Southwest and Midwest counties, the dataset contained a measure of the severity of the crime. In the Southwest, we know whether the arrest was for a technical violation; and in the Midwest, we know whether the arrest was for a misdemeanor, felony, or some other charge.Using these data, we created three distinct periods over the forty-eight months of data\u2014a lookback period, a preexpansion observation period, and a postexpansion observation period. The six-month lookback captures a person\u2019s prior history with the criminal justice system. This period was followed by eighteen months of the preexpansion and twenty-four months of the postexpansion period. Arizona and Minnesota expanded their Medicaid programs beginning January 1, 2014; Louisiana did so beginning July 1, 2016. Observations are at the person-month level.Within each county, people entered the study cohort when first arrested and were followed throughout the study. If someone is arrested in the preexpansion period, we considered them at risk for the remainder of the study. If someone is arrested only in the postexpansion period, we considered them at risk for the remainder of that period only. Those who were arrested only in the lookback period were excluded from the analysis. Arrestees who were booked into the county jail as a transfer from one prison to another  or as a hold for state or federal charges were also excluded.Most of the literature on the relationship between Medicaid coverage or behavioral health services and recidivism relies on a single outcome\u2014rearrest. Evidence from Florida suggests that 30 percent of men and 20 percent of women are rearrested within eighteen months of being released . RelyingDespite the high rate of Medicaid eligibility in the jailed population, our data do not indicate who was eligible for or enrolled in Medicaid coverage after expansion or who received behavioral health services as a result of increased financial access to these services. Therefore, in an intent-to-treat analysis, we compare our outcomes of interest before and after Medicaid expansion between expansion and non-expansion counties for each county pair. Evidence is strong, however, on the likely eligibility for Medicaid of the reentry population in expansion states. The GAO estimated that for two states that expanded Medicaid (New York and Colorado), 80 to 90 percent of people in their prison systems were eligible for Medicaid in 2014 . SimilarWe conducted comparative interrupted time series (CITS) regression analysis. The CITS design takes advantage of an exogenous source of variation (the state\u2019s decision to expand Medicaid) between a treatment and comparison group  and alloIn both a difference-in-differences and CITS design, the counterfactual is constructed by assuming the change seen in the comparison group from before Medicaid expansion to after Medicaid expansion would be the same change seen in the treated group if not for the treatment. However, in a difference-in-differences design, the assumption is constrained so that the average change between the two groups is the same. In CITS, the counterfactual is constructed by assuming that the change in the level and trend from the linear extrapolation of outcomes before expansion in the comparison group is a good stand-in for the unobserved outcomes in the treated group. The only reason for differential deviation from these linear trends is the interruption in the treated group, that is, all other reasons for deviation affect the treated and control group in the same way. Given the drivers of the outcome in this study  and how they may vary between the counties, assuming linearity in the time trend of the outcomes in each of the two groups seems more reasonable than assuming that they change in the same average way over time.Because of heterogeneity in the criminal justice and health-care delivery systems, legal dynamics (such as border policy), and populations across our study pairs, we chose to analyze each county pair separately. First, we computed preexpansion means and variances for the probability of rearrest and number of arrests in both the treatment and comparison counties for the full sample and for stratified samples in each county pair. Because the policing, criminal justice, behavioral health, and health insurance systems treat people differently based on observed gender and race-ethnicity, stratification on these dimensions aims to ensure that we are making apples-to-apples comparisons in our estimation strategy. Next, we computed preexpansion monthly means of each outcome of interest to create preexpansion trends.If Medicaid expansion leads to reduced probability of rearrest and number of arrests, then the composition of the pre-and postexpansion cohorts may differ in each observation period, with people arrested in the latter period being at higher risk, on average, than those arrested in the first period. We thus computed the number of individuals arrested and the number of arrests in both periods to check for compositional shifts in severity.3 For the number of arrests (a count), we used ordinary least squares (OLS).4 We used the following general specification for each of the outcomes:i is an individual in the cohort, s is the county, and t is month. Xi is a vector of time-invariant individual characteristics that are allowed to vary with the outcome over time, Pit is a vector of time-varying individual characteristics with effect, \u03b3 is the CITS estimator for the level shift in the outcome, \u03b7 is the CITS estimator for the trend shift in the outcome and the parameter of interest, QUARTER is a vector of quarter fixed effects.For the probability of rearrest, we used a linear probability model.Time and individual-varying characteristics consist of charge severity\u2014whether the arrest was a misdemeanor or felony (Midwest) or whether for a technical violation (Southwest). Time-invariant, individual-varying characteristics include the arrestee\u2019s gender, race-ethnicity (where available), and prior history with the criminal justice system. We interacted both the time-varying and time-invariant person-level covariates with the monthly time trend to adjust for the possibility that the relationship between the covariates and the outcome of interest is also time varying. Within each county pair, we used a Bonferroni correction for multiple testing.As noted, we stratify the CITS regression analyses in each county pair by gender and major local racial-ethnic groups. If baseline differences in racial or ethnic make-up were driving both arrest patterns and Medicaid expansion, then we would expect the causal estimates for these minority groups to differ significantly from that of the pooled sample.A falsification test often used in CITS artificially places the empirical implementation date within a clean study period . It could be that the outcome is noisy and that estimated effects might appear by chance. We varied the implementation date to three months in the preexpansion period\u2014months seven, ten, and thirteen in the time series. Varying the implementation date within only the preexpansion period is preferable to strategies that vary it within the study period as a whole, because the full study includes the treatment effect and may lead to detecting a spurious effect at a falsified intervention point. Our falsification strategy is also not ideal because it cannot account for any anticipatory effects of Medicaid expansion\u2014particularly in the Southeast county pair, where other coverage provisions of the ACA had been in place for more than two years before expansion. Thus we expect a weakening of the estimated impacts as the intervention date is moved away from the true date.Additionally, because the before and after observation windows are of different length, arrestees are at risk for being arrested longer in the postexpansion period than in the preexpansion. This difference should be handled by the CITS design because the observation periods are shared by the intervention and comparison groups. Nonetheless, we checked for any effect of this discrepancy by conducting sensitivity analyses where we truncate the post-period to eighteen months to balance the exposure time before and after.Finally, case studies raise general concerns about the validity of statistical inference. Given only two clusters, estimating and accounting for intracluster correlation is not possible (both the within and between-group variance cannot be estimated in only two groups). Thus typical econometric procedures for standard error adjustment  are not feasible in this setting. We conducted the full sample CITS at the county-month year as a check on statistical inference. Aggregating to the cluster-time level results in less biased standard errors even in a small number of clusters  but prevIn the preexpansion period, the three Medicaid expansion counties arrested more people than the comparison counties , because the populations of the expansion counties are larger than those of the non-expansion counties. Indeed, the proportion of the total population arrested preexpansion is similar in expansion and non-expansion counties\u20141.6 percent of each county in the Midwest, 3.2 percent of both counties in the Southwest, and 3.6 and 2.6 percent respectively in the Southeast. Similarly, the number of arrests are higher in the Medicaid expansion counties prior to expansion than in the non-expansion counties .In the postexpansion period, the number of people arrested was higher in both the expansion and non-expansion counties of each pair  and arrests were also more numerous . This suggests that the composition of the cohort may be changing from the before to the after period.A primary concern with compositional shifts leading to fewer arrests is the possible implication that the least risky individuals (those with fewer impairing conditions) would be disproportionately enrolled in Medicaid and have greater financial access to behavioral health-care services after expansion because they had the cognitive and functional capacity to do so. If individuals with less severe conditions were more likely to enroll, our cohort would include a larger proportion of people more likely to have higher rates of recidivism and commit potentially more serious crimes. This scenario would lead to bias and overestimation of the effect of expansion on recidivist behavior. However, the direction of the observed change  implies that the marginal individual arrested in the postexpansion period is likely not of higher risk than that in the preexpansion period.Data on the types of arrests supports this interpretation. Of the arrests in the Southwest county pair, 3,933 and 3,394 were for a parole violation in the before and after periods, respectively. Additionally, 22,951 (49.4 percent) of the Midwest arrests in the preexpansion period were for misdemeanors and 17,036 (50 percent) in the postexpansion period; 15,121 (32.5 percent) and 11,191 (32.8 percent) were for felonies. Arrest composition was almost identical, suggesting that the marginal individuals arrested after expansion are at roughly the same risk as those arrested before expansion.In general, arrestees were much more likely to be men in each county pair see . The proIn the Southwest county pair, a preexpansion period arrest in Pima County was nearly five times more likely to be for a parole violation than an arrest in El Paso County . Similar arrests in Dane County, Wisconsin, were more likely than those in Hennepin County, Minnesota, to be for misdemeanors (55.9 to 46.7 percent) or felonies (37.3 to 30.6 percent). Although these baseline differences in arrestee demographics and arrest charge or severity were stable across the study period, they suggest that our comparison counties differ, at least in racial-ethnic composition. We stratified regression analyses by gender and dominant minority group to partially address within-pair county heterogeneity.The probability of rearrest in the preexpansion period was higher in Hennepin County, Minnesota  than in Dane County, Wisconsin  and in Hinds County, Mississippi  than in East Baton Rouge, Louisiana  see . The greWhen stratified by gender and race-ethnicity in each county pair, the pattern of results was the same. The probability of rearrest among male and female arrestees was higher in Hennepin County, Pima County, and Hinds County. Among black arrestees in the Southeast, the probability of rearrest was 3.4 percentage points higher in Hinds County than in East Baton Rouge. Additionally, the probability of rearrest among Hispanic-Latino individuals in the preexpansion period was 8.1 percentage points higher in Pima County than in El Paso County. Although differences in the preexpansion period trend may be cause for concern in a difference-in-differences analysis, baseline or trend differences in the outcomes do not invalidate causal inferences for CITS, which models differential levels and trends in both the pre-to the postexpansion period.After Medicaid expansion, the short-term probability of rearrest (the level change) declined by a statistically significant amount in the Midwest and Southwest county pair see . The larIn the Midwest county pair, the probability of an individual being rearrested declined by 0.87 percentage points in the month after expansion  in Hennepin County relative to Dane County, which amounts to a 2.9 percent decrease in the probability of rearrest. Similar declines were seen among male and female arrestees. In the Southeast county pair, Medicaid expansion was not associated with changes in the probability of rearrest.We also found sustained decreased rates of recidivism per month for arrestees in the Midwest  and Southwest  county pairs see . The effBy extrapolating the level and trend change to the end of the study period, we found that Medicaid expansion led to an average decline in the probability of rearrest of 1.49 percentage points (an 4.92 percent decrease) in Hennepin County relative to Dane County and of 3.6 percentage points (13.1 percent) in Pima County relative to El Paso County. In the Southeast, a differential trend increase resulted in a 1.61 percentage point increase (10.1 percent) in East Baton Rouge relative to Hinds County.Like the descriptive statistics for the probability of rearrest, the average number of arrests in the preexpansion period was higher in Hennepin County, Minnesota, than in Dane County, Wisconsin (1.54 to 1.46). The average number of arrests for men and women in the expansion county was also higher than that of the non-expansion county in the Midwest pair see . SimilarOverall, Medicaid expansion resulted in a decline in the average number of arrests per person in the Midwest  and Southwest  expansion counties relative to the respective non-expansion counties in the month after Medicaid expansion see . In bothMuch as in rearrests, the level change in the number of arrests in the Southeast county pair is close to zero and not statistically significant , suggesting the lack of any immediate impact of Medicaid expansion and recidivism.The longer-term effects of Medicaid expansion on the average number of arrests per person were similar to the pattern in the rearrest analyses. The average number of arrests per person per month decreased more in Hennepin County than in Dane County . The average number decreased for the full sample, male, female, and Hispanic-Latino arrestees (roughly 0.003 per month) in Pima County relative to El Paso County. The average number after Medicaid expansion increased for all groups in East Baton Rouge relative to Hinds County .Via linear extrapolation of the level and trend change, we found that Medicaid expansion led to an average decline of 0.1 arrests per person in Hennepin County relative to Dane County, and an average decline of 0.2 arrests in Pima County over El Paso County two years after Medicaid expansion (the end of the study period). In terms of relative changes, this is a 5.8 percent decrease in the Midwest and a 13.3 percent decrease in the Southwest. Taking into account only the trend increases in the Southeast, Medicaid expansion resulted in 0.2 more arrests per person in East Baton Rouge than in Hinds County, a 12.2 percent increase.As suggested, falsification tests such as these may not be ideal when assessing whether we are isolating the causal effect of Medicaid expansion on recidivist behavior. These tests did not permit specifying any ramp up to Medicaid expansion, particularly in the Southeast, where the ACA\u2019s other coverage provisions had been in place for more than two years before Medicaid expansion. With these caveats, the falsification tests suggested that we were capturing the causal effect see , A3.In all three county pairs, no change was detected in the level at the false implementation points for the probability of rearrest or the number of arrests. However, a change in the slope of the line was apparent just prior to expansion for both outcomes in the Midwest and Southwest.In addition to these falsification tests, we also conducted sensitivity analyses. When we truncate the postexpansion period to be of equal length to the preexpansion period, we find no real differences in our estimates, with the exception of the Southeast county pair see . In the The study has several limitations. First, data consisted of booking data. We did not know who gains health insurance under Medicaid expansion and therefore conducted an intent-to-treat analysis. Additionally, we could not differentiate between individuals who are lost to follow up and those who are never rearrested. We assumed that rates of attrition are similar over time and across the counties in each county pair.Second, from interviews and site visits, we identified changes to the behavioral health and criminal justice systems that add context to the results, but also highlighted that in some cases, changes may not be attributed entirely to Medicaid expansion. Hinds County, Mississippi, discontinued a mental health diversion program in late 2015 after the current sheriff was elected in August 2015 (expansion happened in Louisiana in July 2016), despite evidence that the program saved the county $250,000 in the year after implementation.Third, baseline differences in the composition of each county\u2019s arrested population and the overall arrest activities may suggest that these are not perfect comparison counties. We chose the counties based on their similarity on the county\u2019s full demographic characteristics, rather than the characteristics of the jailed population. To the extent that the characteristics of the jailed populations and differential policing and arrest activity were stable over time, our design netted out these differences.Overall, Medicaid expansion reduced both the probability of rearrest and the number of arrests in two of the three county pairs. In the Midwest and Southwest, the estimated effects at two years after expansion were consistent with estimates from other studies on the relationship between access to health-care services and recidivism (between a 5 and 13 percent decrease). Additionally, the mixed nature of the findings (an increase in the Southeast) is also consistent with prior literature.These estimates are similar to other initiatives to reduce recidivism. Adult drug courts reduce recidivism rates by roughly 8 percent . One metHowever, our estimates might be smaller than the true effect of Medicaid expansion on recidivism. We did not measure first-order effects\u2014health insurance coverage and access to care\u2014of Medicaid expansion in the jail-involved population; nor did we measure the change in recidivism in individuals who obtained Medicaid coverage and subsequent behavioral health treatment. If we were able to conduct a treatment-on-the-treated analysis, then our estimates would scale by the proportion of individuals who enrolled in Medicaid coverage due to expansion. If we were to use the proportion of jail-involved individuals eligible for Medicaid expansion from In the Southeast, we failed to detect a change in the level in East Baton Rouge relative to Hinds County in the implementation of Medicaid expansion, and the change in the slope resulted in an overall increase in the probability of rearrest and number of arrests twenty-four months after expansion. This could be a result of changes to behavioral health and criminal justice practices required by the federal consent decree in Hinds County and the lack of integration and coordination between these two systems in East Baton Rouge Parish.Additionally, we stratified our analyses by race and gender to address within-pair heterogeneity and make comparisons across individuals who are treated more similarly by the policing, health-care, and criminal justice systems. The estimates for these stratified groups were either the same size or larger than the full sample, which strengthens our inferences. Moreover, our qualitative analysis of the efforts occurring in each of the counties allowed us to draw out the contexts that may make Medicaid expansion more or less effective in reducing recidivist behavior. Indeed, our results mirrored the previous literature\u2014enhanced financial access to health-care services contributes to a reduction in recidivism.Other contributing factors include coverage of evidence-based treatment for mental illnesses and substance use disorders; adequate capacity in the community\u2019s behavioral health treatment system; the provision of mental illness and substance use disorder treatment services in jail, both before adjudication and while incarcerated; the coordination and continuity of care across the criminal justice and behavioral health-care systems; the implementation of jail diversion programs that keep individuals with mental illness or substance use disorder from entering the criminal justice system; and the availability of other social programs, such as supportive housing and employment, that improve the social status of individuals with mental illness or substance use disorder. Reducing rates of rearrest, particularly for individuals with severe mental illness or substance use disorders, requires coordinated efforts between multiple social service systems and increased integration of those systems could be an important policy lever to increase time in the community.Supplement"}
+{"text": "Plasmodium vivax or Plasmodium falciparum. However, biological components involved in the development of lung malaria are poorly studied. In experimental models of pulmonary malaria, it was observed that parasitized red blood cell-congested pulmonary capillaries are related to intra-alveolar hemorrhages and inflammatory cell infiltration. Thus, it is very likely that hemolysis participates in malaria-induced acute lung injury. During malaria, heme assumes different biochemical structures such as hemin and hemozoin . Each heme-derived structure triggers a different biological effect: on the one hand, hemozoin found in lung tissue is responsible for the infiltration of inflammatory cells and consequent tissue injury; on the other hand, heme stimulates heme oxygenase-1 (HO-1) expression and CO production, which protect mice from severe malaria. In this review, we discuss the biological mechanism involved in the dual role of heme response in experimental malaria-induced acute lung injury.Malaria is a hemolytic disease that, in severe cases, can compromise multiple organs. Pulmonary distress is a common symptom observed in severe malaria caused by  Plasmodium infecting humans: Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale, Plasmodium knowlesi  rev rev7) rePlasmodium species that infect humans can induce MA-RD  .Heme and its analogs localize differently on erythrocyte membranes and exhibit distinct roles in its partitioning, leakage, and fusion . Under p2O2), and hydroxyl radical (HO\u2219)], reactive nitrogen species (RNS) , nitrogen dioxide (\u2219NO2)], and peroxynitrite (ONOO\u2212)  . These r (ONOO\u2212) \u201344. Ther (ONOO\u2212) , 40.Plasmodium sp. Plasmodium digest ~65% of total erythrocyte hemoglobin during intraerythrocytic development. Part of the hemoglobin's amino acids is incorporated in parasite proteins; however, since free heme released during hemoglobin digestion is a toxic by-product, Plasmodium biocrystallizes heme to hemozoin to store it as a nontoxic molecule in the digestive vacuole  released during hemolysis is engulfed by phagocytes such as macrophages, neutrophils, and dendritic cells . The accpatients . In addipatients . The sampatients . These os influx , 51.in vitro and in vivo . The authors observed that mice with HbS phenotype did not develop cerebral malaria by two different mechanisms, and both pathways depend on low levels of free heme on the bloodstream. The first mechanism involves the stimulation of HO-1 production, and the second one involves heme-induced immunoregulatory roles. The authors suggest that there is a pathogenic and a protective concentration of circulating free heme during malaria  triggered by n tissue , while tnfection . It is nngestion . The aut malaria .P. berghei-infected DBA/2 mice model of MA-ALI, gave hemin to infected mice and observed an increase in HO-1 production correlated with attenuation of lung dysfunction and inflammatory response associated to alteration in lung histoarchitecture. As well, Liu et al. (In recent reviews by Frimat et al.  and Immeu et al.  showed tu et al. .+ T cell activation and migration to lung tissue, reduces the production of inflammatory mediators, and restores endothelial cell barrier integrity.Thus, considering the biological mechanism by which HO-1 induction attenuates brain dysfunction during experimental cerebral malaria, we can speculate that during experimental MA-ALI, the induction of HO-1 downmodulates CD8Free heme and heme derivatives have been widely recognized as pathological molecules in several hemolytic conditions. The participation of heme in malaria is very peculiar because it exerts its effects through different molecular structures as free heme, hemin, and hemozoin. Several studies concerning malaria-induced lung dysfunction show that heme derivatives affect alveolar integrity, induce the production of inflammatory mediators, and accumulate the inflammatory cells in the lung tissue. On the other hand, more recent studies propose that heme exerts a beneficial role during malaria infection by inducing cytoprotective pathways such as HO-1 production. Indeed, more studies are necessary to define the role of heme during malaria-induced lung dysfunction. Overall, we can conclude that the imbalance between free concentration, production/saturation of HO-1, and the activation of coexisting anti-inflammatory pathways dictate if heme is a friend or foe to malaria patients.TP and MS wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "Emojis are frequently used picture characters known as possible surrogates for non-verbal aspects of behavior. Considering the ability of emojis to enhance and facilitate communication, there has been a growing interest in studying their effects in scientific and health-related topics over the past few years. Infection prevention and control (IPC) is a field of medicine that is directly associated with specific behaviors. These include hand hygiene, which is the cornerstone of the prevention of healthcare-associated infections, and essential in stemming the spread of antimicrobial resistance. This paper aims to provide an overview of how emojis have been used in the medical and public health literature and proposes their possible use in IPC and hand hygiene to put forth a vision for the future research. These ideograms have evolved beyond facial expressions, and are increasingly used on digital platforms to demonstrate concepts and ideas. As a Japanese word meaning \u201cpicture character\u201d, emojis were initially created at the end of the twentieth century in order to improve and simplify digital messages , 2. Theythe year . More thAs technology improves and emojis continue to grow in popularity in the digital world, various studies have been conducted in the past few years regarding the impact of these symbols in scientific fields. However, the use of emojis has not yet been highlighted in the medical literature. These surrogates of nonverbal communication have a direct impact on different social interactions that could facilitate communication among healthcare providers and receivers, thus enhancing public health . This pa\u201d is the emoji with the same meaning. More natural in design, emojis have attracted scientific attention as they are able to transmit emotion, attitude and attention when added to text [The ever-evolving medical language, which is globally accepted as medical English, has faced significant changes over time . But des to text , 15. As  to text .Shah et al.  suggesteWith the dramatic increase in public awareness about the significant role of the Internet and social media in disseminating health-related data , InterneImplementing IPC programs is recognized as a global health priority, and it is known that failure to achieve an adequate level of IPC harms patients around the world. That points to the necessity of using a full arsenal of tools and technologies to improve IPC interventions globally . In rece) emoji was proposed with the objective to improve communication and research regarding mosquito-borne diseases and to monitor mosquito-borne disease outbreaks [) and \u201csneezing face\u201d  are among the smileys that may be used to represent a hospital patient, person with a cold or flu or other physical diseases. New health-related emojis in other categories including \u201clab coat\u201d , \u201cmicrobe\u201d , \u201ctest tube\u201d , \u201cpetri dish\u201d  and \u201cDNA\u201d  have also been added as part of Unicode 11.0 in 2018 to facilitate scientific communication in the digital world.A number of emojis have been created that could be related to infectious diseases. In 2017, the \u201cmosquito\u201d  emoji, which makes communication more complicated than using a single emoji. For example, some people may interpret the combination of the \u201cclapping hands\u201d and \u201cbar of soap\u201d  emojis as applauding the use of soap and it may not directly demonstrate hand washing with soap. The development of hand hygiene-related emojis will enable health professionals to communicate more specifically regarding their discoveries and concerns in this field. In addition, the hand hygiene compliance of different places in the world could be better notified by detecting relevant emojis in the social media to predict compliance or even identify problems caused by low adherence. Hand hygiene-related emojis on social media could regularly remind us to protect ourselves against healthcare-associated infections, and the spread of influenza or antimicrobial resistance. More importantly, the general public, including patients suffering from healthcare-associated infections, might be able to easily share and communicate the problems they have faced, as they could be better heard globally and have the possibility of sharing their feelings using different emojis.Hand hygiene-related terms have been transformed over the past years from handwashing and hand disinfection to hand hygiene, underlining the increased performance and use of alcohol-based handrub compared to soap and water, and the prominence of this topic in patient safety , 57. TheEmojis may empower IPC in different aspects such as raising awareness with no language barrier, educating people to adopt healthy behaviors, and enhancing surveillance systems to monitor infectious diseases. It is recommended to evaluate the relevance and appropriateness of the current set of emojis to use for hand hygiene promotion in order to harness the potential beneficial impact of these symbols. Medical emojis that are standardized as a new category of emojis offer much hope for the future, especially in the field of IPC."}
+{"text": "The reduction of postharvest losses in rice and safou is imperative to increase productivity in their respective value chains. In this study, fine broken rice grains were used to produce rice flour and subsequently rice\u2010based biscuits. The biscuits were further fortified with safou powder, and the physical, nutritional, and sensory quality and stability during\u00a0storage of the\u00a0different types of biscuits were analyzed\u00a0using standard methods. Fine or nonsandy biscuits had peak particle size of 500\u00a0\u00b5m, while medium (slightly sandy) and large (sandy) biscuits had peak particle sizes of 1,000\u00a0\u00b5m and 1,400\u00a0\u00b5m, respectively. The hardness varied from 5.7\u00a0\u00b1\u00a02.3\u00a0N for biscuits with large particles to 16.1\u00a0\u00b1\u00a04.4\u00a0N for biscuits with fine particles. Fortification of biscuits with sour safou increased the protein and amino acid content of the biscuits. Tryptophan was absent in both safou and the biscuits produced. There was an increase in phosphorus, potassium, calcium, magnesium, copper, iron, manganese, and aluminum following fortification with safou. Nonsandy biscuits dissolved faster in the mouth (melt) during consumption than the other biscuits although most of the biscuits were perceived to be low in melting and buttery. Nonsandy biscuits were rated as \u201cvery good,\u201d while slightly sandy and sandy were rated as \u201cgood.\u201d Safou rice\u2010based biscuits were perceived as \u201cvery good,\u201d while simple rice biscuits were perceived as \u201cgood.\u201d Fortification of rice biscuits with safou increased the protein, essential amino acid, and mineral contents of the biscuits with very appreciable taste. These biscuits can be used to help fight protein, iron, and zinc malnutrition and in mitigating postharvest losses of underutilized broken rice and safou especially sour safou. In this paper, we show that fine broken rice can be used to produce biscuits which are highly acceptable by consumers. We further demonstrate that such biscuits can be fortified with protein, amino acids, and trace minerals such as iron and zinc by partially substituting butter with safou\u2014an oleaginous fruit widely cultivated in West Africa. The use of fine broken rice and safou to produce biscuits will result in a win\u2010win scenario for both rice and safou value chains through postharvest loss reduction and value addition. IRRIInternational Rice Research InstituteMDAmalondialdehydeNSSRWB\u201050nonsour safou\u2013rice\u2013wheat biscuitRB\u2010100rice biscuitRWB\u201050rice\u2013wheat biscuitSSRWB\u201050sour safou\u2013rice\u2013wheat biscuitWB\u2010100wheat biscuit1Oryza sativa L.) is currently part of the staple foods in Cameroon with close to 896,000 tons consumed in 2018. Milled rice production in Cameroon in 2018 stood at 221,000 tons which is a 2.21% reduction compared to 2016 , Rice (Dacryodes edulis (G. Don H. J. Lam)) fruit is mostly found in forest areas of Central and West Africa and the Gulf of Guinea. Safou is mostly eaten in the roasted or boiled form, as a delicacy alone or in combination with roasted or boiled maize, cassava or plantain. The safou pulp is rich in lipids, and its proportions vary from 33% to 65%. Safou lipids contain a high level of essential fatty acids  before completely drying the flour to 10%\u201312%. Rice flour was produced by sieving ground rice with sieves of different mesh sizes as previously described by Ndindeng, Mbassi, et\u00a0al.\u00a0. Briefly2.1.4Three types of biscuits were produced with rice flour of different particle sizes. Large, medium, and fine particles were used to produce sandy, slightly sandy, and nonsandy simple rice biscuits RB100), respectively. Six different wheat, rice, and safou nonsandy biscuit combinations were produced using fine flour. These were wheat biscuit (WB\u2010100), rice biscuit (RB\u2010100), rice\u2013wheat biscuit (RWB\u201050), sour safou\u2013rice\u2013wheat (SSRWB\u201050), and nonsour safou\u2013rice\u2013wheat (NSSRWB\u201050) biscuits , rice (RB\u2010100), rice safou (RSB\u2010100), rice\u2013wheat (RWB\u201050), sour safou\u2013rice\u2013wheat (SSRWB\u201050), and nonsour safou\u2013rice\u2013wheat (NSSRWB\u201050) biscuits were produced, packaged in either aluminum or polypropylene bags and stored for 5\u00a0months at room conditions . Every month during the storage period, samples were withdrawn and evaluated for hardness, water content, malondialdehyde content, peroxide index, lightness, and aroma.2.22.2.1The particle size distribution of rice biscuits was determined by sieving ground biscuits with sieves of different meshes from 1.4\u00a0\u00b5m to 250\u00a0\u00b5m . The hardness of the biscuits produced was determined through the measurement of the penetration strength with the Texturemeter Stevens Instron3343 at room temperature. The experimental equipment was made up of a borer\u2010like cone, falling at 0.2\u00a0mm/s of speed until the biscuit was broken. The lightness of the biscuit was measured using a Colorimeter (Minolta Color reader CR\u201010) and reported as L*value. This lightness was measured by placing the aperture of the equipment on the sample with white paper as the reference. The L* value was calculated automatically and expressed as a difference , texture , and overall quality. A trained panel of 12 (2 men and 10 women) used a six\u2010point rating scale (\u201c0\u00a0=\u00a0absent\u201d to \u201c5\u00a0=\u00a0very high\u201d) to evaluate the intensity of basic taste, aroma, and texture. A six\u2010point scale was also used to evaluate the overall quality of the biscuits from \u201c0\u00a0=\u00a0very bad\u201d to \u201c5\u00a0=\u00a0excellent.\u201d2.3Radar charts were generated in Excel 2018 software  from the results of sensory evaluation to depict differences in attributes between the formulated biscuits, based on particle size and safou fortification. Analysis of variance (ANOVA) with Fisher's pairwise comparison test was performed to determine the differences between the nutrient composition of dry safou powder and the biscuits. Analysis of covariance was used to study the effect of storage time, type of biscuits and package on hardness, malondialdehyde, moisture, peroxide index, and lightness during storage. Differences between categories of biscuits and packages were analyzed using the Fisher least significant difference. Analysis was done with XLSTAT Premium version 2020.1.2 . Particle size distribution has been shown to depend on the type of milling (wet or dry) and the machine used for grinding .The particle size distribution in each category of biscuits was heterogeneous Table\u00a0. Fine orp\u00a0<\u00a0.05) from nonsandy to sandy biscuits. The hardness was higher for nonsandy biscuits (16. 1\u00a0\u00b1\u00a04.4) and lower for sandy biscuits (5.7\u00a0\u00b1\u00a02.3). The cohesiveness varied from 7.18\u00a0\u00b1\u00a02.68 for nonsandy biscuits to 1.17\u00a0\u00b1\u00a00.70 for sandy biscuits. These observations demonstrate that the smaller the particles, the greater the cohesion of the particles. Lai  than that of nonsour safou (14.07\u00a0\u00b1\u00a01.24). The safou biscuits had lower carbohydrate contents (53%\u201360.9%) than that of simple rice\u2013wheat biscuit (62.87\u00a0\u00b1\u00a01.55%). However, the difference between the carbohydrate level of the sour safou rice biscuit and the simple rice\u2013wheat biscuit was not significant. The crude fiber content of nonsour safou (7.6\u00a0\u00b1\u00a00.18%) was higher (p\u00a0<\u00a0.05) than that of sour safou (1.9\u00a0\u00b1\u00a00.19%). The crude fiber content of simple biscuits was slightly higher than those of safou\u2010fortified biscuits, but with no significant difference from that of nonsour safou biscuits. These observations are like those made by Noor Aziah, Mohamad Noor, and Ho . Flour particle size is an important factor affecting protein quality, damaged starch content, and quality of bakery products  based on particle size distribution is shown in Figure\u00a0p\u00a0<\u00a0.05). Safou (sour or nonsour) fortified biscuits were perceived as \u201cvery good,\u201d while unfortified biscuits were perceived as \u201cgood.\u201d This was probably due to the presence of the safou powder which enhanced the flavor with the characteristic safou aroma. Results from earlier studies in food fortification hold that consumers favorably embrace the incorporation of new and innovative nutrient rich supplements into biscuits. These supplements among others include protein\u2010rich palm weevil larvae (Rhynchophorus phoenicis Fabricius), orange\u2010fleshed sweet potato  composite rice\u2013wheat flour is shown in Figure\u00a03.4p\u00a0<\u00a0.05). MDA was also lower in biscuits stored in aluminum packages compared to those stored in polypropylene. The increase in MDA with duration of storage observed in this study confirms an earlier observation by Fotouo\u2010M, Vorster, du Toit, and Robbertse . Biscuits stored in aluminum packages recorded lower moisture content compared to those stored in polypropylene bags (p\u00a0<\u00a0.05). The increase in moisture content with the duration of storage might be due to the hydroscopic nature of the rice and wheat flours, temperature, relative humidity, and nature of the packaging material  are harder and more cohesive than those produced with larger particles (sandy). Nonsandy biscuits recorded a higher overall acceptability score than sandy biscuits. The essential amino acids\u2014phenylalanine, isoleucine, histidine, tyrosine, lysine\u2014and nonessential amino acids\u2014aspartate, glycine, alanine, gamma\u2010amino butyrate (GABA), proline, and arginine\u2014contents were also higher in nonsour than sour safou. Although Leucine and aspartate were the most abundant essential and nonessential amino acids in safou, the addition of safou to rice\u2013wheat biscuits combinations resulted in histidine increase and lysine decrease in NSRWB\u201050. In addition, aspartate and glycine were increased in SSRWB\u201050 and NSRWB\u201050. Potassium is the most abundant mineral in safou followed by magnesium, phosphorus and calcium in that order with sodium being the least. Iron was the most abundant trace mineral in safou followed by copper, manganese, aluminum, and zinc. Potassium was still the most abundant mineral in rice biscuits produced followed by phosphorus. Although the trace minerals iron and zinc were higher in rice biscuits than in safou, there was higher quantity of aluminum in those biscuits than in safou but this quantity is comparable with data in literature. Thus, the biscuits produced can be a good source for potassium, phosphorus, iron, and zinc for different age and sex groups. Highly acceptable biscuits could be obtained when 30% of sour or nonsour safou powder was used in the formulation. The small variation of peroxide and MDA values in fortified biscuit indicated slight oxidative activity that changed little during storage irrespective of the packaging material used. The contribution of safou powder in making biscuits enabled an increase in protein and mineral content that would contribute in the fight against malnutrition within vulnerable groups of the population. The use of broken rice and safou could reduce postharvest losses recorded in these crops and increase the income of those involved in this sector especially women processors.The authors declare that they do not have any conflict of interest.\u201cThis study was approved by the Institutional Review Board of Yaounde\u20101 University.\u201dWritten informed consent was obtained from all study participants.Supplementary MaterialClick here for additional data file."}
+{"text": "Acute central nervous system (CNS) injuries, such as stroke, traumatic brain injury (TBI), and spinal cord injury (SCI) present a grave health care challenge worldwide due to high morbidity and mortality, as well as limited clinical therapeutic strategies. Established literature has shown that oxidative stress (OS), inflammation, excitotoxicity, and apoptosis play important roles in the pathophysiological processes of acute CNS injuries. Recently, there have been many studies on the topic of ferroptosis, a form of regulated cell death characterized by the accumulation of iron-dependent lipid peroxidation. Some studies have revealed an emerging connection between acute CNS injuries and ferroptosis. Ferroptosis, induced by the abnormal metabolism of lipids, glutathione (GSH), and iron, can accelerate acute CNS injuries. However, pharmaceutical agents, such as iron chelators, ferrostatin-1 (Fer-1), and liproxstatin-1 (Lip-1), can inhibit ferroptosis and may have neuroprotective effects after acute CNS injuries. However, the specific mechanisms underlying this connection has not yet been clearly elucidated. In this paper, we discuss the general mechanisms of ferroptosis and its role in stroke, TBI, and SCI. We also summarize ferroptosis-related drugs and highlight the potential therapeutic strategies in treating various acute CNS injuries. Additionally, this paper suggests a testable hypothesis that ferroptosis may be a novel direction for further research of acute CNS injuries by providing corresponding evidence. Acute CNS injuries, including stroke, TBI, and SCI, are a major burden of morbidity and mortality worldwide , 2019. EFerroptosis, first observed in response to treatment of tumor cells via small-molecule chemical probes, is a newly identified form of regulated cell death characterized by the accumulation of iron-mediated lipid peroxides . It diffSmall-molecule probes are valuable tools for studying different types of regulated cell death . During in vitro . In the endosome, Fe3+ is reduced to Fe2+ by STEAP3, and Fe2+ is then released into unstable iron pools mediated by DMT1, or stored in ferritin, which is composed of FTL and FTH1  to reduce cell death [for review see and vivo . Recentland vivo , TBI  with stroke . In addiIn traumatic SCI, the primary injury causes immediate cellular damage and initiates a continuous secondary injury cascade to induce ischemia, inflammation, and cell death [for review see As ferroptosis may be a significant pathogenic pathway in acute CNS injuries, its therapeutic potential should be taken into consideration. Ferroptosis inhibitors  may prevent iron accumulation or lipid peroxidation, thus offering therapeutic options for treating acute CNS injuries .The mainstay of treatment for acute ischemic stroke is rapid recanalization by mechanical thrombectomy or recombinant tissue plasminogen activator, the only approved thrombolytic agent. It is important to salvage the penumbra, which surrounds the region of the infarct and promotes functional recovery. However, the overall efficacy is limited due to the narrow window of opportunity , but eveAs mentioned above, Lip-1 and Fer-1 are both compounds with specific anti-ferroptotic activity. Intranasal administration of Lip-1 and Fer-1, either immediately or 6 h after reperfusion, significantly reduced neuronal damage and functional deficits in MCAO mice, indicating the possible translational value of special exogenous ferroptosis inhibitors .in vivo studies have reported that oral CoQ10 administration markedly improved neurological outcomes in both rat MCAO models and acute ischemic stroke patients , also requires explication.In this article, we primarily focus on the roles and therapeutic potential of ferroptosis in various acute CNS injury processes, including stroke, TBI, and SCI. Pharmacological effects of multiple inducers and inhibitors of ferroptosis lie at the intersection of lipid, amino acid, and iron metabolism. Although some progress has been made in ferroptosis, there are still controversial questions that have not been fully studied. First, the relationship between ferroptosis and other forms of cell death remains unknown. For example, p53 is an important regulator, both in apoptosis and ferroptosis, while autophagy plays a role in the process of ferroptosis via ferritinophagy. As ferroptosis is involved in acute CNS injuries complicated by necrosis, apoptosis, and autophagy, a head-to-head comparison of individual inhibitors or various combinations of inhibitors is required in further studies. Second, the special molecular markers  for identifying ferroptosis are still lacking. While the increased mRNA levels of PTGS2 were found in cells undergoing ferroptosis, it did not affect ferroptosis progress . The spePotential treatment options targeting ferroptosis  have shown neuroprotective effects in acute CNS injuries. However, these benefits are largely based on animal models and have not yet translated into clinical application. Furthermore, studies are necessary to clarify the appropriate therapeutic window, clinically feasible routes of administration, and BBB penetration ability of anti-ferroptotic agents. Among the above-mentioned agents, edaravone is the only approved drug with proven clinical efficacy and safety, while others should be explored in further clinical studies. More in-depth and comprehensive research on ferroptosis should be conducted to develop therapeutic methods and eventually alleviate the burden of acute CNS injuries in the future.All the authors participated in analyzing and discussing the literature, commenting on, and read and approved the final manuscript. AS and YC supervised the research, led the discussion, and wrote and revised 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."}
+{"text": "The prevalence of nuclear cataracts was observed to be significantly higher among residents of tropical and subtropical regions compared to those of temperate and subarctic regions. We hypothesized that elevated environmental temperatures may pose a risk of nuclear cataract development. The results of our in silico simulation revealed that in temperate and tropical regions, the human lens temperature ranges from 35.0 \u00b0C to 37.5 \u00b0C depending on the environmental temperature. The medium temperature changes during the replacement regularly in the cell culture experiment were carefully monitored using a sensor connected to a thermometer and showed a decrease of 1.9 \u00b0C, 3.0 \u00b0C, 1.7 \u00b0C, and 0.1 \u00b0C, after 5 min when setting the temperature of the heat plate device at 35.0 \u00b0C, 37.5 \u00b0C, 40.0 \u00b0C, and 42.5 \u00b0C, respectively. In the newly created immortalized human lens epithelial cell line clone NY2 (iHLEC-NY2), the amounts of RNA synthesis of \u03b1A crystallin, protein expression, and amyloid \u03b2 (A\u03b2)1-40 secreted into the medium were increased at the culture temperature of 37.5 \u00b0C compared to 35.0 \u00b0C. In short-term culture experiments, the secretion of A\u03b21-40 observed in cataracts was increased at 37.5 \u00b0C compared to 35.0 \u00b0C, suggesting that the long-term exposure to a high-temperature environment may increase the risk of cataracts. The crystalline lens is surrounded by a lens capsule containing a large amount of type IV collagen; this capsule functions as the basement membrane of lens epithelial cells (LECs) arranged in a single layer at the anterior zone. In vivo, the tissue stem cells of LECs at the germinative zone proliferate at a low level in the bow area near the equatorial segment of the crystalline lens , and theThe human crystalline lens with the fetal nucleus in the central zone maintains the transparency and flexibility for focus adjustment over the course of decades. Presbyopia occurs due to a decrease in accommodation power by sclerosis of the crystalline lens nucleus with aging; this is also thought to be a precursor of nuclear cataract. In their investigation of the relationship between environmental temperature and the age at presbyopia onset, Miranda et al. observed that individuals who live in geographic regions with higher temperatures develop presbyopia earlier . Thus, iIn an animal model, 6-month exposure to low-dose UV-A induced nuclear cataracts with opacification in the central zone of the crystalline lens . HoweverWe thus hypothesized that the incidence of cataracts is related to the environmental temperature. In the present study, we evaluated the relationship between the environmental temperature and the lens temperature by conducting a computer simulation in silico. We also examined the effects of different temperatures on the lens in vitro using LECs at the lens temperature of residents of tropical and temperate regions. As LECs are extremely difficult to maintain and culture for a long period, immortalized LEC lines have been used in many experimental reports. One of the existing immortalized LEC lines, SRA01/04, was obtained from infants who underwent surgery for retinopathy of prematurity . Herein,Our computational techniques integrated the thermodynamics and the thermoregulatory response for increased temperature. When we considered the solar radiation, we also took into account its deposition in the human body by using electromagnetic computation. Detailed explanations and validations of our computational code are provided in our previous reports ,14. The A three-dimensional Japanese male model created based on magnetic resonance images was used in our computation . The heiAt the time of cataract surgery in patients who agreed to participate in this study, LECs that had adhered to the collected anterior capsule pieces were subjected to primary culture by the explant method. The composition of the culture medium was as follows: 10% fetal bovine serum (FBS), 10 ng/mL basic fibroblast growth factor (bFGF), \u00d7100 minimum essential medium non-essential amino acids (MEM-NEAA), \u00d7100 GlutaMAX\u2122 , Dulbecco\u2019s modified Eagle\u2019s medium (DMEM) high-glucose medium, and penicillin-streptomycin solution . This study was approved by the Ethics Review Committee of Fujita Health University (No. 004) and was conducted in accord with the provisions of the Declaration of Helsinki for research involving human tissue. As a control for immortalized human LECs, SRA01/04 strain cells  were used and cultured in the medium as instructed by the cell bank.attL1 and attL2 was replaced with the Elongation Factor-1\u03b1 (EF1\u03b1) promoter . The pJTITM Fast DEST plasmid vector carrying phiC31 with attR1, attR2, and the hygromycin resistance gene was then subjected to the Gateway LR reaction using LR clonase, and the Jump-In expression clone plasmid was introduced in Escherichia coli DH5\u03b1 competent cell (Takara Bio). The growth and expression clone plasmids were then purified and transfected into primary human LECs by electroporation. The selection was performed with 200 \u03bcg/mL hygromycin B, and drug-resistant cells were cloned to establish an immortalized human lens epithelial cell line clone NY2 (iHLEC-NY2). The composition of the iHLEC-NY2 medium was as follows: 10% FBS, 2 ng/mL bFGF, \u00d750 GlutaMAXTM I, Advanced DMEM/F12 medium (Thermo Fisher Scientific), and penicillin-streptomycin solution. This gene-recombination experiment was approved by the Kanazawa Medical University Biosafety Committee for Recombinant DNA Research .As the immortalizing gene, we used the modified SV40 Large T antigen-GFP (mSV40-GFP) synthesized for the production of the immortalized human corneal epithelial cell line as reported . We usedWhile replacing the medium for the cells cultured at 37.5 \u00b0C and 35.0 \u00b0C, pre-warmed medium was used so as not to subject the cells to temperature changes. A culture dish was placed on a thin-type thermostatic plate  inside a clean bench to maintain the temperature of the medium. Then, a digital thermometer  connected with a sensor  with a diameter of 2 mm  was used4 cells per cell culture dish . The cells were cultured for 7 days at 37.5 \u00b0C, 35.0 \u00b0C, or 31.0 \u00b0C in 5% CO2 in a humidified incubator, and the medium was replaced every other day. The cells were counted in a B\u00fcrker\u2013T\u00fcrk counting chamber at 1:1 dilution with trypan blue solution (Sigma-Aldrich).The cells were seeded at a density of 2.5 \u00d7 10TM Mycoplasma Test Kit, Biological Industries, Cromwell, CT, USA) according to the manufacturer\u2019s instructions.As iHLEC-NY2 are primary cultured cells of human LECs, the detection of Mycoplasma infection was accomplished with a mycoplasma detection kit . The RNA concentrations were measured using a spectrophotometer  [Total RNA of cells was extracted using an RNA extraction kit  .\u00ae polymerase chain reaction (PCR) system 9700 Thermal Cycler (Thermo Fisher Scientific) to synthesize complementary DNA (cDNA) [\u00ae 7900 HT Sequence Detection System (Thermo Fisher Scientific) with the Primer and Probe for the crystalline lens-marker genes \u03b1A crystalline  and \u03b2B2 crystalline , as well as glyceraldehyde-3-phosphate dehydrogenase  as an internal positive control.Total RNA was reverse-transcribed using the GeneAmpA (cDNA) . Subsequ\u00ae Green I  and specific primers as listed in reference [The qPCR of the A\u03b2 marker genes amyloid precursor protein (APP), a disintegrin and metalloproteinase domain 10 (ADAM10), \u03b2-site APP-cleaving enzyme 1 (BACE1), presenilin 1 (PS1), presenilin 2 (PS2), neprilysin (NEP), and endothelin converting enzyme 1 (ECE-1) was performed using SYBReference .We used the differences in the threshold cycles for GAPDH and target genes  to calculate the levels of mRNA.Cell lysates were prepared in ice-cold radioimmune precipitation (RIPA) buffer with the protease inhibitor phenylmethylsulfonyl fluoride  and cOmplete\u2122 Mini (Sigma-Aldrich) as described . Equal ag for 5 min, and the supernatant was stored at \u221280 \u00b0C until measurement. The concentrations of A\u03b21-40 and A\u03b21-42 were measured using a Quantikine\u00ae Enzyme-Linked Immunosorbent Assay (ELISA) kit  according to the manufacturer\u2019s manual.The culture medium obtained by culturing iHLEC-NY2 and SRA01/04 cells for 2 days was collected and then centrifuged at 1500\u00d7 t-test. The Statistical Package for Social Science (SPSS) Statistics 24  was used to perform the statistical analyses.Each experiment was performed in triplicate and repeated at least three times. Data are presented as the mean \u00b1 standard deviation (SD) and were analyzed by Welch\u2019s 2 [2.The eye temperatures in younger and elderly adults with and without solar radiation were evaluated at ambient temperatures of 19\u201335 \u00b0C. The relative humidity was set at 50% considering the typical ambient condition. The effect of relative humidity is not high in a resting condition ,24. For 2 . ConsideThe temperature distribution in the whole-body model is given at the steady-state in each scenario. The time required to reach a steady-state was 30 min (90% saturation). Measurement of the medium temperature was performed under the following conditions: laboratory temperature, 25.3 \u00b0C; the temperature inside the clean bench, 29.3 \u00b0C, and surface temperature of the working table, 25.0 \u00b0C.On the other hand, the surface temperature of the heat plate was 36.9 \u00b0C when the device temperature was set at 42.5 \u00b0C, and the medium temperature at 5 min was 37.0 \u00b0C, representing a decrease of only 0.1 \u00b0C, which showed that the plate stayed warm even inside the clean bench.p < 0.005). The proliferation of SRA01/04 cells was also significantly greater at 37.5 \u00b0C (p < 0.05). Moreover, the iHLEC-NY2 cells proliferated more actively compared to the SRA01/04 cells at the culture temperature of 37.5 \u00b0C (p < 0.05). The iHLEC-NY2 cells hardly proliferated when the culture temperature was lowered to 31.0 \u00b0C  of each captured band was analyzed and corrected with \u03b2-actin by the ImageJ analysis software. Most notably, CRYAA  was increased by ~1.37-fold in iHLEC-NY2 cells and by ~1.25-fold in SRA01/04 cells cultured at 37.5 \u00b0C compared to 35.0 \u00b0C .p < 0.05, A\u03b21-40 and A\u03b21-42 measured by ELISA are the protein concentrations contained in the medium . Their values were therefore corrected by the number of cells when the medium was collected. The cell number-corrected concentration of A\u03b21-40 was significantly increased when the cells were cultured at 37.5 \u00b0C compared to 35.0 \u00b0C  at culture temperatures of 35.0 \u00b0C and 37.5 \u00b0C, the gene expression at 37.5 \u00b0C was higher than that at 35.0 \u00b0C, and the amount of the synthesis of CRYAA (one of the major constituent proteins of the lens) increased. The A\u03b2-related genes and A\u03b2 proteins secreted on the culture medium were also increased in iHLEC-NY2.The lens temperature has been estimated as 35\u201337.5 \u00b0C depending on the ambient temperature of the eyeball; however, when the ambient temperature exceeds 30 \u00b0C, the estimated temperature of the lens differs depending on the body\u2019s age, and it is higher with aging . This maIn this study, the culture temperature was the key factor. Because it was necessary to replace the medium regularly in the cell culture experiment, any temperature changes were carefully monitored using a sensor connected to a thermometer. Since the room temperature in the laboratory and that inside the clean bench were lower than the culture temperature, even the dish replacement from the incubator into the clean bench caused a slight decrease in the dish temperature. Moreover, the downflow of clean air in the clean bench also caused a decrease in the dish temperature. The equipment used (LABPAD H) has a two-stage structure, consisting of an adapter for the aluminum-block culture dish placed on a control unit with the sensor. The temperature difference between the set temperature of the device and the surface of the heat plate is related to the air downflow and the position of the sensor in the clean bench. In this study, when setting the temperature of the device at 42.5 \u00b0C, it was possible to maintain a temperature of around 37 \u00b0C even while working on the clean bench. To investigate the effect of temperature on the cells, it was important to set the environmental temperature during the experiment using the sensor connected to the thermometer.In our epidemiological studies, the prevalence of nuclear cataracts was significantly increased in tropical and subtropical regions . StudiesThe factors that have been reported to affect the prevalence of nuclear cataracts include smoking , tempera2+) at the molecular level is disrupted, scattered particles with a high molecular weight fraction are formed [The heat shock proteins (HSPs) are created by a typical reaction of a protein to heat (temperature). Among them, the low-molecular-weight HSPs (15\u201330 kDa) are proteins induced by heat stress, and typical HSPs are Hsp27 (HSPB1) and \u03b1B crystallin (HSPB5), the latter of which is a protein consisting of 175 residues of amino acids, with the molecular weight of approx. 20 kDa. The crystalline lens contains a large amount of CRYAA (HSPB4), which has approximately 55% amino acid homology and similar functions and structures, in addition to \u03b1B crystallin. The heat stability and chaperone function of the aggregation of the \u03b1-crystallin protein composed of both subunits is deeply involved in maintaining the transparency of the lens ,40,41,42e formed ,44, resue formed . The fire formed . At thise formed ,48.Intracellular homeostasis is disturbed by slight changes in environmental temperature, and the \u03b1A crystallin expression level may increase as one of the suppression mechanisms. The expressed \u03b1A crystallin is likely to be responsible for suppressing the intracellular dysfunction caused by temperature changes. For investigations of the temperature responsiveness at the molecular level, a lens-derived cultured cell model that is particularly susceptible to heat stress and has a defense mechanism would be optimal.It was reported that A\u03b2 was detected in the lens nucleus and deep lens fiber cells ,49,50,51\u00ae-3Zf (+) vector using a calcium phosphate transfection system. Ibaraki et al. reported that SV40 Large T antigen was not transferred into the regions of CRYAA and CRYBB2 because the SRA01/04 line was created by the gene transfer method at its establishment [The SRA01/04 cells , an immolishment , howeverpseudo-attP site, a non-functional site on the genomic sequence. In addition, modified SV40 Large T antigen is a non-viral vector and its recombination is single-copied. Compared to SRA01/04 cells produced from the LECs of infants with retinopathy of prematurity, the original cells that created iHLEC-NY2 are human adult LECs, in which A\u03b2 observed in age-related cataracts was detected, suggesting that iHLEC-NY2 cells may be the immortalized human LECs that retain the metabolic environment of adult human LECs. We are continuing to analyze the genes and proteins of iHLEC-NY2.It is quite difficult to culture adult human primary LECs, and even if the primary culture is successful, it is difficult to use it for basic research that requires reproducibility, as the cell expression is limited. The newly created cell line iHLEC-NY2 is an immortalized human LEC line that does not affect the existing genomic sequence due to the recombination of a modified SV40 Large T antigen that retains the original cell function , specifiiHLEC-NY2 is a human immortalized lens epithelial cell line created by gene transfer in a safe manner. As the immortalizing gene is introduced into the pseudo-attP site rather than randomly inserted into a gene originally present in the lens epithelial cells, the genetic characteristics of the human lens epithelial cell are preserved. This is an important feature compared to the more conventionally used SRA01/04 cells. In a comparison of iHLEC-NY2 and SRA01/04, iHLEC-NY2 cells had more active cell proliferation, and iHLEC-NY2 cells, but not SRA01/04 cells, secreted A\u03b21-40 protein into the culture medium. In the medium of SRA01/04, A\u03b21-40 protein was below the level of detection sensitivity. Although it could not be conclusively determined in the present study, our results suggested that the mechanism underlying the expression of A\u03b21-40 protein was as follows: A\u03b2 is peptide-degraded from APP, and PS1 and PS2 produce A\u03b21-40. In iHLEC-NY2, the gene expressions of PS1 and PS2 were significantly lower than that of SRA01/04, the concentration of A\u03b21-40 was low, and the expressions of all these proteins were detectable by a commercially available ELISA. Based on our findings, we speculate that iHLEC-NY2 cells are established from adult human lens epithelial cells, and may retain the mechanism of A\u03b2 production in the original human lens epithelial cells. Because it has been reported that the A\u03b21-40 protein is detected in the crystalline lens with age-related cataract  and A\u03b21-Several factors may be involved in age-related nuclear cataracts: connexins and aquaporins (AQPs) related to metabolite transport in the lens, transient receptor potential cation channel subfamily V member 1 (TRPV1) and transient receptor potential cation channel subfamily V member 4 (TRPV4) proteins involved in lens ion transport , the posIt has been speculated that in vivo, the lens temperature rises due to a high ambient temperature, and the HLECs, which originally express very slowly , expressIn computer simulations, when the ambient temperature was 19\u201335 \u00b0C, the estimated lens temperature was 35\u201337.5 \u00b0C. When the newly prepared iHLEC-NY2 cells were cultured at 37.5 \u00b0C and 35.0 \u00b0C, the cells\u2019 proliferation and lens-specific protein gene and protein expressions increased, and higher cell activity was observed with higher temperature. The expressions of proteins and some genes of A\u03b2 detected in human cataracts were also increased at 37.5 \u00b0C, suggesting that HLECs and lens aging may progress longitudinally with higher temperatures. Our present findings thus indicated that the ambient temperature was one of the contributing factors of nuclear cataracts observed in our epidemiological studies."}
+{"text": "Glucocorticoids rapidly provoke serotonin (5\u2010HT) release in vivo. We aimed to investigate molecular mechanisms of glucocorticoid receptor (GR)\u2010triggered 5\u2010HT release.Employing 1C11 cells to model 5\u2010HT neurotransmission, immunofluorescence and Pearson's Correlation Coefficient were used to analyze colocalization of GR, 5\u2010HT, vesicle membrane protein synaptotagmin 1 and vesicle dye FM4\u201064FX. FFN511 and FM4\u201064FX dyes as well as calcium imaging were used to visualize vesicular 5\u2010HT release upon application of GR agonist dexamethasone, GR antagonist mifepristone and voltage\u2010gated calcium channel (VGCC) inhibitors.GR, 5\u2010HT, synaptotagmin 1 and FM4\u201064FX showed overlapping staining patterns, with Pearson's Correlation Coefficient indicating colocalization. Similarly to potassium chloride, dexamethasone caused a release of FFN511 and uptake of FM4\u201064FX, indicating vesicular 5\u2010HT release. Mifepristone, calcium depletion and inhibition of L\u2010type VGCC significantly diminished dexamethasone\u2010induced vesicular 5\u2010HT release.In close proximity to 5\u2010HT releasing sites, activated GR rapidly triggers L\u2010type VGCC\u2010dependent vesicular 5\u2010HT release. These findings provide a better understanding of the interrelationship between glucocorticoids and 5\u2010HT release. In proximity to serotonin\u2010containing vesicles, glucocorticoids (dexamethasone) bind to glucocorticoid receptors (1) and mediate opening of L\u2010type voltage\u2010gated calcium channels . Subsequent calcium influx (3) triggers rapid vesicular serotonin release (4). This mechanism is similar to depolarization\u2010induced serotonin release, which also depends on L\u2010type voltage\u2010gated calcium channels (5). Alterations of the 5\u2010HT system are believed to play a pivotal role in the pathogenesis of many psychiatric diseases such as depression.In serotonergic neurons, 5\u2010HT is synthesized from L\u2010tryptophan and packed into vesicles.Administration of glucocorticoids triggers a rapid increase in extracellular 5\u2010HT concentration in vivo, which is blocked by the glucocorticoid receptor (GR) antagonist mifepristone.We employed murine stem cell\u2010derived 5\u2010HT neurons22.12. For differentiation to serotonergic 1C11 cells (1C115\u2010HT), 40,000 cells were transferred to a 3.5\u00a0cm plate (Sarstedt) containing 3 coverslips or slide chambers (Ibidi) for live cell imaging. Then, culture medium was supplemented with 1\u00a0mM dibutyryl cyclic adenosine monophosphate (cAMP) and 0.05% cyclohexanecarboxylic acid for 4\u00a0days. On day 4, 1C115\u2010HT were shown to display a complete 5\u2010HT metabolism.2B and 5\u2010HT1B/D autoreceptors.1C11 cells were cultured according to standard protocol.2.25\u2010HT were fixed in 1% paraformaldehyde and subsequently permeabilized using 0.1% saponin in blocking solution . Antibodies were incubated in blocking solution supplemented with 0.01% saponin for 60\u00a0min at room temperature . Primary antibodies were: GR ; 1:200; specificity assessed by Weber et al.t\u2010test was used to determine if PCC was significantly greater than 0 (no correlation).For immunostainings, 1C112.35\u2010HT grown on coverslips were kept in imaging buffer  before and after application of either imaging buffer supplemented with 2\u00a0\u00b5M FM4\u201064FX (Thermo Fisher Scientific (F34653); control condition) or reaction buffer (treatment condition). Reaction buffer contained either high potassium or dexamethasone in absence or presence of calcium ions  and was also supplemented with 2\u00a0\u00b5M FM4\u201064FX. Cells were kept in the imaging buffer or reaction buffer supplemented with 2\u00a0\u00b5M FM4\u201064FX for up to 5\u00a0min at 37\u00b0C. After washing out the fluorescence dye with imaging buffer, cells were fixed with 1% paraformaldehyde and mounted using Dako Fluorescence Mounting Medium (Dako Cytomation) after counterstaining for synaptotagmin 1 or GR (see Section 2.2). Data Fluorescent images were acquired using a Leica TCS SP5 Confocal imaging system on a DM IRE2 microscope, equipped with an acusto\u2010optical beam splitter, an argon ion laser (458\u2013514\u00a0nm), a diode\u2010pumped solid\u2010state laser (561\u00a0nm), and a helium neon laser (633\u00a0nm). Laser lines were used as recommended for the applied dyes. For FM4\u201064FX staining, 1C115\u2010HT grown in Ibidi imaging chambers were kept in imaging buffer at 37\u00b0C, which was supplemented with 10\u00a0\u00b5M FFN511 (Abcam (ab120331)) for 10\u00a0min. Release of FFN511 was induced by exchanging imaging with reaction buffer for 5\u00a0min at 37\u00b0C. For buffer exchange, imaging chambers were kept on the heated microscope stage (Ibidi). The GR antagonist mifepristone (10\u00a0\u00b5M) or VGCC inhibitors in the following concentrations were applied for 30\u00a0min prior to loading with FFN511 in presence of the respective inhibitor: GVIA ; MVIIC ; nifedipine ; SNX482 . Each cell was exposed to one VGCC inhibitor only. For calcium imaging, cells were maintained as for FFN511 live cell imaging. Imaging buffer was supplemented with 2\u00a0\u00b5M Fluo4\u2010AM (Thermo Fisher Scientific (F14201)).5\u2010HT.n\u00a0=\u00a024 with at least n\u00a0=\u00a04 in each group was required (G*Power V3.1). Image analysis was performed using ImageJ as described above. All experiments were independently conducted at least three times on different days using different batches of 1C11. For each experimental condition, multiple coverslips or wells were used, and each cell was captured once only. All data points were included in the analysis and no outliers were defined. To generate charts of normalized data and perform statistical analysis with raw data, data files were imported into GraphPad Prism7 . Data were assessed for normality using a normal QQ plot and the Shapiro\u2010Wilk test. For comparisons of quantifications of FM4\u201064FX, FFN511 and Fluo4\u2010AM signals, Kruskal\u2010Wallis test and Dunn's multiple comparisons test was performed using the raw data acquired during the experiments. The significance cut\u2010off (p) was 0.05. Data were expressed as median with limits of the interquartile range (25th and 75th percentile).For FFN511 live cell imaging, 1C1133.15\u2010HT \u00a0=\u00a036.41, p\u00a0<\u00a00.001). We further analyzed colocalization of GR with synaptotagmin 1 using double\u2010immunofluorescence labelling \u00a0=\u00a077.95, p\u00a0<\u00a00.001). Within the limits of conventional confocal microscopy's spatial resolution, our analysis shows that GR immunofluorescence signals localized to cellular structures that also harbor 5\u2010HT and synaptotagmin 1 immunofluorescence signals, indicating that GR may be localized in such close proximity to 5\u2010HT release sites to be directly involved in the vesicular release process.To investigate a possible contribution of GR to 5\u2010HT release, we first analyzed the localization of GR in comparison to 5\u2010HT itself and to synaptotagmin 1, a protein involved in vesicular 5\u2010HT release, with immunofluorescence analysis performed in 1C11T Figure\u00a0. On the g Figure\u00a0. Synapto3.25\u2010HT. As reported previously, high potassium\u2010induced vesicular 5\u2010HT release from 1C115\u2010HT can be visualized directly either by release of fluorescent false neurotransmitters (FFN)5\u2010HT. After exposure to high potassium concentrations, FM4\u201064FX was found to localize in globular structures on the cell soma as well as on neurites. Subsequent immunostaining for the vesicle\u2010associated synaptotagmin 1 revealed that both fluorescence signals overlapped \u00a0=\u00a060.46, p\u00a0<\u00a00.001), emphasizing that the GR resides in proximity to neurotransmitter release sites.Up to date, the molecular framework how GR activation causes a rapid release of 5\u2010HT on the cellular level is not completely understood. To provide more insight into this process, we employed 5\u2010HT neuron\u2010mimicking 1C11d Figure\u00a0, verifyid Figure\u00a0, colocal5\u2010HT. After exposure to high potassium concentrations, FFN511 fluorescence intensities significantly dropped due to vesicular dye release \u00a0=\u00a090.88, p\u00a0<\u00a00.001; post\u2010hoc Dunn's multiple comparison test: p\u00a0<\u00a00.001). As expected, high potassium concentrations also induced calcium influx required for neurotransmitter release, which was visualized using Fluo4\u2010AM \u00a0=\u00a021.57, p\u00a0<\u00a00.001; post\u2010hoc Dunn's multiple comparison test: p\u00a0=\u00a00.001). In order to verify that FFN511 release was calcium\u2010dependent, we employed selective inhibitors for VGCCs and quantified their effect on FFN511 release. As a prerequisite for vesicular release, we also visualized the required calcium influx using Fluo4\u2010AM. Compared to control conditions, selective inhibitors for the N\u2010, P/Q\u2010 and R\u2010type VGCCs did not diminish potassium chloride\u2010induced FFN511 release from 1C115\u2010HT , R\u2010type (p\u00a0=\u00a00.03), or P/Q\u2010type (p\u00a0=\u00a00.011) VGCC inhibitor). In line with the observed effect on FFN511 release, calcium influx was not significantly diminished when N\u2010type or R\u2010type VGCC inhibitors were applied  or R\u2010type (p\u00a0>.999) VGCC inhibitor; for the P/Q\u2010type VGCC inhibitor, only descriptive data are presented). In contrast to the latter findings, inhibition of L\u2010type VGCCs with nifedipine resulted in a significant reduction of FFN511 release from 1C115\u2010HT  and versus 60\u00a0mM potassium chloride without VGCC (p\u00a0=.006). Furthermore, a dose\u2010dependent reduction of Fluo4\u2010AM fluorescence intensity indicated a reduced calcium ion influx when trying to evoke 5\u2010HT release in the presence of an L\u2010type VGCC inhibitor ). Taken together, these observations show that L\u2010type VGCCs mediated calcium ion influx upon depolarization with potassium chloride required to elicit vesicular 5\u2010HT release in vitro.As shown for FM4\u201064FX stainings, FFN511\u2010containing vesicles were located on neurites and cell bodies of 1C113.3\u03c72\u00a0=\u00a0449, p\u00a0<\u00a00.001; post\u2010hoc Dunn's multiple comparison test compared to control: p\u00a0<\u00a00.001), which was blocked when calcium was removed from the reaction buffer \u00a0=\u00a040.29, p\u00a0<\u00a00.001; post\u2010hoc Dunn's multiple comparison test compared to resting cells: p\u00a0<\u00a00.001). Moreover, dexamethasone\u2010induced FFN511 release occurred within seconds after application, likely indicating a rapid, non\u2010genomic action of GR . In the next step, we incubated 1C115\u2010HT with 10\u00a0\u00b5M nifedipine to selectively inhibit L\u2010type VGCCs prior to application of dexamethasone. Comparable to our observation for high potassium\u2010induced FFN511 release, the inhibition of L\u2010type VGCCs blocked dexamethasone\u2010induced FFN511 release from 1C115\u2010HT \u00a0=\u00a088.78, p\u00a0<\u00a00.001; post\u2010hoc Dunn's multiple comparison test of 20\u00a0nM dexamethasone plus 10\u00a0\u00b5M nifedipine compared to resting cells: p\u00a0>\u00a00.999). This suggests that calcium influx via L\u2010type VGCCs contributes to GR\u2010dependent, vesicular 5\u2010HT release in 5\u2010HT neuron\u2010mimicking 1C115\u2010HT.To simulate GR activation in vitro, we applied the selective agonist dexamethasone. Vesicular 5\u2010HT release upon GR activation with dexamethasone was assessed by performing vesicular uptake experiments with FM4\u201064FX. Dexamethasone induced a significant vesicular uptake of FM4\u201064FX (Kruskal\u2013Wallis test (e Figure\u00a0. Our pre45\u2010HT.Previous research suggests that glucocorticoids cause an immediate release of 5\u2010HT,5\u2010HT express most properties of 5\u2010HT neurons, such as a complete 5\u2010HT metabolism, and thus mimic the neurochemistry of adult rodent 5\u2010HT raph\u00e9 neurons.5\u2010HT provide the opportunity to conduct experiments on the molecular level of 5\u2010HT neurotransmission that are difficult to accomplish in vivo. For example, the arborization of 5\u2010HT neurons from the raph\u00e9 nuclei to various brain regions including the hippocampus and prefrontal cortex has constituted a barrier for live cell imaging to track release of 5\u2010HT vesicles in vivo.5\u2010HT. Additionally, our in vitro model creates an isolated, controlled environment of networks of interacting 5\u2010HT neurons. Thus, we can exclude the possibility that the effects of dexamethasone on 5\u2010HT neurons are mediated by other, interacting cells. We analyzed individual, monoclonal 1C115\u2010HT grown in networks in wells or on coverslips. While we cannot completely exclude the risk of erroneous external factors impacting batches of 1C11 or individual wells, we mitigated this risk by conducting each experiment on at least three days with new batches of 1C11, and by performing each experiment using multiple wells or coverslips for each condition. However, the 1C115\u2010HT in vitro model does not resemble the physiological human or rodent brain environment, which imposes the main limitation on this work. Thus, future experimental work should employ in vivo rodent models to confirm our results.A strength of the applied in vitro model is that 1C115\u2010HT, as both dyes are well\u2010established agents to visualize synaptic, exocytotic events.5\u2010HT.We employed FM4\u201064FX as well as FFN511 to visualize GR\u2010induced vesicular release in 1C115\u2010HT, calcium influx via L\u2010type is primarily responsible for depolarization\u2010induced and GR\u2010induced 5\u2010HT vesicle release. These results are in line with experimental data by Trueta at al.,5\u2010HT. While inhibition of the L\u2010type VGCC significantly reduced vesicular release and calcium influx, it did not completely abolish it. Thus, future research needs to compare the contribution of different VGCCs to vesicular 5\u2010HT release.Our results show that calcium influx is at the center of GR\u2010induced vesicular 5\u2010HT release, similar to depolarization\u2010induced 5\u2010HT release.The colocalization between GR, 5\u2010HT, synaptotagmin 1, which resides in the membrane of neurotransmitter vesicles, and the styryl vesicle dye FM4\u201064FX suggests that GR might have a relatively direct, non\u2010genomic effect on the opening of L\u2010type VGCC in proximity to vesicle release sites, as it is hypothesized for the melatonin receptor.5This work extends our understanding how glucocorticoids interact with 5\u2010HT neurons. In murine stem cell\u2010derived 5\u2010HT neurons, the proximity of extranuclear GR, 5\u2010HT and synaptotagmin 1 suggests that GR might be directly involved in rapid 5\u2010HT release. Glucocorticoid binding to GR initiates a rapid vesicular 5\u2010HT release, which depends on extracellular calcium influx via L\u2010type VGCCs. Extending the knowledge of the acute and chronic effects of glucocorticoids on 5\u2010HT neurons might ultimately contribute to the understanding of the underlying mechanisms of stress\u2010related psychiatric disorders.The authors declare that the research was conducted in the absence of any financial support or relationships that could be construed as a potential conflict of interest.TL, NP, TW and DB contributed to the conception of the study. TL, NP, JR and SL conducted the experiments and analyzed the data. TL and NP prepared the manuscript. All authors contributed to critical revision of the manuscript and approved the submitted version.Data S1Click here for additional data file.Data S2Click here for additional data file."}
+{"text": "Thus, sophisticated and expensive separation methods are required after the reaction step. Alternatively, several types of materials have been used to support powder catalysts, so that fixed or fluidized bed reactors may be used. In this context, the objective of this work is to systematize and analyze the results of research inherent to the application of ceramic materials as support of TiO2 in the photocatalytic CEC removal from liquid effluents. Firstly, an overview is given about the treatment processes able to degrade CEC. In particular, the photocatalysts supported in ceramic materials are analyzed, namely the immobilization techniques applied to support TiO2 in these materials. Finally, a critical review of the literature dedicated to photocatalysis with supported TiO2 is presented, where the performance of the catalyst is considered as well as the main drivers and barriers for implementing this process. A focal point in the future is to investigate the possibility of depurating effluents and promote water reuse in safe conditions, and the supported TiO2 in ceramic materials may play a role in this scope.The application of TiO Wastewater reclamation for multiple purposes is now seen as a central aspect of sustainable water resources management. This procedure has a strong impact both quantitatively, by relieving the pressure of decreasing volumes of freshwater required, and qualitatively, by reducing discharges of treated wastewater into sensitive areas ,2. Howev\u22121 to \u00b5g L\u22121. In this view, promising processes have been tested, such as photocatalysis, ozonation, and biofiltration [The contaminants of emerging concern (CEC) are compounds difficult to remove by the technologies implemented in conventional wastewater treatment plants (WWTP). These compounds are found in residual concentrations in liquid effluents and generally encompass pharmaceuticals and personal care products, pesticides, steroids hormones among others ,5. The pltration .2) with the ability to absorb ultraviolet/visible light. In the presence of water molecules, these catalysts can promote the generation of hydroxyl radicals (HO\u25aa) and superoxide ions (O2\u2022\u2212), which are highly oxidizing leading to the degradation of pollutants [Heterogeneous photocatalysis is a low-cost and versatile advanced oxidation process (AOP), based on semiconductors . This material is usually used in suspension systems (slurry reactors). Thus, after the effluent treatment, methods of solid/liquid separation are required for its recovery. Here, technologies such as filtration, decantation, or centrifugation may be applied, which increases the complexity of the treatment operation and reduces its economic viability. Moreover, the application of powder catalyst makes difficult its reuse in continuous treatment processes.According to Jabri and Feroz  TiO2 is 2, which demonstrates high chemical stability [According to the literature, the environmental application of supported photocatalytic semiconductors in the degradation of CEC in liquid effluents can bring short and long-term benefits in the use and sustainable management of water resources. In fact, the utilization of supported catalysts would allow the application of continuous operation treatment systems without the need for sophisticated solid/liquid separation units. This would overcome the major drawback associated to slurry photocatalytic systems. Clays, zeolites, and silica-based materials are common materials for incorporating TiOtability ,12.2 in the degradation of emerging contaminants in liquid effluents. To the best of our knowledge, there is a lack in the literature of a systematic overview regarding the use of supported catalysts in photocatalytic processes which highlights the novelty of the present work. Firstly, an overview of the presence of emerging contaminants in wastewater is given. Afterwards, the relevance of photocatalytic processes on the removal of these contaminants is highlighted and the theoretical foundations of this process are given. The disadvantages of using powder photocatalysts will be discussed and an overview of ceramic materials as TiO2 supports will be given. The application of such technology on the abatement of emerging contaminants and water disinfection is discussed with a special focus on water reuse.In this context, the objective of this work is to systematize and analyze the results of research inherent to the application of ceramic materials as support of TiOCEC includes a diversity of compounds found in trace concentrations in industrial or municipal wastewater effluents. These pollutants comprise pharmaceuticals and personal care products (PPCP), polycyclic aromatic hydrocarbons, polychlorinated biphenyls, pesticides, microplastics, steroid hormones, and disinfection by-products, as well as the resulting processing products ,13. Inde\u22121 for diazinon. Moreover, they observed the environmental relevance of 22 pesticides for species of algae, daphnia, and fish. From the toxicity point of view, it was concluded, that the most problematic compounds were diazinon and diuron, followed by atrazine, simazine, malation, chlorotoluron, terbuthylazine, and isoproturon. Furthermore, Baalbaki et al. [\u22121 in 31 water samples and from 28.1 to 2728.8 ng g\u22121 in 31 sediment samples. In the Amazon estuary recognized as a Ramsar site, 11 PPCP were detected in water samples and 14 in sediments. Caffeine was the most frequent compound, reaching 13,798 ng L\u22121 [\u22121), lincomycin (624 ng L\u22121), flumequin (331 ng L\u22121), caffeine (276 ng L\u22121), ifosfamide (220 ng L\u22121), and cephalexin (172 ng L\u22121). Moreover, cross-contamination was found between these two environments. According to Zhang et al. [Gavrillescu et al.  highlighi et al.  emphasizi et al. . In facti et al.  detectedi et al.  analyzed8 ng L\u22121 . Likewis8 ng L\u22121  tracked g et al. .2) and natural or artificial radiation. The results obtained in experiments with the photocatalytic treatment of liquid effluents showed quite satisfactory performance of TiO2 supported catalysts. Even though, the performance of this methodology must be evaluated in terms of its impact on the toxicity and disinfection capacity of the water [It is important to note that CEC are not normally regulated in environmental legislation . Howeverhe water . Moreovehe water . As thes2 has been extensively studied for wastewater treatment due to its high photocatalytic activity, chemical stability, non-toxicity, and low cost [2 reveals photocatalytic capabilities to absorb photons, and then promote the disintegration of different contaminants, by generating hydroxyl radicals among other moieties. The ability to degrade organic and inorganic pollutants comes from the redox environment generated by the photoactivation of TiO2, which is a semiconductor material [Photocatalytic processes are interesting technologies for the CEC abatement. In this scope, TiOlow cost . Indeed,material .A semiconductor is characterized by the possibility of excitation of electrons from valence bands (VB) to the conduction bands (CB), originating the appearance of holes in the valence band, and occupied states in the conduction band . Thus, t2 as a catalyst, which generates hydroxyl and superoxide radicals. The VB promotes oxidation reactions for degradation of OM and CB aids reduction reactions. The presence of dissolved O2 is important as it can hinder the recombination of e\u2212(CB) and h+(VB), maintaining the electroneutrality of the catalyst.Equations (1)\u2013(8) describe the reactions that may occur during the heterogeneous photocatalysis process of organic molecules (OM) using TiO2 conduction and valence bands are separated by different energy bandgaps dependent upon the crystalline phase. The energy bandgap (Eg) goes from 3.2 eV for anatase and 3.0 eV for the rutile phase, corresponding to the absorption of radiation at 387.5 and 400 nm in the ultraviolet region, respectively [TiOectively ,31. Silvectively  showed tectively .2 used, with a mixture of rutile (30%) and anatase (70%) showing to be highly reactive [2 at lab-scale already proved high potential for wastewater treatment. However, its application at the industrial scale is far from proving this success rate. In part, the application of TiO2 in the wastewater treatment industry as a powder in nanoparticles needs a separation step after photocatalytic oxidation reaction which can represent relevant costs of installation and operation. Therefore, the immobilization of such nanoparticles in suitable supports as ceramic materials can eliminate the need for this separation stage. Considering this, the application of supported TiO2 instead of powder can be a key aspect for this material to be widely used in wastewater treatment at an industrial scale.The effectiveness of the photocatalytic reaction depends on the type of TiOreactive . The comreactive . The app\u22122, part of which (about 5%) corresponds to UV-A and UV-B radiation with wavelengths from 290 to 400 nm [2 (Equations (1)\u2013(8)) [The radiation source is an important point while designing a photocatalytic treatment process for depurating liquid effluents. The average solar energy incident on the earth\u2019s surface is about 240 W mo 400 nm . Thus, U(1)\u2013(8)) . Usually(1)\u2013(8)) .Ultraviolet (UV) radiation is generally defined as radiation with a wavelength between 100 and 400 nm. UV-C is more energetic and can be absorbed by water according to the following reaction:2O2, UV/O3, UV/TiO2. These processes allow obtaining good results in the secondary treatment of effluents enhancing the action capacities of the UV radiation [Equation (9) indicates that the hydroxyl radical can be produced in the presence of this type of radiation without a catalyst. Lamps of different radiation wavelengths, especially of ultraviolet nature , have been applied in the photocatalytic process . This diadiation .2 in different supports [2/UV-A/LED) for the degradation of acetaminophen [2 is applied.Reactors with lamps that emit UV-A, UV-B, and UV-C radiation have been applied for the degradation of different classes of pollutants in aqueous solutions by immobilization of TiOsupports ,39. The supports . Alternasupports ,42,43. Cminophen . Other rminophen . The aut2 and UV-C radiation. The removal of NPX by photolysis was 83% after 3 h, with an 11% reduction in chemical oxygen demand (COD), while the photocatalytic oxidation led to higher removal of NPX (98%) and COD (25%) after the same time.To reduce energy costs, the application of higher wavelength radiation may be a solution. However, for this, it is important to use a suitable semiconductor so that the production of reactive oxidative species may be possible . For exa2 oxidation is a promising process for treating CEC in water [2 and UV-C. The combination UV-C/TiO2/GAC was feasible and under optimal operational conditions, the furfural removal was 95% [3, H2O2) to investigate the degradation of sulfolane in water [\u25aa (and/or other reactive oxygen species). Regarding TiO2 activation, UV-B is more efficient than UV-A [2 as a photocatalyst. This partly reinforces the importance of doping it with appropriate materials to enhance its photoactivity at low-energy radiation sources [2 under UV-C. However, using UV-C radiation with bimetallic materials a remarkable increase in photodegradation of the antibiotic compared to pristine TiO2, which required 90 min for the complete degradation of ciprofloxacin [2 including radiation sources UV-A and UV-C range was also evaluated [2 was applied to substrates such as plastic Petri dishes and glass bottles for degradation of MB under the action of sunlight and UV-B [2 photocatalyst can also be applied for efficient inactivation of Escherichia coli [2 and UV-B light leads to the disruption of the outer membrane which causes the effective inactivation of E. coli bacteria.The results obtained from the kinetic analysis showed that UV-C/TiOin water . The exp was 95% . Other sin water . The authan UV-A ,49. Slow sources . For exa sources ) found gfloxacin . Other sfloxacin . The UV-floxacin . The degvaluated . The mosand UV-B . Under Uhia coli . In this2 catalysts for the abatement of CEC from water [It is important to note that some studies have demonstrated that photocatalytic processes are not characterized by a linear evolution of efficiency-irradiation. In view of this, to optimize the design of the processes it is important to adjust the flow of photons and sources of irradiation to the catalyst substrate and type of pollutant . Globallom water . Martinsom water  verified2 under visible light radiation depends on the electronic properties of TiO2  and the molecular structure of the organic compounds to be degraded [2 enhanced the photocatalytic oxidation of phenol compared to the bare TiO2 under visible light radiation [2 doped with rare metals can degraded phenol under a weak visible light radiation. Moreover, some noble metals, due to their plasmonic resonance, can also efficiently absorb visible light [2-Ni for the degradation of p-Arsanylic Acid  in aqueous solutions under visible radiation. The authors found that TiO2 -Ni showed higher photocatalytic activity than pure TiO2, promoting the degradation of 76% of p-ASA, while 60% of the degradation was achieved with non-doped TiO2. Some authors verified the successful deposition of Pd and Pt atoms on the surface of TiO2 that allowed the absorption of light in the visible region causing the efficient removal of sulfamethoxazole (SMX) in all tested conditions. The effect of different radiation sources for the CEC mixture degradation using noble metals doped onto TiO2 as a catalyst has also been addressed [Aiming industrial processes applications, the photocatalyst should be active under the visible light spectrum since this is the largest fraction of sunlight radiation ,58. The degraded ,60. To idegraded ,61. The adiation  proved tle light ,64,65. Vle light  using doddressed . Despiteddressed . In factCryptosporidium, which are resistant to disinfectants such as chlorine [2 performance under natural sunlight is very limited [2 surface modifications are required to increase its efficiency in the absorption of natural sunlight. In this ambit, catalyst surface doping can be an interesting option [Sunlight radiation is a clean and inexhaustible source of cheap energy that can be applied in the treatment of liquid effluents. In this view, the number of publications using solar radiation for photocatalytic detoxification and disinfection of water and wastewater is increasing ,69. As mchlorine . Althoug limited . Therefog option . Moreoveg option  avoiding2. Several photoreactor projects for solar energy absorption have been tested for water and wastewater treatment [To use sunlight radiation, a suitable reactor with a light collection system is often required. Abdel-Maksoud et al.  emphasizreatment ,74.2 with activated carbon (AC). As a result, more than 90% removal has occurred, and the MO degradation with TiO2/AC using visible or sunlight was similar [Degradation efficiency is a powerful indicator to compare the performance of photocatalytic reactors of different types and geometries . Some st similar . Non-con similar . Composi similar ,77. Alth similar ,79. Seve similar .Regarding the sunlight radiation, it is important to consider the time of the year and the place of the globe where the experiments were performed since the radiation flux and spectrum are different . Gomes eThe selection of a suitable catalyst is a key point while designing a photocatalytic system. In the following sections, the types of materials tested in literature are discussed and their advantages and shortcomings are overviewed. In the photocatalytic processes for the wastewater treatment application, catalysts can be used as dispersed powder and coated or immobilized onto different supports.2, ZnO, Fe2O3, WO3, CdS, ZrO2, MoS2, have been tested for photocatalytic applications [2O3 are semiconductors with unstable photocatalytic activity, in the whole pH range and undergo photocorrosion processes [2 are UV light absorbers, often considered the most efficient semiconductors for promoting photocatalysis in aqueous solution [Numerous materials, such as TiOications . CdS androcesses . ZnO andsolution ,83.2 is non-flammable, poorly soluble, thermally stable, and non-hazardous metallic oxide [2 (3.2 eV) [2 as photocatalyst seems to be the most suitable option for these processes comparing the chemical properties. Furthermore, TiO2 has been reported as one of the most used semiconductors in the degradation of CEC [2 under UV light was considered the most effective for the hydroxyl radicals generation and applicable in a wide range of experimental conditions [2 occur at the catalyst-substrate interface and thus it exhibits properties strongly dependent on the structure, surface, and morphology of nanocrystals [2 surfaces with metals and non-metals according to morphologies or scales allows an improvement in the adsorption capacity of contaminants, which is useful for water treatment [2 with a higher specific surface area led to better results than the use of larger quantities of semiconductors. Pereira et al. [\u22121 of the antibiotic Oxytetratracycline with TiO2 charges ranging from 0.1 to 0.5 g L\u22121 using simulated solar radiation, by an experiment carried out in a pilot plant equipped with CPC. The results proved that 0.5 g L\u22121 of TiO2 at pH 4.4 was the best combination to achieve 100% removal after 40 min of irradiation under 7.5 kJ L\u22121 UV dose. The photocatalytic degradation of the herbicide Metolachlor with TiO2 powder was studied and a degradation rate of 88% was achieved, despite the high toxicity of the byproducts generated [2 (anatase) is attributed to differences in the Fermi level and the superficial hydroxylation extensions of the solid [TiOic oxide . ZnO is (3.2 eV) ,86. Howe(3.2 eV) . In thisn of CEC ,89. In fnditions ,91. The crystals . A previreatment . These aa et al.  evaluateenerated . Indeed,he solid .2, involves the mass transfer of pollutants from the liquid phase to the surface of the catalyst; Adsorption of reactants onto the surface (chemisorption or physisorption); Reactions at the adsorbed phase; Desorption of the product/intermediates from the surface of the catalyst and removal of the product from the interface to the solution [2 powder are related to the possibility of sedimentation and the agglomerations formed in slurry reaction systems [\u22121 of TiO2 (P-25) under sunlight showed significant action in the inactivation of E. coli in water [It must be noted that the degradation of contaminants in heterogeneous processes with TiOsolution . Thus, t systems ,98 whichin water .2 of 2.5 g L\u22121 was found, while for the solar reactor CPC, which is a flow recirculation reactor corresponding to a batch reactor, only 0.2 g L\u22121 was considered as the optimal concentration [2 powder are related to the inefficient exploitation of visible light, the low adsorption capacity of hydrophobic contaminants, and difficulties of separating the catalyst after reaction [Regarding practical applications, the optimal load of catalyst must be chosen to avoid the excess of catalyst and guarantee full absorption of efficient photons. As an exampled, in laboratory experiments using a batch photoreactor, an optimal concentration of TiOntration . The maireaction ,102.2, P-25) has several drawbacks [2 in the removal of CEC, namely ceramic materials, activated carbon, glass, composites, stainless steel, zeolites, among others [2) were successfully immobilized in a composite material made of cement, clay, and wood fibers and demonstrated efficiency in the photocatalytic degradation of phenol [As aforementioned, the utilization of powders as catalysts  strong adhesion between catalyst and support; (ii) no degradation of catalyst reactivity by the bonding process; (iii) offer a high specific surface area; (iv) have a strong affinity for adsorption with pollutants ,107,108.2 exposed to visible light is not very efficient, doping it with other substances may improve catalytic capacity [2.Supported catalysts have gained increasing relevance for the degradation of pollutants ,109. Morcapacity ,111. HowIt is noted that the global demand for ceramic materials with comprehensive applications in the environment and other scientific areas is increasing ,107.2 supports as well as the incorporation methods.In the further sections, a literature overview will be made regarding the most common ceramic materials used as TiO2 both in the degradation of air pollutants as well as in the removal of contaminants in aqueous solutions [2O3, TiO2, SiO2, and ZrO2 that demonstrate good performance in water treatment, turbidity removal, desalination, wastewater treatment, and other important industrial applications, apart from other advantages inherent in energy saving [Ceramic materials have proven to be excellent supports for TiOolutions ,113. Indolutions ,114. Theolutions . Regardiy saving ,116. Howy saving .2 supports [2 photocatalytic activity. Thus, 2:1 clays (bentonite and kunipia-F) were considered better carriers of TiO2 than 1:1 clay (kaolinite) for photocatalytic degradation of MB and Chlorobenzene [2 composite on a photochemically stable clay for degrading herbicides  and ammonia in aqueous solutions were significantly high, with removals ranging from 61 to 85% [2 onto clay beads to promote degradation through UV light and solar radiation.In this literature overview, the focus will be given to the traditional ceramics, since these materials present potential to be applied for the circular economy purpose. For example, clays are ubiquitous constituents of the Earth\u2019s crust that serve as raw materials for traditional ceramics . Clay misupports . Clay teobenzene . Experim1 to 85% ,120. Mor1 to 85%  supporte2 supported in Leca can be an interesting solution for photocatalytic oxidation experiments. Zendehzaban et al., [2 and TiO2/(Leca + UV) photocatalysts and concluded that TiO2/(Leca + UV) composite improved its performance by up to 15%. Furthermore, there is no need to use a separation technology to recover the TiO2 from the liquid media [2 supported in Leca (Cu/TiO2/Leca) activity for the phenol degradation under UV radiation. The CuO load enhances the incorporation of the dopant in the TiO2 mesh which increases the active sites available. In addition, the porosity of the Leca particles allows Cu/TiO2/Leca to be exposed to UV light in a better condition due to the higher surface area.It should be emphasized that clay aggregates are of great interest due to the versatility of applications. In this context, the use of light expanded clay aggregate (Leca) arises interest due to its specific properties inherent to mechanical resistance, low density, and high porosity which means a higher surface area. Leca can enhance the mass transfer and allows proper contact between catalyst, light irradiation, and pollutant in an aqueous solution . For the et al.,  comparinid media . Sohrabiid media  analyzed2 support for photocatalytic degradation of organic pollutants [2 nanoparticles on clay mineral surfaces improves the photocatalytic activity of TiO2 due to high specific surfaces, high adsorption capacity, large pore volumes, chemical stability, and good mechanical properties [2 coated porous substrates  is a medium that effectively prevents bacterial adhesion [2 solution for immobilization leads to a positive effect on the photoinduced surface hydrophilicity of the TiO2 films under UV light [2/fumed silica ceramic material using a 4% phosphoric acid binder was applied. The authors found that the transformation of TiO2 from anatase to rutile started at 725 \u00b0C. Moreover, the substrate exhibited good photocatalytic activity in the complete degradation of a 10 mg L\u22121 methyl orange solution using a radiation flux of 15 W.m\u22122 with ultraviolet light irradiation over 24 h [2 support in the degradation of 5 mg L\u22121 of a Malachite Green solution, providing 86.2% removal after 6 h of ultraviolet irradiation [2 nanoparticles on clay mineral surfaces improves the photocatalytic activity of TiO2, providing more active surface sites and reducing the agglomeration of TiO2 particles in the reaction media. Carneiro et al. [2 nanoparticle layers and found that composites can act both to reduce air pollution and to decompose an aqueous MB solution. The efficiency was related to the porosity and roughness of the material. Recent studies on photocatalytic degradation of organic dye through the TiO2 layer deposited on single-flow ceramic membranes have revealed their photocatalytic efficiency. Convective flow through the pores of the TiO2-coated membrane has improved the mass transfer and, consequently, the photodegradation. The removal of the MB increased by up to 50% with an increase in the thickness of the coating (up to 6 nm) and the intensity of ultraviolet light . This shows that a continuous photocatalysis process associated with membrane filtration has a significant potential for the treatment of polluted water [2-coated alveolar clay foam as a photocatalyst achieving maximum removal of cumene hydroperoxide (CHP) in water using UV-A as a light source. The recyclability has been proven with 94\u201396% of CHP removal during four runs.According to this, the porosity and roughness of support material can be relevant issues to establish good support for photocatalytic activity. Numerous works highlight the properties of ceramic materials as TiOllutants ,119. Typoperties . It shouadhesion . MeanwhiUV light . A new pver 24 h . Porous adiation . For exaadiation  highligho et al.  developeed water ,126. Mored water  applied 2 on clinoptilolite to diclofenac degradation using solar radiation and evaluated the effect of several operating parameters on the oxidation efficiency. Miranda-G\u00e1rcia et al. [2 in glass spheres for degradation of 15 CEC at low concentrations in simulated and real municipal wastewater. The experiments were carried out in a CPC reactor of the solar plant of the Almeria Solar Platform (Spain). The main results indicated 85% of the compounds were degraded within 120 min of lighting time, showing the potential of this technology as a good alternative to suspension systems for the treatment of polluted water.Alternative support materials can be used such as zeolites, glass spheres, and cement-based materials. Casta\u00f1eda-Ju\u00e1rez et al.  supportea et al.  reported2 due to the reduction of specific surface area, low diffusion, and light transmission performance [2) in cementitious materials.Cement materials have limitations in incorporating TiOformance ,130. Zhaformance  synthesi2 is crucial for long-term application to avoid catalyst loss and water secondary contamination with TiO2 nanoparticles [Besides glass beads, other materials such as silica beads have been referred to as excellent supports to the degradation of up to 100% organic compounds in liquid effluents ,127,131.articles .2 nanocomposites immobilized in ceramic materials, namely sol-gel, dip-coating, impregnation method, chemical vapor deposition (CVD), electrophoretic deposition (EPD), and electrochemical treatment [Several methods have been developed for the preparation of TiOreatment ,127,131.This review will focus only on the most used methods for embedding ceramic substrates, namely the ones indicated in 4), inorganic salts, are the precursors generally used for the synthesis of TiO2 nanocomposites using the sol-gel method [The sol-gel method is a process for synthesizing materials from a liquid solution of organometallic precursors . Titaniul method ,107. Amol method ,133. Bell method  comparedl method . Neverthl method ,107.2 powder and does not need precursors like the sol-gel method. This process is very similar to the impregnation method the difference consists in the form of contact between substrate and dispersion solution. The form of the produced substrate by this method is generally based on the use of suspensions, aqueous or alcoholic of TiO2 particles. The material adsorbed on the substrate undergoes a heat treatment to enable the particles to be calcined. Shan et al. [The dip-coating method allows the direct immobilization of the TiOn et al.  mentione2 over several supports [2, which after filtration and drying at 100 \u00b0C must be calcined at 550 \u00b0C for 30 min [2 adsorbed. Alternatively, in the impregnation method, the solvent evaporation stage can be made with microwave assistance, since can result in a more efficient junction between TiO2 and support [The impregnation is one of the most used methodologies for the immobilization of TiOsupports ,135. Typr 30 min ,135. In  support .4) is hydrolyzed by reaction with water vapor at the surface of a substrate to form a film of a titanium oxide precursor. The substrate is then calcined to convert the titanium oxide precursor into titanium oxide. TiCl4 used as a reactant may be replaced by another hydrolyzable titanium compound such as a titanium alkoxide, but from a commercial standpoint, the use of TiCl4 is advantageous since it is less expensive and has a lower boiling point [The chemical vapor deposition (CVD) method is based on the deposition of thin solid oxide films on a heated substrate using vapor phase mixtures of metal-containing precursors  and an oxidant . CVD mayng point . A steamng point  CVD synt2 powder in the form of a film on a metallic substrate has been referenced and with results that demonstrate adhesion of TiO2 in stainless steel, and when used in photocatalytic tests of water purification, no titanium release has been observed [The method of electrophoretic deposition of TiOobserved ,140. Theobserved ,139,140.2 is dispersed in an electrochemical cell that contains the support, solvent, and sacrificial organic substance. This cell is placed in an electrolyte solution as well as the other electrochemical cell containing the sacrificial electrodes [2 doped with Cu, Fe, and Fe/Cu, with relevant efficiency on the diclofenac degradation through photocatalysis. In this work, the sacrificial organic and solvents were water and ethanol, and the sacrificial electrodes were Fe, Cu, or both.The electrochemical method requires an electrolyte solution to promote electroconductivity between the sacrificial organic and sacrificial electrodes. In this method, TiOectrodes . The appectrodes . Casta\u00f1eectrodes  used an 2 onto the support [2 onto Laponite for the photocatalytic oxidation of herbicides. According to the results, the high temperature of hydrothermal treatment promotes the better photoactivity of supported TiO2 [2 anatase immobilization can promote the appearance of the rutile phase or increase their concentration with the duration of hydrothermal treatment [The hydrothermal method consists on the application of pressure and temperature to promote the adhesion of TiO support ,135. Pau support  analyzedted TiO2 . This mereatment  but rece2 onto the support surface.Regardless of the immobilization technology selected, almost all of them comprise the calcination step as a final stage. This step will promote the immobilization of TiO2 [2 in ceramics by different methods has been considered a sustainable alternative in wastewater treatment [Thus, each immobilization technique has advantages and disadvantages, while the preparation procedures and the substrates used directly influence the photocatalytic activity of TiO2 . In thisreatment ,135,141.2 within the support. In fact, the dispersion of TiO2 onto the support is important for the effectiveness of the catalyst in photocatalytic oxidation since the TiO2 can agglomerate, which can lead to an efficiency reduction [2 coating quality control and reduces the TiO2 crystallite size to 18 nm [After a photocatalyst immobilization onto the support, several characterization techniques can be used to analyze the morphology and the homogeneity in the distribution of TiOeduction . In partto 18 nm . The mos2 onto the support. Moreover, Zendehzaban et al. [2 immobilization and concluded that the high porosity of Leca does not suffer significant modification after immobilization. The XRD can be useful to characterize the crystalline structure of materials as well as TiO2 polymorphs supported in ceramic materials [2 supported in structural ceramics and observed that the most photoactive phase (anatase) changed to the less photoactive phase (rutile) with the increase in sintering temperature. However, concluded that red ceramics tend to inhibit this transformation, concluding that at 700 \u00b0C the anatase phase is the major crystalline phase (about 50%). Zendehzaban et al. [2 immobilization onto Leca does not promote considerable changes in the typical XRD patterns of TiO2 alone.The SEM technique makes it possible to analyze the morphology of the composite surface allowing the visualization of the dispersion of TiOn et al.  analyzedaterials ,119. Salaterials  evaluaten et al.  verifiedThe FTIR allows assessing information about the structure of compounds, showing the degree of heterogeneous bonding within the particles ,148,149.2 phases.In addition, transmission electron microscopy (TEM) is a powerful technique to obtain very detailed micrographs . For exa2 instead of powders aims to facilitate the catalyst reuse and recovery. The efforts to improve efficiency in the application of this technology has been stressed by Yang et al. [(i)increasing light-harvesting capacity via defect engineering.(ii)enhancing charge separation via interface engineering.(iii)accelerating surface reaction.The application of supported TiOg et al.  regardin2 photocatalysts for pollutants removal from water is overviewed.In the following sections, the application of supported TiOThe efficiency of the treatment process is in general analyzed based on the removal capacity of the target contaminants . Meanwhi2 supported on expanded perlite, show that pH did not influence the adsorption tendency of SMX. On the contrary, the highest photodegradation occurs in the less adsorbed anionic form. This suggests that the pH-induced changes in reactivity may be related to the changes in the SMX molecule and not in the surface of TiO2 [2 in glass plates together with UV radiation has been effective in removing ciprofloxacin (CIP) in aqueous solutions. The removal efficiency of CIP in synthetic and real effluent was 92.8% and 86.6%, respectively. In addition, the removal of the antibiotic had a direct relation with contact time and an inverse relation to the initial concentration of CIP and pH (3 and 5). Concerning adsorption contribution, [The studies involving the degradation process of sulfamethoxazole (SMX) with TiO of TiO2 . Other s of TiO2  demonstribution,  showed t2 in ceramic substrates has been quantified with respect to several contaminants, and 2 in ceramic substrates has been efficient and, in some cases, superior to powder P-25 [The photocatalytic activity of TiOder P-25 ,143.2 as important parameters for catalytic activity and adsorption of pollutants to the active sites of the catalyst. Indeed, the performance of heterogeneous photocatalysis involves among other parameters the size of the anatase crystals, porosity, and pH [2 surface can be protonated or deprotonated [The mesoporous structure of clay mineral particles allows exposure to light of the crystalline anatase particles and the diffusion of organic molecules to their surfaces ,120. Som, and pH ,155. Hig, and pH ,156 becaotonated .2 supported on ceramic materials under UV or visible light decreases gradually over time according to the Langmuir-Hinshelwood model, according to Equation (10) [Rr is the degradation rate, RC is the concentration, t is the reaction time, Kr and K are reaction and adsorption constants, respectively.The photocatalytic decomposition rate of CEC in liquid effluents using TiOion (10) ,103.. The results showed that with TiO2 suspension 97% of HBsAg (pH 7.2) was destroyed, while with the ceramic plate as support about 94% of disinfection capacity was observed. In both cases, the destruction occurred under UV irradiation (365 nm) for 4 h. Probably suspensions of TiO2 have a higher impact on disinfection in comparison to immobilized catalysts [2 can generate a scavenger effect, which in turn leads to a reduction in the efficiency of the catalyst in inactivating bacteria.Despite few studies involving TiOion rate ,167. Speion rate  comparedatalysts ,169. Howatalysts  concludeThe performance of effluent treatment technologies should also be assessed in terms of their impact on water toxicity ,171.In f2 immobilized in glass beads. The consensus method was used to evaluate the CIP toxicological data, including LC50 . The results showed that most of the 17 degradation products detected had lower toxicity than CIP, but some of the degradation products were more toxic.Xing et al.  evaluate2 mixture namely PC-500 in ceramic plates by the sol-gel method. The results of the TOC  and ecotoxicological experiments with Spirodela polyrrhiza showed that UV/TiO2 could mineralize and effectively reduce the ecotoxicity of pharmaceutical products. Similar results were obtained by Alfred et al. [2 on natural kaolinite clay, Na2WO4, and biomass. The photodegradation of these compounds was higher than 90% under sunlight. In addition, the concentrations of by-products of the mineralization process after photo-degradation are safely below WHO standard limits for drinking water.Other studies  evaluated et al.  on the dThe use of organisms for testing , among others, can serve as a model indicator of the total toxic effect ,172. In 2 supported by ceramic materials. Although heterogeneous photocatalysis has been studied for the removal of pollutants in aqueous matrices for two decades, its use in wastewater treatment is still at a stage of technological research [2O2 and aided by TiO2 catalysts have promoted the reduction of cytotoxicity and mutagenicity within the permitted limits, ensuring that treated wastewater could eventually be suitable for industrial reuse, irrigation, and aquaculture [2 in the treatment of textile wastewater, revealed the potential to remove color and other physical and biological properties that guarantee the quality for its re-use for agricultural and domestic purposes [\u22121 of TiO2 in a CPC-type pilot solar plant to treat effluent from a municipal wastewater treatment plant contaminated with 22 pharmaceutical compounds at moderate concentrations  good efficiencies were found [V. fischeri bioluminescence after 15 and 30 min. The results showed that the inhibition percentages were already low (<15%) from the beginning of the experiment, thus indicating that the effluent was not significantly toxic. Also, the efficiency of a CPC reactor [2 coated on glass beads for the treatment of wastewater with pesticide mixtures , 100 \u03bcg L\u22121 each was evaluated. It was found that the degradation rate of Imazalil during the photocatalytic experiments was very high and was not affected by the presence of the other contaminants. Preliminary results show that the composite in question proved to be photocatalytically active and mechanically stable during the experiments. Therefore, the optimization and application for tertiary effluent treatment in citrus processing industries are recommended. Moreover, Sousa et al. [2 assisted photolytic and photocatalytic oxidation of the anxiolytic drug Lorazepam under artificial UV light and natural sunlight. They compared the photolytic and photocatalytic degradation kinetics of Lorazepam using two experimental systems: a laboratory-scale photochemical reactor supplied with a medium pressure UV mercury lamp (LsAUVP) and a solar pilot plant with compound parabolic collectors (SPP-CPC). Lorazepam was tested in its most marketed dosage form in Portugal, 1 mg (Wyeth). Preliminary results showed that using the LsAUVP apparatus, the highest degradation performance of Lorazepam was obtained by photolysis, while using the SPP-CPC system, the best degradation performance was obtained by photocatalysis with 200 mg L\u22121 of TiO2. Other efficient wastewater treatment alternatives involve combining two or more physico-chemical processes to maximize the removal of recalcitrant organic compounds [To the best of our knowledge, the literature does not contain specific information on the reuse of treated wastewater by TiOresearch . Howeveraculture ,178. Forpurposes . Water rpurposes . In thisre found . Moreove reactor  with TiOa et al.  studied ompounds .2 supported by ceramic materials can be a future contribution to conserving valuable water sources and thus mitigate the effects of climate change.The great interest in researching approaches to process optimization and integration to solve ecological risks and enhance removal efficiency should be highlighted. Thus, the recovery and reuse of wastewater by AOP in particular with TiOThe reuse of recovered water in practice is lagging behind the political ambition. Concern about its impact on human and environmental health has been identified as a key barrier to its full recovery ,185. AmoAlthough effluents in some cases have presented toxic intermediates, numerous publications show compatible levels of toxicity for the discharge of treated effluents as well as their reuse ,183.\u22121, 50 mg L\u22121, and 25 mg L\u22121 of TiO2 suspensions in wastewater treatment. The adverse effects of treated wastewater on maize growth attributes were significantly improved with a load of 25 mg L\u22121 (p < 0.05), whereas 100 mg L\u22121 load of TiO2 significantly inhibited seed germination, seedling growth and caused the accumulation of phenolic in maize plants (p < 0.05). In other studies [\u22121 TiO2 under UV-A, in acid conditions (pH = 3) completely discoloring textile effluents was observed and a reduction of the chemical oxygen demand (COD) between 40% and 90% after 4 h treatment. A comparison of the photocatalyst (TiO2) synthesized by the sol-gel technique and coated on different substrates  was carried out [2 coated glass under UV-C irradiation. Some studies have demonstrated good results regarding adhesion and duration of TiO2 coating on ceramic materials in several test cycles, thus with positive perspectives to implement this approach on a larger scale for wastewater treatments containing CEC [Yaqoob et al.  evaluate studies  by usingried out . The coaning CEC . In this2 as a slurry catalyst for the degradation of CEC in liquid effluents has some disadvantages due to the tendency of sedimentation with a decrease in the amount of light absorbed by the catalyst, and difficulties in avoiding the loss of catalyst that requires sophisticated and expensive methods of separation. In view of this, ceramic materials reveal excellent characteristics and properties as substrates for the incorporation of TiO2, exhibiting high levels of removal both in the degradation of air pollutants and contaminants in aqueous solutions. This is associated in part with the efficiency of light absorption as well as the porosity of ceramic substrates which effectively influence the intrinsic properties of TiO2, inhibiting energy dissipation and increasing the active sites of the catalyst. Several incorporation methods improve TiO2 adhesion to the substrate providing composite stability (TiO2 + substrate).The application of TiOIn this perspective, in the future, this treatment technique may become competitive to boost the reuse of water for different purposes due to its low cost and contribute to the conservation of valuable water sources and mitigate the effects of climate change. In this literature overview, it was possible to conclude that there is a knowledge gap related to the associated costs to immobilize nanoparticles onto ceramic materials. In fact, this can be one of the factors that need to be considered in such an approach. It is important to highlight that the cost associated with this technology can be decreased with the number of cycles of reuse without loose relevant efficiency. If the supported catalyst can be reused many times, the immobilization costs can be insignificant comparing with the operational costs powder nanoparticles catalyst recovering from the reaction medium. Moreover, the usage of supported ceramic materials over different cycles is relevant from the circular economy perspective due to the reduction of waste that will be produced in this case.In relation to the recognized potential risks associated with the presence of emerging contaminants in the environment, assessments of the ecotoxicity of treated effluents serve as an indicator for monitoring the quality of wastewater for re-use purposes. Several aspects must be solved in the application of the technology with emphasis on assessments and reduction of ecotoxicity of treated effluents, energy dissipation, reactor configuration, increase of the surface area of the catalyst.The appropriate load of the catalyst that prevents its agglomeration, concentration of contaminants, and pH should be optimized on a laboratory scale to increase the photocatalytic process on an industrial scale. Overall, it is noticeable that from an energy cost point of view, the technology presents advantages for the equatorial regions due to the higher incidence of sunlight per year.2 through the sunlight radiation. Regarding this, the main challenge is to find a suitable ceramic substrate, since this kind of material can have reliable properties as support, even from a circular economy perspective. Indeed, ceramic materials can be considered for other applications at the end of their life cycle. Moreover, the reduction of the bandgap to make TiO2 more active under visible sunlight radiation is another relevant feature for the photocatalytic oxidation performance. This reduction of bandgap can be achieved with metallic or non-metallic species. In this way, one of the best solutions for wastewater treatment is the immobilization of doped TiO2 onto suitable ceramic support to be used in reactors with higher solar exposure areas. This solution may have a higher lifetime without a significant loss of efficiency.In fact, future industrial wastewater treatment likely encompasses the usage of immobilized TiO"}
+{"text": "RiskProfile, leverages genetic and environmental information to communicate one\u2019s risk for smoking-related diseases. Although prior studies have examined attitudes toward genetic results, little research has investigated these perceptions through a lens of in-vivo testing; that is, user-centered design feedback in response to personalized genetic results being returned contemporaneously. This qualitative study engaged current smokers in usability testing of the RiskProfile within the context of concurrently receiving this personalized, genetically-informed smoking cessation intervention.The use of genetically-informed personalized risk information for behavioral disorders, namely smoking and smoking-related behaviors, is a promising yet understudied area. The Genetics and Smoking Risk Profile, or RiskProfile intervention that they had received moments before. Data were analyzed via the conventional content analysis approach in which themes were allowed to emerge throughout the analysis.Eighty-nine participants who were current smokers responded to open-ended interview questions on perceptions of smoking-related genetic information and the content and format of the RiskProfile. Overall, current smokers perceived the RiskProfile to have high potential utility. Constructive feedback that current smokers offered about the tool centered around suggested improvements to optimize its usability and technical content.Participants were able to reference and offer design input on specific elements of the RiskProfile among current smokers and can play an important role in optimizing the design and implementation of personalized genetic risk interventions moving forward.The detailed and constructive feedback from participants highlights that in-vivo feedback offers a useful design approach that addresses concerns of rigor and relevance when returning genetic results. This unique method demonstrated perceived utility and constructive design feedback for the The online version contains supplementary material available at 10.1186/s12920-021-00976-1. Despite numerous strides in the precision medicine initiative , there rThere is noted public interest in genetic testing for psychiatric disorders, including disorders of addiction. That interest varies based on context  and perceived potential impact and value of the genetic results , 16\u201318. To facilitate optimal implementation of genomics in behavioral health, a working group was recently established to continue bridging this gap between genetic information and behavioral health , 40. TheA key scientific gap is on perspectives from current smokers in the context of receiving personalized genetic information. Methods from the fields of implementation science and design thinking can aid in establishing the consistent use of genetic information for behavioral disorders in real-world, contextually-relevant scenarios by uncovering innovation-, individual-, organization-, and system-level barriers to integration and use of this evidence , 49. An RiskProfile. The RiskProfile was previously developed and initially validated \u201d while another simply wanted to know if there was \u201ca genetic variant that caused you to be more addicted to the tobacco or \u2026 made it harder to quit.\u201dInterestingly, even participants who were not well-versed in the genetics of smoking were often familiar with the influential role that genetics plays in smoking and related behaviors. For example, one participant noted \u201cI wanted to [\u2026] just see more about how the addictive gene affects our family and me personally.\u201d Others discussed genetics in a deterministic manner, implying that they either thought or hoped that it explained their smoking behaviors more than their environment . As one participant stated, \u201cSo I guess I was interested in the genetic aspect of it to see if there were factors working against me that I may not have a lot of control over.\u201dThere was a subset of participants who participated in the study to gain very specific types of information about their health. For example, some participants were interested in their overall risks for disease and smoking-related illnesses and how this could impact their health. Some participants even hoped for a formal diagnosis or prognosis from this study. Others wanted information to support their efforts to quit smoking . As one participant noted, \u201cI wanted to learn how hard it would be to quit and what would be the best way to quit.\u201dRiskProfile\u2019s utility\u2014how it can inform and educate or potentially motivate behavior change.Participants focused on two key themes of the RiskProfile can also help inform individuals about why they smoke and/or struggle with craving nicotine. Although most participants were already aware of the harms of smoking, several were of the mindset that more information and education, particularly when presented in a unique and more personalized way, was useful. One participant stated, \u201cI think it\u2019s just one more bullet in the chamber, one more weapon in your belt, however you want to put it.\u201dParticipants commented on how useful it is for individuals who smoke to know their genetic risk because that can inform future health and behavior decisions. As one participant said, \u201cI wasn\u2019t sure how high of a risk I was [for smoking-related outcomes], but to see I was in the higher risk category\u2014that was pretty eye opening. And if I ever have children too, it would be good to have some kind of knowledge to help make an environment that they wouldn\u2019t want to smoke [in].\u201d The RiskProfile can help participants focus on their health and the long-term goal of quitting because of its motivational impact. For example, one participant stated that the tool was \u201cJust more motivation. It tells you how critical it is to [\u2026] attempt to quit.\u201d As one participant noted, \u201cI think [\u2026] it will help [others] change their lifestyle and I would like to think that it\u2019s going to help me change my lifestyle.\u201dSeveral participants commented on the various ways that the RiskProfile. These included having no concerns, concerns about under-utilizing the tool, and privacy concerns.Participants expressed a range of potential concerns about using the It is worth noting that the majority of participants did not have any concerns about the genetic smoking risk tool. Participants generally remarked on how they did not find anything concerning with the smoking risk profile tool and mentioned that it could be helpful to use. For example, one participant commented \u201cNo, no concerns. I love the idea.\u201dRiskProfile being under-utilized by themselves and others moving forward. Some of these concerns involved personal agency following receipt of the tool, for example, including acknowledgment of now facing the difficulty of cigarette reduction or cessation and fear of such efforts failing. A couple of participants also noted the concern that the tool might not be used after the visit, possibly because it is simply not useful enough for individuals who already know the risks of smoking. For example, one participated noted, \u201cI know the hazards [of smoking] and [\u2026] at a certain point [\u2026] we just don\u2019t care because a lot of [us] figure, \u2018I\u2019ve lived my time.\u2019\u201d Additional concerns were about potential unanticipated consequences of the RiskProfile and whether the tool could be enabling for individuals with a low genetic risk for smoking-related outcomes. As one participant said, \u201cI don\u2019t so much have any concerns besides [someone] being at genetically lower risk and thinking that they have a free pass to smoke now because they\u2019re at low risk.\u201dMany participants were concerned about the RiskProfile itself. These concerns were mitigated by participants\u2019 trust that the research team associated with this project were upholding the highest possible privacy standards, but such standards were not assumed to be upheld by third parties. Participants expressed broad concerns about the safety of their genetic information, third party tracking, distribution, and use of their genetic information .Many participants expressed concerns about the privacy of their information, but not about the information in the RiskProfile, including leaving it as-is, improving the tool\u2019s content, and improving the tool\u2019s impact.Participants provided a range of feedback about the RiskProfile, most had no suggestions and/or voiced approval. As one participant said, \u201cI think that it did what it was designed to do. I don\u2019t know how it could be improved.\u201dAlthough several participants noted areas of improvement for the RiskProfile. Although these comments were quite diverse, they did provide concrete suggestions to improve the tool in order to better motivate cessation attempts. Feedback on key areas of improvement included simplifying the layout, adding more information about the genetic information that went into making the profile risk score, and improving the language because the tool was not intuitive and required explanation. Some participants also suggested to include risk for family members. Finally, participants recommended periodic updates to the tool as new information  are made available.Some participants commented on the formatting, layout, information, and content of the RiskProfile more impactful and meaningful, thus potentially improving motivation to attempt smoking cessation. Some suggestions included expanding what went into the risk profile score, such as asking more information about behaviors, environment, markers, and family history. Other suggestions included ways to make the experience more personalized, such as offering information about personalized smoking cessation. Other suggestions of how to maximize the impact and meaning included increasing the perceived severity of disease risk such as more sensationally displaying the potential consequences of continued smoking  and arranging follow-up professional outreach with a healthcare professional. A few participants also discussed ways to improve the reach of the study, such as more advertisement and targeting younger adults. This feedback highlights the importance of reaching and engaging the right audience with highly personalized and actionable content, all of which is perceived to be essential to more widespread implementation of this tool.There were several responses that provided insight beyond the format or content of the tool itself. Many participants offered improvements that would help make the RiskProfile provided. Many noted that the tool was helpful and motivating and that the information presented was unavoidable and eye-opening. Additional positive feedback about the RiskProfile centered around the fact that the information was relevant and credible, the tool itself would be useful for young people , and the layout was appropriate. Notably, one participant specifically liked that even though it was a personalized tool, there was no identifying information on the tool itself. As they said, \u201cI mean you\u2019re handing me a paper that doesn\u2019t have my name on it so even if I dropped it, nobody\u2019s going to know it was me.\u201dThere was much generalized positive feedback offered by participants that cut across all of the other four domains. Participants seemed happy about the experience as a whole, from the in-person aspect of the study to the personally engaging information that the RiskProfile). As a whole, participants expressed favorable views of the RiskProfile, providing detailed information about specific components of the tool that would not have been available without the in-vivo design. Results across five inter-connected domains provide a richer understanding of current smokers\u2019 motivations to participate, the specific perceived benefits and potential utility of the RiskProfile, and the concerns and suggested opportunities to improve this tool. To date, few studies have studied how to translate responses to personalized genetic susceptibility information into evidence-based interventions designed to communicate disease risk and motivate behavior change [RiskProfile moving forward.The current study qualitatively analyzed in-vivo responses about a personalized, genetically-informed Genetics and Smoking Risk Profile  and engagement factors  , 62. To Challenges and Future Studies below). While these take-aways are specific to the RiskProfile, they offer insights for other teams who are currently working on returning genetically-informed information to participants. First, the overarching positive feedback demonstrated broad support among current smokers for the RiskProfile, a novel genetically- and environmentally-informed risk score that cannot be obtained through any direct to consumer or other third-party genetic testing services. However, the information provided by the RiskProfile may not be sufficiently motivating for all levels of smokers. Reflecting upon some participants\u2019 responses about prevention opportunities and \u201ctargeting youth,\u201d it is possible that this risk profile would have value for prevention programs or early smoking cessation efforts among new smokers. For the tool to be maximally beneficial among adult (and presumably longer-term) smokers, concerns raised in the current study need to be addressed. These include expanding the parameters of the risk profile , addressing privacy concerns, consulting with a smoking cessation expert and genetic counselor, and receiving feedback about participants\u2019 experiences with the RiskProfile after more time has passed and they can reflect more deeply about the tool.Due to the current study\u2019s design, there are three key take-aways that were only made apparent due to the in-vivo design of the study and which can immediately be applied to next steps in this research trajectory  and engagement-related  factors, that may uncover essential intervention targets for future research. Finally, as the focus of this study was on individuals in the community who smoke, it is unknown to what extent this type of tool would or should impact the smoking cessation care that healthcare professionals provide. It is plausible that patient-specific genetic and clinical risk information could facilitate patient-centered discussions and highlight the urgency and potential benefits of smoking cessation for healthcare professionals; however, to change their clinical practice, healthcare professionals must be convinced that this tool provides unique and useful insight. If found to be clinically useful, a future step would be to integrate the RiskProfile into community health agencies or clinical settings with healthcare practitioners delivering the tool to participants. Alternatively, an online version of the RiskProfile could be implemented in the future, such that individuals could access their personalized results and interpretation of those results, without the need to attend in-person counseling.There are three main layers of examination and validation that will drive future directions of this research. First, immediate improvements can be made to the tool based on the current in-vivo feedback, and acceptability and feasibility should be iteratively assessed via in-vivo feedback with qualitative and quantitative metrics in future versions of the This study is not without limitations. First, as a qualitative study, we prioritized saliency of expressed responses to construct thematic content rather than quantitative metrics that often provide more summative evaluations. Relatedly, although we encouraged honest critiques of the usability and usefulness of the tool, it is possible that participants were reluctant to share more harshly negative feedback. Second, the potential confounding effects of the study staff were not examined . Third, it should be noted that organizational and clinical guidelines do not yet support the return of genomic information to participants for clinical reasons. Therefore, the current study was not designed to inform clinical decisions and participants were encouraged to speak with their physician about smoking cessation efforts. For instance, given the large consumer interest in reduced risk nicotine options, such as nicotine vaping products, some participants may have desired clinical recommendations about these alternatives with respect to their specific genetically-informed risks. Although we were unable to include such recommendations in this study, examining the utility of lower risk nicotine products in relation to genetic risk, particularly among individuals with high genetic risk, is an important area of scientific inquiry moving forward. Finally, 23andMe uses genotyping technology that comprises mostly European populations. This is a limitation in the field of genetics for genotyping and sequencing and has been discussed elsewhere . Through detailed in-vivo, user-centered design feedback, we identified five inter-connected domains\u2014the motivations, perceived utility, potential concerns, suggestions for improvement, and positive aspects of the RiskProfile. The RiskProfile was largely well received by participating current smokers and, with modifications that align with end-user feedback described herein, may hold promise as a useful smoking cessation tool in the future. This study highlights the advantages of using an in-vivo design to optimize the design of genetically-informed intervention tools and to maximize the rigor and relevance of end-user feedback when returning genetic information to participants.The current study presents an in-vivo approach to assessing participants\u2019 attitudes and perceived utility about receiving personalized genetic information from a novel Genetics and Smoking Risk Profile ."}
+{"text": "Cutibacterium acnes\u00a0(C. acnes)\u00a0as a cause of bacterial pericarditis.\u00a0This case report highlights C. acnes as a prevalent cause of both pleural and pericardial infections. The diagnosis can be challenging, considering that this bacterium is difficult to isolate, slow growing, and causes indolent illness.\u00a0Prolonged incubation time may be required. In addition to the more traditional causes of bacterial pericarditis, namely Staphylococcus and Streptococcus species, C acnes appears to play an important role. It should not be considered a contaminant as it may require further investigation.Infectious pericarditis does not always present with all the classic findings. Some of the traditional signs of fever, pleuritic chest pain, and frictional rub may be missing. This presents a diagnostic challenge, thus clinical suspicion is important. The most common cause of infectious pericarditis is viral.\u00a0However, bacterial pericarditis may occur with severe complications such as constrictive pericarditis, pericardial effusion, cardiac tamponade, left ventricular pseudoaneurysm, and aortic mycotic aneurysm. The purpose of this presentation is to increase awareness of  Cutibacterium acnes\u00a0(C. acnes) in a 54-year-old female with a previous history of splenectomy due to Hodgkin\u2019s lymphoma. The goal of this\u00a0presentation is to increase awareness of this mycroorganism\u00a0as a cause of bacterial pericarditis.Infectious pericarditis presents a diagnostic challenge in an immunocompromised patient due to poor inflammatory response. In this scenario, fever, pleuritic chest pain, and frictional rub may be missing ,2. The mA 54-year-old female Caucasian patient with past medical history of paroxysmal atrial fibrillation, heart failure with preserved ejection fraction, Hodgkin\u2019s lymphoma in remission, rheumatoid arthritis, and splenectomy was admitted to our hospital with one week of malaise, fever dry cough and shortness of breath. She also noted abdominal bloating and bilateral leg swelling. She denied chest pain. There was no history of trauma or recent surgery. At the time, she was on digoxin, diltiazem, and furosemide. Her vaccines were up to date. She had no history of cigarrete smoking, alcohol drinking comsuption\u00a0or recreational drug use. She reported severe allergic reaction to penicillin. Physical examination on admission showed a frail middle-aged woman in no acute distress. Blood pressure 128/90 mmHg, pulse 130 irregular beats/min, respiratory rate 20 breaths/min, axillary temperature 97.6\u02da Fahrenheit and oxygen saturation of 96% on room air. Body mass index was 16 kg/m2. Cardiovascular exam was remarkable for normal S1 and S2 and irregular rhythm. No murmurs or gallops were appreciated. Jugular venous distension was noticed. Pulmonary auscultation was remarkable for decreased breath sounds at the lung\u00a0bases. The abdomen was mildly distended and there was hepatomegaly on palpation. Bilateral pitting edema 2+ was observed in the lower extremities. White blood count was 9500 cells/ul, hemoglobin 13,6 g/dl, blood urea nitrogen 22 mg/dl, creatinine 0,64 mg/dl, B-type natriuretic peptide 144 pg/ml, troponins negative and electrolytes within normal range.Electrocardiogram (EKG) revealed atrial fibrillation with rapid ventricular response and generalized low voltage QRS Figure . Chest XPatient underwent pericardial window and right chest tube placement. Pleural and pericardial fluid were sent for analysis. The results are summarized in the Table C. acnes.\u00a0Pericardial tissue histology showed pericarditis with fibrinous exudate and organization. Due to penicillin allergy, the patient was treated initially with ceftriaxone and transitioned to oral linezolid at discharge. After two weeks of linezolid, she developed vision loss. Due to concerns about drug-induced optic neuritis, linezolid was switched to doxycycline\u00a0for more two weeks. Her symptoms eventually resolved. New echocardiogram two weeks later showed reduction of pericardial and pleural effusion.\u00a0After seven\u00a0days of incubation, both pleural and pericardial fluid grew\u00a0Streptococcus pneumoniae\u00a0as the prevalent bacterial cause of infectious pericarditis prior to 1943 and\u00a0Staphylococcus aureus\u00a0as the most prevalent after 1943 [Cutibacterium acnes, formerly,\u00a0Propionibacterium acnes, appears to play an important role as a causative agent of pericarditis\u00a0[C. acnes,\u00a0Staphylococci spp, and\u00a0Streptococci\u00a0spp, with\u00a0C. acnes\u00a0being the most prevalent. In this study, some of the predisposing factors were immunosuppression, post-cardiac surgery, pneumonia, dental procedures, and connective tissue disease\u00a0[In an earlier review of the literature between 1889 and 1975, Klacsmann et al. found\u00a0Cutibacterium acnes is considered normal flora of the skin, oral cavity, large intestine, and conjunctiva. It is a slow-growing gram-positive anaerobe that can cause severe infections, including bacteremia [C. acnes\u00a0was unknown, but the lead hypothesis was that it may have occurred as a consequence of a prior thoracentesis or pleuropulmonary seeding from the lymph nodes in patients with an underlying disease [C.acnes pericarditis with calcifications mimicking a pericardial tumor [cteremia , subduracteremia , endocarcteremia ,13, proscteremia ,10. It hcteremia . In one  disease . Iseki eal tumor .C. acnes\u00a0as the cause of simultaneous infections of the pleural and pericardial space. Our patient did not have risk factors such as a recent dental procedure, prior cardiac surgery, or an implanted device. However, she had a history of splenectomy secondary to Hodgkin\u2019s lymphoma, heart failure, and rheumatoid arthritis. One hypothesis is that both fluids accumulated due to acute on chronic heart failure which then served as a medium for a\u00a0C. acnes. Another hypothesis is that\u00a0C. acnes\u00a0may have been the primary reason for the effusions.\u00a0To the best of our knowledge, this is the first case report of\u00a0Cutibacterium spp\u00a0are susceptible to penicillin, carbapenems, cephalosporins, and vancomycin. The duration of treatment depends on the source. They are inherently resistant to metronidazole [Most\u00a0nidazole . There inidazole .\u00a0Table 2C. acnes as a potential cause of both pleural and pericardial space infections. The diagnosis can be challenging, considering that this bacterium is difficult to isolate, slow growing, and causes indolent illness.\u00a0Prolonged incubation time may be required. In addition to the more traditional causes of bacterial pericarditis, namely Staphylococcus and Streptococcus species, C acnes appears to play an important role. It should not be considered the only contaminant and may require further investigation.This case report highlights"}
+{"text": "Pancreatic Ductal Adenocarcinoma (PDAC) constitutes a leading cause of cancer death globally. Its mortality remains unaltered despite the considerable scientific progress made in the fields of diagnostics and treatment. Exosomes comprise of small extracellular vesicles secreted by nearly all cells; their cargo contains a vast array of biomolecules, such as proteins and microRNAs. It is currently established that their role as messengers is central to a plethora of both physiologic and pathologic processes. Accumulating data have shed light on their contributions to carcinogenesis, metastasis, and immunological response. Meanwhile, the advancement of personalized targeted therapies into everyday clinical practice necessitates the development of cost-efficient treatment approaches. The role of exosomes is currently being extensively investigated towards this direction. This review aims to summarize the current pre-clinical and clinical evidence regarding the effects of exosomal applications in the timely diagnosis, prognosis, and therapeutic management of pancreatic cancer. Pancreatic cancer has long been a major public health issue and statistical figures highlight the ever-growing severity of the problem. According to GLOBOCAN 2020, 495,773 new cases and 466,003 deaths were attributed to pancreatic cancer worldwide in the year 2020 . CurrentRegardless of histology, our understanding concerning the etiology of this disease is fundamentally lacking. As is the case with many malignancies, \u201cmodus vivendi\u201d is a major determinant of PDAC risk. Smoking, excess body weight, and diabetes mellitus are all recognized risk factors ,13,14,15Undoubtedly, the pathogenesis of PDAC remains elusive. However, a more pressing matter and a source of great concern is its insidious nature and the lack of effective treatment options. At diagnosis, PDAC is usually at least 2\u20134 cm in diameter and has in many occasions already infiltrated the surrounding structures and lymph nodes . UnfortuExtracellular vesicles (EVs) are membrane-bound lipid particles secreted by all cells and function as a means of communication between the parent and the recipient cell through their biological cargo . With regard to their classification, the International Society of Extracellular Vesicles recommends the division of EVs based on their size: medium/large EV (>150 nm) and small EV (<150 nm) . NonetheSmall EVs or Exosomes: Exosomes are nanoparticles with a diameter measurement in the range of 30\u2013150 nm. The first step in the biogenesis of exosomes is the inward budding of the extracellular membrane, which forms the endosome. The endosome undergoes inward invaginations and thus encloses biological cargo, which produces the intraluminal vesicles. The total number of intraluminal vesicles and the endosome in which they are contained comprise a multivesicular body (MVB). Once formed, these MVBs can either fuse with lysosomes and have their content degraded or release their content to the extracellular matrix through the endosomal sorting complex required for the transport- (ESCRT)-dependent or the ESCRT-independent pathway. Among exosomes, there are several common features such as the tetraspanin family of surface proteins , integrins, heat shock proteins, ESCRT, actin and flotillins, while other molecules are specific to the donor cell, such as type MHC Class I and II. The end result is the activation of a variety of signaling cascades, which are determined by the interconnection between exosome surface proteins and receptors on the recipient cells [nt cells ,40.Microvesicles: The sizes of these vesicles range from 50 nm to 1\u03bcm and are generated by the outward budding of the cell membrane [membrane . Their cmembrane ,43.Apoptotic Bodies: These vesicles comprise large fragments of a cell which has undergone apoptosis. They are the largest subset of the EVs with a size of up to 2 \u03bcm and therefore provide a substantial molecular pool for recipient cells [nt cells . Apoptotnt cells . Similarnt cells .Classically, exosomes were considered as a means for the disposal of nonessential cellular cargo. The first documentation of the term \u201cexosome\u201d in the literature was in a publication delineating the maturation of reticulocytes . The devSimilarly, the participation of exosomes in cancer pathophysiology with applications in diagnosis, prognosis, and treatment has been extensively studied ,58,59.Focusing on PDAC, exosomes have the potential to help determine treatment decisions. One such application is the perspective given by liquid biopsy, as was hinted by Buscail et al. who demonstrated that the level Glypican-1 positive exosomes circulating tumor cells and CA19-9 are able to be used for diagnostic and prognostic purposes in patients with resectable tumors ,61. MoreExosomes possess abilities and characteristics that render them a highly promising drug delivery system. However, the utilization of natural exosomes comes across a variety of technical issues such as difficulties in large-scale production, isolation, drug conjugation, stability, and quality control. To compensate for these deficiencies, a considerable effort is being made that aims to develop artificial exosomes by means of nanobiotechnology. With respect to the production of these vesicles, many different approaches have been suggested, all of which can be summarized into three distinct filter categories, namely, \u201ctop-down\u201d, \u201cbottom-up,\u201d and \u201cbiohybrid\u201d technologies .The \u201ctop-down\u201d strategy aims at the creation of nanovesicles through cell manipulation. This can be achieved with multiple techniques such as forced passage through porous membranes or microfluidic devices, nitrogen cavitation, cell membrane blebbing by virtue of sulfhydryl-blocking, and exposure to alkaline solutions followed by sonication. The major advantage of the \u201ctop-down\u201d methodology is that the produced nanovesicles are fully biological with similar features to natural exosomes. Unfortunately, their production process also involves cell destruction, which limits its production sustainability and is associated with the presence of various contaminants ,66,67,68On the other hand, \u201cbottom-up\u201d methodologies produce exosome-mimetics by supramolecular chemistry, which combines basic components such as lipids or proteins in a progressive manner so as to form complex structures . This apFinally, biohybrids, a form of exosome-mimetic, can be created through the merging of synthetic nanoparticles with natural vesicles, which is achievable by freeze-thawing, incubation, or co-extrusion . This stIn summary, the products of nanobiotechnology are expected to comprise a major drug delivery system in the near future and bring medicine closer to personalized therapy. However, this field is still in its early stages of evolution. Therefore, further research is needed to improve production protocols, devise characterization methods, and achieve a meaningful outcome. The above-mentioned techniques are presented in The PDAC\u2019s microenvironment consists of a wide variety of heterogeneous cell populations, namely, endothelial cells and pericytes comprising the vascular compartment, extracellular matrix (ECM)-producing cells, neuroendocrine cells, and immune and inflammatory cells at various activation states. From a review of the preceding literature, it is apparent that their contribution to tumorigenesis has long been underappreciated. This is reflected by the utilization of nonspecific chemotherapeutic agents in PDAC\u2019s therapy in order to achieve cell-cycle arrest. With the broadening of our understanding of PDAC\u2019s initiation, proliferation, and metastatic potential as well as of the dynamic interconnection between tumor cells and the cells of tumor microenvironment (TME), new target-specific therapeutic approaches are under development. This complex network is interconnected with physical interactions, immune suppression, and pleiotropic crosstalk mechanisms . This inAs far as a tumor\u2019s physiology is concerned, the PDAC cells are crowded in an intense stroma of fibrous tissue, which is characteristic of the disease . The immBeyond mere description of disease pathogenesis, the exploration of exosome-mediated communication might uncover a new basis for potential treatment. Based on this thesis, Zhou et al. created an exosome-based dual delivery biosystem for enhancing PDAC immunotherapy and reversing the tumor immunosuppression of M2 macrophages through the disruption of the galectin-9/dectin 1 axis . TME wasTaking all of this into consideration, PDAC TME is central to pathogenesis and the physical history of the disease. These processes\u2014mediated largely by exosomes\u2014are becoming ever more defined. Thus, the next step is to utilize the attributes of exosomes and to manipulate them in such a way that they become clinically meaningful. A brief synopsis of the above is presented in The urgency regarding the generation of a more personalized approach to medical diagnosis and treatment has placed exosome technology in the spotlight. The main applications that are under investigation concern the reduction of drug resistance, the utilization of exosomes as drug-carries, the therapeutic modification of the immune tumor microenvironment, and the targeting of KRAS. The contributions of exosomes in the therapy of PDAC will be presented below.Resistance to chemotherapy is an appreciable problem in the treatment of pancreatic cancer, and there is growing evidence that exosomes are crucially involved. The underlying mechanisms for nontargeting drugs include the upregulation of multidrug resistance proteins (MDR) which comprise several classes of drug efflux pumps such as P-glycoproteins (P-gp), multidrug-resistant protein-1 (MRP-1), and ATP-binding cassette sub-family A member 3(ABCA-3). They also include the enhancement of proliferating and anti-apoptotic pathways, the downregulation of drug targets, and the mutational alteration of drug targets. The latter also constitutes the leading cause of drug resistance in targeted therapies . ExosomeRadical oxygen species (ROS) generation is a well-documented precipitant of cell death. Patel et al. documented that among the EVs, exosomes contribute the most to gemcitabine (GEM) resistance by conveying superoxide dismutase 2 (SOD2) and catalase (CAT) to detoxify ROS. In parallel, the exosomally transmitted miR-155 inhibits the expression of deoxycytidine kinase (DCK), deescalating the phosphorylation of GEM monophosphate to its active metabolite. The inhibition of mir-155 or the downregulation of DCK counteracts the exosome-induced GEM resistance . MikamorExcepting the metabolic support that Cancer-associated fibroblasts (CAF) provide to the cancerous epithelial cells, Richards et al. documented that CAFs, when exposed to GEM, tend to transmit their innate resistance to GEM with exosomes highly enriched in miR-146a and Snail. Blocking the secretion of exosomes with GW4869 enhances PDAC cell killing by GEM in co-cultures and shrinks the tumor volume . AnalogoThe rationale for using exosomes as drug carriers in the treatment of pancreatic cancer stems from the similarity of their cell membranes to those of human cells. This trait is responsible for some advantageous properties, including favorable bioavailability and biocompatibility, inconsequential immunogenicity, the enhancement of drug release stability, the expansion of a drug\u2019s half-life, and the ability to penetrate biological membranes. \u03a4he end result is the development of more effective drugs with improved profiles of side effects .As mentioned above, the administration of drugs to cell populations of mesenchymal origin  can trigger the generation and secretion of exosomes. This feature is exploited in order to create a model where chemotherapeutics are incorporated in mesenchymal tissues and secreted with exosomes. Another technique utilizes modern nanotechnology to load or pulse drugs directly into exosomes. The discovery of exosomes and the evolution of their isolation and processing technology has transformed the logic of cancer vaccination. The initial vaccine development efforts aimed at stimulating antigen-presenting dendritic cells with cancer-specific antigens in order to create vaccines that targeted distinct cancer cell populations . An incrWang et al. demonstrated that under hypoxic conditions, PDAC cells are urged in an HIF-1a/2a-mediated manner to upregulate the expression of miR-301a-3p. The secretion of miR-301a-3p-enriched exosomes inhibits PTEN expression and potentiates the PI3K signaling pathway to polarize macrophages towards an M2 phenotype. The end result is a statistically significant association of miR-301a-3p with a more aggressive clinicopathological disease regarding the extent of tumor invasion, metastatic potential, perineural infiltration, and TNM staging. The silencing of miR-301a-3p reverses those effects  and coulThese scientific efforts led to a more comprehensive theranostic approach. Yang et al. modified MIA-PaCa-2 cell-derived exosomes loading Chlorin e6 (Ce6), a potent photosensitizer. Ce6 can enhance the photoacoustic signals and trigger ROS generation upon exposure to laser irradiation. Additionally, potentiating the local antigen-presenting cells releases a cytokine reaction which generates an immune reaction by CD8 T cells. This combination of image-guided photodynamic therapy and immunotherapy showed promising results in mice with minimal side effects  and shouKRAS mutations drive carcinogenesis and are existent from early adenomatous lesions to late metastatic disease, there were not until recently any KRAS-targeting therapies. In fact, for decades, KRAS was considered \u201cundruggable\u201d based on the immense affinity of Ras for the highly abundant GDP,GTP in the cell\u2019s cytoplasm [Despite the abovementioned fact that ytoplasm . Numerouytoplasm , and we ytoplasm . Recentlytoplasm , potentiytoplasm . In para\u03a4he production of cutting-edge biotechnological therapeutic products often runs into the inability to produce sufficient quantities to cover patients\u2019 needs. Mendt et al. generated siRNA-loaded exosomes to an extensive scale. The KrasG12D-silencing RNAs were incorporated via electroporation into mesenchymal-derived exosomes inhibiting the PDAC tumor growth in mouse xenografts. After intraperitoneal administration, iExosomes were allocated preferentially in pancreatic tumor tissue in comparison to the neighboring organs with a mechanism which remains unclarified . The KRAThe use of exosomes in clinical practice has the potential to become an important weapon in our therapeutic armory, capable of serving the demands imposed by personalized precision medicine. Although the consolidation of evidence concerning the importance of exosomes is relatively recent, a significant amount of preclinical data has begun to accumulate into the literature and a modest fraction of this is directed into clinical trials in order to test its diagnostic  or thera"}
+{"text": "Exosomes are extracellular vesicles with the diameter ranging from 50 to 100\u00a0nm and are found in different body fluids such as blood, cerebrospinal fluid (CSF), urine and saliva. Like in case of various diseases, based on the parent cells, the content of exosomes  varies and thus can be utilized as potential biomarker for diagnosis and prognosis of the brain diseases. Furthermore, utilizing the natural potential exosomes to cross the blood\u2013brain barrier and by specifically decorating it with the ligand as per the desired brain sites therapeutics can be delivered to brain parenchyma. This review article conveys the importance of exosomes and their use in the treatment and diagnosis of brain/central nervous system diseases. Brain neurological disorder/disease is one of the major causes of disability and death worldwide. It is the second leading cause of death as per 2016 estimate and is the leading cause of disability adjusted life years [Extracellular vesicles are the natural nanocarriers that pack bioactive molecules such as proteins and coding and non-coding RNAs that help in transferring information between cells and tissues and thus playing essential role in cellular communication . They arThe extracellular biogenesis involves:Formation of early endosome from the plasma membrane through a process of inward budding.Formation of late endosome, microvesicles containing intraluminal vesicles (ILVs).On fusion of late multivesicular bodies (MVBs) with a plasma membrane, the content is released and, if it fuses with lysosomes, it is degraded.http://www.exocarta.org). Most of the studies mentioned that the process by which exosomes selectively pack their cargos remains uncertain.Endosomal sorting complex required for transport (ESCRT) such as ESCRT-0, I, II, III and accessory proteins is involved in biogenesis, formation and vesicle scission \u201318. UbiqThe process of exosome trafficking involves the release of exosome in the extracellular space followed by its uptake by the recipient cells, the mechanism of which is still not well known and it remains uncertain. In some of the cellular responses generated by exosomes, it does not require exosome uptake and it is produced by exosome surface protein adhesion to the receptors on cells such as Fas ligand (FasL) or TRAIL (tumour necrosis factor\u2013related apoptosis-inducing ligand) on exosome membrane present on the recipient cells. In the case of ribonucleic acid (RNA) transfer, exosome uptake by recipient cell is important compared to transfer through membrane. Macropinocytosis or phagocytosis, receptor-mediated endocytosis and fusion are different methods of cellular exosome absorption. Exosome absorption is not automatic but relies on the interaction between exosome proteins and recipient cells on the surface. Numerous studies have indicated that exosome surface adhesion\u2013related molecules such as tetraspanin, glycoproteins and integrins decide which cell receives exosome .Exosomes are natural nanoscale transport vesicles of messenger RNA (mRNAs), microRNAs and proteins, receptors and enzymes and thus play an important role in theranostics of brain diseases .This sort of vesicles (40\u2013200\u00a0nm) is secreted by several natural cells, and thus, production becomes easy .Due to the existence of major histocompatibility complex (MHC) molecules and a co-stimulatory cluster of differentiation 86 (CD86) molecule on the surface, these vesicles display low immunogenicity, which can potentially improve immune response, especially with chronic exposure and excellent biocompatibility .Exosomes have demonstrated inherent stability .There may be intrinsic therapeutic benefit in certain unmodified exosomes .Drug delivery efficiency is very high  and has In several drug delivery investigations, it has been exploited as a drug transporter based on which novel therapeutics can be loaded for treatment of brain ailments .The exosome-based approach requires a simple, efficient and accurate biosynthesis and self-assembly mechanism .Another comparison to the advanced and poorly regulated synthesis methods used to integrate peptides and antibodies into targeted vehicles is that exosomes can be genetically engineered to increase delivery capability and target specificity, thus specifically targeting drugs to the brain .Microvesicles (MVs) can also be configured to express particular ligands on the surface of the membrane; then, they can penetrate through the specific tissues with artificially engineered MVs .Exosomes have been reported to be involved in many cellular functions due to their release from many cell types and involvement in biological fluids, including protein secretion, immune response control, antigen presentation, RNA and protein transfer, infectious cargo transmission and cell\u2013cell signalling based on these properties exosomes paves good path for brain targeting .Exosomes can operate at a near range and even at a distance through the transfer of biological fluids such as plasma .As potential diagnostic tools, exosomes also hold great promise as they are released by tumour cells and contain tumour protein biomarkers such as the epidermal growth factor (EGF) receptor variant in glioblastoma .The lack of unnecessary accumulation of therapeutic cargo in the liver and the low homing of exosomes to the liver could explain the favourable toxicity profile as well as the high efficiency of delivery to the brain by these vesicles is the major advantage of the use of exosomes compared to other nanoparticle delivery vehicles .Exosomes are relatively stable in the blood as they avoid opsonin\u2019s, coagulation factors  and the The small exosome size should also be helpful in preventing particles from phagocytosis by the mononuclear phagocyte system (which clears particles\u2009>\u2009100\u00a0nm in size), bypassing lysozyme swallowing and promoting their extravasation by vessel fenestration and passage through the extracellular matrix .The discovery that exosomes bear parent cell\u2013derived cargoes gave rise to the idea that they could provide tissue-specific disease biomarkers, and the diagnostic value of exosome cargoes in cancer, heart disease, infection and pregnancy is now being thoroughly explored . As suchDrug delivery vehicles derived from exosomes have wider distribution of biological fluids, likely to produce longer circulation time and potentially improved efficacy .Research shows that exosomes derived from mesenchymal stem cells (MSC-exo) retain some of the features of their parent MSCs, such as immune system control, neurite outgrowth regulation, angiogenesis promotion and the ability to regenerate damaged tissue, such as after kidney injury .Loading or expressing a therapeutic agent in or on exosomes extends its half-life, achieving impressive effectiveness by delivering the drugs to the intended target .Repeated systemic exosome administration does not cause liver toxicity, which supports its safety profile .Differential ultracentrifugation and density gradient centrifugation: It is regarded as the gold standard method of exosome isolation. It involves the application of centrifugal force to an exosome-containing solution  , for exaImmunoaffinity chromatography: In this technique, antibodies are covalently bonded with beads, filters or other matrices, wherein it will bind to the surface protein or antigen present on the targeted exosomes (buffer is used to collect bound fraction from the stationary phase). The non-targeted exosomes will remain free. It isolates exosomes in pure form as their isolation depends on antibodies recognition . ImmunoaSize exclusion chromatography: In this, different size components are separated based on their size. This method utilizes heterosporous beads packed columns and the elusion time, which is inversely related to the particle size. It helps in maintaining integrity of the exosomes . For exaPolymer precipitation: In this technique, a solution of PEG (8000\u00a0Da) is mixed with bio fluid containing exosomes by incubating overnight at 4\u00a0\u00b0C. Then, composition is centrifuged. Furthermore, it utilizes pre-isolation step (centrifugation) to remove lipoprotein as contaminant and post-isolation step (SephadexG-25 column) to remove polymer . Other pMicrofluidic technology: It is based on immunoaffinity, sieving and trapping exosomes on porous structures. This approach requires a smaller starting material volume and minimal processing time for highly pure exosome preparation .Ultrafiltration: It is a membrane separation technique based on size and molecular weight of membrane used for separation of exosomes .Magnetic separation: The capture and separation of exosomes includes antibody-modified magnetic beads. Due to its contactless, high through performance and precise separation, this method is often used. The exosomes are preserved by immunomagnetic beads while the phosphate buffer washes away other contents. In the chamber, these beads are further lysed, captured and analysed , for exaAcoustic fluid separation: It utilizes the principle of separation based on size. In acoustics, depending on the\u00a0size, particles are\u00a0subjected\u00a0to various acoustic forces and thus differentiating. It is a label free and contactless process, and thus, it is validated and then used for separation .Dielectrophoretic separation (DEP): It operates on the theory of a non-uniform electric field generated dielectric forces experienced by polarized particles. Cell\u2019s intrinsic dielectric properties, size of the particles, magnitude and frequency of electric field determines the magnitude of forces exerted by DEP on exosomes. The larger polarized particles attract towards lower electric field while exosomes are attracted towards higher electric .Deterministic lateral displacement (DLD) separation: It uses devices or the chips and is based on the principle of particle flow path which is greater than the critical size. Researcher faces difficulties with separation and clogging (it is easy to use and label free) .Nanotrapped wire: In this process, a polymer nanoporous membrane is used or porous structures are used to trap exosomes, such as nanowires (silicon based) arranged with micropillars. This technique operates on the concept of exosomes trapping based on the size. Degradation of silicon leads to generation of silicic acid .Researchers are exploring advanced methods for exosome isolation and purification considering its potential to be used as biomarkers and therapeutics. It is isolated by various methods using its physical properties such as size, float density and marker protein presence such as Alix, tumour susceptibility gene (TSG 101), heat shock protein (HSP 70) and CD9 \u00a0surface Exosomes have a liposome like membranous structure that motivates researchers to extend their prior drug loading experience in them . The loaPreloading of exosome is a common technique for achieving desired target specific exosome which is accomplished before exosome formation and isolation by treating or transfecting cells.This is a common technique for specifically targeting exosomes that is achieved before exosome formation and isolation by treating or transfecting cells . This teThis process utilizes hydrophilic or hydrophobic drug or salt solution for incubation of parent cell for the desired time, and the cells will exocytose these substances in the form of loaded vesicles. Packing of these exosomes is depending on interaction between materials and cells .In this method, parent cells are manipulated using commercialized transfecting reagents with MicroRNA (miRNA) or small-interfering RNA (siRNA) or plasmid\u00a0(pDNA) that the cells load into the inner core of the extracellular vesicles (EVs) or stack for excretion on the outer layer using therapeutic application \u201366. A fuExosomes are non-living structures hence substances and reaction conditions are feasible for surface functionalization. Additionally, during cell-based modification only limited amount of content envelops inside the vesicles. Exosomes are isolated and purified using ordinary liquids such as culture medium, serum or breast feed for further processing during post-loading . Post-loThe method of incubation is used for exosome loading by incubating them with cargo of interest, especially hydrophobic interfaces. After incubation with purified exosomes, many small lipophilic drugs, such as curcumin, dopamine, celastrol, porphyrin, cucurbitacin, methotrexate and doxorubicin, were competently stacked at room temperature , 70\u201375.This technique has been developed to expand packaging quality. Sound energy as an automatic force is used in sonication to interrupt the exosome layer so that cargo or theranostic agents can be loaded in them. Shear mechanical power is used as a lipid extruder to connect exosome and agents under controlled temperature in the extrusion technique .Exosomes contain the genomic and proteomic material of their originating cells, whereas on the outside of exosomes, definite antibodies can be bound to a particular antigen called antibody-specific loading. However, through gene transfection, non-native receptors could be added to the exosomes .In order to improve the layer permeability and loading of hydrophilic agents, including miRNAs, siRNAs, smaller drugs and electroporation of superparamagnetic iron oxide nanoparticles (SPIONs), it is often possible to use the approach to make pores on the exosome lipid dual layer film by applying an electrical field (150\u2013700\u00a0V)\u00a0to allow the loads to be stacked inside the exosomes , 76\u201378. Due to the aggregation of sphingomyelin, cholesterol and gangliocytes, exosome shows more rigid lipid bilayer in comparison to the cell membrane. Exosome can be engineered by incubating at room temperature for fixed time with therapeutic agent followed by repeated freeze and thaw period  , 31.It can have a higher internalization level. Saponin is an active compound that forms complex with\u00a0cholesterol\u00a0on the exosome surface and creates holes followed by improving membrane permeability , 80.Based on size, structure, protein and lipid content, exosomes can be characterized . This heThe ability to bypass blood\u2013brain barriers (BBB) is one of the most important core features of exosomes in the treatment of brain diseases or disorders, representing a promising strategy for the treatment of brain diseases . Approxi1. Transcellular route: Exosomes are internalized by endothelial cells of BBB through cell type\u2013specific protein via receptor-mediated endocytosis and then undergoes transcytosis.2. Paracellular route: Exosome cross the intercellular junction of endothelial cells of BBB and then enter the central nervous system (CNS).Evidence showed that Parkinson\u2019s disease was treated by using antioxidant protein catalase loaded exosomes by crossing blood\u2013brain barrier after intranasal administration . In crosFurther research showed that tetraspanin CD9 on the surface of exosomes interacts with surface glycoprotein on target cells and facilitates exosome to fuse with the cell membrane, thus helping in direct cytosolic delivery of gene . It has Exosomes are shed by the cells under both normal and pathological conditions. They carry nucleic acid, proteins, lipids, metabolites and antibodies from their host cells which indicate the pathophysiological conditions and thus are widely considered to be important biomarkers for clinical diagnostics . These aPresent therapeutic methods take advantage of unmodified extracellular vesicles bearing beneficial intrinsic properties for the treatment of Alzheimer\u2019s diseases, such as stem cell\u2013derived exosomes. Lipids, proteins and nucleic acid are loaded during exosome development. Sphingomyelin, cholesterol, phosphatidylserine, de-saturated lipids, ganglioside monosialodihexosylganglioside 3 (GM3) and ceramides are part of a lipid. The proteins distributed throughout the exosome membrane or cytostome includes enzyme linked to the formation of fusion proteins, chaperones and MVBs, such as CD9, CD63, CD81, Alix and TSG101. Antibodies, metabolites, mRNAs, miRNAs and other coding as well as non-coding RNAs and DNAs are found in the nucleic acid. Exosome derived from neural cells houses about 6000 known proteins and more than 85% of known miRNAs. Furthermore, the content of exosome is dependent on the type and state of parent cells , 98, 99.Globally, injuries to the central nervous system, particularly brain strokes, are the principal cause of death in people, which may otherwise result in long-term impairment. Although, to some extent, medically approved treatment for the brain injury owing to cerebral stroke is available to break up the blood clot by administering tissue plasminogen activator (tPA). A diagnostic marker to predict the austerity of the stroke would be immensely useful. Recent research suggests the potential use of exosomes as biomarkers for identifying patients at risk of haemorrhage or other severe complications following a brain stroke, as well as a possible neuroprotective agent as an approach from the treatment angle. Many studies describe groups of mRNA targets as promising non-invasive biological markers for the diagnosis of stroke . It is kStudies propose that the delivery of MSC exosomes has restorative effects in patients with stroke and improves post-stroke neurodegeneration and prevents post-ischemic immunosuppression . In anotHypoxia\u2013ischemia is the primary cause of brain damage in premature and full-term neonates, which is eliciting higher morbidity and mortality rates worldwide. Perinatal hypoxic-ischemic brain injury in preterm new borns has led to long-term neurological complications, and so far, no conclusive therapeutic strategies are available. A study was conducted wherein ovine foetuses with hypoxic-ischemic encephalopathy (HIE) was studied and it demonstrated the neuroprotective properties of MSC-derived exosomes in preterm brain injury. Cerebral hypoxia\u2013ischemia was inflicted upon the ovine foetuses by brief umbilical cord occlusion to mimic the conditions under which hypoxic-ischemic brain injury occurs in neonates. In-utero intravenous MSC exosomes were administered, and its therapeutic efficacy was analysed by determining the changes in structural injury by microscopical examination or biopsy of the brain, evaluating the seizure burden and anti-inflammatory effects. It was reported that the systemic administration of MSC exosomes improved functional recovery, reduced cognitive impairments, induced long-term neuroprotection and stimulated neurogenesis and angiogenesis . It was The incidence of traumatic brain injuries (TBI) in young adults (15\u201324\u00a0years) and older adults (\u2265\u200975\u00a0years) in the world, especially in the United States (US), is expanding exponentially and diagnostic strategies for assessing the degree of neurological damage and to timely prevent complications related to brain injury, as well as to predict the response to therapy are imperative. Because of the inability to biopsy neurological components, injury-linked biological markers are essential to define the pathophysiological mechanisms and predict the neurological outcomes. One such approach to a circulating biomarker is the use of exosomes, i.e. \u201cliquid biopsy\u201d. Several researchers have reported changes in exosomes in the plasma and CSF of patients with TBI. In 2008, a study initially demonstrated the potential of circulating exosomal RNAs as a diagnostic tool for patients with TBI. Furthermore, investigations demonstrated that exosome release after injury mediates the production of pro-inflammatory cytokines and elevations in the interleukin-1 cytokines, especially, interleukin-1\u03b1, interleukin-1\u03b2 and interleukin-18 after a TBI was observed . As docuEpilepsy is a complex disorder which has many possible clinical presentations and its diagnosis primarily depends on clinical examination and the patient\u2019s medical history. Since the diagnostic strategies for epilepsy are limited, this poses a great challenge and thus, rapid and non-invasive approaches like biomarkers would be of great significance. In a recent study they demonstrated the use of exosomal proteins to study epilepsy. The group originally identified three major proteins, a blood clotting factor viz Christmas factor (F9), an adhesive glycoprotein thrombospondin-1 (THBS1) and an integral membrane protein amyloid-\u03b2 precursor protein (APP), and analysed their levels in plasma exosomes . The stuStudies utilizing MSCs as therapy have currently gained significance for various neurological diseases, and one such study reported the therapeutic potential of MSC-derived exosomes in pilocarpine-induced epilepsy models. The study group treated 1\u20138-week-old C57 black male mice with exosomes derived from pluripotent stem cells, and the results showed that the native exosomes from bone marrow MSCs have robust anti-inflammatory and neuroprotective properties. The treatment alleviated inflammation as well as reduced neuronal loss, helped in normalization of neurogenesis and significantly improved spatial learning in mice by targeting the astrocytic glial cells in the hippocampus . AdditioThe casualties due to drug dependence are steadily on the rise, and the world is facing a devastating health crisis because of drug abuse and overdose. Society is being crushed by the toll of the opioid epidemic. The growing health problem has called on the urgent need to identify potential therapies to combat opioid addiction. A study was conducted to examine the therapeutic potential of exosome delivered small-interfering RNAs (siRNAs) in case of opioid addiction. A researcher made use of a cell penetrating peptide namely rabies virus glycoprotein (RVG) peptide attached to exosomes, to allow them to pass through the BBB efficiently. These RVG-modified exosomes were loaded with opioid receptor Mu (MOR) siRNA. A study group of C57 black male mice was intravenously injected with the modified exosomes, and siRNA levels were assayed in plasma after 6\u00a0h. The siRNA, MOR mRNA and protein levels were also assessed in brain tissues after 24\u00a0h. Following a morphine-administered relapse, a conditioned place preference (CPP) test was conducted, and it showed that mice treated with RVG exosomes containing MOR-siRNA exhibited behaviours corresponding to restrained drug addiction in contrast with control subjects administered with saline . The resp\u2009<\u20090.05) upregulation and under regulation in sixteen and eleven exosomal miRNAs, respectively, in the CSF. Considerable overexpression was observed in miR-153, miR-409-3p, miR-10a-5p and let-7\u00a0g-3p in the Parkinson\u2019s CSF exosomes, whereas miR-1 and miR-19b-3p displayed a significant reduction in the independent samples. Bioinformatic analysis conducted by DIANA-mirPath revealed that the significant pathways that possessed great quantities of the patterns of miRNA were neurotrophin signaling, mechanistic target of rapamycin , ubiquitin mediated proteolysis, dopaminergic synapse and glutamatergic synapse. Messenger RNA transcripts such as amyloid precursor protein, \u03b1-synuclein, Tau, neurofilament and RP11-462G22.1 and prostate cancer antigen 3 (PCA3) which are long-coding RNAs were found to be expressed dissimilarly in the CSF exosomes of Parkinson\u2019s as well as Alzheimer subjects. The results obtained from the study revealed that the exosomes with the encapsulated RNA molecules isolated from the CSF were valuable biomarkers with remarkable robustness with respect to the sensitivity and specificity to differentiate Parkinson\u2019s disease from healthy and disease controls [Neurodegenerative disorders are currently one of the prime reasons for dysfunction and in worst cases death and continue to pose a major challenge for its present-day medical management. However, with the emergence of newer modes of drug delivery, exosomes offer great potential not only as a therapeutic method, but also as an invaluable prognostic biomarker for the treatment of various brain pathologies including neurodegenerative disorders . The difcontrols . Figure\u00a0It was also attempted to determine the expression of miRNAs in exosomes present in the serum and to measure the expression of circulating miRNA in Parkinson\u2019s patients. In the current study, the serum of 109 candidates with Parkinson\u2019s disease as well as 40 healthy volunteers was collected and the expression of 24 candidate\u2019s human miRNAs which are clinical biomarkers of Parkinson\u2019s disease was investigated. The exosomes in the serum containing encapsulated RNAs were extracted and subjected to reverse transcription following which the miRNAs present in the serum were quantitatively analysed using the reverse transcription polymerase chain reaction (qRT-PCR), and the analysis of the characteristic curves of the operating receiver was carried out in order to determine the ability of the miRNAs to appropriately distinguish Parkinson\u2019s. Furthermore, validation of the down regulation of miR-19b as well as upregulation of miR-195 and miR-24 in candidates with Parkinson\u2019s were conducted. In comparison to the control subjects, the values for area under the curve for miR-19b, miR-24 and miR-195 were found to be 0.753, 0.908 and 0.697, respectively, thereby indicating that the level of expression of miR-19b, miR-24 and miR-195 in the serum could be extremely beneficial in diagnosing Parkinson\u2019s disease . A studyIn an attempt to identify the pathogenic role of long non-coding RNAs (lncRNAs) in the development of Parkinson\u2019s, the dissimilarities in the expression of these lncRNAs in the peripheral blood exosomes of patients with Parkinson\u2019s were studied. The level of lncRNAs isolated from the plasma exosomes was determined by next-generation sequencing along with real time PCR. The results revealed the upregulation and downregulation of 15 and 24 exosomal lncRNAs, respectively, in the patients with Parkinson\u2019s and also indicated the involvement of lnc-MKRN2-42:1 in the development and progression of Parkinson\u2019s .Due to the absence of an appropriate blood test to distinguish Parkinson\u2019s disease from atypical Parkinsonian syndrome, researchers assessed the practicality of serum neuronal exosomes as a biomarker. The results of the study indicated a twofold increase in the \u03b1-synuclein present in the neuronal exosomes in Parkinson\u2019s disease patients in comparison to other neurodegenerative pathologies. The exosomal \u03b1-synuclein demonstrated an excellent capacity in the differentiation of Parkinson\u2019s disease across various populations. In patients with non \u03b1-synuclein proteinopathies, there was also found to be an elevation in the clusterin exosomes. Thus, the findings of the study indicated the effectiveness of determining the presence of \u03b1-synuclein and clusterin exosomes as an exceptional method for the differentiation of Parkinson\u2019s disease from atypical parkinsonism for at-risk populations . AnotherIn order to develop a more biocompatible method for the treatment of Parkinson\u2019s, researchers utilized blood exosomes for the delivery of drugs across the BBB. The exosomes displayed a nanosize and were deftly loaded into the blood exosomes through a saturated solution incubation technique. The distribution of dopamine in the brain as well as the therapeutic efficacy of the blood exosomes were greatly enhanced in the mouse model of Parkinson\u2019s, thereby indicating the effectiveness of the exosomes for the treatment of Parkinson\u2019s disease . The intThe researchers also investigated the small RNA contents of the exosomes derived from the brain and their effective use for the determination of pathological changes in the onset of Alzheimer\u2019s disease and its use as an early diagnostic blood test. The exosomes which were derived from the frontal cortex of the brain of Alzheimer\u2019s patients were found to contain an elevated level of miRNA, which could provide a pattern of the biological pathways affected in the course of the disease . It was A group of researchers isolated exosomes from the astrocytes of Alzheimer\u2019s disease patients through immunochemical methods and were further matched with controls for the quantification of complement proteins by enzyme-linked immunosorbent assay to determine the mechanism of astrocyte inflammation. Astrocytes are abundantly present in glial cells of the CNS that possess a very important neuronal trophic function through a variety of homeostatic mechanisms. Many neurodegenerative, inflammatory as well as ischemic conditions of the nervous system are known to produce well synchronized multicellular reactions which cause a rise in the number of astrocyte cells, thereby affecting their differentiation into either inflammatory type or ischemia-related type. From the results obtained, it was deduced that the production of antibody-dependent enhancement (ADE) complement effector proteins could be attributed to dysregulated systems in patients with Alzheimer\u2019s disease and were found to be elevated as compared to the controls and also had the potential to cause neuron damage in the late inflammatory stages of Alzheimer\u2019s disease. The current work proposed the pathogenic role of A1 type astrocytes in Alzheimer\u2019s disease due to the presence of inflammatory complement proteins which attained high levels in Alzheimer\u2019s patients in comparison to the matched controls. The reduced amount of various complement regulatory proteins in the early phases of Alzheimer\u2019s, points towards the inhibitory loss of the classical and alternative complement pathways and could be a major reason for complement-mediated neuroinflammation in Alzheimer\u2019s disease. The data obtained also indicated that the current complement-directed therapies could be beneficial to the patients with Alzheimer\u2019s, having elevated levels of complement-mediated neuroinflammation .The researchers suggested a novel approach to pack miR-29 in exosomes, and to further administer the modified vesicles to avoid certain memory deficiencies in A\u03b2-treated model rats. In order to prevent the flare up of an immune response, the exosomes were derived from the stromal cells of rat bone marrow. The results indicated an increase in the amount of miR-29b, thereby resulting in the downregulation of its target genes beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) and BIM M (Bcl-2 interacting mediator of cell death (BCL2-like 11)), after treatment of the U87 cells with miR-29b-based exosomes. Based on the data obtained, injection of the exosomes, containing miR-29b, conferred protective properties against amyloid pathogenesis, thereby proving the successful outcome of this miRNA-based therapy in the pathogenesis of Alzheimer\u2019s disease .The avenues for the treatment of Alzheimer\u2019s disease were further enhanced by the study which attempted to investigate the regulatory role of exosomes which were isolated from human umbilical cord MSC developed in a 3D culture. The supernatants from these 3D cultures were procured to isolate the exosomes. From the results obtained from the study, it was deduced that the 3D cultured exosomes upregulated the expression of a secretase and downregulated that of b-secretase, thereby reducing the production of A\u03b2 in the pathogenic Alzheimer\u2019s cells as well as in transgenic mice, thereby enhancing the therapeutic response on the improvement of memory and cognitive deficiencies in mice with Alzheimer\u2019s .Exosomes derived from the MSC were also used to enhance the neurogenesis as well as the cognitive capabilities in a mouse model for Alzheimer\u2019s disease. The results of the study indicated that the exosomes isolated from the stem cells were able to augment neurogenesis in addition to restoring the cognitive function which was decreased on administration of A\u03b21-42 aggregates . SimilarAnother novel method was developed which utilized the concentration of salivary exosomes as a correlation measure for the degree of cognitive impairment using nanoparticle tracking analysis technique in Alzheimer\u2019s patients. On measurement of the total exosomes present in the saliva, the results indicated significant differences in the concentration of the exosomes between patients suffering from Alzheimer\u2019s and the healthy controls. A similar result was also observed upon validation using an exosome surface marker, i.e. CD63. The findings were further confirmed by correlation with the expression levels of oligomeric amyloid-beta and phosphorylated-tau protein obtained from salivary exosomes. It was also observed that the phospho-tau, A\u03b2 oligomer/fibril and A\u03b2 protein was abundantly present in patients with Alzheimer\u2019s and the cognitive impairment as opposed to their healthy counterparts. Therefore, the results of the study indicated that the quantity of salivary exosomes, obtained through nanotracking method, presents prospective use as a cost-effective method in the early diagnosis of Alzheimer\u2019s .A novel technique for the longitudinal and quantitative in vivo neuroimaging of exosomes was recently developed by group of researchers using gold nanoparticles as a labelling agent in combination with the superior visualizing capability of the classical X-ray computed tomography. This method was utilized to track the patterns of homing and migration after the intranasal delivery of the MSC-derived exosomes of the bone marrow in various CNS diseases. It was observed that the exosomes were specifically targeted in the pathological areas of murine model brain regions and were accumulated up to a time period of 96\u00a0h post-administration, whereas the healthy counterparts exhibited a more diffused migratory route and clearance within 24\u00a0h. There was an extremely high correlation between the neuroinflammatory signal in pathological brains and exosomal accumulation, thereby proposing the mechanism to be inflammatory driven. Moreover, the MSC-derived exosomes exhibited selective uptake by the neuronal cells in the pathological sites but not glial cells. All in all, the results of this study contribute significantly to the use of exosomes in the diagnosis and therapy of numerous CNS disorders including Alzheimer\u2019s and Parkinson\u2019s diseases .The study also involves proteomic analysis of the exosomes derived from the CSF of patients suffering from amyotrophic lateral sclerosis in order to identify new biomarkers associated with the disease. Liquid chromatography-tandem mass spectrometry of the fluid fractions of amyotrophic lateral sclerosis (ALS) subjects was conducted utilizing gel filtration chromatography. Novel INHAT repressor (NIR) protein was found to be greatly elevated in ALS patients whose dysfunction could possibly contribute to the nucleolar stress in sporadic ALS pathogenesis .Researchers investigated therapeutic effect of exosomes derived from adipose stem cells in the SOD1(G93A) murine model of mice. The effectiveness of the exosomes through the intravenous and intranasal route was also determined. The findings of the study revealed an improvement in the motor capacity as well as protection of the lumbar motoneurons and muscle cells. There was significant reduction in the activation of glial cells in the murine models thereby proving it as a potential treatment for amyotrophic lateral sclerosis in humans .In order to determine the extent of inflammation in patients with ALS, a study conducted involves the concentration of interleukin-6 (IL-6) in exosomes that were derived from astrocytes. An increase in the levels of IL-6 in the exosomes was indicative to sporadic ALS but was limited to subjects having the disease for less than a year . It was Another genetic neurodegenerative disease that has been treated through the use of exosomes is Huntington\u2019s disease (HD). HD is caused due to an abnormality in the expansion of cytosine, adenine and guanine (CAG) repeats that is responsible for encoding the gene huntingtin. This results in a myriad of neurological symptoms including the cognitive impairment, involuntary choreiform movements and neuropsychiatric conditions. Though the exact mechanism of action of neurodegeneration in Huntington\u2019s disease remains unclear, it could be attributed to alterations in RE1-silencing transcription factor (REST) which is a transcription regulator. This results in inability of the mutant huntingtin to silence the activity of REST, thereby causing enhanced binding of REST to a neuron restrictive silencer element which results in transcriptional dysfunction. Other researchers used exosomes as a means of delivery in the treatment of this degenerative condition. One of the prime miRNAs, i.e. miR-124, which is repressed in Huntington\u2019s disease was observed to be overexpressed in a stable cell line from which the exosomes were further harvested. The exosomes were found to be greatly express miR-124 expression, which later were taken up by the recipient cells. After injecting the exosomes into the striatum of R6/2 transgenic HD mice, the expression of the target gene, RE1-silencing transcription factor, was found to decline. Although, the miR-124 based exosomes did not result in significant behaviour improvement. The current investigation served as conceptual evidence for the delivery of miRNA using the exosomal method of administration for neurodegenerative diseases . Table 2Exosomes are rich in cargos as they contain higher amounts of nucleic acids and proteins, which directly reflect the metabolic state of the cells. Differential expression and disrupted homeostatic features of exosomes help in cargo trafficking against several diseases like cancer. Increased release of exosomes leads to oncogenic progression and metastases. Exo-miRNA can communicate with the adjacent cells of the same tissue or neighbouring tissue through gap junctions. This is termed as the \u201cbystander effect\u201d. This effect leads to autophagy of the cells and makes the cells more cancerous , 153. ReGlioblastoma (GBM) is a malignant tumour with several cells protruding deep into the cerebral lobes of the brain. Exosomes play a very prominent part in glioblastoma. They remain enclosed with oncogenic proteins which cause the spreading of the tumour to the neighbouring tissues. These tumours also show miRNA upregulation . ExosomeIn the last few years, it has been discovered that milk, blood, urine and saliva contain exosomes, and as they have higher amounts of RNA, lipid and specific protein content, they became useful in the diagnosis of several diseases. Blood borne miRNAs are very imported biomarkers as reported by some groups in 2008. As naked RNA was found degrading faster and faster in blood, it was supposed that these RNAs must be protected by few macromolecular complexes which later came to be the \u201cexosomes\u201d . NanoscaMUT) samples. It was reported that free-circulating miRNAs of GBM patients help in diagnostic purposes rather than exosomal miRNAs [CSF contains large amounts of tumoural exosomes, because they need not cross the BBB to enter the CSF and so it is less contaminated with platelet-derived exosomes. So, a more convenient means of sample collection is blood. Therefore, the most specific sample for diagnosis of GBM is still in difficulty . It was l miRNAs . It was l miRNAs . DiffereMany phase I studies were conducted in the 2000s for exosomes. The first study was the vaccination of metastatic melanoma patients with autologous dendritic cell (DC)\u2013derived exosomes (DEX). These were generated by adding functional major histocompatibility complexes (MHC) capable of promoting T cell immune responses along with tumour rejection . The preNeuroinflammation is an innate immune response induced by microglia and astroglia when they are triggered by various damage stimuli. Neuroinflammation results in the production of reactive oxygen species, chemokines, secondary messengers and cytokines . NeuroinExosomes play a very pivotal role in triggering the inflammatory cascade. This is mainly favoured by proteins such as amyloid \u03b2, \u03b1-synuclein and prions, which move from one cell to another cell . Cross-tNumerous pieces of evidence suggested the potential role of exosomes as vectors containing miRNA, mRNA, lncRNA, peptides, siRNA and synthetic drugs for cell therapy against neurological, neurodegenerative and neuroinflammatory diseases and the biomarkers for brain diseases. Recent studies have demonstrated that patients diagnosed with bipolar disorders or schizophrenia showed differentially expressed miRNAs when compared to the matched controls . BesidesFurthermore, hippocampal neurogenesis was impaired in mice by the injection of cultured exosomes consisting of known pathogens into the dentate gyrus . CorticoExosomes possess special features that make them the optimal tools to deliver the molecules therapeutically by crossing BBB. They transfer the brain antigens to the periphery and regulate the peripheral immune system . It was Protection of the brain was shown by the treatment of the animal (mice) model of oligodendrocyte glycoprotein peptides induced experimental autoimmune encephalitis with the use of exosome-loaded curcumin through the intranasal path. This was observed due to the selective uptake by microglial cells of the curcumin entrapped exosome and thus resulted in apoptosis of the cells . In a reHuman stem cell\u2013derived extracellular vesicles such as exosomes offer numerous benefits in comparison to cell-based treatments for the diagnosis and therapy of functionally impaired tissues and organ sites . ExosomeSo far, there are no clear regulatory criteria for pharmaceutical production and clinical application, considering the growing interest in these carriers. However, it is unavoidable that their production should be carried out strictly in accordance with GMP (good manufacturing practice), GLP (good laboratory practice), GDP (good documentary practice), GCP  and GSP (good storage practice) in a specific well-organized structure in order to eliminate any possible contamination and take into consideration both donor and recipient safety . The intSource: Parental cells from where exosomes are sourced are an important consideration in developing therapeutic exosomes. Its critical technologies should be addressed, and associated side effect should be identified , for exaBiodistribution: The natural characteristics of exosomes make it a challenging carrier in brain targeting of therapeutics and diagnostics. For example, exosomes have long retention within the blood due to high CD47 expression that prevents its phagocytosis via macrophages and monocytes by interacting with SIRP\u03b1 , 204Loading: It is done by endogenous techniques like natural biogenesis and exogenous techniques as mentioned in section loading of exosomes. Large loads lead to exosomes aggregation and thus its instability. Furthermore, loading efficiency is also variable in nature , 201.Production: production of exosomes on large scale is one of the major requirements for using exosomes in clinical use. Various methods of its production are mentioned in the section \u201cMethods of exosomes isolation and purification\u201d. Along with its scalability, integrity, functionality and purity are an important consideration for therapy , 205\u2013207Hypoxic cellular conditions: Hypoxia is a severe cellular stress that regulates exosome release and consequently alters their contents and hence it have been studied in several types of hypoxia disorders, reflecting their biological origin and disease status via bioactive cargo, making exosomes helpful as a possible biomarker for diagnosis or prediction of hypoxic diseases. Exosomes produced during hypoxia, for example, ExoHypoxia, serve a crucial function in assisting cancer cells to cross-talk with microenvironment constituents to establish conditions favourable for cancer growth and metastatic propagation , 218.In the past decade, many clinical trials have attempted to use exosomes in clinical therapy, but questions about its optimization remain unanswered, such as how to increase the exosome yield specific to the cell type and maximize output which is to be considered in using exosomes for the brain. In addition, how to store broad therapeutic substances in and on exosomes and how to design a better delivery procedure in clinical practice for particular tissues are uncertain. Following are the factors that affect the clinical use of exosomes:In the near future, more massive and accurate medicine of exosomal material may be used for the treatment of brain disorders by optimizing exosomal nanocarrier formulation. Much advancement in the field is anticipated in the future, given the positive efforts made to verify exosome potential to treat brain diseases, bring them to clinical trials and the pharmaceutical aspects of it will remain to be explored in more detail in the future. In order to understand exosome biogenesis, cargo selection and release in vivo, more sophisticated techniques and methodologies need to be established. There is also a need to consider awareness of how exosomes distinguish the normal part from the damaged part of the brain. Specific proteins and microRNAs responsible for the neuroprotective properties of exosomes should be established. Exosomes is a complex system that raises the problems of protection and predictability, so future research should concentrate on an exosome mimetic delivery system targeting particular cells and the desired therapeutic molecule. It is harder to envision the use of exosomes as a drug product, and only in minimal, personalized medicine configurations can possible applications be realized. In future research, it is important to determine if exosome therapy has a long-term lasting decrease in chronic deficiency of the brain. Future studies are important in order to better identify different aspects of cerebral-targeted exosomes in small and large animal models and to translate the findings into human clinical applications. The challenging task is the use of exosomes for brain targeting and needs close co-operation from multi-disciplinary fields such as academia, industry, clinicians and regulatory agencies. The different studies reported in this paper suggest that in the future, exosome-based formulations may be useful tools for neurodegenerative disorder therapy. Therefore, as the research continues, exosome-based drug delivery to the brain will have a huge opportunity and broader prospect to cure cerebral disease in the near future."}
+{"text": "Among extracellular vesicles, exosomes have gained great attention for their role as therapeutic vehicles for delivering various active pharmaceutical ingredients (APIs). Exosomes \u201carmed\u201d with anti-cancer therapeutics possess great potential for an efficient intracellular delivery of anti-cancer APIs and enhanced targetability to tumor cells. Various technologies are being developed to efficiently incorporate anti-cancer APIs such as genetic materials , chemotherapeutics, and proteins into exosomes and to induce targeted delivery to tumor burden by exosomal surface modification. Exosomes can incorporate the desired therapeutic molecules via direct exogenous methods  or indirect methods by modifying cells to produce \u201carmed\u201d exosomes. The targeted delivery of \u201carmed\u201d exosomes to tumor burden could be accomplished either by \u201cpassive\u201d targeting using the natural tropism of exosomes or by \u201cactive\u201d targeting via the surface engineering of exosomal membranes. Although anti-cancer exosome therapeutics demonstrated promising results in preclinical studies, success in clinical trials requires thorough validation in terms of chemistry, manufacturing, and control techniques. While exosomes possess multiple advantages over synthetic nanoparticles, challenges remain in increasing the loading efficiency of anti-cancer agents into exosomes, as well as establishing quantitative and qualitative analytical methods for monitoring the delivery of in vivo administered exosomes and exosome-incorporated anti-cancer agents to the tumor parenchyma. In the past few decades, the field of nanomedicine has experienced unparalleled rapid advances in both disease diagnosis and therapeutic applications . Among nNumerous technologies to efficiently incorporate anti-cancer drugs and APIs into exosomes are actively being developed ,20,21. ENaturally produced exosomes, known as na\u00efve exosomes, inherit the physiological characteristics of the cells from which they originate, and show comparable therapeutic potency with a better safety profile, when compared to the original cell therapy ,25,26. IOne of the attractive therapeutic approaches for intractable cancers is gene therapy using nucleic acids, such as DNA and RNA. However, there is a need for enhancing their stability for clinical use due to the disruption of the structure and function in the biological fluids. RNA is extremely unstable in plasma and demonstrates low intracellular delivery in vivo, requiring efficient delivery vehicles for therapeutic applications. MicroRNAs (miRNAs) and short-interfering RNAs (siRNAs) are short, non-coding, double-stranded RNAs that suppress gene expression by regulating the translation process of messenger RNAs (mRNAs). Exosomes do not contain large quantities of RNAs, but a wide variety of these nucleic acids\u2014miRNA, ribosomal RNA (rRNA), and mRNA\u2014have been previously detected in isolated exosomes ,27. UnmoOne of the technical approaches for loading small RNAs into exosomes is the transfection of exosome-producing cells with small RNAs, which are then endogenously incorporated into exosomes through their natural biogenesis pathway. Katakowski et al. transfected marrow stromal cells with the miR-146b expression plasmid to obtain miR-146b-loaded exosomes, and the intratumoral injection of these exosomes suppressed glioma growth in a rat xenograft model . SimilarG12D into exosomes (iExosome) ,3635,36]Exosome) . Upon syExosome) . While tft model . Exosomeft model . Xu et aft model . HepG2-dft model . Large pft model . The loaft model .Despite the promising anti-tumor effects of RNA-loaded exosomes shown in preclinical studies, the therapeutically effective amount of RNAs per exosome needs to be established through further studies. To achieve that goal, the loading amount of RNAs per exosome should be accurately measured to determine the therapeutically effective dose of RNA-loaded exosomes. Determining the loading amount of RNA requires thorough consideration concerning the potential bias caused by different RNA profiling methods, the perturbations made by different exosome purification methods, and the sample-to-sample heterogeneity caused by technical variability .As exosomes are lowly immunogenic, highly bioavailable, naturally derived lipid nanoparticles, the exosomal incorporation of chemical drugs can enhance the solubility and bioavailability of chemotherapeutics . In addiEncapsulating chemotherapeutic agents into exosomes confers multiple advantages, such as lowering unwanted toxicity and enhancing tissue targeting. Nevertheless, increasing the loading efficiency of chemotherapeutics into exosomes remains a major challenge, as conventional loading methods such as electroporation and sonication demonstrate loading efficiencies as high as 20% . ExogenoMacromolecules such as proteins can be incorporated into exosomes using exogenous methods such as electroporation. However, the susceptibility to the destruction of physical structures of proteins and exosomes limits their usefulness, along with the low loading efficiency of functional proteins ,50. HencArabidopsis thaliana . E. E54]. Ethaliana ,56. By fthaliana ,58. pYSTthaliana . EngineeSTAT3 IB a. In conSTAT3 IB b. To exareatment c. Additireatment d. The coic model e, suggesAnti-cancer therapeutic exosomes could be targeted to cancer cells or tissues in a \u201cpassive\u201d or \u201cactive\u201d manner . The pasNanoparticles ~100 nm in size are known to be delivered to tumor parenchyma by a mechanism called the \u201cenhanced permeability and retention\u201d (EPR) effect ,61. ConsRecent studies have suggested that exosomes can demonstrate natural tumor targetability depending on their cells of origin. Exosomes derived from different parental cells possess different tissue and organ tropisms in vivo ,67. AddiHowever, Smyth et al. suggested that tumor-derived exosomes harbor tumor targetability only when injected locally into the tumor parenchyma . They obActive targeting of anti-cancer therapeutic exosomes to cancer cells or tissues could be achieved by the following two main strategies: one is a non-genetic approach that directly engineers the surface of exosomes via diverse exogenous methods, and the other is a genetic approach that non-directly engineers exosomes by genetically modifying exosome-producing cells  77,78,7,778,79.The surface of exosomes can be directly modified via various chemical or physical engineering methods. Targeting moieties can be labeled to the exosomal surface either by covalent attachment methods such as \u201cclick chemistry\u201d or through non-covalent methods ,80. ClicPTEN mRNA by cellular nanoporation demonstrated a two-fold increased accumulation in orthotopically implanted U87 glioma in nude mice and prolonged survival with a median survival of 49\u2009days, compared with the 37\u2009days median survival reported for non-engineered exosomes [PTEN mRNA showed 1.5-fold higher accumulation in orthotopically implanted U87 glioma in C57BL/6 mice and increased survival with a median survival of 45\u2009days, compared with 31\u2009days for non-engineered exosomes [The surface of exosomes can be engineered indirectly by genetically modifying exosome-producing cells. The indirect engineering of exosomes has several advantages over direct surface modification in terms of the expression yield and stability of targeting moieties displayed on exosomes ,91. The exosomes . In addiexosomes .G12D targeting siRNA-loaded iExosomes expressing high levels of CD47 (CD47high exosomes) by overexpressing CD47 in normal fibroblast-like mesenchymal cells to generate \u201carmored\u201d exosomes that are able to evade phagocytosis by monocytes [high exosomes exhibited a two-fold increased retention in the circulation of mice 3 h after an intraperitoneal injection compared to CD47 knockout exosomes, the retention of which was abrogated by anti-CD47 antibody blocking the interaction of CD47 with SIRP\u03b1 but not with the non-blocking anti-CD47 antibody [Interestingly, Kamerkar et al. generated KRASonocytes . CD47higantibody .Overall, expressing specific cancer-targeting moieties on the surface of exosomes by conjugation with exosomal membrane-associated domains such as GPI, C1C2 domain, Lamp2b, and tetraspanins, could serve as a promising strategy for the active targeting of anti-cancer therapeutic exosomes to cancer cells and tissues.As exosomes are cell-derived nanoparticles for the delivery of therapeutics, considerations including the selection of proper exosome-producing cells, the development of efficient cargo loading technology, and the establishment of large-scale production and purification systems should be made for the successful entry of exosomes onto the clinical stage. The choice of exosome-producing cells should be considered very carefully to minimize the incorporation of unwanted cellular bioactive cargoes and maximize the loading of the desired therapeutic payloads on a large scale. To increase the cell culture capacity and exosome productivity, suspension cells are preferred for exosome-producing cells because of their feasibility in applying large-scale culture technologies such as various types of bioreactors ,104. MorAmong anti-cancer nanomedicines, lipid-based nanoparticles such as liposomes and polymeric micelles constitute the majority of clinically approved therapeutics . Liposom"}
+{"text": "Extracellular vesicles (EVs) are nano-sized membrane-enclosed particles released by cells that participate in intercellular communication through the transfer of biologic material. EVs include exosomes that are small vesicles that were initially associated with the disposal of cellular garbage; however, recent findings point toward a function as natural carriers of a wide variety of genetic material and proteins. Indeed, exosomes are vesicle mediators of intercellular communication and maintenance of cellular homeostasis. The role of exosomes in health and age-associated diseases is far from being understood, but recent evidence implicates exosomes as causative players in the spread of neurodegenerative diseases. Cells from the central nervous system (CNS) use exosomes as a strategy not only to eliminate membranes, toxic proteins, and RNA species but also to mediate short and long cell-to-cell communication as carriers of important messengers and signals. The accumulation of protein aggregates is a common pathological hallmark in many neurodegenerative diseases, including Alzheimer\u2019s disease, Parkinson\u2019s disease, Huntington\u2019s disease, amyotrophic lateral sclerosis, and prion diseases. Protein aggregates can be removed and delivered to degradation by the endo-lysosomal pathway or can be incorporated in multivesicular bodies (MVBs) that are further released to the extracellular space as exosomes. Because exosome transport damaged cellular material, this eventually contributes to the spread of pathological misfolded proteins within the brain, thus promoting the neurodegeneration process. In this review, we focus on the role of exosomes in CNS homeostasis, their possible contribution to the development of neurodegenerative diseases, the usefulness of exosome cargo as biomarkers of disease, and the potential benefits of plasma circulating CNS-derived exosomes. Extracellular vesicles (EVs) are secreted by all types of cells, including neuronal cells, and can be found in almost all body fluids, including urine, saliva, blood, and cerebrospinal fluid (CSF) . EVs canExosomes originate during the maturation of early endosomes through the inward budding of the endosomal membrane that generates intraluminal vesicles (ILVs), resulting in the formation of late endosomes (LEs) with multiple small vesicles termed MVB . During Exosome biogenesis can occur through an Endosomal Sorting Complex Required for Transport (ESCRT)-dependent mechanism or by an ESCRT-independent pathway. The first relies on multiple proteins that assemble into four ESCRT complexes  and accessory proteins, such as an AAA-ATPase, VPS4 complex, and ALIX that are involved in MVB budding and loading .The exosome biogenesis also occurs through an ESCRT-independent pathway mediated by tetraspanins and ceramide-enriched lipid rafts . TetraspCeramide and its derived metabolites are organized in raft-based microdomains that interact with proteins, such as flotillins. These lipid-enriched structures are involved not only in endosomal membrane invagination for ILV formation  but alsoProteins that are commonly found in exosomes are the ones involved in their biogenesis and are located in the endosomal membrane. The proteins detected in exosome fractions comprise components of the ESCRT machinery such as ALIX, TSG101, and flotillins , proteinUbiquitinated proteins were detected in exosomes  and mighMost of the proteins used as exosome markers are found in exosomes from different sources and are not exclusive to neuronal cells. Despite the enrichment of exosomes with some of these proteins, they can also be found in other types of EVs, as they are membrane-associated proteins . This raLipids in exosomes share similarities with plasma membrane composition, particularly lipids that are normally found in lipid rafts such as plasma membrane composition cholesterol, sphingomyelin, glycosphingolipids, and ceramide . The rafExosomes are natural carriers of genetic material, with almost any type of RNA being found inside, and to some extent also nuclear and mitochondrial DNA (mtDNA) . ExosomeOne of the most important exosome cargoes are miRNAs, which are non-coding RNAs with 17\u201321 nucleotides, that regulate protein expression by binding to the untranslated regions (UTR) of mRNAs thus inhibiting their translation. Several studies have shown enrichment of selective miRNAs in exosomes, which suggests a selective mechanism of miRNA loading during the vesicle biogenesis .The packaging of miRNAs into MVBs is favored by the presence of specific exosome-sorting motifs in the 3\u2032 portion of miRNA that is recognized by RBPs such as the chaperone hnRNPA2B1 (Heterogeneous Nuclear Ribonucleoprotein A2/B1), by a ceramide-dependent mechanism . Also, 3The miRNAs are also players involved in the loading mechanism of mRNA into exosomes. Specific sequences in 3\u2032UTR of mRNA, with approximately 25 nucleotides and a CTGCC motif within a stem-loop structure, act as a sorting sequence to exosomes through a mechanism mediated by miR-1289 .Exosomes also contain mtDNA  and singIn a recent report, the authors employed a novel approach for exosome isolation with a combined methodology of high-resolution density gradient fractionation followed by a direct immunoaffinity capture to selectively isolate exosomes . The stuThe interaction of MVBs with actin and microtubules is essential for their transport to the plasma membrane . The traAfter secretion, the exosomes will dock into the membrane of the target cells and activate signaling events or will be internalized through specific receptor\u2013ligand interactions . The traThe uptake of exosomes by brain cells seems to be cell-type dependent. For instance, neurons and glial cells seem to uptake exosomes by clathrin-mediated endocytosis . Some neIf originally exosomes were considered to be disposable vehicles for the elimination of cellular components, now multiple studies demonstrated that exosomes play multiple physiological roles in the nervous system. Exosomes can be released from all brain cells, including neural stem/progenitor cells , neuronsNeural stem/neural progenitor cells (NSCs/NPCs), found in neurogenic niches, can secrete or uptake exosomes, thus playing key roles in neuronal development . ExosomeExosomes are also important mediators of neuron-to-neuron communication. The secreted exosomes enter the recipient cells and can be re-secreted along with the recipient neuron endogenously formed exosomes, which facilitates specialized long-distance communication within a cell type . NeuronaTrophic support of neurons is assisted by exosomes released from oligodendrocytes and astrocytes . GlutamaExosome-mediated signaling by astrocytes seems to be involved in neurite growth and survival . SynapsiMicroglia, which constitutes the first line of defense during brain injury or infection, releases exosomes enriched in aminopeptidase CD13 involved in neuropeptide catabolism . ExosomeExosomes are also released by the peripheral nervous system, namely, Schwann cells, which were demonstrated to further support local axonal maintenance and regeneration . The preEVs have been a recent object of research interest, resulting until now in several studies that search for connections between their physiological and/or pathological role and the development of neurodegenerative diseases. Several neurodegenerative disorders share other mechanistic features besides the accumulation of insoluble proteins. Exosomes are known to participate in these processes. Finding how the structure, biogenesis, cargo, communication, and final target of exosomes are altered in disease will give insights into their role in disease modulation and importantly enlighten their potential as a source of disease biomarkers. In this section, we describe the latest advances in exosome research in neurodegenerative disorders, as summarized in HTT gene  led to the uptake of RNA but curiously not the protein (HD140Q KI)  . Huntingminase 2 . In HD aminase 2 . On the minase 2 . The exominase 2 . In contminase 2 . AltogetAlzheimer\u2019s disease (AD), the most common form of dementia, has, as a causative mechanism, the accumulation of aggregated \u03b2-amyloid (A\u03b2) in the brain and phosphorylated tau in neurofibrillary tangles . The accAfter evidence of existing A\u03b2 in association with exosomes, the concern was to understand whether these vesicles contribute, as a vehicle, to spreading the pathology or can be protective by clearing the toxic elements associated with AD pathophysiology. Exosomes derived from neuroblastoma cells injected into the brain of the hAPP-J20 AD mouse model were able to trap A\u03b2 bound to the glycosphingolipid surface and later transport to microglia for degradation. These findings supporting the exosome mentioned \u201cclearance role\u201d through the decrease in the levels of A\u03b2 and A\u03b2-mediated synaptotoxicity . AltogetThe levels of certain enzymes and structural components in exosomes promote A\u03b2 elimination by microglial and lysosomal processes, including the insulin-degrading enzyme, ceramide, or the ganglioside GM1 .C)  incubated with exosomes were sequestered to their superficial structure by interacting with surface proteins including the prion protein (PrPC) . The seqC) . Indeed,r for A\u03b2 . Other er for A\u03b2 , 2014. Hr for A\u03b2 .Although the abovementioned studies reveal an eliminatory role for exosomes to discard A\u03b2, C-terminal fragments, and full-length APP, recent data suggest that these vesicles can also transport A\u03b2o, further contributing to the neurotoxicity seen in AD. The use of biological material/samples from AD patients can more accurately help to unravel what specifically happens in the development of the disease other than cellular or animal models. Recently, a study in post-mortem tissue from the temporal neocortex of AD patients showed that flotilin-1 and A\u03b2o co-labeled and there were increased levels of oligomers in exosomes compared to controls . Incubattau phosphorylation and formation of inclusions  and decreased levels of synaptic proteins, as synaptophysin, synaptotagmin, and 25-kDa synaptosomal-associated protein (SNAP-25), in NDEs from AD patients  patients, and AD dementia patients and predicting the risk of MCI progressing to AD dementia  of the striatum and the accumulation of \u03b1-synuclein in cytoplasmic inclusions called Lewy Bodies (Parkinson\u2019s disease (PD) is the most common neurodegenerative movement disorder affecting 0.3% of the general population. The pathological hallmark is the loss of dopaminergic neurons in the substantia nigra y Bodies . The fory Bodies .in vivo caused the enhancement of \u03b1-synuclein release in association with exosomes and the levels of \u03b1-synuclein oligomers  affects both upper motor neurons and lower motor neurons leading to motor and extra-motor symptoms. The majority of cases is related to alterations in four genes, namely, C9orf72, TARDBP , SOD1 (encoding superoxide dismutase), and FUS (encoding RNA binding protein FUS), which results in aggregation and accumulation of ubiquitinated protein inclusions in motor neurons .G93A mouse model) (ALS was first associated with exosome trafficking in 2007 when a study showed the transport of both WT and mutant Cu/Zn superoxide dismutase (SOD1) by exosomes in a cell model for ALS (mouse neuron-like NSC-34 overexpressing human mutant SOD1) . Furthere model) . Moreovee model) . In accoe model) . Additioe model) . These eThe continuous search for the role of exosomes in ALS motivated concerns about the plausible spreading of other pathological agents in the disease than the mutant protein mainly implied, SOD1. Further analysis showed that TDP-43 protein was transported through exosomes isolated from the CSF of ALS patients  to U251 human glioma cells . The uptThe identification of miRNA  with altered levels in both neuronal-derived and total exosomes from ALS patients (CSF and plasma) gave some insight into novel targets to overcome the pathology . NonetheIn the last decade, the research into the exosome\u2019s biological characteristics has seen exponential growth. Exosomes are secreted from virtually all types of cells acting as mediators of intercellular communication physiologically. Interestingly, evidence has been reported that exosomes are involved in the development of human neural circuits by regulating signaling pathways. The maturation and function of neurogenic niches are highly regulated by miRNA  and most of them were found in exosome cargo from different types of cells . MoreoveIn contrast, exosomes have been suggested to be important players in the propagation of neurodegenerative diseases by providing a mechanism for the spreading of toxic content such as misfolded, aggregated forms of proteins. In the last few years, a causal connection between exosomes and the development and progression of CNS diseases such as AD, PD, prion disease, multiple sclerosis, traumatic encephalopathy, and stroke, among others, has been explored . These dA major pitfall in the exosome field is the technical difficulties in the isolation and characterization of \u201cpure\u201d exosomes. The exosome cargo is enriched in nucleic acids, proteins, lipids, amino acids, and metabolites that vary according to the cell of origin. The heterogeneity among preparations and the possible contamination of EVs/exosome fraction with body fluid contaminants might interfere with the characterization of vesicular cargo. Our knowledge about exosome research is challenged due to the lack of reliable and accurate isolation protocols, the lack of selective biomarkers, and the lack of high-resolution characterization techniques. Routine isolation methods include ultracentrifugation, density-gradient centrifugation, and ultrafiltration with all having their own advantages and disadvantages, and a universally accepted methodology for exosome isolation is still an intense debate. Identifying and separating different subpopulations of exosomes from specific cellular types will provide tremendous advantages in defining an exosome biosignature to act as a diagnostic biomarker. Isolation of neural-derived exosomes from human plasma is a particularly attractive method to improve the specificity of disease fingerprints. The immunoprecipitation of neuronal exosomes by antibodies targeting the marker proteins NCAM or L1CAM has proven to significantly increase the accuracy of exosome content as a disease biomarker. NCAM and L1CAM are not exclusive from neurons, despite their enrichment, impelling the search for more precise markers that will allow to obtain purer exosome populations from different brain cell types and eventually compare exosome biomarkers in the same sample. Importantly, there is an urge for more robust biomarkers obtained through a minimally invasive method for diagnostic purposes, preferably in the early stages of the disease, to monitor disease progression and to evaluate treatment response.Exosomes are a promising tool with potential clinical application both as a diagnostic biomarker and as a therapeutic agent, particularly as drug/gene delivery systems. For a long time, mesenchymal stem cell-derived exosomes have been used in a myriad of pathological conditions with success . UndiffeExosomes have natural cargo transportation properties, turning them into an excellent vehicle for transferring bioactive molecules such as drugs, proteins, non-coding miRNA, and gene therapeutic agents to specific cells involved in CNS diseases. Exosomes combine a myriad of advantages as nanocarriers including stability, biocompatibility, low immunogenicity, ability to cross the blood\u2013brain barrier, and efficient cellular uptake that can be modulated to target specific cells according to appropriate membrane proteins. The field of nanotherapy centered on nanoparticle-based exosome mimetics has evolved rapidly and can be a promising tool for drug delivery. Of major interest is the incorporation of miRNA into exosomes to be delivered into recipient cells for protective effects. However, despite the advances stressed, there are many challenging points to consider. One is related to the low yield of exosomes obtained from cells since large-scale production of exosomes for clinical use must be achieved. Furthermore, the use of exosomes for therapeutic purposes should clarify the dose\u2013response output and the distribution and most importantly increase the specificity for the target cells. The lack of an efficient method for cargo loading into the exosomes is also a limitation. The current strategies include conjugation of harvested exosomes with drugs by different methods , incubating cells with compounds to be incorporated into the exosomes, or transfection of donor cells with active molecules before the collection of the exosomes. So far, none of the strategies have been validated for clinical use. The short run is expected to have exosome-based therapeutics treating a wide range of maladies. Meanwhile, fully exploiting the exosomes\u2019 potential is critical to define the safest and more efficient modes of delivery into the target tissue, either by systemic administration or by local delivery. Exosomes in circulation are rapidly sequestered by organ or cleared, remaining in the serum less than 5% at 5 min upon intravenous injection .Importantly, the examples discussed above have demonstrated that exosomes comprise a great potential for the diagnosis and treatment of neurodegenerative disorders. Currently, the efforts in the field are focused on not only obtaining exosomes from specific brain cell types but also exploring the possibility of targeting exosomes to a specific neuronal type in the brain. These findings are expected to open up new avenues for pharmacological treatments with minor side effects. Overall, we have come a long way in exosome research, although several obstacles remain to be overcome before transitioning from innovation to widespread clinical use.MB, RV, and CL conceived, wrote, and participated in the revising of the manuscript. All authors have approved the final 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."}
+{"text": "Exosomes derived from various sources can deliver therapeutic agents such as small molecule drugs, nucleic acids, and proteins to cancer cells by passive or active targeting. These exosomes can encapsulate drugs inside the exosomes, extending drug half-life and increasing drug release stability. In addition, exosomes are highly biocompatible due to their endogenous origin and can be used as nanocarriers for tissue-specific targeted delivery. This review discusses recent advances in exosome-based drug delivery for cancer therapy.Exosomes are a class of extracellular vesicles, with a size of about 100 nm, secreted by most cells and carrying various bioactive molecules such as nucleic acids, proteins, and lipids, and reflect the biological status of parent cells. Exosomes have natural advantages such as high biocompatibility and low immunogenicity for efficient delivery of therapeutic agents such as chemotherapeutic drugs, nucleic acids, and proteins. In this review, we introduce the latest explorations of exosome-based drug delivery systems for cancer therapy, with particular focus on the targeted delivery of various types of cargoes. Exosomes, which are membrane structures surrounded by a lipid bilayer with a diameter of about 100 nm, have recently attracted much attention as novel drug delivery nanoplatforms. Exosomes are known as transporters that carry cargos such as nucleic acids, proteins, and lipids as part of the interaction between the parent cell and the distant or adjacent cells to respond to the external environment ,3. As thThe composition of exosomes can represent the biological state of the cells from which they are derived and has been implicated in the physiological and pathological processes of various diseases. In particular, it is known to play an important role in the pathogenesis of cancer, including the communication between multiple cells such as cancer cells and immune cells ,5.Despite their involvement in tumor progression and metastasis, exosomes obtained from both healthy and diseased cells could be utilized as drug delivery vehicles, immunomodulators, etc. ,7,8. ExoExosomes show very high cell uptake efficiency via directly interacting with the extracellular proteins or the direct membrane fusion or internalization . ExosomeBesides membrane proteins, exosomes inherit bioactive molecules that may have therapeutic benefits from donor cells. For example, M2 macrophage-derived exosomes accelerated wound healing by delivering anti-inflammatory cytokines derived from their parental cells to a mouse wound healing model . Milk-deSources of exosomes widely used as drug carriers include cells, blood (body fluids), and food. Therapeutic cargos for cancer therapy such as anticancer drugs, nucleic acids, and proteins are loaded into exosomes harvested from these sources through various loading methods. In addition, for efficient drug delivery, exosomes are being optimized through various exosome surface engineering. In this review, we discuss a state-of-the-art exosome-based drug delivery system for cancer treatment depending on the exosome source, types of cargoes, and exosomal surface engineering .As the field of research on effective drug delivery systems (DDS) development has been grown eagerly, the exosomes possessing low immunogenicity and a high bio-absorption rate are being focused as potent drug carriers. The main purpose of the DDS is to allow the drug to overcome biological barriers  to have high bioavailability in target regions ,26,27,28The physicochemical properties affecting pharmacokinetics of exosomes may differ depending on the type of source of exosomes ,30. TherBasically, exosomes, which are a type of nano-sized extracellular vesicle (EV), are released by almost any type of cell ,52,53. CThe HEK cell line (HEK293T) is the most commonly applicated cell line in the field of biopharmaceutical manufacturing due to its advantageous properties such as ease of growth, non-demanding maintenance conditions, and high transfection efficiency .According to some previous studies, exosomes isolated from HEK293T have membrane resemblances to various tissues in our body  ,56,57,58Since cancer cell lines overexpressed two subtypes of Rab proteins (Rab27a and Rab27b) involved in the process of exosome release , cancer Dendritic cells (DCs) that present tumor antigens to naive T cells are widely used for T cell-mediated immunotherapy but have the disadvantage of a short lifespan after activation . HoweverAmong various cell types known to secret exosomes, mesenchymal stem cells (MSCs) are also considered an ideal source to prepare exosomes for clinical application in that they can be isolated from a variety of human tissues and have a large capacity for ex vivo expansion ,65. The Basically, exosomes contain many types of biological molecules characterized by their parent cells, such as nucleic acids, proteins, and lipids ,68. Thus12 cells/L) exists in each blood unit [TM3000 (41.0%) and INTERFERin\u00ae (31.3%) which have a lower toxicity [According to the previous study from the Le group, red blood cell (RBC)-derived exosomes were suggested as a versatile delivery vehicle for therapeutic RNAs. RBC-derived exosomes have several advantageous properties for clinical application. Briefly, (1) blood units, the source of exosomes, can be readily obtained from blood banks and patients, themselves, as required. (2) Considering a relatively large amount of RBC  are also considered as promising candidates of an exosome source. First, it has been reported that PDEs per se have protective effects on inflammatory disease ,85,86. FExosomes are broadly utilized as drug vehicles for various cancer therapeutic cargoes with the advantages described above. As a delivery system, exosomes with lipid bilayer membranes can safely protect and deliver different cargoes, including anticancer small molecule drugs, nucleic acids, and proteins, in a sustained release manner. In this section, the unique advantages and several examples of exosomes for delivering each cancer therapeutic cargo are presented .Hydrophilic and hydrophobic chemotherapeutic drugs including Dox and PTX have been reported to be loaded in exosomes. A number of accumulating studies have shown that exosome-mediated chemotherapeutic delivery can enhance anti-cancer effects ,89,90,91Dox, one of the most effective anti-cancer drugs, is used for the treatment of leukemia, lymphoma, and many types of solid tumors. However, the clinical use of Dox is very limited due to their poor biocompatibility and serious side effects such as bone marrow suppression and cardiotoxicity. Although many efforts are being made to enhance the biocompatibility and anti-cancer effects of Dox through various nanoparticle technology, there have also been nanoparticle-associated side effects that must be overcome, such as immune response and oxidative stress ,93.As Dox can be easily tracked by its intrinsic fluorescence, it has been well studied in exosome-mediated anti-cancer therapy. Exosomes generated through serial extrusion from doxorubicin-pretreated macrophages show superior anti-cancer effects than free Dox or Dox-loaded liposome groups in the colon adenocarcinoma mouse model . Compare50 than free PTX [PTX is another widely used anti-mitotic agent for malignant tumors such as glioblastoma multiforme and breast cancer ,98. PTX free PTX . In addiBesides PTX, hydrophobic natural compounds such as curcumin are also being studied clinically. Among hydrophobic anti-cancer drugs, curcumin is readily incorporated into the exosomal membranes. According to a recent study, loading curcumin into exosomes derived from intestinal epithelial cells showed potential as an oral drug with improved cellular uptake and intestinal permeability .Gene therapy using nucleic acids such as DNA and RNA is an attractive and promising strategy for cancer treatment. In particular, small RNAs such as siRNA or miRNA generally bind to mRNAs and increase or decrease their activity to regulate gene expression ,117. NanRecently, exosomes are attracting attention as vehicles for gene delivery due to their unique characteristics that can overcome these difficulties. Abundant miRNAs involved in intercellular communication were detected in exosomes , some ofIn addition to the pre-overexpression method, as nucleic acids are highly hydrophilic and membrane impermeable, the electroporation method can be used to form pores in the exosomal membranes to facilitate nucleic acids\u2019 entry into exosomes. In particular, 293T cells were transfected with a plasmid expressing the transferrin receptor-binding peptide, T7, to decorate exosomes and antisense miR-21 was loaded by electroporation . The oncSimilar to natural exosomes, exosome-mimicking technology for nucleic acid delivery has been developed. Nano-sized exosome-mimics were generated by extruding MCF-10A cells through filters of various pore sizes . The yieThe membrane surface composition of exosomes contributes to their high cellular uptake ,123,124.Besides delivering small RNAs (miRNAs and siRNAs), exosomes could also carry long non-coding RNAs, mRNAs, and circular RNAs. Long non-coding RNAs have attracted much attention due to their high stability and role as miRNA sponges for regulating gene expression. For example, long non-coding circular RNAs, which exhibit a relatively low expression rate in hepatocellular carcinoma patients, were loaded into exosomes derived from HL 7702 cells via a pre-overexpression method . The cirNote that the yield of exosomes is one of the major obstacles in the development of therapeutic exosomes. Most exosome studies are conducted with exosomes derived from primary mesenchymal stem cells or immortalized cells. In contrast, RBCs, which make up about 84% of our body cells, feature high exosome yield. The RBC-derived exosomes have the advantage of avoiding immune responses. Usma et al. have recently reported that anti-miR-125 and Cas9 mRNAs were loaded into exosomes derived from RBCs obtained from a type O donor . EngineeOne of the most promising ways to deliver macromolecular proteins is to utilize exosomes. Proteins can be encapsulated into exosomes via the genetic engineering of donor cells or by direct physical loading such as electroporation.Donor cells are transfected with a gene encoding the protein of interest. As a result, the cell synthesizes proteins encoded by the inserted genes, which are subsequently secreted into exosomes. The anti-apoptotic protein survivin plays an important role in the viability in multiple cancer cells. The inactive mutant survivin-T34A acts as an inhibitor of this survivin, initiating the mitochondrial apoptotic pathway in cancer cells. It was demonstrated that exosomes derived from melanoma cells overexpressing survivin-T34A by plasmid transfection induce apoptosis and enhance gemcitabine sensitivity in various pancreatic adenocarcinoma cell lines . ExosomeProteins could also be loaded directly into exosomes. To increase membrane permeability, tyrosinase-related protein-2 (TRP2) was loaded into serum-derived exosomes through a detergent such as saponin or by the electroporation method . ExosomeBesides loading proteins into the hydrophilic inner space, exosomes could also transport therapeutic membrane proteins in the form of their native structures . CD47, aConsidering that exosomes are secreted by cells, they contain cell adhesion molecules, ligands, and lipids that are endogenously expressed in parent cells. Several studies suggest that exosomes have the ability to target their parent cells ,128. HowPassive tumor targeting is the result of the enhanced permeability and retention (EPR) effect due to increased permeability of the tumor vasculature and ineffective lymphatic drainage. However, EPR effects are a controversial concept, with drug delivery efficiencies of less than 1% even in xenograft tumor models . The hetv integrin-specific iRGD (CRGDKGPDC) peptide, which is highly expressed in endothelial cells, was designed to be expressed with the exosomal membrane protein (Lamp2b) on exosomes [Therefore, efficient passive tumor targeting may be an important prerequisite for tumor cell-targeting systems via systemic administration. Strategies to enhance the EPR effect include prolonging the circulation time of exosomes and promoting their extravasation . As in texosomes . These DModification of the exosomal membrane affects their biodistribution and specific targeting ability in vivo. Exosomes can be modified to express various extrinsic targeting ligands and stimuli-responsive components, as well as intrinsic targeting ligands such as glycans and integrins.A fusion protein-based exosome engineering strategy for the treatment of chronic myelogenous leukemia expressing extrinsic targeting ligands has been developed . Althoug4 construct [Another engineering strategy to incorporate targeting ligands into exosome membranes is to use click chemistry such as copper-catalyzed azide-alkyne cycloaddition. The specific ligands of interest can be attached to the exosomal surfaces without the use of solutions which can damage the biological features of exosomes by click chemistry that can attach ligands ,146. Moronstruct . These eBesides adding targeting ligands to the exosome surface, it is also possible to alter the targeting ability of exosomes by removing endogenous surface molecules. Glycosyltransferases, which control the composition of glycome, are strongly associated with cancer progression ,149. GlyEnvironmentally sensitive functional peptides as stimuli-responsive components could also be added to the exosome surface to confer targeting functions. The tumor microenvironment is mildly acidic (pH 5.6 to 6.8) due to glycolysis, hypoxia, and insufficient blood perfusion ,153. TheExosomes offer promising aspects to enhance the delivery of therapeutics due to their unique properties such as their endogenous origin and tissue tropism. Moreover, exosomes could be easily engineered to improve drug loading efficiency and targeting capabilities. Despite these advantages of exosomes, detailed understanding related to the exosome biology is still in its infancy and there is much work to be done in the future.For the therapeutic applications, the choice of exosome source must be made very carefully. Tumor-derived exosomes show remarkable targeting ability against cancer cells and they are loaded with bioactive cargoes that have potential to directly or indirectly promote cancer growth ,156. In Recently, various strategies such as incubation, transfection, sonication, and electroporation have been developed for loading therapeutic cargoes into exosomes. However, current exosome-cargo-loading strategies are not sufficient to satisfy the loading efficiency required for clinical applications. In particular, the simple incubation method is very limited in the type of cargo to be loaded and the efficiency is too low to be utilized in clinical applications. Transfection methods should further simplify the process and reduce the cost of mass production. The current physical treatment, such as electroporation, is the best method for loading nucleic acids such as siRNA or miRNA into exosomes. However, as this process could induce the aggregation and degradation of charged nucleic acids, as well as could change the properties of exosomes, new approaches are needed .Another major obstacle for the clinical application of exosomes is their low yields. In most preclinical experimental studies, exosomes are obtained through cell culture. Although there are some differences depending on the type of donor cells, less than 1 \u03bcg of exosomal protein is produced per ml of culture, which is a level that requires the culturing of huge amounts of cells for clinical trials ,158. To In addition to their potential as drug carriers, exosomes have unlimited potential as biomarkers for cancer diagnosis and prognosis. Indeed, numerous studies have been attempted to explore the various profiles and functions of exosomes and to facilitate their clinical applications ,174,175.Recently, numerous studies have demonstrated that exosomes could be utilized in cancer immunotherapy. Tumor-derived exosomes are considered as a double-edged sword considering they contain both antigens to induce anticancer immunity and factors that can induce cancer progression. Recently, to overcome the risk of such tumor-derived exosomes, exosomes extracted from antigen-presenting DCs have also been utilized. ,179. ForWith their low immunogenicity and strong biocompatibility, exosomes have heralded a new chapter in drug delivery. Conventional delivery methods of anticancer agents, nucleic acids, and proteins for cancer treatment often fail to achieve desired effects due to in vivo degradation of the therapeutic agent and the lack of targeting ability. Taking advantage of the intrinsic advantageous properties of exosomes, numerous studies have demonstrated that exosomes can be used as carriers for drugs or engineered for anticancer therapy. Although several challenges and obstacles in building a commercial exosome-based drug delivery system remain to be elucidated, an understanding of the detailed biological mechanisms of exosomes and further clinical studies will allow them to emerge as a next-generation nanoplatform for cancer therapy."}
+{"text": "The emergence of multiple variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) highlights the importance of possible animal-to-human (zoonotic) and human-to-animal (zooanthroponotic) transmission and potential spread within animal species. A range of animal species have been verified for SARS-CoV-2 susceptibility, either in vitro or in vivo. However, the molecular bases of such a broad host spectrum for the SARS-CoV-2 remains elusive. Here, we structurally and genetically analysed the interaction between the spike protein, with a particular focus on receptor binding domains (RBDs), of SARS-CoV-2 and its receptor angiotensin-converting enzyme 2 (ACE2) for all conceivably susceptible groups of animals to gauge the structural bases of the SARS-CoV-2 host spectrum. We describe our findings in the context of existing animal infection-based models to provide a foundation on the possible virus persistence in animals and their implications in the future eradication of COVID-19. Coronaviridae family, which is part of the largest group of viruses known as the Nidovirales order [During late 2019, a novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged as the causative agent of the current coronavirus disease 19 (COVID-19) pandemic. SARS-CoV-2 is a member of the SARS-CoV-2 is the most recent zoonotic coronaviruses to cause devastation in humans compared to previous coronavirus outbreaks including SARS and MERS-CoV . The gen1 subunit contains the receptor-binding domain (RBD), which enables binding to the host cell receptor, namely angiotensin converting enzyme 2 (ACE2) [Invasion of a host cell is the initial phase in SARS-CoV-2 infection, which is mediated by the spike (S) glycoprotein. S1 and S2 are two subunits of the S-protein, which is a glycosylated type I membrane protein . The S-p2 (ACE2) . Binding2 (ACE2) ,7. Both SARS-CoV-2 and SARS-CoV enter host cells via the interaction with the ACE2 receptor that is expressed on the surface of many pulmonary and extra-pulmonary cell types, including renal, cardiac, intestinal, and endothelial cells ,9. ExpreThe emergence of SARS-CoV-2 variants, particularly those with critical mutations in the RBD of the S-protein fuels the global spread of SARS-CoV-2. Several of these novel variants can spread more easily, particularly those recognized as Variants of Concern (VOCs) by the World Health Organization . In addiDue to a large and diverse number of species possessing ACE2 receptors , it is iFor viral attachment and entry, SARS-CoV-2 efficiently uses various host factors in humans. The SARS-CoV-2\u2019s S-protein binds to the hACE2 receptor more effectively than that of closely related coronaviruses . In addiSARS-like coronaviruses rely on the S-protein for cell entry . The genOn the verge of the S-ACE2 receptor interface, it appeared that SARS-CoV-2 has acquired advantageous mutations in the RBM, resulting in successful cellular entry and higher transmissibility rates, when compared to SARS-CoV. A total of seven unique residues have been attributed to the natural selection of SARS-CoV-2 for human ,22. Out The affinity for the RBM by ACE2 is determined by complimentary charges of the interacting structures. The surface deep channel of the ACE2 receptor and its surrounding ridges are highly negative, containing residues D136, E150, N154, D157, D292, D295, and D299, which have high areas of solvent accessibility (ASA) values. These ridges may provide a possible binding site for the positively charged RBD of the S glycoprotein . Thus faAlthough the overall spatial structure of the ACE2 varies slightly, there is evidence that variations within a selection of the residues are involved in binding with the S-protein. The majority of these variants display a similar binding affinity for SARS-CoV-2; however, the alleles rs73635825 and rs143936283 demonstrate noticeable variations in their intermolecular interactions with the S-protein. These variations adversely affect the stability of the encoded protein complex, resulting in a low binding affinity with the SARS-CoV-2 S-protein. This could possibly cause a change in the overall negativity of the ACE2 surface deep channel, suggesting the possibility of a natural, intrinsic resistance of a certain degree against SARS-CoV-2 [Homo sapiens populations, indicating that binding sites may be specific to taxon groups. Homo sapiens were found to possess the same amino acids as Neanderthals and Denisovans at each corresponding binding site. Conservation of these ACE2 binding sites over time may have provided a survival benefit, perhaps due to the essential role of ACE2 in cardiovascular physiology [We have performed genomic analysis of the hACE2 receptors to assess and compare the amino acids at 30 crucial variable binding sites of the ACE2 receptors based on the analysis of 70 different species. No polymorphisms were detected within ysiology . HoweverIntriguingly, the ACE2 receptor in children demonstrates a reduced binding affinity for the S-protein and exhibits an alternate tissue distribution to adults, which reduces viral entry. The ACE2 receptor expression and affinity increases within the respiratory epithelium with age but has not yet proven to increase the infection susceptibility . HoweverNumerous non-human primates, ranging from our closest ancestors the Great Apes to our more distant relatives the Catarrhine (Old-World) Monkeys, are susceptible to infection with SARS-CoV-2 and are likely to develop mild respiratory symptoms similar to those observed in humans with mild COVID-19. This is anticipated due to the high degree of ACE2 conservation across most non-human primate species. Firstly, the regions of ACE2 which interact with the S-protein of SARS-CoV-2, namely residues 30\u201341, 82\u201384, and 353\u2013357 and contain five critical binding positions, are completely homologous between humans and most non-human primates  have disAlthough susceptibility is undoubtable, the severity of the disease caused by SARS-CoV-2 in non-human primates is less clear. Infections have been established in non-human primates in infection model experiments, and there have also been reports of small outbreaks of mild respiratory disease caused by SARS-CoV-2 in multiple zoos across the world . Unlike Our sequence alignment analyses confirmed existing findings that humans, Great Apes, and Old-World Monkeys share the same 12 critical binding residues in ACE2 and that ACE2 is overall highly conserved across these species, indicating that all of the aforementioned primates will display a certain degree of susceptibility to SARS-CoV-2 infection ,32,36. NRhinolophidae bats, which have been observed to harbour SARS-related coronaviruses [Rhinolophus sinicus, Rhinolophus ferrumequinum, Miniopterus magnate, Pipistrellus abramus, Pipistrellus pipistrellus, Tylonycteris pachypus, Myotis ricketti, and Scotophilus kuhlii [Rhinolophus ferrumequinum and Rhinolophus sinicus [Rhinolophus affinis (horseshoe bats) located in the Yunnan Province in China [Since the SARS and MERS outbreaks in 2002 and 2012, respectively; studies have suggested a link between bats and coronaviruses, specifically aviruses ,41. Diffs kuhlii . In part sinicus . Uniquel sinicus . A recenin China ,43.Bats are recognised for having a low rate of tumorigenesis and the remarkable ability to achieve a bat\u2013virus equilibrium . StudiesRhinolophus ferrumequinum) had a relatively low binding energy (\u221244.47 EEU), implicating their heightened susceptibility to SARS-CoV-2. Bats that were found to possess a specific Y41H/Q42E substitution within the ACE2 receptor achieved a binding energy score of less than \u221247 EEU, making them more susceptible to SARS-CoV-2 infection. This is attributed to the fact that H41 in place of Y41 can no longer form a hydrogen bond to T500, lowering the van der Waals packing energy, and that substitution of Q42 with glutamic acid cannot form hydrogen bonds with G446 and Y449, disrupting the high affinity S-ACE2 interaction and hence clarifying the permissiveness of bats to SARS-CoV-2 [As mentioned before, horseshoe bats are very likely the natural reservoir of SARS-CoV-2, given that its genome shares a high degree of conservation with the bat coronavirus RaTG13) genome . Phyloge genome .Sequence comparison between SARS-CoV-2 and RaTG13 revealed high sequence homology in the S-protein ectodomain, with a low degree of conservation in the S-RBD. Such changes are likely to contribute to the low binding energy required for S-ACE2 binding in bats. Structural analyses of the bat virus (RaTG13) S protein identified a tyrosine substitution instead of Gln493 is unable to form a hydrogen bond with ACE2 Glu35. In addition, Glu484 substitution with threonine cannot form an intramolecular salt bridge with ACE2 Lys31 and a Tyr498 instead of Gln498 leading to inability to form a hydrogen bond with ACE2 Tyr41 .28 and Arg357, were identified to have 10 or more potential amino acids [The ACE2 protein contains 24 essential amino acid sites that facilitate stabilisation of the interaction occurring between the RBD within S1 of SARS-CoV-2 and the protease domain (PD) of ACE2 . Frank eno acids . A totalno acids . Importano acids . Furtherno acids . Notablyno acids .Rhinolophidae family are known to exhibit a high degree of polymorphism. When utilising R. sinicus as a model, Guo et al. [R. sinicus ACE2, whereby nonsynonymous single-nucleotide polymorphisms (SNPs) identified eight variable residues  and the combination of these eight residues result in eight distinct alleles  [ACE2 sequences in the o et al.  reportedles 1\u20138) . Upon stles 1\u20138) . The plales 1\u20138) .Mustela lutreola) and American (Neovison vison) minks have been proven to be susceptible to SARS-CoV-2 infection, resulting in the implementation of mass culling in several countries to prevent transmission of the virus between both mink\u2013mink and mink\u2013human populations. Case studies from 2020 in Danish and Dutch farms revealed that minks can be infected with SARS-CoV-2 and transmit the virus back to humans [European ; Mustela lutreola Protein ID\u2014QNC68911.1 (full length sequence)) revealed that the European mink ACE2 protein is 805 aa similar to hACE2, while the American mink ACE2 protein  is only 471 amino acids long. Investigation of 22 of the documented residues of hACE2 known to interact with SARS-CoV-2 were aligned with those of both European and American mink ACE2 sequences. It was evident that multiple ACE2 residues differed between minks and humans but importantly were still found to interact with the same residues on the S -protein of SARS-CoV-2 [The RBD of SARS-CoV-2 S-protein interacts with the human ACE2 receptor . The seqRS-CoV-2 ,55,56. TRS-CoV-2 ,56. WithRS-CoV-2 , highligRS-CoV-2  suggesteRS-CoV-2 .The interaction of SARS-CoV-2 S1 with the hACE2 protein is associated with five amino acid residues located between positions 353 and 357 . StudiesSeveral variants of SARS-CoV-2 have emerged as a result of mink infections. One variant, known as Cluster 5, was detected in 12 people in Denmark . ClusterOryctolagus cuniculus) were inoculated with SARS-CoV-2 and observed to excrete infectious virus from their airways [Rabbits have been identified to exhibit a strong and consistent ACE2 binding to the S1 subunit of the S protein of SARS-CoV-2, thus implying efficient viral entry . In a re airways , highligMesocricetus auratus), whereby aerosol transmission and direct contact was proven to cause infection in other hamsters [50) to be only five infectious particles [Phodopus roborovskii) has been described as highly susceptible to SARS-CoV-2 and even exhibits severe lung damage, reflective of that observed in human patients [There are several reports suggesting that hamsters have varying susceptibility to SARS-CoV-2. Pathogenesis and transmission have been reported in Golden hamsters  can allow SARS-CoV-2 S-protein binding in rodents [The structures of the SARS-CoV-2 RBD, ACE2 N-terminal helix, and their interactions have now been well characterised . Based o rodents . Therefo rodents .SARS-CoV-2 is able to replicate in the nose and throat of cats, whilst causing pathology in the upper respiratory tract that is associated with inflammation. Additionally, airborne transmission has been documented between cats. Similar to cats, SARS-CoV-2 can also be found in the upper respiratory tracts of ferrets; however, only a poor transmission was documented between individuals . It is hOne of the leading factors mediating the transmission of SARS-CoV-2 from humans to animals is the human activity, which results in ramifications for animal and human health management alongside wildlife conservation. With the viral origin and causative agent of the COVID-19 pandemic having arisen from a natural animal reservoir, the health implications to humans and other animals remains tremendously high . The firAt present, it is plausible to assume that tigers and lions can re-infect humans due to close interactions and a very strong evolutionary association between SARS-CoV-2 in humans and big cat groups . McAloos\u22121) than humans (\u221250.1 KJ\u00b7mol\u22121), signifying that the binding ability of the dog ACE2 receptor to the S-protein on SARS-CoV-2 is weaker than that of hACE2 [Domestic animals such as cats and dogs that are in regular close contact with humans can be susceptible to SARS-CoV-2 infection. Analysis of SARS-CoV-2 infected beagle dogs concluded that replication of SARS-CoV-2 was poor, and no viral particles were detected in any major organs or tissues. Furthermore, the transmission of SARS-CoV-2 in beagle dogs was also poor, indicating that dogs possess a low susceptibility to SARS-CoV-2 infection . The ACEof hACE2 . This reof hACE2 . It is iof hACE2 . HoweverPaguma larvata) were identified and confirmed as the main intermediate host for the previous coronavirus outbreak, caused by SARS-CoV. Unsurprisingly, research has determined that palm civets are also susceptible for infection by SARS-CoV-2. The palm civet ACE2 receptor displays a binding affinity to the S-protein (\u221237.1641 KJ\u00b7mol\u22121) based on the free binding energy analysis compared to dogs (37.389 KJ\u00b7mol\u22121) and humans (\u221237.389 KJ\u00b7mol\u22121) [Paguma larvata ACE2 amino acid sequence only shows 83.48% similarity to the hACE2 receptor sequence and differs in six of the key amino acid residues required for S-protein binding [Palm civets (J\u00b7mol\u22121) . However binding .As previously mentioned, cats and dogs are both susceptible to SARS-CoV-2, as evidenced via human-to-animal transmission ,73. DespThe types of animals used as livestock vary globally, but livestock are typically always in close proximity with humans. If these animals were susceptible to infection with SARS-CoV-2 and are able to transmit the virus, this could pose a huge threat to food security. Viruses have previously spread via the clinical infection of livestock and subsequently via their meat and dairy products, such as tick-borne encephalitis . HoweverComputer modelling and analysis indicates that ACE2 proteins of different livestock show a high similarity to hACE2 . NotablyLimited studies have been performed on other livestock species. Alpacas have shown sero-reactivity following inoculation with SARS-CoV-2, but it is unclear if they could naturally be infected with the virus . Camels,Investigation into the ACE2 sequence of livestock species and comparison of sequence similarity to hACE2 has been undertaken. Investigation of all livestock species demonstrated variations in amino acid structure sequence from that of the hACE2 receptor. Two substitutions were observed in goats and sheep, whereby a methionine to threonine substitution was identified as semi-conservative with a potential impact on ACE2 structure, feasibly as a result of an increased capacity to form hydrogen bonds. The same may apply to cows because, even though they exhibited three substitutions, only the methionine to threonine substitution would appear to have a structural impact, again through changes to the ability to form side chain hydrogen bonds. Pigs, camels, and alpacas all exhibit variation from hACE2 at four amino acid sites. Substitutions in pigs were associated with either complete loss of the ability to form H-bond side chains at that site, as well as changes in hydrophobicity, side chain flexibility, or from nonpolar to polar (and vice versa) amino acids. In pigs, the H34L substitution may be of particular significance, leading to a loss of ability for ionic bonds, H-bonds, and aromatic stacking. Similarly, in camels, one of these substitutions is associated with a complete loss of H-bond potential, whilst others are associated with a gain from 0 to 3 side chain H-bonds. Substitutions in alpacas are principally associated with changes in H-bond side chain formation and side chain flexibility. These modulations are potentially sufficient to adapt ACE2 structure and change binding affinity with the SARS-CoV-2 S-protein. Overall, it can be determined that there is no direct evidence that livestock currently possess the potential to act as the intermediate host for SARS-CoV-2. However, observation should be maintained to prevent any future risks that could arise from mutations in the S-protein of SARS-CoV-2 variants.Gallus gallus), turkeys , and ducks (Anas platyrhinchos). After inoculating the birds with SARS-CoV-2, they were monitored for virus presence for 7 days post-infection and for antibodies 14 days post-infection. Results showed that SARS-CoV-2 could not replicate within the birds and that no antibodies were generated in response to the inoculation. Studies by Schlottau et al. [Alpha- and Betacoronaviruses are known to have the capacity to infect mammals but are typically unable to infect birds, whereas Delta- and Gammacoronaviruses are usually able to infect birds and subsequently cause disease . Howeveru et al.  have fouu et al. .Alexander et al.  have ideOther key substitutions between human and bird ACE2 residues include position 79 in hACE2, which is a positively charged lysine, whilst all bird species have polar asparagine residues. In hACE2 Tyr83, a nonpolar aromatic tyrosine residue binds with Asp83 in the RBD of the S-protein through hydrogen bonding. However, in five analysed birds, this is substituted with an aromatic phenylalanine residue . AnalysiThere is a relatively high conservation of ACE2 receptors, with 65% identity of the key residues involved in binding the S-protein of SARS-CoV-2 across five different bird species. Discrepancies in bird ACE2 conservation include duck and vultures possessing a negative Glu24 residue, whilst chickens and turkeys have a polar Gln24 residue. Despite having a different amino acid at this position, these residues are similar in size and, in some cases, polar residues can substitute for charged residues with minimal impact on its function. Additionally, ducks have an aliphatic Met27, chicken and turkeys have a polar threonine at this position, and vultures have an aliphatic isoleucine. These residue alterations exhibit major changes, as two are non-polar and one is polar, which will impact on the electrostatic interactions and therefore could change the ACE2 protein conformation. Markedly, vultures are unusual in that they possess a negative Glu30, yet ducks, turkeys, and chickens all possess aliphatic alanine at this position, which is a much smaller residue lacking charged groups. Similarly, most birds have an aliphatic Val34, whilst vultures have polar proline at this position, which can influence protein conformations due to its rigid ringed structure. Position 38 of the ACE2 receptor displays similar trends, whereby most birds have a negative aspartate, but vultures have a polar asparagine, although this alteration may have less of an impact as it is similar in size and charge. Another important residue in which birds display polymorphism is residue 82. Ducks have a polar asparagine at this site, vultures have a polar serine, while turkeys and chicken possess a positive arginine. These residues significantly differ in size, which has the potential to influence ACE2 receptor-S binding, although they display similar charged properties, which may reduce its impact on the protein. Furthermore, vultures have a positive Lys322, whilst other bird species have a polar asparagine, and these residues share similar bonding properties. Most birds possess a negative Glu325, but turkeys have an aliphatic alanine. The final key polymorphic residue worth mentioning is 329, whereby ducks possess a positive lysine, chickens and turkeys have a polar threonine, and vultures have a polar asparagine. Despite sizing differences, overall the displayed properties here are quite similar and as such the binding capabilities of ACE2 receptors are unlikely to be majorly impacted by these amino acid substitutions. Overall, the majority of examined residues in avian receptors appear to be largely conserved, and where they are not, polymorphic substitutions often retain similar binding properties and are hence unlikely to affect the binding ability of ACE2 to the S-protein ,91.Overall, amongst the bird species studied, there is relatively little difference between the key amino acid residues in their ACE2 receptors, with turkeys and vultures markedly differing the most in their residues. However, there is a significant difference in the amino acid sequence between hACE2 and the ACE2 receptor of birds that is worth noting because there are many substitutions at the residues involved in binding the S-protein, and little to no conservation is retained of the binding properties, size, and charges of these residues. This suggests that, despite some homology within the overall sequence of the ACE2 receptor, birds are unlikely to be susceptible to SARS-CoV-2 infection as their key S binding residues are unlikely to bind as efficiently to SARS-CoV-2, although it is difficult to completely guarantee this as the amino acid sequence is not the only determining factor in ACE2-S binding .Manis javanica) have been previously reported as feasible intermediate hosts due to their known capability to host a betacoronavirus that shares a 90% sequence identity with SARS-CoV-2 [Pangolins . Alterations to these key binding residues are likely to contribute to the relatively low binding energy observed for pangolin ACE2 to SARS-CoV-2 S-protein. However, favorable interactions are extensively formed, including thatE38 could form two hydrogen bonds with Q498 and Y449, E30 and E24 could form a hydrogen bond with K417 and N487, respectively and S34 could form a hydrogen bond with Y453. It can be concluded that although pangolin ACE2 does facilitate binding with SARS-CoV-2, it only does so at a very low capacity, and additional research is required to identify the intermediate host of the SARS-CoV-2.Snakes had once been considered as the original source, or an intermediate host, of SARS-CoV-2, but this was proven not to be the case . StudiesThe Root Mean Square Deviation (RMSD) of 14 key residues influencing the binding of ACE2 in different species to SARS-CoV-2 has been investigated by Fang et al.  to deterStructural and genetic analyses highlight the potential of different animals in harbouring SARS-CoV-2 infections and potential back and forward transmission of the virus. These insights suggest that the transmission of SARS-CoV-2, particularly its emerging variants, to previously unknown animals, poses the risk of the generation of alternate viral reservoirs. Similar to mink-associated re-emergence of infection in human, the zooanthroponotic transmission of SARS-CoV-2 is a likely event, particularly at the tail-end of the pandemic. Owing to myriads of anthropogenic factors, including climate change, deforestation, and urbanization, the emergence of future viruses is inevitable, and stopping zoonotic and zooanthroponotic spillover of viruses warrants global efforts in reducing the contact between human populations and wildlife. In addition, health should be considered as multifactorial system that can be influenced by the environment, pathogens, and human activity."}
+{"text": "Dysregulated epigenetic modifications play a critical role in cancer development where TRMT112 is a member of the transfer RNA (tRNA) methyltransferase family. Till now, no studies have revealed the linkage between TRMT112 expression and diverse types of tumors. Based on TCGA data, we first probed into the relation between TRMT112 and prognosis and the potential role of TRMT112 in tumor microenvironment across 33 types of tumor. TRMT112 presented with increased expression in most cancers, which was significantly prognostic. Furthermore, TRMT112 was associated with tumor-associated fibroblasts in a variety of cancers. Additionally, a positive relationship was identified between TRMT112 expression and multiple tumor-related immune infiltrations, such as dendritic cells, CD8+ T cells, macrophages, CD4+ T cells, neutrophils, and B cells in lung adenocarcinoma and breast invasive carcinoma. In summary, our results suggest that TRMT112 might be a potential prognostic predictor of cancers and involved in regulating multiple cancer-related immune responses to some extent. Epigenetic modifications have been demonstrated to play an important role in cancer development, whose dysregulation can alter the expression of tumor suppressors and activators . For exaTransfer RNA (tRNA) methyltransferase subunit 11-2 (TRMT112), a small evolutionarily conserved protein, is a cofactor of diverse methyltransferases implicated in ribosomal RNA (rRNA), DNA, tRNA, and protein methylation \u20136. THUMPGiven that tumorigenesis is a complex process, pan-cancer analysis appears to be critical in analyzing any target gene. In the meantime, the associations with clinical outcomes and the underlying mechanisms are also being placed in a significant position. The Cancer Genome Atlas (TCGA), funded by the government, contains functional genomic data for a variety of tumors , which eHere, we initially explored the pan-cancer patterns of TRMT112 expression by using the TCGA project and GTEx database . Furtherhttps://www.oncomine.org) with P < 0.001 and fold change >1.5. Besides, RNAseq data in TPM format from tumor and matching normal samples, respectively, documented in TCGA  and GTEx databases, which were unified and processed by the Toil process [https://xenabrowser.net/datapages/) and then transformed by base-2 logarithms (log2). Subsequently, TRMT112 expression in tumor and normal samples was examined and compared in R (version 3.6.3) software (Student's t-test). Moreover, TRMT112 expression data corresponding to various pathologic stages  were extracted from the GEPIA2 (v2) pane \u201cPathological Stage Plot\u201d  [The TRMT112 mRNA expression pattern in diverse tumors was examined utilizing the Oncomine database  . The TRMhttp://ualcan.path.uab.edu/index.html) is a platform available for cancer data analysis, and it provides protein expression analysis for the dataset from the National Cancer Institute's Clinical Proteomic Tumor Analysis Consortium (CPTAC) [http://vip.sangerbox.com/) [UALCAN ( (CPTAC) . In thisox.com/) .The survival data of TRMT112 for disease-free survival (DFS) and overall survival (OS) in all TCGA tumors were extracted from the \u201cSurvival Plots\u201d pane of GEPIA2. Cancer patients were assigned to the high and low-expression cohorts according to the median TRMT112 expression level. The hypothesis was tested utilizing a log-rank test. The \u201cSurvival Analysis\u201d function of GEPIA2 was utilized to construct the survival plots. Besides, further COX regression models were established to determine the link between clinical characteristics and survival outcomes in head and neck squamous cell carcinoma (HNSC) RNAseq data in the TCGA database.https://cistrome.shinyapps.io/timer/) website was visited to extract the link of TRMT112 expression with tumor-related fibroblasts in all TCGA tumors, followed by the Spearman rank correlation test with calculations of partial correlation (cor) values and P values. Scatter plots and heat maps were utilized to provide a visual representation of the data. Additionally, the link of TRMT112 expression with the infiltration status of 6 immune cells, namely, dendritic cells, CD4+ T cells, macrophages, CD8+ T cells, neutrophils, and B cells, was examined. In the meantime, the association with tumor purity was identified as well.The \u201cGene Module\u201d under \u201cImmune Association\u201d of the TIMER (https://string-db.org/) was visited for protein-protein interaction (PPI) analysis. The following criteria were used to search for TRMT112's target-binding proteins: active interaction sources (\u201cExperimentes\u201d), the maximum number of interactors to show (\u201cno more than 50 interactors\u201d in the 1st shell), the meaning of network edges (\u201cEvidence\u201d), and the minimum required interaction score (\u201cLow Confidence (0.150)\u201d).The STRING web platform (http://timer.cistrome.org/)module \u201cGene_Corr.\u201d The corr values and P values were derived with the help of the Spearman rank test.The \u201cSimilar Genes Detection\u201d function of GEPIA2 was applied to acquire the topmost 100 TRMT112-related target genes across all datasets of TCGA tumors and normal samples, and the \u201cCorrelation Analysis\u201d function was utilized to characterize the correlation of TRMT112 expression with selected genes using the Pearson analysis. Additionally, heat map data for the chosen genes were acquired using the TIMER2 (R package (https://cran.r-project.org/web/packages/VennDiagram/index.html) was used to construct a Venn diagram for cross-analysis.To obtain the genes that both bind and interact with TRMT112, the VennDiagram https://david.ncifcrf.gov/) tool was utilized for analyses of Gene Ontology (GO) annotation and the Kyoto Encyclopedia of Genes and Genome (KEGG) pathways, followed by the ggplot2 package applied for visualization.The DAVID .Results of the CPTAC data showed that TRMT112 proteins in colon cancer, ovarian cancer, and clear cell renal cell carcinoma samples were more abundant relative to that in the respective normal control , and kidney renal clear cell carcinoma (KIRC) .P=0.046), HNSC (P=0.0014), lower-grade brain glioma , LIHC (P=0.0028), and pancreatic adenocarcinoma  (P=0.021).To assess the survival significance of TRMT112, the patients were assigned to comprise two groups according to TRMT112 expression from TCGA data. Higher TRMT112 expression predicted poorer OS among patients with adrenocortical carcinoma  . AdditioP=0.042), KIRC (P=0.042), kidney renal papillary cell carcinoma , HNSC (P=0.01), LGG (P=0.029), and prostate adenocarcinoma   .R package was utilized to detect the link between TRMT112 expression, clinical characteristics, and survival in TCGA-HNSC data. In the univariate COX analysis, TRMT112 expression, radiotherapy, initial treatment effect, and lymphatic vascular infiltration were related to OS; and TRMT112 expression and initial treatment effect were related to disease-specific survival (DSS)   Tables  and S2. ) Tables .R\u2009=\u20090.104), ESCA (R\u2009=\u20090.263), UCEC (R\u2009=\u20090.259), and COAD (R\u2009=\u20090.186); while TRMT112 level was negatively linked to tumor-related fibroblasts in OV (R\u2009=\u2009\u22120.177), PCPG (R\u2009=\u2009\u22120.434), skin cutaneous melanoma (SKCM) (R\u2009=\u2009\u22120.119), PRAD (R\u2009=\u2009\u22120.159), thymoma (THYM) (R\u2009=\u2009\u22120.212), and lymphoid neoplasm diffuse large B cell lymphoma (DLBC) (R\u2009=\u2009\u22120.350) (Being important in the tumor microenvironment (TME), immune infiltrates are significantly involved in the initiation, development, or metastasis of cancer , 21. In \u2009\u22120.350) . The aboR\u2009=\u20090.085, P=5.94E \u2212 2) in LUAD, but presented with a positive link to tumor purity  in BRCA. These results imply that TRMT112 might influence the survival of patients with LUAD and BRCA by interacting with tumor-infiltrating immune cells.Six typical immune cells were selected to evaluate the link between TRMT112 and the infiltration status of immune cells in LUAD and BRCA by the TIMER tool. Positive associations were indicated between the TRMT112 expression and the infiltration status of dendritic cells, neutrophils, CD4+ T cells, macrophages, CD8+ T cells, and B cells in LUAD and BRCA . MoreoveR\u2009=\u20090.45), SCYL1 (R\u2009=\u20090.5), ZNHIT2 (R\u2009=\u20090.5), FAU (R\u2009=\u20090.54), and PRDX5 (R\u2009=\u20090.52) in a positive manner , serves as a cofactor for diverse methyltransferases implicated in rRNA, tRNA, and protein methylation [Human TRMT112 protein, a homolog of hylation , 25. As hylation , 27. Earhylation . NonetheWe observed that TRMT112 showed upregulated expression in many types of tumors. High TRMT112 expression was linked to the adverse OS in HNSC, PAAD, ACC, LGG, and LIHC. Additionally, high TRMT112 expression was also a risk indicator for unfavorable DFS in CHOL, HNSC, KIRC, KIRP, LGG, and PRAD. Notably, the expression of TRMT112 was found to correlate with both OS and DFS in HNSC. In this context, the relationship between TRMT112 and survival of HNSC patients was further investigated with clinical characteristics included in univariate and multivariate COX analyses, which indicated the independent prognostic significance of high TRMT112 expression for poor OS and DSS.The TME exerts crucial roles in tumor proliferation, invasion, metastasis, angiogenesis, metabolism, immunosuppression, and drug resistance , 29. BeiTo discuss the molecular mechanism of TRMT112 in tumor occurrence, we combined data on TRMT112-binding protein and TRMT112 expression-associated genes in all cancers. The data revealed that in the majority types of cancers, TRMT112 expression was positively linked to SART1 expression, which was the only common member in the above two groups. As a bicistronic gene, SART1 participates in the initiation and development of HNSC  and coloIn summary, this is the first study devoted to TRMT112 in pan-cancer, reporting increased expression of TRMT112 in a variety of tumors. TRMT112 may be a potential prognostic predictor. Moreover, our findings also suggest the potential of TRMT112 as an immunomodulatory factor in cancer."}
+{"text": "Lung squamous cell carcinoma (LUSC) has a poor clinical prognosis and lacks effective targeted therapy. This study is aimed at investigating the role of PSMA1  in LUSC. The differential expression genes (DEGs) in LUSC were retrieved from The Cancer Genome Atlas (TCGA) by \u201cedgR\u201d algorithm and by \u201climma\u201d R package. Then, the relationship between genes and overall survival (OS) was explored by the least absolute shrinkage and selection operator (LASSO) and multivariate Cox (multi-Cox) regression. Next, the PSMA1 expression in tissues of LUSC was detected by IHC, qRT-PCR, and western blot (WB). Moreover, the effects of PSMA1 on the proliferation and viability of LUSC cell were explored by cell counting kit 8 (CCK-8) assays, colony formation assays, and flow cytometry (FCM) analysis. All 4421 DEGs were screened by TCGA database, and 26 genes associated with OS were selected by multi-Cox. Based on TCGA database, PSMA1 was highly expressed in tissues of LUSC patients, and OS and FP of patients with PSMA1 overexpression were significantly lower than those of patients with low PSMA1 expression. Furthermore, PSMA1 knockdown significantly decreased the proliferation of LUSC cells and promoted the apoptosis of LUSC cells, and these effects were reversed by PSMA1 overexpression. The results of this project supported that PSMA1 might be a critical gene regulating the development of LUSC and has the potential to be explored as a prognostic biomarker of LUSC. Lung squamous cell carcinoma (LUSC), the second common histologic subtype of non-small-cell lung cancer (NSCLC), is associated with poor clinical prognosis and limited treatment progress . Driver PSMA1 is one of the 17 essential subunits of the 20S proteasome, a multicatalytic proteinase complex with a highly ordered ring-shaped core structure, which is relevant to the survival of MDS and AML cells and plays a critical role in multiple neoplasms . PSMA1 hhttp://xena.ucsc.edu/). TIMER is a database that is capable of analyzing the clinical impact of different immune cells in diverse cancer types [https://cistrome.shinyapps.io/timer). P < 0.05 was deemed statistically significant. UALCAN  is used to export results of gene expression and survival analysis based on TCGA [http://kmplot.com/analysis), which is able to assess the correlation between the expression of genes  and survival [The gene expression data from TCGA and GTEx were all Trans Per Million (TPM) normalized from the raw RNA-Seq data, which belongs to the UCSC Xena project based on a uniform pipeline | > 1. According to the detected prognostic genes, the significant signature associated with survival was further investigated by the LASSO regression model. Then, in order to filter out the independent prognostic factors from these robust genes, multi-Cox regression analysis was performed with the \u201csurvival\u201d R-package.Differential RNA expression between the LUSC group and normal group was identified by \u201cedgR\u201d algorithm and by \u201climma\u201d R package . The criIn this study, all specimens were collected by the Sixth Affiliated Hospital of Xinjiang Medical University, in compliance with institutional consent and Institutional Review Board (IRB) protocols . All 30 pairs of the samples were obtained with a diagnosis of LUSC. Our study was performed in accordance with appropriate data protection legislation and the provisions of the Declaration of Helsinki. Written informed consent was obtained from each patient for the use of tissue samples. Fresh tissues were dissected immediately after surgical removal from patients, flash frozen in liquid nitrogen, and stored at \u221280\u00b0C.2 humidified incubator at 37\u00b0C.The human LUSC cell lines including LTEP-s, NCI-H596, NCI-H520 cells, and the normal lung cell line BEAS-2B were obtained from the National Infrastructure of Cell Line Resource , and the cells were cultured in complete Dulbecco's modified Eagle's medium (DMEM)  supplemented with 10% fetal bovine serum (FBS)  and 1% penicillin/streptomycin (1\u2009:\u2009100) . The cells were preserved in a 5% CO\u03bcg/mL puromycin. The sequences of shRNAs are listed in Following the manufacturer's instructions, the pHB-Basic vector  was used to subclone PSMA1. The PSMA1-overexpressed plasmid was selected with penicillin and verified by sequencing. Moreover, two lentivirus-mediated short hairpin RNAs (shRNAs) were used to silence PSMA1. The qRT-PCR was applied to examine the efficiency; shRNA-1 and shRNA-2 were recognized as the effective shRNAs. shRNA-1 and shRNA-2 were synthesized by RiboBio . We use electrotransfection to transfect cells with PSMA1-overexpressed plasmid by a NEPA 21 electroporator . LTEP-s and NCI-H596 were infected with lentivirus carrying PSMA1-overexpressed vector according to the manufacturer's instructions and then selected with 1\u20094 cells/well for 0\u2009h, 24\u2009h, 48\u2009h, 72\u2009h, and 96\u2009h. Cell viability was detected by CCK-8 reagent (100\u2009\u03bcL/mL medium) with incubation for 2\u2009h, and the absorbance was detected at 450\u2009nm using a microplate reader .BEAS-2B, LTEP-s, NCI-H596, and NCI-H520 cells were plated into 96-well plates with a density of 3 \u00d7 10\u0394\u0394Ct\u2212 method and presented as mean \u00b1 SD of replicates. mRNA was reversed transcribed to synthesize cDNA according to the manufacturer's instructions through PrimeScript\u2122 RT Reagent Kit  on an iCycler iQ system . The primers for PSMA1 and GAPDH primers are shown in The RNAprotect Cell Reagent was applied to isolated total RNA. The qRT-PCR was performed by a QuantStudio\u2122 7 Flex Real-Time PCR System  and NovoStart\u00aeSYBR qPCR SuperMix Plus. The expression of PSMA1 was normalized against that of GAPDH using the 2\u03b2-actin , at 4\u00b0C overnight. Then, a secondary rabbit anti-rabbit antibody (1\u2009:\u20091000) was used to incubate the membranes at the following day for 1\u2009h at room temperature. The ImageJ software was used for quantitation of the immunoreactive bands.The total protein was isolated by modified RIPA buffer  and was quantitated by BCA Protein Assay Kit . Then, the total protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Next, the proteins were transferred to polyvinylidene fluoride (PVDF) membranes and then blocked in 5% milk diluted with TBST for 1\u2009h at room temperature and probed with primary antibodies, PSMA1  and \u03bcm thickness) were heated overnight at 60\u00b0C. The slides were washed with TBS (pH\u20097.4), and antigens were retrieved with Tris/borate/EDTA buffer (pH\u20098-8.5) by heating at 95\u00b0C for 8\u2009min and boiling at 100\u00b0C for 1.5\u2009h. Tissue sections were incubated with anti-PSMA1 antibody (1\u2009:\u2009400 dilution) in TBS containing 1% BSA at 37\u00b0C for 1\u2009h. Antibody reaction was detected with the UltraView Universal DAB Detection Kit (Ventana). Hydrogen peroxide substrate and 3, 3\u2032-diaminobenzidine tetrahydrochloride (DAB) chromogen were used to visualize the complex, because they could produce a dark brown precipitate which was able to be detected by light microscopy. In line with the average stain intensity, immunohistochemical was scored as follows: 3 for dark brown color, 2 for medium brown, 1 for weak brown, and 0 for no staining.Before staining, paraffin sections  conjugated Annexin V and propidium iodide (PI) which is referred to the manufacturer's protocols. The cells were run on either a three-laser BD Canto-II or a four-laser BD LSRFortessa X-20, using FACS Diva software (BD Biosciences). Acquired data files were analyzed using FlowJo 10.01 (BD Biosciences).t test within the 2 groups for statistical significance. The Spearman correlation analysis and distance correlation analysis were conducted to calculate correlation coefficient. In order to assess whether the difference is statistically significant, the log-rank test was used in a calculated way. The difference is deemed to be significant, when the two-tailed P value must be <0.05.SPSS version 21.0 software was applied to analyze data. And the data comparison used unpaired Student's P value < 0.05 and |log\u2009FC| > 1. A total of 4866 differential genes were obtained, including 2437 upregulated genes and 2429 downregulated genes .The merge function in R was adopted to integrate the expression profiles of the 12,912 module genes with the corresponding 360 LUSC patients' survival time and status information. Notably, 40 genes associated with overall survival (OS) were then identified by uni-Cox analysis . AccordiIn this study, the mRNA expression of PSMA1 in LUSC samples and normal samples was compared in the TIMER database  and UALCThe expression of PSMA1 in 30 pairs of LUSC tissues and adjacent normal tissues was detected through qRT-PCR. As shown in The expression level of PSMA1 was further validated in LTEP-s, NCI-H596, and NCI-H520 cells. The qRT-PCR results suggested that the mRNA expression of PSMA1 in LUSC cells was abnormally increased compared with that in the control group . The resIn order to investigate the potential biological functions of PSMA1 in LUSC, the expression of PSMA1 was silenced through transfecting LTEP-s and NCI-H596 cells with shPSMA1-1# and shPSMA1-2#. PSMA1 overexpression was achieved by transfecting LTEP-s and NCI-H596 cells with vector-PSMA1. The expression level of PSMA1 was increased in the vector-PSMA1 group and significantly decreased after PSMA1 knockdown . Then, CLUSC is one main cause of cancer-related mortality all over the word, which was identified to be driven by mutated genes . The theIn our study, the result showed that the PSMA1 expression level was notably accumulated in both LUSC patients' tissue and available data based on TCGA dataset. The expression levels of mRNA and protein of PSMA1 were particularly high in clinical tumor tissues compared to normal tissues. These results suggested that PSMA1 might play a role in the development and progression of LUSC. Next, the functions of PSMA1 were explored to uncover the potential mechanism of its action in LUSC.Patients with LUSC were divided into a PSMA1 low-expression group and PSMA1 high-expression group by the model. The Kaplan-Meier analysis indicated that PSMA1 expression level could be a guide in predicting prognoses and systemic treatments. To further explore the biological function of PSMA1 affecting LUSC processions, the PSMA1 was knocked down and overexpressed in two LUSC cell lines (LTEP-s and NCI-H596) using a lentivirus-mediated shRNA and vector RNA, respectively. Our result demonstrated that PSMA1 overexpression increased the viability and reproduction of tumor cells, suggesting that PSMA1 exerted an oncogenic activity in LUSC procession. Unfortunately, the underlying mechanism of PSMA1 in LUSC was not elucidated in this study, which will be investigated in further studies. It is worth mentioning that collecting more clinical datasets will help to reaffirm the value of PSMA1, which would be in our plans.In summary, we verified that PSMA1 was overexpressed in LUSC patient's tissues. Functionally, PSMA1 overexpression promoted the growth of LUSC cells by promoting the viability and inhibition of apoptosis of the cells, while PSMA1 knockdown had the opposite effects. In the following research, the investigation would be focused on elucidating the mechanism of PSMA1 in the development of LUSC, which will contribute to comprehensive understanding of tumor progression of LUSC and add the extra direction of therapeutic targets for LUSC."}
+{"text": "The intervertebral disc is designated the most important cartilaginous articulation of the vertebral column that functions to withstand compressive biomechanical forces and confer strength and flexibility to the spine. A thorough study of the complex fine structure and anatomic relationships of the intervertebral disc is essential for the characterization of the integrity of each individual structure in the discovertebral segment.\u00a0This elaborate work in human cadavers explores the sophisticated internal structure of the normal intervertebral disc and the discovertebral segment, providing detailed data derived from the dissection of specimens through imaging and close anatomic-histologic correlation.\u00a0Familiarity with the normal appearances and basic functional properties of the lumbar intervertebral disc and discovertebral segment is fundamental for the recognition of aberrations that may have important clinical implications in patients with low back pain.\u00a0In Part I of this article, the anatomic structure and features of the discovertebral complex in adults will be described. The intervertebral disc\u00a0is a fibrocartilaginous structure that forms a symphysis with the adjacent vertebra, comprising the discovertebral segment.\u00a0Although intuitively deemed a simple, bi-component structure that is comprised of the centrally located nucleus pulposus surrounded by the annulus fibrosus, in virtue it is a highly sophisticated structure with intricate hydraulic biomechanical properties  that enaStudies in human cadavers provide solid evidence of a highly sophisticated internal structure of the intervertebral disc .\u00a0This imCadavers: specimens and preparationLumbar spine columns were harvested from 65 fresh-frozen cadavers in one piece with a saw.\u00a0Ages at death ranged from 29 weeks of gestation to 90 years (mean 70 years).\u00a0All specimens contained the vertebral bodies from L1 through S1, resulting in a total of 325 intervertebral spaces, of which 320 were intact.\u00a0Excessive paraspinal soft tissues were removed.\u00a0All cadaveric spines were examined with conventional radiography to determine the presence or absence of surgical intervention or instrumentation, degenerative changes, rheumatologic disorders, vertebral fractures, malignancy, and gross destruction of the vertebral end plate.\u00a0The specimens were deep-frozen at \u221280\u00b0C for 48 hours and then allowed to thaw for 24 hours at room temperature prior to the multimodal imaging studies.MR imaging assessment\u00a0MR images were obtained with 1.5-T superconducting MR units .\u00a0A standard receive-only head or knee coil was used for imaging the entire lumbar spine in the supine position, and three-inch surface dual coils were used for imaging individual intervertebral discs.\u00a0MR images were acquired in the sagittal and axial planes.\u00a0For MR discography, images with fat suppression were acquired with the same technical parameters as those used in pre-contrast MR imaging studies.\u00a0CT images were obtained in the axial- and were reformatted in the sagittal plane.\u00a0Gray scale sonography was performed at the level of each intervertebral disc in axial and sagittal planes with 10-MHz transducers .After imaging, cadaveric specimens were deep-frozen again for 72 hours and subsequently cut into 3 mm-thick sagittal slabs with a band saw so that anatomic sections corresponded closely to MR scans.\u00a0Low-kilovoltage contact radiographs  of each section were obtained with an x-ray unit with fine grain film processed in a normal manner.\u00a0The images derived from each of the methods  were evaluated in a random order and in close correlation to the findings on macroscopic inspection of the anatomic slices.\u00a0Finally, the slices were decalcified in 20% formic acid, and histologic sections were prepared with hematoxylin-eosin (HE), Elastica van Gieson, toluidine blue (pH=7), and Mallory-Azan staining.\u00a0For each of the discovertebral segments, data derived from multimodality imaging were closely correlated to those of the anatomic sections.The intervertebral disc: biochemical considerationsDifferences in the biochemical and histologic composition of the two distinct disc components grant unique biomechanical properties to each of these parts.\u00a0The annulus fibrosus contains a rich network of collagen fibrils arranged in a lamellar pattern.\u00a0Regional differences in the structure of the annulus account for the stiffness of its peripheral portion enduring resistance to axial and torsional stress .\u00a0The nucWith aging, a cascade of biochemical alterations including dehydration of the matrix, changes in proteoglycan composition with an accumulation of metalloproteinases from the degrading collagen, and cellular senescence lead to appreciable morphologic changes in the discovertebral segment associated with alterations in the mechanical properties of tissue ,10,12.\u00a0WThe intervertebral disc and discovertebral segment: anatomic considerationsFormation of the embryonic intervertebral disc is a highly-coordinated developmental process that is ruled by numerous genes and regulatory molecules.\u00a0At the embryonic level, the annulus fibrosus and the cartilaginous end plate are derived from the sclerotome, whereas the nucleus pulposus originates from the endoderm, a remnant of the notochord .\u00a0In the The nucleus pulposus is a semi-liquid structure that occupies an eccentric position with regard to the vertical midline axis of the vertebral body, closer to the posterior border of the intervertebral disc.\u00a0The annulus fibrosus is a multi-lamellar structure (contains 15 to 25 lamellae) engulfing the nucleus pulposus in an elliptical, reniform arrangement ,12.\u00a0The Less thickening of the outer fibers in the anterolateral surface of the annulus fibrosus is seen at the site of wide insertion of the anterior longitudinal ligament.\u00a0We have noted overt pathologic changes of degeneration, calcification, laxity, and rupture of the longitudinal ligaments on anatomic inspection of the specimens in our study.\u00a0Such abnormal morphologic alterations, resulting in structurally weaker ligaments, may play an indisputable role in the pathogenesis of disc displacement.At the discovertebral junction, the outermost anterior fibers of the annulus fibrosus are fixed to the anterior surface of the vertebral body by firm Sharpey fibers ,17.\u00a0In tAn increased frequency of degenerative lesions with advancing age was documented, in our specimens.\u00a0The concept of progression of degenerative change to breach in the cortical bone underscores the difficulty encountered when trying to distinguish among degenerative, traumatic, or malignant invasion of the end plates and, moreover, indicates that a spectrum of pathologic conditions may exist in many cases.\u00a0The vertebral bone marrow varies in cellular composition and appearance according to age.\u00a0At birth, metabolically active red marrow (composed of 60% hematopoietic cells and 40% fat cells) occupies the entire vertebral body, and with aging, it is progressively replaced by metabolically inactive yellow marrow (composed of 95% fat cells and 5% nonfat cells)  and a small field of view (8-10 cm).\u00a0Because of the fine structure of the cartilaginous end plates, improved resolution and detailed sharp definition of the contours of the vertebral end plates were achieved with the use of a surface coil and gradient-echo sequences, in reference to previous postmortem studies  Figure .The value of MR discography  in evaluating disc morphology and abnormalities directly related to discogenic pain has been studied .\u00a0ComplemWe realized that no other imaging method affords direct visualization of the nucleus pulposus itself, a finding that may indeed prove of diagnostic value in the investigation of discogenic pain in living subjects.Sagittal high-resolution sonographic images (n=35) acquired with 10-MHz linear array transducers demonstrate the normal echogenic structure of the disc, sandwiched between the vertebral bodies.\u00a0The nucleus pulposus appears relatively isoechoic or hyperechoic to the annulus fibrosus.\u00a0On the axial plane, the echogenic nucleus pulposus is situated in the center of the disc, and a distinction between the nucleus and the annulus is feasible.\u00a0Axial images show numerous, concentric fine echoes in the outer portion of the annulus fibrosus, corresponding to its multilayered architectural structure  Figure .Although post-mortem studies are fundamental to further understanding of the imaging findings as reflected by the anatomic sections, this study in cadavers has some weaknesses.\u00a0First, we used standard MR imaging sequences to delineate basic anatomy in great detail, similar to that revealed in the anatomic studies.\u00a0Dynamic contrast-enhanced imaging, spectroscopy, diffusion-weighted imaging, or other MR imaging techniques that are utilized in living subjects aimed at biochemical tissue analysis and functional assessment of anatomy in the spine are not actually applicable in our study material ,25,27-30The anatomy of the intervertebral disc and the discovertebral segment is quite complex.\u00a0The results of our imaging-anatomic-pathologic correlative study provide insights into the internal structure of the intervertebral disc.\u00a0We have concluded that good-quality radiographs and CT images are useful in the depiction of the bony end plates.\u00a0Distinct components comprising the intervertebral disc can be depicted with MR imaging in detail similar to that revealed in anatomic studies of cadavers.\u00a0MR discography is the only imaging technique that allows a dedicated depiction of the nucleus pulposus.\u00a0An understanding of the anatomy of the intervertebral disc may provide a basis for further characterization of alterations in discal structure, igniting symptoms and disability."}
+{"text": "Rapastinel as the allosteric modulator of N-methyl-D-aspartate receptor (NMDAR) produces rapid antidepressant-like effects dependent on brain-derived neurotrophic factor (BDNF) and VGF (nonacryonimic) release. Herein, we further explore the molecular mechanisms of the antidepressant effects of repeated administration with rapastinel in mice. Our results showed that continuous 3-day rapastinel  produced antidepressant-like actions dependent on the increase in extracellular regulated protein kinase (ERK)/mammalian target of rapamycin (mTOR) signaling and downstream substrates p70S6 kinase (p70S6k) and the eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), which may induce the expression of VGF and BDNF in the hippocampus and prefrontal cortex of mice. Furthermore, compared with a single treatment, our data indicated that 3-day repeated rapastinel treatment produced antidepressant-like actions accompanied by potentiation of ERK/mTOR/VGF/BDNF/tropomyosin-related kinase receptor B (TrkB) signaling. Based on previous and our supplementary data that showed the pivotal role of on \u03b1-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) in the rapid release of VGF and BDNF and activation of TrkB by a single dose of rapastinel, we postulate that the antidepressant-like effects of single or repeated administration of rapastinel may result in the rapid release of VGF and BDNF or ERK/mTOR signaling pathway-mediated VGF/BDNF/TrkB autoregulatory feedback loop respectively. Our current work adds new knowledge to the molecular mechanisms that underlie the antidepressant-like actions of rapastinel in mice. Major depressive disorder (MDD) is a chronic and devastating mental disorder with a high prevalence and incidence . DelayedPrevious research had indicated that ketamine has a rapid antidepressant effect in patients with drug-resistant MDD \u20139. KetamIt used to be thought that rapastinel (formerly GLYX-13) is a novel glutamatergic compound that acts as an NMDAR modulator with glycine-like partial agonist properties \u201314, likeAn increasing number of studies have demonstrated that BDNF and VGF (nonacryonimic) proteins are decreased in animal models of depression , 29\u201332. Adult male C57BL/6J mice  were reared in the animal facility of Ningbo University School of Medicine, China. All animals were maintained at 22\u2009\u00b0C\u2009\u00b1\u20092\u2009\u00b0C and 60%\u2009\u00b1\u20095% relative humidity under a 12-h-light/-dark cycle (lights on at 7:00 am) with ad libitum access to food and water when the stressors were not applied. Required animals were estimated based on our past experience of performing similar experiments and animals were randomly assigned to treatment groups. All stressors were applied to animals outside their housing areas in a separate procedure room. All procedures involving animals were conducted in accordance with the National Institute of Health Guidelines for the Care and Use of Laboratory Animals  and the European Community Council Directive for the Care and Use of Laboratory Animals of September 22, 2010 (2010/63/EU). All experiments were approved by the Institutional Animal Care and Use Committee of Ningbo University, School of Medicine.The following drugs were used: rapastinel  and PD98059 . The doses of rapastinel were based on previously reported clinical and preclinical studies , 14 and The first set of experiments were performed to compare the differences between repeated and single treatment with rapastinel in mice  that was divided by two cross black lines drawn on the floor. Mice were placed in the arena and allowed to explore for 5\u2009min. The number of the line crossings and rearings were considered as parameters of locomotor activity and were recorded over the 5-min period by a digital system.SPT is a method to evaluate anhedonic-like behavior in animals based on sucrose taste preference. All mice were trained to adapt to the two bottles, one containing 1% sucrose solution and the other with fresh water for 24\u2009h. The positions of the two bottles were randomly placed and were exchanged to prevent the formation of position preference. After adaptation, the mice were deprived of water for 24\u2009h. The mice were housed in individual cages and had free access to two bottles containing sucrose solution and water. After 16\u2009h, the volumes of the sucrose and water were measured and the sucrose preference rate was calculated as a percentage of the amount of sucrose intake/(water intake\u2009+\u2009sucrose intake) \u00d7\u2009100%.NSFT was conducted over a 6-min period. Briefly, mice were food deprived for 24\u2009h and then placed in a novel environment (50\u2009\u00d7\u200950\u2009\u00d7\u200940\u2009cm Plexiglas chamber). The latency to approach and feed on a single food pellet  placed on a white paper platform positioned at the center of the brightly lit box was measured.The mice were placed individually for 6\u2009min into a clear plastic cylinder  filled with 10-cm depth of water (maintained at 23\u2009\u00b1\u20092\u2009\u00b0C). The immobility time was recorded over the following 4\u2009min of the 6-min testing duration. The immobility time was defined as time when mouse floated with only active activity necessary to keep their heads above the water.After the last behavioral task, the animals were immediately sacrificed for biochemical assays.g for 30\u2009min at 4\u2009\u00b0C. The protein concentration of each sample lysate was determined using a BCA kit . Each sample  was separated on 10% SDS polyacrylamide gel electrophoresis gels and transferred to PVDF membranes . The membranes were then incubated with anti-phospho-ERK1/2 (Thr204/187) , anti-ERK1/2 , anti-phospho-mTOR (Ser2448) , anti-phospho-p70S6K (Thr389) , anti-phospho-4E-BP1 (Thr37/46) , and anti-GAPDH  at 4\u2009\u00b0C overnight. The membranes were then incubated with an Alexa Fluor 800-conjugated antibody  for 60\u2009min. Target bands were captured and quantified using a fluorescence scanner .Frozen hippocampal and PFC tissues in each group of mice were homogenized in ice-cold radioimmunoprecipitation assay lysis (RIPA) buffer containing protease and phosphatase inhibitor cocktail . Lysates were centrifuged at 12,000\u2009\u00d7\u2009Immunofluorescence was performed to quantify the expressions of BDNF and VGF in the hippocampus and PFC, respectively. Mice (5 per group) were anesthetized with pentobarbital and then transcardially perfused with cold 0.9% saline, and their brains were removed and fixed in phosphate-buffered 4% paraformaldehyde. Mouse brain coronal sections of the hippocampus and PFC (30-\u00b5m thick) were collected using a cryostat  and were first permeabilized with 2% Triton X-100 in PBS for 30\u2009min, then incubated with PBS containing 5% donkey serum for 1\u2009h at room temperature, followed by incubation in diluted primary antibodies used for immunostaining including VGF  and BDNF  overnight at 4\u2009\u00b0C. Twenty-four hours later, the primary antibodies were removed and washed in PBS and then incubated with secondary antibodies  in blocking buffer for 1\u2009h at room temperature. Brain sections were then stained with 4,6-diamidino-2-phenylindole (DAPI) for 15\u2009min, mounted onto slides, and coverslipped with ProLong Gold Antifade Mountant (Invitrogen). The images were analyzed using a confocal laser scanning microscope .P\u2009<\u20090.05 was considered statistically significant.All data statistical analyses were performed using GraphPad Prism  and are presented as means \u00b1 standard errors of the mean (SEM). The significance was assessed by one-way ANOVA followed by Bonferroni multiple comparison. P\u2009<\u20090.01), 7 (P\u2009<\u20090.01), and 14 days (P\u2009<\u20090.01) after withdrawal of rapastinel \u2009=\u20099.088, P\u2009<\u20090.01, Fig. F \u2009=\u20097.005, P\u2009<\u20090.01, Fig. F \u2009=\u200934.04, P\u2009<\u20090.01, Fig. F \u2009=\u200912.76, P\u2009<\u20090.01, Fig. F \u2009=\u20094.362, P\u2009<\u20090.05, Fig. Our data showed that CUS paradigm substantially decreased the line crossings \u2009=\u200962.32, P\u2009<\u20090.01, Fig. F \u2009=\u2009119.5, P\u2009<\u20090.01, Fig. F \u2009=\u200948.25, P\u2009<\u20090.01, Fig. F \u2009=\u2009147.9, P\u2009<\u20090.01, Fig. F \u2009=\u200938.30, P\u2009<\u20090.01, Fig. F \u2009=\u200960.29, P\u2009<\u20090.01, Fig. F \u2009=\u200979.96, P\u2009<\u20090.01, Fig. F \u2009=\u200986.71, P\u2009<\u20090.01, Fig. F \u2009=\u200940.42, P\u2009<\u20090.01; CA2/3, F \u2009=\u200931.75, P\u2009<\u20090.01; DG, F \u2009=\u200918.39, P\u2009<\u20090.01; As shown in 3O, PFC: F \u2009=\u200938.42, P\u2009<\u20090.01) and VGF \u2009=\u200919.92, P\u2009<\u20090.01; CA2/3, F \u2009=\u200929.44, P\u2009<\u20090.01; DG, F \u2009=\u200927.72, P\u2009<\u20090.01; As shown in 3O, PFC: F \u2009=\u200991.80, P\u2009<\u20090.01) were significantly reversed by repeated rapastinel treatment in mice.The effects of rapastinel on the expressions of p-ERK1/2, ERK1/2, p-mTOR, p-p70S6k, p-4E-BP1, BDNF, and VGF in the CUS-exposed mice were observed in the hippocampus Fig.  and PFC F \u2009=\u200919.07, P\u2009<\u20090.01, Fig. F \u2009=\u200914.02, P\u2009<\u20090.01, Fig. P\u2009<\u20090.01) and rearings (P\u2009<\u20090.05). Additionally, for sucrose preference in the SPT, the increase in sucrose consumption by rapastinel was completely blocked by pretreatment with the antagonist PD98059 \u2009=\u200973.17, P\u2009<\u20090.01, Fig. F \u2009=\u20097.151, P\u2009<\u20090.05, Fig. F \u2009=\u200917.87, P\u2009<\u20090.01, Fig. We explored whether ERK-mediated signaling was required for repeated treatment with rapastinel-produced rapid antidepressant-like effects and the expression of BDNF and VGF in mice \u2009=\u200969.28, P\u2009<\u20090.01, Fig. F \u2009=\u200936.07, P\u2009<\u20090.01, Fig. pocampus, F \u2009=\u200931.93, P\u2009<\u20090.01, Fig. F \u2009=\u200963.17, P\u2009<\u20090.01, Fig. F \u2009=\u200920.25, P\u2009<\u20090.01, Fig. F \u2009=\u200940.35, P\u2009<\u20090.01, Fig. F \u2009=\u200929.22, P\u2009<\u20090.01, Fig. F \u2009=\u200941.44, P\u2009<\u20090.01, Fig. P\u2009<\u20090.01). No significant alteration was noted in total ERK1/2 levels in any group (data was not shown). Additionally, the immunohistochemical results revealed that the downregulation of BDNF and VGF in the CA1 \u2009=\u200955.98, P\u2009<\u20090.01, Fig. F \u2009=\u200988.99, P\u2009<\u20090.01, Fig. F \u2009=\u200924.12, P\u2009<\u20090.01, Fig. F \u2009=\u200916.33, P\u2009<\u20090.01, Fig. F \u2009=\u200978.43, P\u2009<\u20090.01, Fig. F \u2009=\u200943.77, P\u2009<\u20090.01, Fig. F \u2009=\u200959.42, P\u2009<\u20090.01, Fig. F \u2009=\u2009137.3, P\u2009<\u20090.01, Fig. Western blot results indicate that rapastinel (10\u2009mg/kg) significantly reversed the decrease in p-ERK1/2 , accompanied by potentiation of ERK/mTOR/VGF/BDNF/TrkB signaling. However, a single-dose treatment with rapastinel induced antidepressant-like actions only within 1 week. Comprehensive analysis of our findings, in the short-term, revealed that repeated rapastinel therapy may be a better dosage regimen to maintain and to extend its antidepressant effects dependent on ERK/mTOR/VGF/BDNF/TrkB signaling. Our results shown that continuous 3-day rapastinel produced significantly antidepressant-like actions. In consistent with the recently work, which found that repeated administration of rapastinel produces exceptionally prolonged rescue of memory deficits in phencyclidine-treated mice . Given tTo confirm the dose-dependent effect of rapastinel on the antidepressant actions and enhancing of the ERK/mTOR/VGF/BDNF/TrkB signaling, we aimed to investigate the antidepressant effect of rapastinel in the CUS model after 3-day repeated administrations. We examined the antidepressant-like effects of rapastinel, used as a reference compound at three doses , based on a previous report for testing rapid antidepressant effects in mice. Consistent with our previous study , our preAs reported previously, the exceptional profiles of the antidepressant-like activity of rapid antidepressants were related to the immediate activation of molecular processes that lead to enhanced neuroplasticity, mainly in the PFC and hippocampus , 48, 49,Increasing evidence supports a pivotal role of the ERK subclass of mitogen-activated protein kinase (MAPK), especially ERK1/2, which has been most thoroughly investigated and characterized in the PFC and hippocampus of depressed animals and has played a pivotal role in various neuropsychiatric disorders, including MDD \u201352. HereIn line with our expectations, the effects of rapastinel were significantly blocked by intracerebroventricular injection of the MEK/ERK inhibitor PD98059. The upregulation of phosphorylation of mTOR, ERK1/2, p70S6K, and 4EBP1 after repeated treatment with rapastinel in the hippocampus and PFC was abolished by PD98059. Additionally, the increase in VGF and BDNF levels by repeated rapastinel treatment was blocked by PD98059. The rapid and long-lasting antidepressant effects of rapastinel demonstrated that ERK/mTOR signaling cascades led to a significantly increase in protein translation and protein synthesis  and strengthened VGF/BDNF/TrkB signaling in a self-reinforcing autoregulatory loop that could be required for one potential mechanism underlying the efficacy of rapid-acting antidepressants . ImportaA growing body of evidence demonstrates that NMDAR-mediated synaptic plasticity and metaplasticity play important role in the pathophysiology and for antidepressant activity outlasting drug presence , 69, 70.In conclusion, our results indicated that repeated rapastinel treatment exerts significantly antidepressant-like effects in mice and the effects may be mediated partly through the ability of rapastinel to enhance VGF and BDNF expression and then the VGF/BDNF/TrkB autoregulatory feedback loop by increasing the ERK/mTOR pathway (The working model is shown in Fig. Supplementary data"}
+{"text": "Drosophila melanogaster. These neurons have a morphology where they innervate a patchwork of glomeruli. We used electrophysiology to determine whether their nonspiking characteristic is because of a lack of sodium current. We then used immunohistochemsitry and in situ hybridization to show this is likely achieved through translational regulation of the voltage-gated sodium channel gene, para. Using in vivo calcium imaging, we explored how these cells respond to odors, finding regional isolation in their responses\u2019 spatial patterns. Further, their response patterns were dependent on both odor identity and concentration. Thus, we surmise these neurons are electrotonically compartmentalized such that activation of the neurites in one region does not propagate across the whole antennal lobe. We propose these neurons may be the source of intraglomerular inhibition in the AL and may contribute to regulation of spontaneous activity within glomeruli.Inhibitory interneurons are important for neuronal circuit function. They regulate sensory inputs and enhance output discriminability . Often,  Drosophila melanogaster. The role of the nonspiking characteristic of similar interneurons in other species is not fully understood. Further, the sources of specific regulatory mechanisms such as intraglomerular inhibition in the fly are unclear. The characterization of nonspiking interneurons in Drosophila begins to explain these mechanisms and provides an avenue for further study into the roles of similar cells across species.These findings are a novel discovery of nonspiking interneurons specifically in the olfactory system of adult Drosophila melanogaster. The AL is the first olfactory relay of the fly, where olfactory receptor neurons (ORNs) with the same receptor converge  are prevalent throughout animal nervous systems and often serve to regulate circuit function. One such example are the LNs in the antennal lobe (AL) of the fruit fly converge  and synaconverge  in neuroconverge . Odor liconverge . LNs regconverge .Drosophila. By inhibiting ORNs, LNs allow the full dynamic range of ORN spiking to be used across a broad range of odor concentrations : 103 NaCl, 3 KCl, 5 N-tris(hydroxymethyl)methyl-2-aminoethane-sulfonic acid, 8 trehalose, 10 glucose, 26 NaHCO3, 1 NaH2PO4, 1.5 CaCl2, and 4 MgCl2 (adjusted to 270\u2013275 mOsm). This solution was bubbled with 95% O2/5% CO2 and adjusted to a pH of 7.3. Pipettes were pulled to 7\u201311 M\u03a9 resistance from thin-walled borosilicate glass . For current clamp recordings, pipettes were filled with an internal solution containing (in mm) 140 potassium aspartate, 10 HEPES, 4 MgATP, 0.5 Na3GTP, 1 EGTA, and 1 KCl. For voltage clamp recordings, the internal solution contained (in mm) 140 CsOH, 140 aspartic acid, 10 HEPES, 1 EGTA, 1 CsCl, four MgATP, and 0.5 Na3GTP. Internal solution osmolarities were adjusted to 265 mOsm with a pH of 7.2. Preparations were illuminated during patching via a fiber-optic oblique infrared LED (Thorlabs), and cells were targeted by their expression of mCD8::GFP under binary expression systems. All recordings were digitized at 10\u2009kHz. Current clamp recording data were low pass filtered at 5\u2009kHz with an AM Systems model 2400 amplifier (AM Systems). After breaking in, resting potentials of spiking cells were adjusted to elicit baseline firing rates observed in previous cell-attached recordings. Nonspiking cells were adjusted to similar potentials. Voltage clamp data were low pass filtered at 1\u2009kHz and cells were held at \u221260\u2009mV. Current signals were conditioned using positive/negative subtraction  was used to specifically block voltage-gated sodium channels. During voltage clamp recordings, synaptic transmission was blocked with CGP-54\u2009626 , mecamylamine , and picrotoxin . Experiments involving these drugs were done using a recirculating perfusion system.To determine sodium-dependent spikes and currents, TTX  was used to detect RNA transcripts. Probes for n MATLAB .GCAMP7b  was exprFor temporal analysis of calcium imaging data, we calculated four metrics; response latency, half rise time, half decay time, and response duration. The response latency was defined as the time between odor onset and the first peak in the derivative of the \u0394F/F trace during the odor period. Half rise time was the time between the response onset as determined in the latency calculation and when the \u0394F/F value was half of the peak \u0394F/F value. Half decay time was the duration in which the \u0394F/F value remained above half of the peak value after reaching the peak. Response duration was calculated as the time between the \u0394F/F values determined as the half rise and half decay.z scores to reduce the contribution of response strength to the correlation, thus biasing the result to focus on activation patterns.Because the focal plane was not always consistent between preparations, principal components analysis (PCA) and correlation coefficients were calculated within individual fly responses and then compared across flies. For input into principal components analysis and correlation calculations, the average peak \u0394F/F frames were concatenated row-by-row from the frame\u2019s pixels into 1-dimensional vectors for each fly. The resulting vectors each had the same number of elements as pixels in the average frames. These vectors were then inserted into a matrix for analysis for each individual fly, with columns representing trials and rows representing corresponding pixels. The explained variance in PCA and correlation coefficients were then compared between flies. The images are sample PC scores reshaped into the dimensions of the input images. For correlation coefficients, \u0394F/F values were normalized as Drosophila LNs are not well suited to achieve strong space clamp, we were still able to observe TTX-sensitive sodium currents reliably in spiking R70A09-Gal4 LNs  to amplify and detect potentially weak transcript signals. We verified our probe\u2019s specificity for para transcripts by staining Drosophila larval central nervous systems  and odors which activate a restricted subset of ORs , with varying degrees of similarity in activated glomeruli. Peak responses were selected as the highest \u0394F/F value during the odor presentation period and averaged from the three frames centered around the peak. We hypothesized that within each fly, spiking lines would respond with the same spatial pattern for each odor, as action potentials would propagate their activity across the entire antennal lobe. For nonspiking LNs, we hypothesized that activation may be spatially restricted as the graded potentials would not propagate as actively. This would result in activation patterns which would spatially vary across odors within a given fly. Consistent with previous literature, the NP3056-Gal4 and R70A09-Gal4 spiking lines produced calcium responses that largely activated the entire visible region of the AL  on the odor response \u0394F/F activation patterns. As the focal plane in wide-field imaging is not consistent between preparations, we ran PCA on individual fly response patterns on a pixel-by-pixel basis. The percentage of variance explained by each principal component (PC) could then be averaged across flies. For individual flies, we saw only one major pattern in PC1 when PC scores were projected back onto the AL for the spiking lines A,B whileNext, we compared how similar odor response patterns were within each fly by calculating the spatial pattern correlation coefficients between odors for each fly. Intensities were normalized to calculate only the difference in pattern regardless of the response strength. For spiking LN lines, we saw the response patterns had high correlation between odors, meaning the responses were all spatially similar E,F. Coef\u22124 (Stronger concentrations of an odor activate more ORs  and beca\u22124 D. Howeve\u22124 D.Drosophila AL. While not uncommon in other insects, nonspiking LNs were not previously described in the early olfactory circuitry of the fly. We have demonstrated their unique nonspiking characteristic through electrophysiology, immunohistochemistry, and in situ hybridization. Further, we have investigated the potential physiology of these neurons through calcium imaging during odor responses.In this work, we report the observation of nonspiking patchy LNs in the Drosophila, it was previously assumed the entire population of LNs in the adult AL were spiking (Drosophila AL LNs which fail to fire action potentials. This contrasts with other insects, such as the locust in which nonspiking cells are the prevalent olfactory LN type (Drosophila as most neurons of the larval brain are para negative (Drosophila brain have also been identified as nonspiking. For instance, a variety of neurons, including interneurons, in the fly visual system are nonspiking (para transcript level is low (While neurons which fire action potentials are considered the norm, nonspiking olfactory interneurons are present across many insect species . In Dros spiking . Here, w LN type . Other s LN type . Early inegative . Other nnspiking . The lacnspiking , and arenspiking . Each APnspiking . The APLnspiking  and its l is low , supportDrosophila neurons only start spiking in the adult, some amacrine cells start as spiking cells during development then lose spiking when the retina reaches maturity (Nonspiking cells are also common in noninsect organisms. For example, several amacrine cells of the vertebrate retina are nonspiking . Like olmaturity . They armaturity  as well maturity , and thematurity .Patch-clamp electrophysiology revealed these cells lack detectable voltage-gated sodium current, and immunohistochemistry showed this is because of a downregulation of the voltage-gated sodium channel Para. Under strong odor stimulation and current injection, we did not observe action potentials in R32F10-Gal4 LNs. This was true for multiple recordings within flies, across flies, and between the Gal4 and LexA versions of the R32F10 promoter fragment driver. Taken together, these data confirm that our observation of nonspiking cells was not an artifact of our recording setup or technique but rather a reproducible result.Drosophila express a single voltage-gated sodium channel, Para (Drosophila LNs are particularly susceptible to poor space clamp as their processes are small and spike initiation sites are often located far from the soma. Despite this, we were consistently able to see TTX-sensitive currents from spiking cells which have similar physical attributes to nonspiking LNs despite poor space clamp, and therefore we attribute our result to the physiology of the cells and not the recording technique.el, Para , which iel, Para . We did para expression in R32F10-Gal4 patchy LNs. The results from this experiment further validate the absence of sodium current. Para has been previously shown to localize in distal axon segments which resemble mammalian action potential initiation sites (para translation may be a common theme in nonspiking neurons in Drosophila. Similar to the R32F10-Gal4 LNs, multiple classes of nonspiking cells in the Drosophila visual system continue to express para transcript but fail to generate typical sodium action potentials (Consistent with the nonspiking phenotype, we also used the FlpTag technique  to demonon sites , and ourtentials .para transcript. It is important to note transcription and translation are not necessarily correlated. In developing Drosophila, it has been shown that genes which have downregulated transcripts may have upregulated protein products and vice versa (para expression in these cells is subject to post-transcriptional regulation. In larval flies, translational repressor Pumilio works with Nanos and Brain Tumor to regulate para transcripts in motoneurons (Although R32F10-Gal4 LNs lack sodium channels and current, they still produce ce versa . Therefooneurons , and we Drosophila AL. In addition to their nonspiking characteristic, this population of LNs has the patchy morphologic pattern. This morphology is distinct from other anatomic patterns of LNs, as the processes innervating a given glomerulus tend to be isolated from the neurites in other glomeruli (Nonspiking LNs could serve a variety of functions in the lomeruli . Furtherlomeruli . From oulomeruli .Drosophila LNs innervate many glomeruli (One potential role for nonspiking LNs is intraglomerular inhibition . This islomeruli  and therDuring odor responses, the population of nonspiking LNs showed patterns of activation which did not correlate between odors. This contrasts with spiking LNs, which have been shown both in this work and in previous literature to have pan-glomerular, odor-identity independent activation during responses . This wiDrosophila AL.Increasing concentrations of odorants activate more ORNs . We obse"}
+{"text": "Food fortification with synthetic folic acid (FA), along with supplementation, results in a marked increase in the population total of serum folates and unmetabolized folic acid (UMFA). Despite the success in reducing neural tube defects at birth in the intended target population (women of childbearing age), the potential deleterious effects of chronically high levels of UMFA in susceptible segments of the population require further investigation. In this study, we examine the effects of FA concentrations, ranging from depletion to supraphysiological levels, on markers of proliferation, DNA methylation, and DNA damage and repair in a human lymphoblastoid cell line (LCL). We note that both low and high levels of FA similarly impact global DNA methylation, cytome biomarkers measured through the CBMN assay, DNA damage induced by oxidative stress, and DNA base excision repair gene expression. An optimal intake of dietary folate is essential as mammalian cells lack the enzyme required for folate biosynthesis ,2. FolatC. elegans revealed that high doses of FA induce severe oxidative stress and an accumulation of homocysteine (Hcy) [While folic acid was previously considered safe, with little or no known systemic toxicities, emerging evidence reveals that chronically elevated levels of FA are associated with deleterious effects ,10. Somene (Hcy) , while one (Hcy) . In a mone (Hcy) . In this study, we tested whether excessive folic acid exposure to a human lymphoblastoid cell line can mimic folate restriction and induce functional folate deficiency in vitro. Folate deficiency leads to uracil misincorporation into DNA, generating point mutations, single- and double-strand breaks, and chromosome breakage ,30,31. F2 at 37 \u00b0C. Cells were initially grown in a medium containing a final concentration of 300 nM FA for 3 passages before seeding cells in either 12, 180, 300, 2300, or 10,000 nM FA.The human lymphoblastoid cell line (GM16113) , was cultured in a folate-free RPMI-1640 medium , supplemented with 10% dialyzed FBS, 1% penicillin/streptomycin (5000 IU penicillin/5 mg streptomycin), 1% glutamax, and 1% sodium pyruvate, and incubated in a humidified atmosphere with 5% COCells were seeded at the selected concentrations in a 12-well culture plate, and counted after 4 h, as T = 0, and then after 24, 48, and 72 h using trypan blue and an automated cell counter, TC20 TM . The cell population doubling time (DT) is reported as the mean of 3 days and was calculated using the formula: DT = In2/In (Xe/Xb), where T is the incubation time in any units, Xb is the cell number at the beginning of incubation time, and Xe is the cell number at the end of the incubation time. g for 10 min at 4 \u00b0C. The cell pellet was homogenized on ice in 1 mL ice-cold 1\u00d7 PBS, then centrifuged at 10,000\u00d7 g for 15 min at 4 \u00b0C. The supernatant was removed and stored on ice. The homocysteine level was determined using a commercially available Homocysteine ELISA kit , according to the manufacturer\u2019s instructions.Cells were washed twice with ice-cold 1\u00d7 PBS and then collected by centrifugation at 2000\u00d7 \u00ae Genomic DNA Mini Kit , following the manufacture protocols. LINE-1 methylation assay was performed using a Global DNA Methylation LINE-1 kit . Briefly, 100 ng Msel-digested genomic DNA was hybridized to the LINE-1 probe and immobilized onto a streptavidin-coated plate. After the binding of primary and secondary antibodies, data were obtained and analyzed through a colorimetric plate reader reaction by comparison to a standard curve of methylated and nonmethylated DNA.Genomic DNA was isolated using a PureLinkThis assay was adopted from protocols published by Thomas and Fenech . Cells wFor the second part of the experiment involving the exposure of LCLs to hydrogen peroxide and the CBMN assay, we followed the protocols published by Main et al. ,41. Brie\u00ae Reagent . First-strand cDNA synthesis was performed using an ImProm-IITM Reverse Transcription System . The mRNA expression level of selected genes was assessed with quantitative real-time PCR (qPCR) , and normalized to the geometric mean of HPRT1 and \u00df-actin using the 2\u2212(\u2206\u2206Cq) method. Primers were validated and tested using external standards for each gene and prepared by subcloning using a TOPO\u00ae TA Cloning\u00ae kit . Primer sequences are provided in Total RNA was extracted from LCLs using TRIzolp-values of less than 0.05 were considered statistically significant.Statistical significance between means was determined using one-way and two-way ANOVA, followed by Tukey\u2019s post hoc test when appropriate . Any p < 0.0001). There was no significant difference in the measured doubling time across all other concentrations . Supraphysiological supplementation at 10,000 nM FA also reduced methylation levels (p < 0.05). There was no significant difference in methylation across the 180, 300, and 2300 nM FA concentrations (Long interspersed nuclear elements (LINE-1) are mobile parasitic genetic elements that comprise 17% of the human genome, and their methylation levels are considered a surrogate marker of global genomic DNA methylation. Folate depletion has been associated with decreased lymphocyte methylation . Supplemtrations d.p < 0.05). Supplementation with the lowest level of FA (12 nM), representing a folate-deficient state, resulted in the highest score across the three scoring criteria, indicating extensive genomic damage (p < 0.0001). Supraphysiological FA supplementation  resulted in a significant increase in MNi compared to the midrange FA concentrations , an increase in NPBs, compared to 300 nM FA (p < 0.005), and an increase in NBUDs, compared to 300 and 2300 nM FA (p < 0.05) . The CBMN assay measures endpoints for DNA damage, such as micronuclei (MNi), which are a biomarker of chromosome breakage and/or whole chromosome loss; nucleoplasmic bridges (NPBs), which are a biomarker of DNA misrepair and/or telomere end-fusions; and nuclear buds (NBUDs), which are a biomarker of elimination of amplified DNA and/or DNA repair complexes. MNi in lymphocytes are very sensitive to small changes in micronutrient status, including folate, which makes them a robust biomarker to identify the impact of folate status on genome stability ,47,48. C < 0.05) b\u2013d. FurtUNG, or UDG), followed by a multistep process culminating in the repair of the damaged sites by the rate-limiting enzyme DNA polymerase \u03b2 (Pol\u03b2). Our previous work revealed that folate deficiency can impact and regulate the base excision repair pathway [RAD21 is a key gene involved in cellular S-phase arrest and the repair of double-strand breaks by homologous recombination (HR) and sister chromatid cohesion [POL\u03b2, UNG, and RAD21, compared to all other levels of FA supplementation (p < 0.05)   a\u2013c. Howe < 0.05) a,b. Lymphocytes are accepted as a sensitive model with which to examine the effects of folate status on genomic stability markers such as strand breakage, microsatellite instability, methylation, and uracil misincorporation ,53,54,55C. elegans revealed more intricate dynamics: both folate deficiency and over-supplementation disrupted folate homeostasis by favoring thymidylate synthase over the methionine synthase cycle [C. elegans [Folate is crucial for nucleotide synthesis and DNA methylation, and it is involved in multiple pathways that are essential for cellular proliferation and homeostasis. We noted that folate depletion (12 nM FA) resulted in a significant increase in doubling time in our LCLs. However, no significant difference in the cellular proliferation rate was observed across the rest of FA-supplemented cell cultures. Some suggested that excess FA can cause an increase in UMFA, leading to saturation of DHFR and impacting the dynamics of the one carbon cycle . A studyse cycle . This im elegans . AlthougAssessment of DNA methylation through the LINE-1 assay revealed that both FA depletion (12 nM) and supraphysiological FA  produced a decrease in global methylation compared to 180 nM FA. While folate depletion is known to be linked to global hypomethylation of DNA , the linUNG and POL\u03b2, revealed a similar trend where FA excess mimicked the effect of FA depletion , is one of the standard methods of cytogenetic and genetic toxicology testing . The MNiepletion a,b, and ome loss ,70. Thesome loss ,69,71. Etrations c, thoughOur analysis revealed that FA supplementation can differentially impact how LCLs react to genotoxic stress within the optimal range of FA concentration. These concentrations  are representative of proposed optimal culture conditions. Exposure to hydrogen peroxide revealed that cells incubated in 300 nM FA media experienced the least damage e\u2013g. ThisIn summary, our findings corroborate what others have uncovered in various models. Deficient and excessive levels of FA similarly impact global DNA methylation, cytome biomarkers, and DNA repair gene expression. While the specific mechanism underlying the observed effects requires additional research, the accumulation of evidence from recent reports warrants a fresh perspective on the role of folic acid fortification in health and disease."}
+{"text": "Triticum aestivum L.) belonging to one of the most diverse and substantial families, Poaceae, is the principal cereal crop for the majority of the world\u2019s population. This cereal is polyploidy in nature and domestically grown worldwide. Wheat is the source of approximately half of the food calories consumed worldwide and is rich in proteins (gluten), minerals , vitamins (B-group and E), riboflavin, niacin, thiamine, and dietary fiber. Wheat seed-storage proteins represent an important source of food and energy and play a major role in the determination of bread-making quality. The two groups of wheat grain proteins, i.e., gliadins and glutenins, have been widely studied using SDS-PAGE and other techniques. Sustainable production with little input of chemicals along with high nutritional quality for its precise ultimate uses in the human diet are major focus areas for wheat improvement. An expansion in the hereditary base of wheat varieties must be considered in the wheat breeding program. It may be accomplished in several ways, such as the use of plant genetic resources, comprising wild relatives and landraces, germplasm-assisted breeding through advanced genomic tools, and the application of modern methods, such as genome editing. In this review, we critically focus on phytochemical composition, reproduction growth, types, quality, seed storage protein, and recent challenges in wheat breeding and discuss possible ways forward to combat those issues.Wheat ( It isWheat is of supreme importance among cereals mainly because of its grains, which comprise protein with exclusive physical and chemical attributes. It also encompasses other useful components, such as minerals , protein, and vitamins , and is also a valuable source of carbohydrates . HoweverWheat production and quality could possibly be enhanced through the development of new and improved varieties that are able to produce a superior yield and perform better under various agro-climatic stresses and conditions . It is t1.1.Wheat was first cultivated approximately ten thousand years ago during the \u2018Neolithic Revolution\u2019, which saw a shift from hunting and collecting food to stable land management. Diploid, i.e., genome AA, einkorn, and tetraploid, i.e., genome AABB, emmer, were the first types of wheat to be grown and, according to their hereditary relationship, they originated in the southeastern regions of Turkey , 19. CulThe previously grown forms of wheat were essentially landraces from wild populations that were carefully chosen by farmers, probably because of their higher yields. However, domestication is also linked with the genetic trait selection of wheat, which is detached from that of its wild ancestors. Two characteristics are of significant importance, the first being spike-shattering loss at maturity, which causes a loss of seeds at the time of harvest . It is aTriticum aestivum L. em Thell, 2n\u2009=\u200942, AABBDD) is almost 1.7\u2009\u00d7\u20091,010 base pair. It is approximately 100\u00d7 greater than that of the genome of Arabidopsis, 40\u00d7 that of rice, and nearly 6\u00d7 that of maize . The modern name, Triticum aestivum, represents hexaploid bread wheat with genomes A, B, and D, differentiating it from tetraploid macaroni wheat, which is Triticum durum, comprising genomes A and B, and is consumed predominantly for the production of pasta. Nowadays, bread wheat (Triticum aestivum) is the most extensively grown wheat. It is a hexaploid type of free-threshing wheat (genome AABBDD). According to Nesbitt and Samuel, it stemmed from the recent hybridization of the diploid (DD) Aegilops tauschii var. strangulate and an allotetraploid wheat (AABB) no longer than 8,000\u2009years ago . Wheat has been cultivated in the form of spring or winter crops. In extremely cold areas, spring varieties of wheat have been propagated during spring so that they can grow and ripen rapidly and can be harvested before the arrival of the autumn snowfall. Within more temperate areas, winter wheat is propagated prior to the onset of the winter snowfall that otherwise covers the saplings, resulting in vernalization and allowing quick growth when the snow thaws in the spring. In warm environments, peculiarity in spring and winter wheat is almost futile. The point of significant difference is early or delayed maturity , such as Vrn-B1, Vrn-A1, and Vrn-D1, and their superficial adjustment by negligible flower-inducing genes . Spring Wheat development is largely determined by temperature, the requirement of a cold phase, variety, and plant responses to the corresponding lengths of dark and light periods during their developmental phase. As previously stated, winter wheat variety maturation was found to be accelerated due to the flowering process in plants, i.e., with low-temperature exposure, usually 3\u201310\u00b0C, for 6 to 8\u2009weeks. Growth has also been enhanced through long day exposure which meaning growth is enhanced through longer period of light as the days lengthen in spring. As the varieties differ in their responses to vernalization, temperature, photoperiod, and the extent of interaction between certain factors, they vary continuously in their maturation rate and, therefore, contribute to the broader distribution and adaptation of wheat in agriculture globally , 43.1.4.1.4.1.Wheat seeds require moisture levels of 35\u201345% for germination , 45. Dur1.4.2.More than one node can grow in the soil based on sowing depth, all exhibiting roots . The roo1.4.3.After germination, the apex of a vegetative shoot gives rise to secondary leaf primordia. Leaf primordial count can differ from seven to fifteen  and is i1.4.4.Stem elongation overlaps with the growth of tillers, leaves, inflorescence, and roots . Stem elThe peduncle is the last segment to extend. The height of the wheat plant extends from 30 to 150\u2009cm depending on the variety and the propagating state. Alterations in plant stature are generally attributed to the differences in internode dimensions and not to the internode number , 50.1.4.5.The first lateral branches to arise are formed between the coleoptile axils and the first true leaf. Generally, three intervals between two successive leaves divide the leaf emergence and its subtended tiller. In winter wheat, small numbers of tillers develop in winter or autumn if circumstances are moderate. The main shoot and initially developed tillers fulfill their growth and develop granules in spring and winter wheat .1.5.Wheat is principally an intra-floral pollinated crop. However, the rate of outcrossing is up to 10% or greater on the basis of genotype, population density, and environmental conditions. Cross-fertilization due to wind depends greatly on physical aspects such as excessive humidity and warm climates . Dry, wa1.5.1.The shift to propagative growth takes place close to apical cupola elongation, once the core shoot has almost three complete leaves. Floret division initiates in the crucial portion of the spike and continues both up and down as spikelet induction is completed. It creates a growth pyramid inside the prickle, which continues throughout grain growth and anthesis. Terminal spikelet instigation indicates the completion of spikelet formation .During pre-anthesis, various developmental phases synchronize with one another . Kirby i1.5.2.The ratio of multiplication of the endosperm cell is affected by water stress, light intensity, genotype, and temperature , 59. The1.6.Wheat grain is divided into three main segments, all structurally and chemically distinguished from erstwhile. These are: the germ, also called the embryo, which is located at a single end of the grain in the form of a tiny, yellow mound, simply differentiated from the rest of the kernel; the endosperm, which covers a larger part of the whole grain and supplies nutrition to the developing plant as the kernel evolves; and the external seed crust and cover lying underneath, which contains protein cells that cover the whole kernel and protects the embryo and the endosperm on or after injury during the grain\u2019s subsistence (latent phase) . RegardiWheat kernels are usually elliptical, though different types of wheat have kernels that vary from virtually long, trampled, slender, and spherical in shape. The length and mass of the kernel are typically around 5\u20139\u2009mm and 35\u201350\u2009mg. It features a crinkle below the lateral side and it was therefore initially associated with the wheat flower. The wheat kernel  65) enc enc65) eWheat fiber is made up of several layers of cells which, when the seed is dry, adhere so firmly to one another that they are removed by the milling process in comparatively large pieces. By shifting and other mechanical means, almost all the embryo and endosperm are removed. However, as the separation is never perfect, even the purest commercial bran always contains a little endosperm and possibly traces of embryo . ChemicaThe bran, also known as the seed coat, which is present on the external layers of the wheat kernel and composed of numerous layers, is responsible for providing protection to the central part of the kernel. The seed coat is enriched with minerals and vitamin B . The braWheat fiber has a complicated chemical configuration; however, it comprises pentosans and cellulose, polymers founded on arabinose, and xylose firmly attached to the proteins. These elements are typical polymers found in the cell layers, like the aleuronic layer and the wheat cell wall. Carbohydrates and proteins both signify 16% of the bran\u2019s entire dry mass. The value of minerals is somewhat high, at 72%.The two outer strata of the grain, the pericarp and the seed cover, are composed of inactive hollow cells. The internal bran sheet, the aleuronic sheet, is packed with active contents of plant cells . This soThe endosperm forms approximately 84% of the entire seed, its proportions varying with the plumpness of the grain. This part of the seed provides food for the growing embryo. Unlike the constituents of the embryo, those of the endosperm are relatively stable substances, designed by nature to remain unchanged until the germinating embryo draws on them for its first supply of food .via the fused seed coat and the pericarp. In the external endosperm, the aleuronic sheet holds a unique configuration . Wheat germ is obtained as a by product during wheat milling .1.7.The main chemical components of wheat are given below.1.7.1.Vitamin B5, B1, B6, B3, B8, B2, B12, K, E, and A; ascorbic acid; boran; dry ascorbic acid; iodine; sodium; carotene; group 2 metals of the periodic table; magnesium; molybdenum; potassium; zinc; aluminium; copper; phosphorus; sulfur; Iron; and selenium .1.7.2.Superoxide dismutase, protease, amylase, lipase, transhydrogenase, cytochrome oxidase .1.7.3.Amino acids, e.g., valine, asparagine, aspartic acid, alanine, glutamine, proline, methionine, glycine, phenylalanine, threonine, leucine, arginine, tryptophan, isoleucine, tyrosine, serine, histidine, and lysine; mucopolysaccharides; chlorophyll; P4D1, i.e., glucoprotein; bioflavonoids, such as apigenin, luteolin, and quercitin; laetrile; indole complexes; and choline, i.e., amygdalin \u201376.1.8.Wheat grains and their products are significant constituents of our daily diet. The average wheat consumption is 318 grams per person each day, making up 83% of the overall cereal consumption .Wheat contributes a larger percentage of protein than energy to the nutritional requirement of an adult male. Alone, it can fulfill the daily requirement of niacin and thiamine. The majority of the daily riboflavin and iron requirement is fulfilled by the quantity of wheat recommended for an adult male .Wheat is predominantly considered a source of protein, vitamins, calories, and minerals. It is comparable with various cereals in nutritional content. Its protein content is higher than sorghum, rice, and maize and about equivalent to that of other cereals. The protein content is influenced by a variety of cultural and environmental conditions, such as soil temperature, moisture, availability of nitrogen, and method of cultivation. The percentage of protein in wheat can be influenced to a certain extent by the time of fertilizer application and fertilizer type .The nutritional content of protein is estimated not simply based on the concentration of protein but also the amino acid equilibrium within the protein. During human digestion, protein is broken down into its constituents, absorbed by the bloodstream, and then assembled again to form different types of protein required by the body for growth, maintenance, and repair . Eight aThe biological significance of wheat is determined by limiting essential amino acids. These amino acids become deficient due to the body\u2019s increased requirements. Lysine is the deficient amino acid in wheat . During 1.9.Wheat quality has two main characteristics: external quality and internal quality. External quality involves freedom from foreign material and weather damage, type, and purity of color. These factors are used to separate wheat into visual grades . Interna1.9.1.Protein is regarded as the most significant nutrient for animals and humans, as the name of its origin indicates . The protein content varies from 10\u201318% of the entire dry mass of the wheat grain . ProteinWheat proteins have been classified  81) by  by 81) bWater soluble albuminsGlobulins, not soluble in natural water but soluble in diluted solution of sodium chloride whileinsoluble at high NaCl concentrationsGliadins, soluble in 70% ethanolGlutenins, soluble in diluted NaOH or acid solutionsAlbumins and globulins are the smallest wheat proteins. The partition of globulins and albumins was not clear as originally recommended by Osborne. Gliadins and glutenins represent complex proteins of high molecular weight . The maxTraditionally, protein in wheat grains has been divided into prolamins and non-prolamins. The prolamins consist of gliadins and glutenins, while the non-prolamins include salt and water-soluble globulins. Albumin and globulin proteins concentrate during the initial phase of grain development, after which, the content of these proteins remains constant from 10\u201315 days after flowering (DAF) onwards, the albumins and globulins tend to accumulate in the emerging starchy endosperm from 10 to 15 DAF, involving primarily trypsin inhibitors, \u03b1, \u03b2-amylase, and triticins. The characteristics of wheat flour quality depend on the prolamin content and composition in the endosperm, whereas the role of albumins and globulins in the development of flour quality is not defined as well as that of prolamins .Albumins and globulins are primarily metabolic enzymes, which have a role in numerous metabolic events during the course of grain filling, comprising starch synthesis, protein synthesis, folding, and energy metabolism. Storage proteins (gliadins and glutenins) constitute approximately 75% of the overall protein content. Wheat crops accumulate proteins in this way for seedling usage in advance. They are usually found in the starchy endosperm, not in the germ or seed coat sheet. Wheat storage proteins are technically dynamic. They lack enzyme action, but they perform a role in dough development; for example, these proteins are able to hold gas, generating soft baked foodstuff .Albumins and the wheat endosperm\u2019s globulin cover 20\u201325% of the total grain protein. Globulins and albumins have an excellent amino acid equilibrium with regard to nutrition. Several of these such proteins (enzymes) are involved in metabolic actions .Wheat is exclusive among the palatable kernels, as its flour possesses a complex protein known as gluten, which, when prepared as dough, has viscous and elastic characteristics and is essential for manufacturing leavened bread. The rheological characteristics of gluten are required not merely for bread manufacture but for a broad range of foodstuff that is only prepared using wheat, like cookies, pastries, pitta bread, pasta, noodles, etc. The proteins in gluten include monomeric and polymeric gliadins and glutenins. Glutenins and gliadins are considered the main storing proteins of wheat, representing around 75\u201385% of the overall seed proteins, with a ratio of nearly 1:1 in bread and common wheat. They are enriched with proline, glutamine, asparagine, and arginine, but nutritionally significant amino acids, like tryptophan, lysine, and methionine, are present in small amounts .The gliadins, which represent 30\u201340% of all total proteins in flour, are a polymorphic blend of proteins that are soluble in 70% alcohol. They are separated into alpha, beta, gamma, and omega gliadins, with an MW of 30\u201380 kilo Daltons, as defined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The omega gliadin MW is in the range of 46\u201374 kilo Daltons, while alpha, beta, and gamma are low molecular weight gliadins, ranging from 30\u201345 kDa by amino acid sequencing and SDS-PAGE. Recent methodology has revealed a close link between alpha and beta gliadins and, thus, these are frequently called alpha-type gliadins .Gliadins are freely soluble in dilute alcohols, except glutenin polymers; however, their subunits have the ability to be dissolved in a similar way to the gliadins. The subunits of glutenin can be acquired through the treatment of glutenin using a disulfide reducing agent; for example, \u03b2-mercapto-ethanol or dithiothreitol. Gliadins and glutenin subunits both have unexpectedly high levels of glutamine and proline. Hence, it has been suggested that these storing proteins be called \u2018prolamins\u2019, as they display strong similarities with most storage proteins in associated cereals, such as rye or barley. Residues of cysteine have an important role in the structure of gliadins and glutenin subunits. These residues have a role in either disulfide bonds inside similar or different polypeptides, i.e., intra-chain disulfide bonds, or inter-chain disulfide bonds .Gliadins have shown an extremely varied fusion of a monomeric form of gluten proteins. Three anatomically different gliadins, alpha, gamma, and omega, can be illustrated. The evaluation of amino acid sequences has shown that \u03b1-and \u03b3-gliadins are linked to low-molecular-weight glutenin subunits. For that reason, they have been categorized as \u2018prolamins enriched with sulfur\u2019. Residues of cysteines are situated at alpha type six remains of cysteine, and gamma type eight remains of cysteine gliadins have been found at extremely preserved sites and have a role in preserved intra-chain bonds of disulfide. Conversely, cysteine residues are absent in \u03c9-type gliadins and possess a very small amount of methionine. So, these gliadins have been termed \u2018sulfur-poor prolamins\u2019 .Triticum aestivum, the Glu-A1 locus encodes null (N) subunit and 1Ax, while the Glu-B1 locus commonly codes for 1Bx and 1By. Occasionally, Glu-B1 codes for 1Bx or 1By subunits, whereas the Glu-D1 locus codes for 1Dx and 1Dy subunits  at the Glu-B1, Glu-D1, and Glu-A1 positions, respectively. Tightly linked genes (two) are found in each Glu-1 locus encoding x or y subunit types. In subunits . As a resubunits .Electrophoretic studies have shown a significant alteration in mobility and the number of HMW-G subunits in pasta and bread wheat. LMW-GS constitutes around one-third of all the protein in the seeds and 60% of all the gluten protein . The LMWLow molecular weight proteins that are abundant in cysteine might affect the viscoelastic characteristics of dough through disulfide or sulfhydryl exchange reactions with the proteins of gluten. Proteins capable of binding lipids can influence gluten-lipid protein interactions, and consequently, the functionality of protein in gluten . Accordi1.9.2.Proteins of gluten principally define the bread-making capability of wheat flour. A gluten protein allows the formation of cohesive viscoelastic dough when the flour is mixed with water and is able to retain gas produced in the process of fermentation or baking, forming bread\u2019s exposed form configuration after baking. The viscoelastic attributes of dough that are crucial for bread manufacture are mainly regulated by the gluten proteins of wheat, but the collaboration between the gluten protein medium and the additional constituents of flour, such as lipids in flour , arabinoThe bread manufacturing ability of wheat flour is directly associated with the protein content in flour  and, theA sufficient viscoelasticity equilibrium or the potential thereof is mandatory for excellent breadmaking. Inadequately flexible gluten will lead to decreased loaf size. Improved elasticity indicates increased loaf volume; however, excessively elastic gluten inhibits gas cell expansion , also ca1.10.An increase in the world\u2019s population of almost 10 billion is expected by 2050, which will result in an increase in wheat demand at a rate of approximately 1.7 annually . TherefoFusarium head blight) severely hamper wheat productivity  can result in a yield loss of up to 100%, and the sudden rise and spread of stem rust in Africa, known as Ug99, to Iran, the Middle East, and other countries is a severe concern for wheat production worldwide. Tilletia indica  disease is not only responsible for yield loss but also affects the quality of grain due to the infection of kernels. This disease was detected in various other countries, such as Mexico, Pakistan, India, Iran, Nepal, Afghanistan, Iraq, South Africa, and the United States , deoxynivalenol (DON), and zearalenone (ZON). Yield losses occur due to shriveled grain, low test weight, and failure of seed formation , which is a universally occurring wheat foliar disease responsible for terrible yield loss  or QTL mapping can be employed to find genes with drought resistance in unexplored germplasm. The use of genes/transcription factors from wheat germplasm like DREB, NHX2, AVP1, and SHN1 and their associated markers is a viable method for producing salt-tolerant wheat genotypes . QTLs inPrecise pre-breeding and selection approaches need to be carefully designed and followed for the identification and exploitation of the most effective and resilient loci, pyramiding, and partially tolerant gene accumulation. Identification, cloning and modification, and transfer of various R genes to diverse crop species through conventional breeding methods, molecular Marker Assisted Selection (MAS), and biotechnological tools, such as OMICS , can be used to combat pests and diseases and achieve long-lasting resistance .Tools in tissue culture techniques, like micropropagation, gametic embryogenesis , 109, soin vitro approaches, like protoplast fusion (The incorporation of t fusion , gametict fusion , somatict fusion , OMICS tt fusion , 117, cat fusion . Anothert fusion , applyint fusion , 118, ant fusion  and imprt fusion .in silico breeding and phenomics, etc. Therefore, public sectors must incorporate novel technologies into Mendelian genetics and the principles of quantitative genetics in order to make dynamic alterations in crop production (Gene stacking can be used to combat disease-resistant genes and inherit them as a sole trait . The incoduction .AK: write-up and revision of manuscript. AH: planning, finalization of basic idea, and revision. MT: revision of manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "Triticum aestivum, genome BBAADD) is a young hexaploid species formed only 8,500\u20139,000\u2009years ago through hybridization between a domesticated free-threshing tetraploid progenitor, genome BBAA, and Aegilops tauschii, the diploid donor of the D subgenome. Very soon after its formation, it spread globally from its cradle in the fertile crescent into new habitats and climates, to become a staple food of humanity. This extraordinary global expansion was probably enabled by allopolyploidy that accelerated genetic novelty through the acquisition of new traits, new intergenomic interactions, and buffering of mutations, and by the attractiveness of bread wheat\u2019s large, tasty, and nutritious grain with high baking quality. New genome sequences suggest that the elusive donor of the B subgenome is a distinct (unknown or extinct) species rather than a mosaic genome. We discuss the origin of the diploid and tetraploid progenitors of bread wheat and the conflicting genetic and archaeological evidence on where it was formed and which species was its free-threshing tetraploid progenitor. Wheat experienced many environmental changes throughout its evolution, therefore, while it might adapt to current climatic changes, efforts are needed to better use and conserve the vast gene pool of wheat biodiversity on which our food security depends.Bread wheat ( We describe the evolution of bread wheat in nature and under human selection with an emphasis on the donors of its subgenomes, evolution under polyploidy, and the \u201cwhere when and how\u201d of its domestication. Triticum aestivum L.), also known as common wheat, is an annual, predominantly autogamous species belonging to the Triticeae tribe of the grasses (Poaceae) family. It is an allohexaploid species, composed of 21 chromosome pairs organized in three subgenomes, A, B, and D, Genome BBAADD, 2n\u2009=\u20096x\u2009=\u200942 . Here, we focus on T. aestivum ssp. aestivum, which is of global importance, the other subspecies being only locally cultivated, and we refer to it as bread wheat. In this review, we follow Mac Key\u2019s classification, yet additional classifications were proposed as summarized by Bread wheat  and recent advances, major questions regarding bread wheat evolution remain: Which species are its progenitors? What was the impact of hybridization and allopolyploidization on the genome structure and stability? What were the main genetic changes during recent evolution under cultivation, domestication, and modern breeding? We present our current understanding of wheat evolution, starting from early discoveries to recent achievements and we discuss open questions.n\u2009=\u20092x\u2009=\u200914), tetraploids (2n\u2009=\u20094x\u2009=\u200928), and hexaploids including bread wheat (2n\u2009=\u20096x\u2009=\u200942). Soon after this discovery, T. turgidum ssp. dicoccon (genome BBAA 2n\u2009=\u20094x\u2009=\u200928), analysis of chromosomal pairing at meiosis of the F1 pentaploid hybrids indicated that hexaploid wheat, T.\u2002aestivum, contains the B and A subgenomes of tetraploid wheat with an additional diploid subgenome , is the donor of the third subgenome of T. aestivum. When the genome of Ae. tauschii was sequenced, first as a draft  (T. aestivum. Whole-genome sequencing confirmed the high synteny between ssp. dicoccoides (. durum (The wild progenitor of tetraploid wheat that contributed to the A and B subgenomes of Aaronson . The ferccoides) , as wellccoides)  indicateccoides)  and thusoccoides  and ssp. and Ae. tauschii (D subgenome), is well established, that of the B subgenome, the cytoplasm donor of bread wheat, remains elusive. There is no extant diploid species whose chromosomes have a high affinity for the B subgenome, suggesting that the progenitor is either extinct or remains to be discovered, or that the B subgenome has a polyphyletic origin and evolved at the polyploid level through multiple hybridizations with close extant species, for example, the five species from the Sitopsis section: Ae. speltoides; Ae. bicornis; Ae. sharonensis; Ae. Longissima; and Ae. searsii. Recent insight from whole-genome sequences suggests that Ae. speltoides diverged from the B subgenome \u223c4.5 MYA  \u223c6.4 MYA  5.5 MYA, then, 800,000 YA, A and B reunited again, but this time to form an allotetraploid species . And to close the cycle of \u201cdiverge-and-merge,\u201d the D genome, which is itself an ancient homoploid hybrid between B and A, merged with the BBAA genome approximately 9,000 YA to form allohexaploid bread wheat , one fission, and two telomeric ancestral chromosome fusions  as well as an additional lineage-specific translocation (between chromosomes 4A and 7B) to reach the modern 21 chromosomes  where nucleoli form is seen as secondary constrictions on chromosomes . NORs coTriticum . The dipd on 5AS . In the d on 5AS . Newly sd on 5AS . Guo andd on 5AS .Allopolyploidy involves genetic redundancy, even in an allohexaploid where the different subgenomes diverged from each other millions of years ago. This was shown in a milestone study by Ernie Sears with the demonstration that each of the seven pairs of chromosomes of a subgenome could compensate for the absence of a specific pair of another subgenome , 1959. TCS genes identified 107,891 \u201chigh confidence\u201d genes distributed almost equally between the A, B, and D subgenomes. Synteny was highly conserved in the three subgenomes. Synteny was more prominent, with larger syntenic blocks, in interstitial regions compared to sub-telomeric regions or pericentric regions . As shown by Pioneering molecular studies revealed a high level of gene synteny and collinearity in the homoeologous chromosomes of bread wheat . The natAe. tauschii and bread wheat D subgenome, only 87.4% of the genes were present at the expected orthologous position, highlighting a dynamic gene copy-number variation that is not related to whole-genome duplication . Interestingly, there were significant differences between subgenomes when functions were assigned to the families that expanded: there was an over-representation of seed-related genes (embryo and endosperm) in the A subgenome and of vegetative growth and development in the B subgenome. Families that expanded in all three genomes were enriched in genes that were hypothesized to play an important role in wheat breeding for trait related to yield or biotic and abiotic stress resistance ; however, a causal relationship has not been shown to date.Genome merging in hybrids or allopolyploids generates both cytological and genetic challenges to the nascent species. For example, homoeologous pairing may lead to partial sterility and multisomic inheritance, and genomic clashes between regulatory elements of different subgenomes can lead to hybrid necrosis . RewirinUpon allopolyploidization, the immediate triggering of a variety of cardinal genetic and epigenetic changes that affect genome structure and gene expression might be essential to bring about genetic and cytological diploidization. Such rapid changes have been reported in newly formed allopolyploids, including elimination of coding and noncoding DNA sequences, transposable element (TE) and tandem repeat elimination or amplification, and gene expression modifications . RemarkaTEs activity can be triggered by allopolyploidization, affecting gene expression and inducing many structural rearrangements, including deletions or duplications . Due to T. turgidum, having the same BBAA genome. Moreover, the initial process of domestication might have taken approximately 2\u20133,000\u2009years . First described by Ph1 is located on chromosome arm 5BL and suppresses homoeologous chromosome pairing without affecting homologous chromosome pairing. A deletion of Ph1 was induced by ph1b locus). In the absence of Ph1, pairing occurs between the homoeologues of the A, B, and D subgenomes of bread wheat or between homoeologous chromosomes in hybrids between hexaploid wheat and wild relatives  and a small deletion induced by clustered regularly interspaced short palindromic repeats (CRISPR)\u2013Cas9 yielded high levels of homoeologous pairing  and has fascinated archeologists, anthropologists, plant geneticists, and evolutionary biologists, including The phases of wheat evolution under human selection are: (1) the gatherers  period when wheat was harvested from wild stands; (2) a predomestication cultivation period when wild wheat was grown in small plots; (3) the domestication of wild emmer wheat, namely, the period when mutations in genes affecting spike morphology appeared and became fixed. These mutations, which hindered seed dispersal and facilitated threshing, suited the farmer rather than the wild plant and turned wheat into an organism dependent on humans for its dissemination; (4) the formation of bread wheat under domestication; (5) the spread of bread wheat and the accumulation of landraces; and (6) the green revolution and modern breeding.We know little of the predomestication period, when human were gatherers and brought grains near their dwellings, due to a limited amount of available data. The earliest data relevant to wheat comes from archeological records from the upper paleolithic Last Glacial Maximum period approximately 23,000\u2009years ago in the hunter\u2013gatherer sedentary camp of Ohalo II on the shores of the lake of Galilee , suggestT. turgidum ssp. dicoccon which had a hulled grain and a nonbrittle rachis. Free-threshing tetraploid wheat appeared soon following the nonfragile types, in the Near east, late Pre-Pottery Neolithic B (PPNB)  .Ae. tauschii to form hexaploid wheat  from Transcaucasia also contributed \u223c1% on average to the current wheat D subgenome  locus in several wheat species, that the BBAA subgenomes of hexaploid wheat originated from a free-threshing tetraploid form and therefore it could not be T. turgidum ssp. dicoccon, the most ancient domesticated hulled grain tetraploid wheat. This leaves us with the question of the identity of the domesticated tetraploid parent of hexaploid wheat. The free-threshing tetraploid macaroni wheat T. turgidum ssp. durum is also an unlikely donor since there is no evidence that it existed approximately 8,500 YA when T. aestivum was formed. The domesticated free-threshing tetraploid wheat that was cultivated approximately 9,000 YA, starting in the Levant and spreading throughout the fertile crescent, when T. aestivum was formed is the primitive tetraploid T. turgidum ssp. parvicoccum . The earliest archeological findings of hexaploid wheat are from an 8,400-year-old, free-threshing type in Cafer Hoyuk, upper Euphrates  leads to nonbrittleness  led to partial fragility while the combination of both was needed for the full nonbrittle rachis phenotype. A panel of 113 wild emmer, 85 domestic emmer, and 9 durum lines showed that all domesticated forms carried both mutations  fragility locus in 3D, Br-D2  remains unknown. Tg loci and showed that dominant homoeoalleles on chromosomes 2A and 2B of emmer wheat (Tg-A1 and Tg-B1) contribute together with the q WT allele, to the nonfree-threshing phenotype  examined so far did not have this genotype but rather had a recessive tg-D1 allele and a dominant Tg-B1 and q allele and are thus thought to be derived from hybridization between free threshing hexaploid wheat with tetraploid hulled emmer wheat , addition of the D subgenome from the central Asiatic Ae. Tauschii greatly extended the range of adaptation of hexaploid wheats to a more continental climate over the continental plateaus of Asia and the colder temperate areas in eastern, central, and northern Europe  that enabled growth under harsh winter conditions or photoperiod sensitivity (e.g. PPD-D1) that enabled growth under a broad range of latitude, enabled the expansion of wheat to new areas. The selection of reduced height (Rht genes) enabled the green revolution through reduction of lodging under chemical fertilization and high yield.Major milestones in wheat breeding during the past century, including the green revolution, were recently reviewed . The phy2 concentration by \u223c70\u2009ppm  mutant  mutant  or in thndidates . During"}
+{"text": "Triticum aestivum) has decreased over time while the starch content increased. In addition, it was shown that, compared to bread wheat, ancient wheats contain more protein and gluten and greater contents of many CD\u2010active epitopes. Consequently, no single wheat type can be recommended as better for reducing the risks of or mitigating the severity of CD. An estimated 10% of the population of Western countries suffers from gastrointestinal symptoms that lack a clear organic cause and is often referred to as irritable bowel syndrome (IBS). Many of these patients consider themselves gluten sensitive, but in most cases this is not confirmed when tested in a medical setting. Instead, it may be caused by gas formation due to fermentation of fructans present in wheat or, in some patients, effects of non\u2010gluten proteins. A significant overlap of symptoms with those of CD, IBS and inflammatory bowel disease makes a medical diagnosis a priority. This critical narrative review examines the suggestion that \u2018ancient\u2019 wheat types are preferred for health and better tolerance.Popular media messaging has led to increased public perception that gluten\u2010containing foods are bad for health. In parallel, \u2018ancient grains\u2019 have been promoted with claims that they contain less gluten. There appears to be no clear definition of \u2018ancient grains\u2019 but the term usually includes einkorn, emmer, spelt and Khorasan wheat. Gluten is present in all wheat grains and all can induce coeliac disease (CD) in genetically susceptible individuals. Analyses of \u2018ancient\u2019 and \u2018modern\u2019 wheats show that the protein content of modern bread wheat ( Triticum aestivum), which is sometimes referred to as \u2018common wheat\u2019, have higher gluten contents compared to so\u2010called \u2018ancient wheats\u2019. It has also been claimed that the gluten present in modern wheat is poorly digested, leading to the presence of partially digested protein fragments in the small intestine that play a role in the intestinal pathology of coeliac disease (CD) and are linked to neurodegenerative conditions (gluten ataxia). In addition, it has been suggested that gluten is linked to the exacerbation of chronic brain disorders such as schizophrenia, depression, Alzheimer's disease and autism  wheats results in a range of adverse effects and contributes to chronic diseases including obesity. In addition, it has been suggested that modern varieties of bread wheat , intensWikipedia , defining modern as the most recently bred and widely cultivated and \u2018ancient\u2019 as older types that are no longer widely cultivated . These older types of bread and durum wheats, which may date back to more than a century, may correspond to unselected land races or the products of early breeding and should therefore be considered as \u2018heritage\u2019 or \u2018heirloom\u2019 wheats.The term \u2018ancient grains\u2019 is more widely applied, and therefore used here to describe types of wheat which are suggested to correspond to types which were grown in antiquity but are only currently grown in small volumes today: einkorn, emmer, spelt and Khorasan wheat. To understand the relationships between these and modern types of bread and durum wheats it is necessary to briefly discuss the origin and evolution of wheat.Aegilops speltoides) gave rise to a single tetraploid  wheat species which exists in three major forms  and minor forms (notably Khorasan wheat). Finally, the crossing of wild emmer with a different related grass species  gave rise to a single hexaploid  species with two main forms: spelt\u2010like hulled wheat and bread wheat.In simple terms, the history of wheat (as far as we currently know) extends some 500 000\u00a0years  grown today appear to be descended from wheat types grown during the earliest cultivation, although both have undoubtedly been subjected to selection during their period of cultivation. Hence, both may be correctly termed \u2018ancient\u2019. This does not apply to spelt as the forms that are currently grown appear to have been derived from more recent hybridisations between hexaploid bread wheat and wild emmer, rather than from ancient spelt\u2010like hulled wheat, which may be extinct. Furthermore, modern commercial types of spelt have been subjected to plant breeding in the same way as modern bread and pasta wheats.The types of einkorn and emmer and also Khorasan wheat  that remain undigested in our intestine (and therefore may induce inflammatory and immune responses) differ between modern bread wheat and ancient wheats. This question is relevant because these peptides are known to pose a risk for the development of coeliac disease (CD) in individuals who are genetically predisposed (this is the case in the part of the population [5%\u201340%] who express the haplotypes HLA\u2010DQ2 or DQ8). The proportion of susceptible individuals differs in different regions, as reviewed by  that may cause adverse reactions in the gut. These fragments may contain core amino acid sequences which are able to trigger immune reactivity and the onset of CD in genetically susceptible individuals. These specific sequences to which antibodies bind are referred to as CD\u2010active epitopes. Although preliminary research suggested that wheat breeding had resulted in a higher content of some CD\u2010active peptides , which were grown before the introduction of modern plant breeding technologies, but are still grown today on small scale by local farmers in some countries, including Italy, compared with modern types of bread wheat which are grown on a very large scale. Although these older and modern types of durum and bread wheats are genetically similar, small differences in grain composition and in the effects of digestion and gastrointestinal symptoms were reported , more robust studies are required to conclude whether this would result in significant impacts on health.It is estimated that based on confirmed diagnosis, ~1%\u20137% of the total population may suffer from adverse reactions due to wheat consumption , while variable percentages (1%\u20136%) have been reported for non\u2010coeliac wheat sensitivity (NCWS), the values reported being strongly influenced by self\u2010perception and criteria of evaluation , inflammation and small intestinal tissue damage, known as coeliac disease, which develops only in genetically predisposed individuals  associated with the intake of wheat was attributed to the presence of gluten and described as non\u2010coeliac gluten sensitivity (NCGS). Avoidance of foods containing gluten was shown to induce a relief of symptoms, which was taken as confirmation that gluten was the causative factor , which correlated with markers of systemic immune activation. Thus, in the absence of CD, several nonspecific symptoms and factors were present, which overlap with similar symptoms observed in IBS and limit an accurate diagnosis  but the value may differ depending on the Salerno criteria used and country of study  and modern types of bread wheat grown and analysed under identical conditions have shown that older types contain more protein, including total gluten and gliadin (which is the major source of CD\u2010active gluten peptides). Einkorn, emmer and durum do not contain the D genome encoding the \u03b1\u2010gliadin which is digested to release the \u201833\u2010mer\u2019 peptide (\u03b12\u2010gliadin p56\u201388) and is a strong CD trigger in genetically susceptible individuals. However, they do contain other CD\u2010active peptides and their contents clearly overlap between ancient and modern wheat cultivars. It can therefore be concluded that no single wheat type can be recommended as better or safer for reducing or mitigating CD. Although older and modern types of durum and bread wheats are genetically similar, small differences in the compositions of grain samples occur, some of which may result from differences in the environment and agronomic conditions under which the grains are grown. However, the relevance of these to impacts on health has not been established. Although most of the literature on CD and WA focuses on the role of gluten proteins it should be noted that other wheat proteins may trigger allergic, immune and inflammatory responses in susceptible individuals, including the ATIs. The initial suggestion that gluten was the prime cause of self\u2010reported wheat sensitivity has been disproved in more recent studies which have shown a role of fermentation of FODMAPs resulting in gas formation and bloating. Despite this observation, FODMAPs cannot explain inflammatory and immune reactions observed in some individuals, which indicate that other components are inducing factors. Finally, the impact of gluten\u2010free diets on health and quality of life warrants that accurate medical diagnosis is essential before deciding to exclude gluten\u2010containing grains from diets, especially because strict adherence to GF foods is challenging, costly and may be socially isolating. In addition, GF foods are often of less favourable nutritional composition, have been linked to poor micronutrient and fibre intakes, which was shown to reduce the bacterial richness and microbiota composition in non\u2010celiac individuals in an unfavourable way, and require dietetic guidance to help avoid undesired effects on health outcomes (Caio et al., The authors report no conflicts of interest to declare that are relevant to the content of this article.All authors contributed to the preparation of this manuscript and have read and approved the final text."}
+{"text": "Ovarian lesions are rare but frequent in children. Patients could present with abdominal pain, but ovarian lesions could also be incidentally found on ultrasound. Awareness is required in cases with acute, severe lower abdominal pain, as ovarian torsion could be the cause. Other lesions can be cysts or benign or malignant ovarian tumors. Thus, the aim of this paper is to review typical ovarian lesions according to age, imaging and laboratory findings, and surgical management.We retrospectively analysed the patient charts of 39 patients aged 10.4\u00a0\u00b1\u00a06.1 years (from 3\u00a0months to 18 years) with ovarian lesions treated in our institution between 01/2009 and 08/2020. All clinical and pathological findings of infants and children operated on for ovarian lesions were included.Ovarian lesions in children younger than 2 years of age were typically ovarian cysts, and ovarian tumors were not observed in this age group. In older children over 10 years of age, tumors were more common\u00a0\u2013 with mostly teratoma or other germ cell tumors, followed by epithelial tumors. Moreover, acute or chronic ovarian torsion was observed in all age groups. In general, ovarian tumors were much larger in size than ovarian cysts or twisted ovaries and eventually showed tumor marker expression of AFP or \u00df-HCG. Simple ovarian cysts or twisted ovaries were smaller in size. Surgery for all ovarian lesions should aim to preserve healthy ovarian tissue by performing partial ovariectomy.In adolescent girls with acute abdominal pain, immediate laparoscopy should be performed to rule out ovarian torsion. Careful imaging evaluation and the assessment of tumor markers should be performed in painless ovarian lesions to indicate an adequate surgical ovarian-sparing approach. Ovarian lesions are not too rare in children. They could present with abdominal pain but could also be incidentally found on ultrasound. Awareness is required in cases with acute, severe lower abdominal pain, as ovarian torsion could be the cause. Other lesions can be cysts or benign or malignant ovarian tumors.Cystic ovarian lesions are more common in infants and in older girls due to either maternal hormonal stimulation in newborns or either onset of hormonal activity in prepubertal age. Ovarian lesions in newborns and infants are mostly cystic in nature. Asymptomatic ovarian cysts are occasionally seen on ultrasound prenatally. The incidence of prenatal ovarian cysts is 1:2.500 . In addiOvarian torsion is a rare condition in infants and children with an incidence of approximately 5 per 100.000 . AlthougOvarian tumors can be of benign or malignant nature. Benign ovarian neoplasms are often asymptomatic. Gynaecologic malignant tumors account for only 2% of all types of malignancies in children, and 60\u201370% of these are ovarian tumors . Acute aAlpha fetoprotein (AFP) and \u00df-human chorion gonadotropin (\u00df-HCG) are markers of tumorous lesions of the ovary . AFP canOvarian lesions can be diagnosed by ultrasound examination. MRI should be preferred to CT scan because the diagnosis is very accurate and to minimize radiation exposure . TeratomThus, the aim of this paper is to review typical ovarian lesions according to age, imaging and laboratory findings, and surgical management.We retrospectively analysed the patient charts of 39 female patients aged 10.4\u00a0\u00b1\u00a06.1 years (from 3\u00a0months to 18 years) with ovarian lesions treated in our institution between 01/2009 and 08/2020. All clinical and pathological findings of infants and children operated on for ovarian lesions were included. From the patient charts, age, primary symptoms, sonographic findings, MRI or CT scan findings, and laboratory findings, including tumor markers , were assessed. The operative procedure, intraoperative findings and pathological diagnosis were also retrieved from the patient charts. All retrospective clinical data were obtained after written informed consent was obtained\u00a0from the parents, and the study was approved by the ethical council of the University Hospital Frankfurt .2 test (for two variables). Significance statements refer to p-values of two-tailed tests that were less than 0.05.SPSS for Windows  was used for statistical analysis. Cross table statistics were performed using the XThe data of 39 patients were available. The median age of the patients was 10.4\u00a0\u00b1\u00a06.1 years, with a range from 2.5\u00a0months to 18.1 years of age. The patients were grouped according to age as follows: age group <2 years, age group 2\u201310 years, and age group >10 years. A total of 8/39 (20.5%) patients were <2 years old, 5/39 (12.8%) patients were 2\u201310 years old, and 26/39 (66.6%) patients were >10 years of age. In total, 14/39 (35.9%) patients underwent laparoscopic surgery, 14/39 (35.9%) patients underwent combined laparoscopic and open surgery, and 11/39 (28.2%) patients underwent primary open surgery. In 20/39 (51.3%) patients, a total ovariectomy was performed, and in 8/39 (20.5%) patients, partial ovariectomy was performed. Other procedures included detorsion of the ovary , cyst excision , and cyst fenestration . In 13/39 patients (33.3%), ovarian tumors were found. Tumors were in 6 cases mature teratoma, in 2 cases immature teratoma, yolk sac tumor in 1 case, Sertoli-cell tumor in 1 case, cystadenoma in 2 cases, and adenocarcinoma of the ovary in 1 case. In the age group <2 years, eight patients were identified; of these patients, 5/8 (62.5%) had ovarian torsion, 7/8 (87.5%) had an ovarian cyst, and no tumors were found in patients <2 years . In the Tumor size and longest tumor axis were assessed. In patients with tumors, the size (axis) was larger with a 15.9\u00a0\u00b1\u00a08.0\u00a0cm\u00a0(n=13) longest axis measure compared to patients with a pure cystic lesion  or patients with ovarian torsion .Expression of tumor markers are shown in In our own patient series, we observed no tumorous ovarian lesions in children younger than 2 years of age, but an increasing number of ovarian tumors in older children . This isMostly, the volume of torsed ovaries was greater than that of simple ovarian cysts or normal ovaries . In adolIn adolescent girls, benign or malignant ovarian tumors were more frequent than in younger girls or infants . CarefulLaboratory examinations should include tumor markers of ovarian tumors, namely, AFP, \u00df-HCG and Ca-125 . In our Ovarian lesions in newborns and infants are typically ovarian\u00a0cysts, and ovarian tumors were not observed in this age group. In older children and adolescents, tumors were more common\u00a0\u2013 with mostly teratoma or other germ cell tumors, followed by epithelial tumors. In general, ovarian tumors were much larger in size and eventually showed expression of tumor markers AFP or \u00df-HCG. Simple ovarian cysts or twisted ovaries were smaller in size. Surgery for mature ovarian teratoma should aim to preserve healthy ovarian tissue by partial ovariectomy. Moreover, acute or old ovarian torsion was observed in all age groups. In adolescent girls with acute abdominal pain, immediate laparoscopy should be performed to rule out ovarian torsion.Supplementary MaterialClick here for additional data file."}
+{"text": "Hexaploid triticale results from crosses between durum wheat and rye. Despite its high agronomic potential, triticale is mainly used for livestock feed. Triticale surpasses their parental species in adaptability and tolerance to abiotic and biotic stresses, being able to grow in acidic soils where a high amount of iron (Fe) and zinc (Zn) is typical. On the other hand, high amounts of these essential trace elements can be cytotoxic to bread wheat. The cytotoxicity induced by seed priming with a high concentration of Fe and Zn impaired root cell division and induced nucleolar changes in bread wheat. Such cytogenetic approaches were expedited and successfully determined cytotoxic and suited micronutrient dosages for wheat nutripriming. With this study, we intended to analyse the hexaploid triticale cv \u2018Douro\u2019 root mitotic cell cycle and nucleolar activity after seed priming performed with aqueous solutions of iron (Fe) and/or zinc (Zn), containing a concentration that was previously considered cytotoxic, to bread wheat and to infer the higher tolerance of triticale to these treatments. The overall cytogenetic data allowed us to conclude that the Fe + Zn treatment enhanced the root mitotic index (MI), mitosis regularity and nucleolar activity of \u2018Douro\u2019 relative to the control and the individual treatments performed with Fe or Zn alone. The Fe + Zn treatment might suit triticale biofortification through seed priming. Biofortification can increase the Fe and Zn contents of edible parts of plants, contributing to the eradication of the hidden hunger caused by the deficiency of these essential trace elements in the human diet . StangouTriticosecale Wittmack; AABBRR; 2n = 6\u00d7 = 42) is a synthetic amphiploid that results from crosses between durum wheat  and rye . Triticale grain is mainly used as livestock feed, given its high biomass and yield  in normal interphase cells with one to four nucleoli with regular shapes using the Digimizer Image Analysis software .In each seed priming treatment, we also measured the nucleolar area [A  using a CCD digital camera XC-10  and the software cellSens .n = 3), and the other three for nucleolar activity evaluation (n = 3). A variable number of cells were scored per preparation. The results are presented as mean \u00b1 standard error (S.E.) values per treatment. The statistical analyses of variance (ANOVA) and the post hoc Tukey tests were made with IBM SPSS Statistics v23 . The p-value significance, due to the different effects and their interaction, was established for probabilities lower than 5% (p < 0.05).Six mitotic spreads per seed priming treatment were performed. Each mitotic spread was done using a single root apex from a different plant. Three mitotic preparations were used for the mitotic cell cycle analysis .In this work, the seed priming treatment performed with Fe + Zn increased the mitotic index (MI) and decreased the percentage of dividing cells with anomalies (DCA) relative to the control and the seed priming treatments with Fe or Zn alone. The increase in the MI indicated enhancement of cell division and, hence, root growth, which is particularly needed in dry soils and for seedling growth and establishment. Despite the higher average number of cells in prophase that suggest cell cycle arresting, a reduced DCA was also verified, revealing a higher regularity of mitosis.The overall cytogenetic data gathered with the cell cycle and nucleolar activity evaluation allowed us to conclude that among the tested seed priming treatments, the combination of Fe + Zn was the most suitable and less cytotoxic to triticale cv. \u2018Douro\u2019 could be helpful in its biofortification. Additionally, the Fe + Zn treatment was not as cytotoxic as it was to bread wheat, confirming the higher tolerance of hexaploid triticale to high amounts of these two micronutrients. Nonetheless, as referred before, micronutrient dosages to be used in priming seeds of any crop should be previously tested using the same analyses performed in this work and/or additional and complementary approaches focusing on the agronomic and physiological performances and nutritional content of the biofortified plants and resulting food products. Interestingly, the present findings highlight that triticale accumulates a high amount of Fe and Zn and could be used as bridge material in wheat breeding programmes through interspecific crosses with wheat. Triticale can be used as a Fe- and Zn-dense progenitor in such interspecific crosses to ensure the maintenance of high amounts of these micronutrients in the ensuing generations, constituting one of the major challenges to agronomic biofortification."}
+{"text": "Expectations are often dynamic: sports fans know that expectations are rapidly updated as games unfold. Yet expectations have traditionally been studied as static. Here we present behavioral and electrophysiological evidence of sub-second changes in expectations using slot machines as a case study. In Study 1, we demonstrate that EEG signal before the slot machine stops varies based on proximity to winning. Study 2 introduces a behavioral paradigm to measure dynamic expectations via betting, and shows that expectation trajectories vary as a function of winning proximity. Notably, these expectation trajectories parallel Study 1\u2019s EEG activity. Studies 3 (EEG) and 4  replicate these findings in the loss domain. These four studies provide compelling evidence that dynamic sub-second updates in expectations can be behaviorally and electrophysiologically measured. Our research opens promising avenues for understanding the dynamic nature of reward expectations and their impact on cognitive processes. A series of 4 experiments  involving slot machine interfaces demonstrates that sub-second changes in reward expectations can be behaviorally and electrophysiologically measured. On December 18, 2022, 90\u2009min into the final World Cup game with France opposing Argentina, soccer fans all over the world were holding their breath. The game had started with a clear advantage for Argentina. At halftime, Argentina led by two goals. But with less than 10\u2009min left in the game, France brought the score to a tie. During the next 30\u2009min of overtime, Argentina got the lead back, but France once again leveled the score after a late penalty. The game went to a penalty shootout, a best-of-ten tie-breaking method, that finally ended with Argentina winning the cup. For Argentinean and French supporters alike, the game was a roller-coaster ride, with the expectations of seeing their team winning going up and down, and up again.8. Much of this research has examined expectations as static, that is, fixed in time. However, as our soccer example illustrates, we intuitively know that expectations can vary rapidly even in the sub-second time range. Here using a slot machine task that elicits rapid changes in expectation, we provide behavioral and electrophysiological evidence of moment-to-moment changes in expectations.Reward expectations play a critical role in how people process outcomes and make decisions, and have been extensively researched in psychology, economics and neuroscience11. Expectations enhance preparatory attention and reduce stimulus conflict12, prioritize which information should be stored in working memory13 and guide cognitive control allocation14. Expectations also have a strong effect on affect, as illustrated by the placebo effect15, or the findings that enjoyment of a film, a vacation2, a beer3 or a wine4 is influenced by expectations about their quality. In risky decision-making, unexpected outcomes were found to have greater emotional impact than expected outcomes16, and a recent computational analysis showed that happiness ratings in response to a wheel of fortune\u2019s outcomes are better predicted by reward expectations and reward prediction errors than by earnings17. Expectations can also have substantial effects on choice behavior, for example by encouraging individuals to participate in a lottery18 or to engage in exploration vs. exploitation19.Reward expectations have a central role in cognition with powerful effects on learning and performance20, or an image22. In risky decision-making research, studies have used an array of methods to convey reward probability in order to manipulate expectations, such as varying the position of a horizontal line 23, the area of a wheel of fortune associated with a gain17, or the colors of cues24. In all these studies, the assumption is that expectations stay constant, with little attention paid to the dynamic evolution of expectations leading up to an outcome. However, as our soccer example illustrates, expectations are often dynamic, especially in situations where new information about the odds of receiving a reward is provided.Expectations have traditionally been studied as static predictions about future outcomes. Reward predictions are typically elicited by a single predictive cue, such as a sound, an odorInvestigating dynamic expectations requires finding an appropriate task and the right methodology. Here we use slot machines as a case study to assess moment-to-moment changes in expectations. In the simplest casino slot machines, players start games either by pushing a button or pulling the handle. The reels spin, then decelerate to a stop, and players are rewarded if matching symbols align on the payline Fig.\u00a0. The con26. To increase their ecological validity, some of these studies have used computerized slot machine paradigms that mimic casino settings, with 3D graphics, sounds and realistic spinning and deceleration phases29. Notably, because it was not their focus of interest, these studies did not examine the dynamic aspect of slot machines and mostly ignored the spinning and deceleration phases. The few studies investigating these phases either looked at the overall brain activity during the spinning independently of the final outcome30, focused on a single moment during the deceleration31 or used a static, sequential slot machine game with no spinning32.Slot machines have been extensively used and validated in the gambling literature29  a methodology with a sub-second temporal resolution and 2) a method that does not rely on self-report, as participants might not be able to accurately describe their sub-second internal beliefs. In addition, repeatedly asking participants to report their expectations during a slot machine game would interrupt the experience flow. Here we sought to overcome these challenges using a combination of electroencephalography (EEG) and behavioral methods to investigate the dynamics of expectations.35. The exception is a study by Alicart et al.31. Although the authors emphasize that the stronger effects were observed post outcome, they report that one second before the machine stopped, activity in theta and alpha frequency-bands was higher for Near Wins than Full Misses. However, the paper did not look separately at NWB and NWA, while we predict that these two conditions should show different expectation trajectories. Further, the analyses focus on one timepoint of the deceleration phase (1\u2009s before outcome), and do not examine how the signal evolves during the deceleration phase as expectations are continuously updated. Here we focused on the continuous EEG activity during the deceleration phase. While there is no known EEG metric of dynamic expectations, the prefrontal-dependent contingent negative variation (CNV) event-related potential (ERP) is well known to be linked to static expectations38. It is characterized by a fronto-central scalp distribution and is maximal at the vertex (electrode Cz)34. We predicted that CNV-like EEG activity changes would distinguish between the different outcomes.EEG has an excellent temporal resolution, detecting millisecond changes in brain activity, and it does not rely on self-report nor does it require overt responses. Past EEG studies looked at Near Wins in slot machine games, however they mostly focused on the outcome evaluation phase (once the machine stops) and did not examine the period preceding feedbackIn parallel, we designed a paradigm, \u201cSlot or Not\u201d, to behaviorally measure moment-to-moment changes in expectations and statistically relate them to those observed in EEG responses. This task was inspired by live betting  which refers to gambling that occurs after a sport or gaming event has started. It offers gamblers the opportunity to identify and capitalize on changing odds during the course of the game. Here we implemented live betting into a slot machine game to track expectations via betting behavior. We are not aware of other behavioral tasks investigating the sub-second dynamics of expectations.In this paper, we present four studies investigating the dynamics of expectations. In Study 1, we used EEG to define the sub-second electrophysiological correlates of moment-to-moment changes in expectations elicited by the deceleration phase of a slot machine. In Study 2, we introduced a paradigm to measure moment-to-moment changes in expectations from behavior. We examined the relationship between the EEG findings of Study 1 and the behavioral findings of Study 2. Finally, in Studies 3 (EEG) and four , we replicated Studies 1 and 2 in the loss domain, using a modified version of the slot machine where a match is associated with a loss of money, and mismatches with gains. Our results provide evidence that reward expectations are rapid and dynamic, and that they can be tracked in EEG activity and in choice behavior.35. Notably, previous Near-Wins studies baselined the EEG activity to the period preceding the outcome reveal41. However, using the 100 or 200\u2009ms preceding the wheel stop is problematic because this is precisely when expectations might differ the most between the different conditions. Here we baselined EEG activity to the 200\u2009ms period preceding the spinning onset.We first examined EEG activity during the computerized slot machine game Fig.\u00a0 to test p\u2009=\u20090.008 to account for the six time-windows . Greenhouse\u2013Geisser correction for analysis of variance ANOVA tests was used whenever appropriate. If the ANOVA was significant, we performed pairwise comparisons using Tukey tests. Here we report the findings of interest. Full results are presented in the Supplementary materials . For each of these time-windows we ran a one-way repeated-Measure ANOVA analysis , with a significance threshold set at p\u2019s\u2009>\u20090.15). As the deceleration progressed, the effect of Outcome emerged in the [\u22122000 \u22121500] time-window \u2009=\u20095.72, p\u2009=\u20090.003). This effect increased in the [\u22121500 \u22121000] time-window \u2009=\u200914.87, p\u2009=\u20090.001). Here we detail these effects for the last second before the machine stopped.At deceleration onset ([\u22123000 \u22122500] and [\u22122500 \u22122000] time windows) there was no effect of Outcome \u2009=\u200927.50, p\u2009<\u20090.001). Pairwise comparisons revealed that EEG amplitude was smaller for Win (\u22127.26\u2009\u00b1\u20096.57\u2009\u03bcV) compared to NWB  and Full Miss , but not compared to NWA . NWA were also smaller than NWB  and FM . There was no significant difference between FM and NWB . As can be seen on Fig.\u00a0In the [\u22121000 \u2212500\u2009ms] time window, pre-wheel stop EEG activity differed depending on Outcome \u2009=\u200913.37, p\u2009<\u20090.001). The pairwise comparisons revealed a different pattern of results than in the prior deceleration time window. EEG amplitudes were smaller for Win (\u22126.83\u2009\u00b1\u20095.67\u2009\u03bcV) than for NWA  and FM , but not compared to NWB . NWB were also smaller than NWA  and FM . There was no significant difference between FM and NWA . As can be seen in Fig.\u00a0In the [\u2212500 0\u2009ms] time window EEG activity also differed depending on Outcome  and the P3 (see Methods). The full results are presented in Supplementary Tables\u00a07,p\u2019s\u2009<, p\u2009<\u20090.001)44,48. Notably, NWB elicited larger P3 than the other No-Wins . Given that P3 has been associated with surprise and reward prediction errors50, these results reinforce the claim that NWB create higher expectations than NWA and FM right before outcome onset.In addition, in line with the EEG literature on slot machinesStudy 1 assessed sub-second changes in expectations: we found evidence that EEG activity tracked expectations while the slot machine decelerated and participants accumulated information. In Study 2, we measured moment-to-moment changes in expectations via behavior.t . Greenhouse\u2013Geisser correction for analysis of variance ANOVA tests was used whenever appropriate. If the ANOVA was significant, we performed pairwise comparisons using Tukey tests. Full results are presented in Supplementary Tables\u00a0We used the same approach as for the EEG data: we divided the deceleration phase into six 500\u2009ms time-windows, and ran a one-way repeated-Measure ANOVA analysis  for each time-window. The significance threshold was set at p\u2019s\u2009>\u20090.2).We found no main effect of Outcome for the [\u22123000 \u22122500], [\u22122500 \u22122000] and [\u22122000 \u22121500] time windows \u2009=\u200911.34, p\u2009<\u20090.001). In that time window, NWA were associated with a higher tendency to bet on the slot machine  than Wins , NWB , or FM . All other comparisons were not significant.A significant effect of Outcome was found for the [\u22121500 \u22121000] time window \u2009=\u200933.63, p\u2009<\u20090.001). In that time window, NWA were still associated with a higher tendency to bet on the slot machine  than Wins , NWB , or FM . In addition, Wins were associated with a higher tendency to bet on the slot machine compared to NWB (p\u2009<\u20090.001) and FM (p\u2009<\u20090.001). There was no difference between NWB and FM (p\u2009=\u20090.987).A significant effect of Outcome was found for the [\u22121000 \u2212500] time window \u2009=\u200919.06, p\u2009<\u20090.001). In that time window, Wins were associated with a higher tendency to bet on the slot machine  than NWB , NWA , and FM . In addition, FM were associated with a lower tendency to bet on the slot machine compared to NWB (p\u2009=\u20090.043) and NWA (p\u2009=\u20090.009). There was no difference between NWB and NWA (p\u2009=\u20090.941). However, visual inspection of the grand averages \u2009=\u20095.53, p\u2009<\u20090.001).A significant effect of Outcome was found for the [\u2212500 0] time window   Fig.\u00a0. In that01) Fig.\u00a0.These results provide evidence that the differences in EEG activity before the machine stops are not due to a visual artifact from wheel deceleration, but reflect different expectations associated with the four outcomes. There are some notable differences between the results of Studies 1 and 2. For example, In Study 1, we observed a change in the trajectory of NWA around 500\u2009ms before the machine stopped, while in Study 2, the decrease in expectations occurred about 500\u2009ms earlier (1000\u2009ms before the machine stopped). However, this divergence is not surprising given the differences between the tasks and methods used in the two studies. EEG tracks cognitive processes with millisecond precision, whereas behavioral tasks require a decision and a button press, which influences their temporal resolution. Further, in Study 1, participants were obligated to bet on the slot machine, and once they chose an item, they were passive viewers of the reel spinning. In Study 2, participants could make betting decisions during the spinning and deceleration phases, making the game more engaging, and potentially increasing attention to the deceleration phase.Taken together, Studies 1 and 2 confirm our predictions that Near Win and Full Miss are perceived differently hundreds of milliseconds before the slot machine stops providing evidence that sub-second changes in expectations can be measured and tracked both at the behavioral and electrophysiological levels.In Study 3, we modified the slot machine game presented in Study 1 so that a match was associated with a \u201cbig\u201d loss of money , and mismatches (\u201cEscapes\u201d) with small gains ($.10). This manipulation had two aims. First, it allowed us to test for the robustness of our findings: do they replicate under different task parameters? Second, the comparison between expectations for gains and expectations for losses might provide insights into what precisely is being tracked in the EEG signal.In this version of the game, participants encountered 25 Losses, 25 Near Loss Before (NLB) (when the machine stops just one item before a loss), 25 Near Loss After (NLA) (when it stops just one item after a loss) and 75 Full Escape (FE) (when it stopped at least two items away from a loss).53 and in the Balloon Analog Risk Task54, but not in a slot machine game. No study has looked at moment-to-moment changes in expectations in Near Losses using either electrophysiology or behavior. We predicted that right before the machine stops, participants should have smaller expectations of losing (higher expectations of winning) for NLA and FE than for NLB and Loss, for which the uncertainty about the outcome of the slot machine is only resolved when the machine stops.Near Losses  are less studied than Near Wins. They have been implemented in lottery paradigmsp\u2019s\u2009>\u20090.028). However, as the deceleration progressed, differences emerged: the effect of Outcome became significant in the [\u22122000 \u22121500] time-window \u2009=\u20097.48, p\u2009<\u20090.001). This effect increased in the [\u22121500 \u22121000] time-window \u2009=\u20099.31, p\u2009<\u20090.001).The analyses presented below followed those performed in Studies 1 and 2. The EEG results during the deceleration phase were similar to those of Study 1 Fig.\u00a0. Full reF\u2009=\u200917.08, p\u2009<\u20090.001). Pairwise comparisons revealed that EEG amplitude was smaller for Loss (\u22123.85\u2009\u00b1\u20095.42\u2009\u03bcV) compared to NLB  and FE , but bigger than NLA . NLA were also more negative than NLB  and FE . There was no significant difference between FE and NLB . As in EEG study 1, we observed two pairs of outcomes: Loss and NLA (with NLA being more negative than Loss) showing a CNV-like enhanced negativity, and NLB and FE with no negative shift.In the [\u22121000 \u2212500\u2009ms] time window, we found that EEG activity differed depending on Outcome \u2009=\u20098.83, p\u2009<\u20090.001). However, the pairwise comparisons revealed a different pattern of results than in the earlier deceleration time window. EEG amplitudes were more negative for Loss (\u22125.34\u2009\u00b1\u20096.06\u2009\u03bcV) than for NLA  and FE , but not compared to NLB . NLB were also more negative than NLA  and FE . There was no significant difference between FE and NLA . Again, we observed two new pairs of outcomes: Loss and NLB showed a CNV-like negativity, while NLA and FE did not. Topographies for the different windows of interest are shown in Fig.\u00a0In the [\u2212500 0\u2009ms] time window, we again found that EEG activity differed depending on Outcome (p\u2019s\u2009<\u20090.001), confirming that participants understood the meaning of a match in this opposite slot machine game. P3 was also larger for Loss than Escapes . Notably, NLB elicited larger P3 than other Escapes (p\u2019s\u2009<=\u20090.001). As for Study 1, these results support the claim that NLB create higher expectations than NLA and FE right before outcome onset. For full FRN and P3 results, see Supplementary Tables\u00a0As predicted, we found a larger FRN for Loss than Escapes . A significant effect of Outcome was found for the [\u22121500 1000] time window \u2009=\u20096.68, p\u2009=\u20090.0023). In that time window, NLA were associated with a lower tendency to bet on the slot machine  than Losses , NLB , or FE . All other comparisons were not significant.We found no main effect of Outcome for the [\u22123000 \u22122500], [\u22122500 \u22122000] and [\u22122000 \u22121500] time windows \u2009=\u200910.99, p\u2009<\u20090.001). In that time window, NLA were still associated with a lower tendency to bet on the slot machine  compared to NLB , or FE . NLA did not differ from Losses . All other comparisons were not significant.A significant effect of Outcome was found for the [\u22121000 500] time window \u2009=\u200911.23, p\u2009<\u20090.001). In that time window, Losses were associated with a lower tendency to bet on the slot machine  than NLB  and FE . The comparison to NLA approached significance . In addition, FE were associated with a higher tendency to bet on the slot machine compared to NLB (p\u2009=\u20090.027 and NLA (p\u2009=\u20090.012). There was no difference between NLB and NLA (p\u2009=\u20090.989). As in Study 1, visual inspection of the trajectories \u2009=\u2009\u22124.04.53, p\u2009<\u20090.001).A significant effect of Outcome was found for the [\u2212500 0] time window   Fig.\u00a0. In thatTaken together, Studies 3 and 4 show that Near Losses and FEs are perceived differently hundreds of milliseconds before the slot machine stops. These findings extend Studies 1 and 2 results to the loss domain, and as discussed below, shed light on what cognitive process is being tracked in the EEG signal.Reward expectations are critical for adjustments in decision-making and reward-seeking behavior. Expectations are likely to evolve in situations in which new information about the odds of receiving a reward becomes available\u2014for example during a horse race or a soccer game. However, little is known about the dynamics of expectations. Here, in four studies , we investigated the sub-second dynamics of reward expectations, using slot machines as a test case. In EEG Study 1, we found that different outcomes elicited different EEG activity during the deceleration phase of the machine, indicating that different expectations were formed before the final outcome was revealed. Similar electrophysiological findings were found in EEG Study 3 in the loss domain. In behavioral Studies 2 and 4, we implemented a paradigm designed to track moment-to-moment changes in expectations via betting behavior. We found that different outcomes elicited different expectation trajectories in the deceleration phase. Moreover, we found that these dynamic expectations correlated with the EEG activity in both Study 1 and 3 in the last second of the machine\u2019s deceleration. Taken together, these findings provide evidence that reward expectations are rapid and dynamic and can be tracked at the electrophysiological and behavioral levels. Below we discuss these findings, as well as their implications for healthy and unhealthy cognition.31. Notably, we found that in that last second, EEG activity changed over time, suggesting that expectations evolved as participants gathered more information about the location of the selected item on the reel and the speed of the reel. This interpretation is supported by the significant correlation between the EEG activity and the expectation trajectories obtained in the behavioral experiments (Studies 2 and 4). Further, in both experiments, there was no difference between the different outcomes 3\u2009s before reel deceleration . As a result, we predicted that Study 3 and 4\u2019s results would be a mirror image of Study 1 and 2\u2019s results. This is what we found when comparing the behavioral curves of Studies 2 and 4. However, the similarity between the EEG deceleration findings of Study 1 and Study 3 is striking. As shown in Figs.\u00a056. The CNV is negative-going ERP deflection traditionally linked to stimulus anticipatory activity. The CNV typically occurs following a stimulus S1 when a motor response to a second stimulus S2 requires maintaining information about S1. In our studies, S1 would be the items passing on the payline during the deceleration, and S2 the machine stopping\u2014which doesn\u2019t require any behavioral response. However, the CNV is not a purely motor process: recent studies showed it represents the neural correlate of expectancy for the S2 stimulus38, and is larger for unpredictable targets36. This is consistent with the fact that just before the machine stopped, we found enhanced negative amplitudes for uncertain outcomes  vs. certain outcomes . In addition, the known fronto-central scalp CNV distribution34 is also consistent with our findings 50. In Study 1, P3 was larger for NWB compared to NWA and FM, suggesting that participants were more surprised by their loss following NWB. Study 3\u2019s results were similar, with larger P3 for NLB vs. NLA and FE. These results are in line with past Near Win studies\u2019 findings41. The combined results of Studies 1 and 3 support the finding that P3 tracks unsigned  reward prediction error magnitude58. Further discussion of the P3 and FRN findings can be found in Supplementary Note\u00a0Finally, the P3 findings strengthen the findings that Near Win Before create higher expectations than Near Win After and Full Miss. Indeed, P3 has been associated with surprise and reward prediction errors60. However, in our slot machine task, such processes should elicit a similar ramping up (or down) of the EEG signal pre-outcome onset for all four outcomes. Thus, while these processes might account for some of the EEG activity, they cannot explain the unique patterns of activity elicited by the different outcomes. Alternatively, attention/arousal could be correlated with expectations. It could be that during deceleration, participants are more aroused/attentive when the outcome is a Near Win Before vs. a Full Miss because of the uncertainty regarding their outcome, and that this extra arousal/attention is what is being tracked by the EEG signal. This possibility does not undermine or contradict our conclusions that differential sub-second changes in expectations can be tracked at the electrophysiological level.One might wonder whether the EEG findings could be accounted by alternative explanations such as basic perceptual features (for example speed of spinning), arousal or attention. Indeed, the deceleration phase is characterized by a change in visual input, as the blurry items presented during the fast-spinning phase become clearer, and stay for longer durations on the payline. Participants might also become more attentive during the deceleration phase, and more aroused, as they know that the trial is about to end. Indeed, cues about the relevance of an upcoming target result in the deployment of selective attention, making it difficult to disentangle the effects of expectations from those of attentionThe \u201cSlot or Not\u201d paradigm used in Studies 2 and 4 was specifically designed to measure sub-second changes in expectations via behavior. Studies 2 and 4\u2019s findings confirm our prediction that expectations get updated during the deceleration phase of the slot machine. We developed a statistical method to identify relationships between timeseries and we found that the EEG and the behavioral findings significantly correlate, providing evidence that what is being tracked in the EEG is indeed dynamic expectations.Reward expectations play a critical role in healthy cognition, and have powerful effects on learning, memory, affect and decision-making. Studies addressing reward expectations typically assume that expectations are static. Our findings confirm what many of us know intuitively: expectations can change from moment to moment. Investigating the temporal dynamics of expectations is crucial if we want to understand how expectations affect us in the real world, when one can accumulate evidence regarding the odds of a certain event and update their expectations, whether it is on the road, at the horse races, or during a romantic date.64. Schizophrenia65 and attention-deficit/hyperactivity disorder66 have been associated with reduced striatal BOLD signal during reward anticipation, while pathological gamblers show the opposite pattern67. The representation of expected value in the orbitofrontal cortex is also altered in manic patients68. Altered expectations likely contribute to some of the clinical features of these disorders, such as enhanced/decreased motivation for seeking rewards, under/overestimation of risks and maladaptive choice behavior. Examining the temporal dynamics of expectations may shed light on what goes awry in these clinical populations. While slot machine games are of particular relevant to problem gambling, they may be useful for assessing expectation trajectories and reward responsivity in clinical and developmental populations because they are easily understood and easy to play32. Our behavioral paradigm, \u201cSlot or Not\u201d, is also easy to implement and can be run online. This is a major advantage for studies aiming to recruit participants with disabilities  or with rare disorders.Aberrations in how people form expectations play an important role in several mental disorders. In major depressive disorder and anxiety disorders neural responses to anticipated gains are different than those of healthy controls69\u2014an effect we also find in Study 1. This effect is so powerful that it is illegal to increase the incidence of Near Misses in Casino slot machines in many jurisdictions70. One account of the Near Miss Effect is that Near Wins are mistakenly interpreted as skill acquisition, and thus foster an \u201cillusion of control\u201d72. Another account of the Near Miss effect is counterfactual thinking: events that almost happened have a stronger emotional impact than events that didn\u2019t74. Our findings point to a third, parsimonious explanation: the Near Miss Effect could be the result of the different expectation trajectories leading to the outcome. Expectations and reward prediction errors are a key factor in satisfaction17. Since NWB and NWA have different expectation trajectories toward the end of the slot machine spinning, different effects on affect are predictable. This interpretation is in line with the finding that the Near Miss Effect disappears in paradigms that lack an anticipation phase52. Expectations could also explain why Near Miss, like wins, activate regions of the reward network including bilateral ventral striatum and right anterior insula75. Given that the ventral striatum and insula are also engaged in expectations formation and RPE77, this activity could reflect the high expectations elicited both by wins and Near Win Before right before the machine stops, or the similar RPEs elicited at outcome onset. These events may be masked by the temporal resolution of the BOLD fMRI response. The specific relationship between changes in expectations, motivation and happiness is a subject of future research. Importantly, the illusion of control, counterfactual thinking and expectations accounts of the Near Miss Effect are not mutually exclusive.The Near Miss Effect illustrates how dynamic expectations can add to our understanding of cognition. In the gambling literature, the Near Miss Effect refers to the finding that Near Wins are experienced as less pleasant than Full Misses, yet paradoxically influence a gamblers\u2019 actual behavior with bigger money bets following near-misses along with an increased desire to continue gamblingTo summarize, in a series of 4 studies, we found that expectations are rapidly updated in the deceleration phase of a slot machine game. These findings confirm that expectations are dynamic, and show that sub-second changes in reward expectations can be tracked in EEG activity and in choice behavior. We further examined the relationship between the behavioral and electrophysiological timeseries, and found that the two were significantly correlated. The results open exciting avenues for studying the ongoing dynamics of reward expectations salient to understanding their role in cognition and affect in healthy and clinical populations.We ran four studies: 2 EEG studies (Studies 1 and 3) and two behavioral studies (Studies 2 and 4). In Study 1, we used EEG to define the sub-second electrophysiological correlates of moment-to-moment changes in expectations elicited by the deceleration phase of a slot machine. In Study 2, we introduce a paradigm to measure moment-to-moment changes in expectations from betting behavior. In Studies 3 (EEG) and 4 , we replicated Studies 1 and 2 in the loss domain using a modified version of the slot machine where a match was associated with a loss of money, and mismatches with gains. Different participants took part in the different studies.Participants: The experiment was conducted on 42 participants. Data from six participants were excluded because too few artifact-free trials were available. The final sample was composed of 36 participants , all undergraduate and graduate students recruited at the University of California, Berkeley. Subjects had normal or corrected-to-normal vision by their self-report, and no history of\u00a0neurological disorders. Participants were paid $12 per hour to participate in the study. Informed consents were obtained after the experimental procedures were explained. This study, like the three other studies presented in this paper, was approved by the Institutional Review Board at the University of California, Berkeley. In all studies, all ethical regulations were followed.Stimuli and Procedure: During the task, participants sat in a dark acoustic room and played a computerized slot machine game on a desktop PC. The task lasted for approximately 35\u2009min. It was composed of 150 trials, divided into 3 blocks of 50 trials. Between blocks, participants were given a break and were offered water.29. The task was programmed using Neurobehavioral Systems Presentation (version 14.1), incorporating sounds and 3D graphics that made the task realistic and engaging. Participants were informed that their gains in the game were hypothetical. Each trial started with a fixation cross (750\u2009ms) and consisted of four phases: choice, fast spinning, deceleration and outcome , and two electrodes placed on the participants\u2019 earlobes for offline re-referencing. Horizontal electrooculogram (EOG) were recorded from electrodes placed at the outer canthi of both eyes. Vertical EOG was recorded from an electrode placed below the right eye, and from the right frontopolar electrode FP2. EEG data preprocessing and analyses were conducted using the Fieldtrip toolbox78 and custom code in MATLAB.EEG recording and preprocessing: EEG data was recorded reference-free using an Active 2 system  with 64 electrodes spread out across the scalp according to the extended 10\u201320 system , combined with manual selection of artifact components based on correlation with the EOG channels and typical component topographies.The EEG was continuously sampled at either 1024 or 512\u2009Hz and stored for offline analysis. Offline, EEG data were down-sampled to 512\u2009Hz (when needed), and re-referenced to the average of both ear lobe channels. EEG data were notch filtered at 60\u2009Hz, bandpass filtered between 0.1 and 256\u2009Hz, demeaned and detrended. Electrodes with excessive noise were replaced with an interpolation from neighboring electrodes using spherical spline interpolationTrials were segmented from \u22123000\u2009ms (beginning of the deceleration phase) to 1000\u2009ms relative to the outcome phase (when the machine stops). Each segment was baseline corrected to the 200\u2009ms preceding the start of the spinning. Trials contaminated by muscle activity, large voltage shifts and amplifier saturation were identified by visual inspection and discarded using the Fieldtrip function\u00a0ft_reject_visual. Trials were then averaged separately for the four types of outcomes: Win, Near Win Before, Near Win After and Full Miss. These averages were digitally filtered with a low-pass filter at 30\u2009Hz.Stimuli and Procedure: The experiment was conducted online, on the Qualtrics platform, using custom JavaScript scripts. Participants were recruited online through Prolific. They received a $3 participation fee, with an option to win a bonus.Participants provided informed consent to take part in the study, and then read the tasks instructions. They were tested on their comprehension of the instructions with six questions. If they made any mistake, they were presented with the instructions again. Participants who failed the comprehension questions three times did not get to do the actual task, and received $1 for their time. Following the comprehension test, participants played three rounds of practice and then moved on to the actual task , and was equal to 1, 5, 10, 15, 20 or 25 points.Participants selected one option using the right and left arrows on their keyboard. The chosen option was highlighted with an orange frame. The slot machine then started spinning, and then decelerated to a stop. Importantly, participants could change their choice (switch from \"slot machine\" to \"safe amount\" and vice versa) as often as they wanted during the trial, up to 750\u2009ms after the machine stopped. To incentivize participants to report their expectations at each given timepoint, we created the following payment scheme. Participants were instructed that one trial, and one timepoint during that trial would be randomly drawn, and that their decision at that timepoint would be implemented. If at that timepoint they chose the sure amount, they would receive the sure amount for this trial as their bonus. If they chose the slot machine, their bonus would depend on the outcome of the slot machine: they would receive 100 points if the slot machine\u2019s outcome was a jackpot, 0 otherwise. Participants were told that the chance that their bonus would\u00a0be determined by the outcome of the Slot Machine (or the Sure Amount)\u00a0is proportional to the time they spent on that option during the round selected for payment.\u00a0For example, if they chose the Sure Amount from the beginning and didn\u2019t switch, they would always receive this amount. On the other hand, if they spent half on the round on the Slot Machine, there would be a 50% chance that their bonus would be determined by the outcome of the Slot Machine. In other words, we wrote, \u201ceach moment you spend on one option increases the chances that that option will determine your bonus\u201d. Participants were provided with an illustration of how their bonus would be determined Fig.\u00a0. Points Over the 36 trials of the study, participants encountered 6 jackpots (same items on the payline), 6 Near Win Before, 6 Near Win After, and 18 Full Miss. The order of these trials was randomized for each participant. The position of the slot machine on the screen (right/left) was randomized for each trial.The slot machine stimuli were the same used in Study 1: we extracted videos from the Presentation experiment and integrated them into the Qualtrics experiment, in order for Study 1 and Study 2 to be comparable. We changed the written feedback that appeared at the outcome phase: instead of \u201cYou win $0.25\u201d and \u201cNo Win\u201d, the words \u201cJackpot\u201d and \u201cMiss\u201d were displayed.Participants and exclusion criteria: 51 participants were recruited online. 16 participants failed three times on the comprehension test and did not play the game. We also excluded five participants who did not switch at all in 90% or more of the trials, including in the 750\u2009ms time window following the stop of the machine . Our final sample was thus composed of 30 participants .Studies 3 (EEG) and 4  aimed to extend the understanding of moment-to-moment changes in expectations to the loss domain. To investigate people\u2019s expectations of losing, we modified the slot machine game presented in Study 1. In Study 3, a match was associated with a loss of money, and mismatches with gains. In this mirror-image of a classical slot machine, players could thus encounter Losses, NLB (when the machine stops just one item before a loss), NLA (when it stops just one item after a loss) and FE.Participants: The experiment was conducted on 41 new participants. Data from 6 participants were excluded because too few artifact-free trials were available. The final sample was thus composed of 35 participants , undergraduate and graduate students recruited at the University of California, Berkeley. Subjects had normal or corrected-to-normal vision by their report, and no history of\u00a0neurological disorders. Participants were paid $12 per hour to participate in the study. Informed consents were obtained after the experimental procedures were explained.Stimuli and Procedure: We used the exact same slot machine game as in Study 1. The only difference was the meaning of a match. In this version of the game, if the two items on the payline matched, there was a buzzer sound, and participants lost $0.25. In all other cases, participants won $0.10 and heard a cash register sound. We introduced gains in this paradigm for two reasons: 1) to keep participants motivated during the task, 2) so that the slot machine would have the same expected value as in Study 1. As in Study 1, the monetary gains and losses were hypothetical. Participants encountered 25 Losses (the two items on the payline matched), 25 NLB (the machine stopped one symbol before a match), 25 NLA (the machine stopped one symbol after a match), and 75 FE (the machine stopped two or three positions away from a match).EEG recording: Recordings settings and preprocessing steps were identical to those of Study 1. As in Study 1, the amplitudes of the FRN and P3 elicited by the outcome phase were measured as their mean value in an 80\u2009ms window around their peak. The FRN elicited by the outcome phase peaked at 248\u2009ms, and we measured it as the mean value in the 210\u2013290\u2009ms window. The P3 peaked at 424\u2009ms, and we measured it as the mean value in the 385\u2013465\u2009ms window. For the deceleration phase, we used the same time windows as in Study 1: \u22121000 to \u2212500\u2009ms, and \u2212500\u2009ms to 0.Study 4 was a replication of Study 2 in the loss domain.Stimuli and procedure: We used the exact same paradigm (\u201cSlot or Not\u201d) as in Study 2, but changed the meaning of matches. In this version of the game, if the two items on the payline matched, this was a miss, associated with the potential loss of 50 points. If the two items on the payline did not match, this was a jackpot, associated with a potential win of 30 points. Over the 36 trials of the study, participants encountered 6 Losses (same items on the payline), 6 Narrow Escape Before, 6 Narrow Escape After, and 18 FE.Participants and exclusion criteria: 61 participants were recruited online. 32 participants failed three times on the comprehension test and did not play the game (we suspect some failed on purpose to get the $1 compensation fee). We also excluded 8 participants who did not switch at all in 90% or more of the trials, including in the 750\u2009ms time window following the stop of the machine, that is, when the outcome was already known. Our final sample was thus composed of 21 participants .55. We averaged the data into six 500\u2009ms time windows from \u22123000\u2009ms (beginning of the deceleration) to the stop of the machine . Then, for each time window we ran a one-way repeated measures ANOVA . The significance level was set at 0.008 to account for the fact that we tested 6 time-windows, following the formula\u03b1\u2032\u2009=\u2009Bonferroni correction, \u03b1\u2009=\u2009critical p value (0.05) and k\u2009=\u2009number of tests (6). Greenhouse\u2013Geisser correction for analysis of variance ANOVA tests was used whenever appropriate. If the ANOVA was significant, we performed pairwise comparisons using Tukey tests with a significance level at 0.05.Study 1: For the analysis of the deceleration phase, we used electrode Cz, where the CNV is maximal81. The amplitude of the FRN elicited by the outcome phase was measured as its mean value in an 80\u2009ms window centered around its peak. Peaks were determined as follows. Since the FRN has been shown to be maximal at fronto-central midline sites83, we averaged the data across the three frontocentral midline electrodes . The peak of the FRN was then defined as the most negative point in the 200\u2013350\u2009ms time window of the grand average across subjects. The FRN elicited by the outcome phase peaked at 251\u2009ms, and was measured as the mean value in a 210\u2013290\u2009ms window.Post-outcome, we examined the Feedback Related Negativity (FRN) and P3. The FRN  peaks over the scalp\u2019s midline within 250\u2013350\u2009ms after feedback is provided, and is generated in part in the anterior cingulate cortex84 and reflects the summed activity of multiple intracranial sources85. Since P3 is maximal at the centro-parietal area86, we averaged the data across Cz, CPz, Pz, POz, and Oz. The peak of the P3 was then defined as the most positive point in the 300\u2013500\u2009ms time window of the grand average. The P3 elicited by the outcome peaked at 373\u2009ms, and was measured as the mean value in a 330\u2013410\u2009ms window. For both the FRN and the P3, we ran a one-way repeated measures ANOVA . The significance level was set at 0.05. Greenhouse\u2013Geisser correction for analysis of variance ANOVA tests was used whenever appropriate. If the ANOVA was significant, we performed pairwise comparisons using Tukey tests.The P3 is a large positive component occurring in the 300\u2013600\u2009ms time window after stimulus onset when the stimulus has behavioral consequences. It is generated in frontal and temporo-parietal sitesStudy 2: The behavioral analyses followed the same approach as Study 1\u2019s ERPs analyses. For each trial, we obtained a timeseries of 0 and 1 values  sampled every 50\u2009ms. We defined six 500ms-time windows of interest from \u22123000ms (beginning of the deceleration) to the stop of the machine . For each subject, in each one of these time windows, we averaged the number of 0\u2009s and 1\u2009s separately for the four outcomes . Then, for each time window we ran a one-way repeated-measure ANOVA (four outcomes). The significance level was set at 0.008 (0.05 divided by 6) to account for the fact that we tested six time-windows. Greenhouse\u2013Geisser correction for analysis of variance ANOVA tests was used whenever appropriate. If the ANOVA was significant, we performed pairwise comparisons using Tukey tests with a significance level at 0.05. Figure\u00a0Do the different outcomes expectations\u2019 time course found in Study 2 relate to Study 1\u2019s individual subjects EEG activity prior to outcome onset? We will first present an overview of the method we adopted to examine the relationship between the two timeseries; technical details appear below. Figure\u00a0Our analysis required us to select a time window on which to calculate the correlation between EEG and behavioral data. Rather than selecting one based on visual inspection, we performed a data-driven search and looked for the time window (t seconds before outcome) that maximizes the absolute average correlation between the behavioral curves and the EEG activity. For each possible time window (t seconds before outcome), we calculated the correlations between each participant\u2019s EEG (averaged per condition) and the corresponding group-level behavioral curves. Note that time-windows were of varying lengths depending on t. We then averaged these correlations across participants, and took this average\u2019s absolute value. The time window that maximized this absolute correlation was selected. Second, a permutation test was performed to assess the significance of this correlation. Under the null hypothesis that the relationship between the EEG and the behavioral data is not outcome-specific, we permuted the data by randomly shuffled the labels of the four outcomes attached to the individual subjects\u2019 EEG signal. For each permutation, we identified the time window maximizing the absolute correlation between the behavioral curves and the EEG activity and noted the value of this correlation. The permutation was performed 10000 times to create a null distribution against which the actual correlation was compared to.The parameter search was performed using fmincon in MATLAB using sequential quadratic programming with bounds at \u22123 and \u22120.5\u2009s. We set the minimum duration of time-windows to 500\u2009ms. For each subject, for a given time window -t to 0\u2009s, we interpolated the four behavioral curves and the four EEG curves in 100 timepoints between -t and 0\u2009s to hold the number of data points constant throughout the parameter search process. The correlation coefficient was then obtained by concatenating the four behavioral curves (100 time points each) into one vector and concatenating the four EEG curves  into one vector and calculating the Pearson correlation between the two vectors (each with 400 time points).Study 3: EEG analyses were identical to those performed in Study 1.Study 4: Analyses were identical to those performed in Study 2.Further information on research design is available in the\u00a0Peer Review FileSupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Reporting summary"}
+{"text": "Neodothiora populina CPC 39399T, the closest species with strain JAF-11, showed a sequence similarity of 97.75% for LSU and 94.27% for ITS, respectively. The result suggests that the strain JAF-11 represents a distinct species that cannot be assigned to any existing genus or species in the family Dothideaceae. Strain JAF-11 produced a biosurfactant reducing the surface tension of water from 72 mN/m to 34.5 mN/m on the sixth day of culture and the result of measuring the critical micelle concentration (CMC) by extracting the crude biosurfactant was found to be 24 mg/l. The molecular weight 502 of the purified biosurfactant was confirmed by measuring the fast atom bombardment mass spectrum. The chemical structure was analyzed by measuring 1H nuclear magnetic resonance (NMR), 13C NMR, and two-dimensional NMRs of the compound. The molecular formula was C26H46O9, and it was composed of one octanoyl group and two hexanoyl groups to myo-inositol moiety. The new biosurfactant is the first report of a compound produced by a new yeast strain, JAF-11.Biosurfactants reduce surface and interfacial tension due to their amphiphilic properties and are an eco-friendly alternative for chemical surfactants. In this study, a new yeast strain JAF-11 that produces a biosurfactant was selected using drop collapse method, and the properties of the extracts were investigated. The nucleotide sequences of the strain were compared with closely related strains and identified based on the D1/D2 domain of the large subunit ribosomal DNA (LSU) and internal transcribed spacer (ITS) regions.  The surfactants have amphiphilic properties possessing both non-polar (hydrophobic) and polar (hydrophilic) moieties that allow reducing the surface and interfacial tension between biphasic systems as a liquid-liquid interface or solid-liquid boundaries , 2. Ther7Pseudomonas aeruginosa  at 25\u00b0C for three days. The biosurfactant production of the yeast isolates was tested using a modified drop collapse method as follows: 100 \u03bcl of the culture supernatant and water  was pipetted and placed on parafilm , 21. Strhttp://www.megasoftware.net).The identification of strain JAF-11 was conducted based on multigene phylogenetic analysis of the nucleotide sequences combined with the D1/D2 region of the large subunit (LSU) and the internal transcribed spacer (ITS) region ribosomal DNA (rDNA) genes. Macrogen Inc. (Korea) performed the DNA sequencing, and the gene sequences of related species were retrieved from the GenBank database. The phylogenetic tree was inferred by using the maximum likelihood method with 1,000 bootstrap replicates and sequences analysis was performed in MEGA X software  every day. For ST measurement, the Wilhelmy plate method was used at room temperature with a force tensiometer K11 (Germany). All tests were performed in triplicates.3 : MeOH, 50:1 to 1:1 (v/v). The crude biosurfactant was dissolved in distilled water and serially diluted to concentrations of 0-250 mg/l. The CMC was determined by plotting the surface tension against the log of the biosurfactant concentration + , indicating four degrees of unsaturation. The 1H NMR spectrum of 1 , 5.28  , 4.93 , 3.66 , 3.65 , and 3.45 . It also showed signals attributable to 14 methylenes at \u03b4H 2.45 /2.42 , 2.36 /2.31 , 2.18 , 1.68 , 1.59 , 1.55 , and 1.35-1.25 (overlapped) and three methyls at \u03b4H 0.90 (overlapped). The 13C NMR spectrum (C 175.0 (C-1\u00a2), 174.6 (C-1\u00a2\u00a2), and 174.2 (C-1\u00a2\u00a2\u00a2), six oxygenated methine carbons at \u03b4C 74.6 (C-6), 74.2 (C-5), 73.2 (C-4), 72.6 (C-2), 71.7 (C-3), and 70.9 (C-1), 14 methylene carbons at \u03b4C 23.4\u201235.2, and three methyl carbons at \u03b4C 14.4 (C-6\u00a2\u00a2\u00a2) and 14.2 (C-8\u00a2 and C-6\u00a2\u00a2). The 1H-1H COSY correlations among six oxygenated methine protons established the presence of an inositol moiety. Inositol moiety was identified as a myo-inositol by the proton coupling constant. Except for an equatorial proton at \u03b4H 5.50 (H-2) with a coupling constant of 2.7 Hz, other protons occupied an axial position based on their proton coupling constants. The 1H-1H COSY spectrum also established six partial structures in three acyl chains, as shown in H 0.90 (H-6\u00a2\u00a2 and H-6\u00a2\u00a2\u00a2) to two methylene carbons at \u03b4C 32.4 (C-4\u00a2\u00a2 and C-4\u00a2\u00a2\u00a2), from the methylene protons at \u03b4H 1.59 to the carbonyl carbon at \u03b4C 174.6 and the methylene carbons at \u03b4C 32.4 and 23.4, and from the methylene proton at \u03b4H 1.55 to the carbonyl carbon at \u03b4C 174.2 and the methylene carbons at \u03b4C 32.4 and 23.4  pers. + . The molrum of 1  showed sspectrum  in combiand 23.4 . The pre\u03b4C 175.0  and frommyo-inositol lipid type was isolated from yeast strain JAF-11 from Prunus mume Sieb. et Zucc. The newly isolated biosurfactant has the potential to be used in industrial applications such as cosmetics, medicine, and health functional foods.In conclusion, a new biosurfactant of http://jmb.or.kr.Supplementary data for this paper are available on-line only at"}
+{"text": "CeGdO2\u2212x NPs possess an ultrasmall size, high colloidal stability, and pronounced antioxidant properties. A comprehensive analysis of LbL capsules by TEM, SEM, LCSM, and EDX techniques was carried out. The research demonstrated a high level of biocompatibility and cellular uptake efficiency of CeGdO2\u2212x-loaded capsules by cancer (human osteosarcoma and adenocarcinoma) cells and normal  cells. The LbL-based delivery platform can also be used for other imaging modalities and theranostic applications.Layer-by-layer (LbL) self-assembled polyelectrolyte capsules have demonstrated their unique advantages and capability in drug delivery applications. These ordered micro/nanostructures are also promising candidates as imaging contrast agents for diagnostic and theranostic applications. Magnetic resonance imaging (MRI), one of the most powerful clinical imaging modalities, is moving forward to the molecular imaging field and requires advanced imaging probes. This paper reports on a new design of MRI-visible LbL capsules, loaded with redox-active gadolinium-doped cerium oxide nanoparticles (CeGdO Gadolinium in ionic form is very toxic and can potentially cause nephrogenic systemic fibrosis (NSF) [\u00ae, OptiMark\u00ae, MultiHance\u00ae, Primovist\u00ae, and Vasovist\u00ae). At the same time, poorly soluble gadolinium oxide is a more stable compound than its ionic form and does not have toxic effects either in vitro or in vivo [3+ chelate complexes [Medical imaging has long served as an important tool for diagnosis and therapeutic efficacy monitoring. MRI is safe and has a very high spatial resolution of around 25\u2013100 \u03bcm in different magnetic fields. Today, gadolinium-containing compounds are widely used as MRI contrast agents [oment 7.9 BM, whicomplexes .2) is the most promising inorganic nanozyme, and it is used in various areas of biomedicine. It has strong antioxidant [3+ ions increases its oxygen nonstoichiometry and, consequently, enhances the antioxidant activity of the nanoparticles [The cerium oxide nanoparticle , sodium carbonate , ethylenediaminetetraacetic acid disodium salt dihydrate , citric acid (HOC(COOH)(CH2COOH)2, #C0759), cerium (III) chloride heptahydrate , gadolinium(III) nitrate hexahydrate (#451134), dextran sulfate sodium salt , 4-iodophenol (#I10201), and poly-L-arginine hydrochloride , were purchased from Sigma-Aldrich. All chemicals were used as received. Ultrapure water with a resistance greater than 18.2 M\u03a9 cm\u22121 was used for all experiments. MTT reagent and Hoechst 33,342 dye were purchased from PanEKO . A live/dead assay kit, LDH assay kit, rhodamine B isothiocyanate, and phalloidin-FITC were purchased from Thermo Fisher Scientific, Cambridge, UK.Calcium chloride (CaClAn aqueous solution containing cerium (III) chloride and gadolinium (III) nitrate was prepared, with a total concentration of rare earth elements of 2 mM. The molar ratio of cerium to gadolinium was 4:1. An anion exchange resin in the OH form was added to the resulting solution until pH 10.0 was reached. The resulting solution was separated from the anion exchange resin by filtration and then subjected to hydrothermal treatment at 150 \u00b0C for 1.5 h, after which it was cooled to room temperature. The sol was stabilised using sodium citrate (cerium/citrate molar ratio was 1:4). Then, the pH of the sol was adjusted to 7\u20138 by dropwise addition of aqueous ammonia.2\u2212x NPs were measured on a Lambda 950 UV/VIS spectrometer . TEM images were acquired using a JEOL-JEM 2010 transmission electron microscope . EDX analysis was performed using an FEI Inspect F microscope. The hydrodynamic diameter and the zeta potential values were measured using a Zetasizer Nano ZS analyzer . The aggregative stability of the CeGdO2\u2212x NPs was studied by dynamic light scattering 1 h after the formation of the CeGdO2\u2212x NPs\u2019 suspension in a phosphate buffer (pH 7.4) , DMEM/F12 culture medium , and DMEM/F12 culture medium containing 10% fetal bovine serum .The absorption spectra of the CeGdO3) was used as a template. The CaCO3 synthesis was initiated by rapid mixing of equal volumes of CaCl2 and Na2CO3 aqueous solutions at room temperature. After intensive stirring, with a magnetic stirrer for 30 s, the precipitate was separated by centrifugation at 1000 rpms for 1 min and washed three times with water. As a result, an aqueous suspension was formed containing spherical CaCO3 microparticles with an average diameter of 3 to 4 \u00b5m. The first polyelectrolyte layer was deposited on the microparticles\u2019 surface by adsorption of positively charged poly-L-arginine hydrochloride (PARg) using a 1 mg/mL\u22121 PARg solution in 0.15 M NaCl (15 min incubation and shaking). The second layer was deposited by absorbing a negatively charged sodium dextran sulfate (DS) from a 1 mg/mL\u22121 DS solution in 0.15 M NaCl (15 min incubation and shaking). The core/polyelectrolyte particles were washed three times with deionised water after each adsorption step. A colloidal solution of CeGdO2\u2212x NPs was taken at a concentration of 0.5 mg/mL. The calcium carbonate nuclei were dissolved in ethylenediaminetetraacetic acid (EDTA) for 30 min, then centrifuged and washed three times with EDTA and then three times with water. The capsule consisted of a biodegradable polyelectrolyte PArg and DS with cerium gadolinium oxide nanoparticles in the middle layer (PArg/DS) (PArg/CeGdO2\u2212x NPs) (PArg/DS).Calcium carbonate (CaCO2\u2212x NPs-loaded capsules were measured on a Perkin-Elmer Lambda 950 UV/VIS spectrometer. TEM images were acquired using a JEOL-JEM 2010 transmission electron microscope. Scanning electron microscopy (SEM) and EDX analyses were performed using an FEI Inspect F microscope. Laser scanning confocal microscopy (LSCM) images were acquired using a Leica TS laser scanning confocal microscope with a 63\u00d7 f/1.4 oil immersion lens. Rhodamine B isothiocyanate (RBITC) labeled dextran was used as a fluorescent marker for the synthesis of the capsules and to study their intracellular localization.The absorption UV spectra of the CeGdO2 flasks  in a CO2 incubator .Cytotoxicity and cellular uptake analyses were carried out on three types of cell cultures: human osteosarcoma cells (MNNG/Hos), human adenocarcinoma cells (MCF-7), and human mesenchymal stem cells (hMSc) isolated from the dental pulp of a healthy orthodontics patient (with his written consent). All cells were deposited in the cryobank of the Theranostics and Nuclear Medicine Laboratory in ITEB RAS. Cells were cultured in a DMEM/F12 cultural medium containing 10% fetal calf serum  and a mixture of antibiotics (penicillin-streptomycin) . The cells were cultured in 75 cm4/cm2 in a DMEM/F12 culture medium  containing 10% fetal calf serum . After 8 h, capsules  were added to the cells. Then, after 24, 48, and 72 h, the medium was replaced with a solution of the MTT reagent (0.5 mg/mL). After 3 h of incubation with the MTT reagent, 100 \u03bcL of DMSO  was added. The optical density of the formazan solution in DMSO was determined using a BioRad plate reader 680  at 540 nm wavelength.Cytotoxicity was assessed using a standard MTT assay . Cells w2, at 37 \u00b0C. Six hours after cell seeding, the medium was replaced with the similar medium containing 1, 10, or 100 capsules per cell. Triton X-100 was used as a positive control. Within 72 h after the addition of the CeGdO2\u2212x NP-loaded capsules, the level of lactate dehydrogenase in the culture medium was determined, according to the manufacturer\u2019s protocol . Absorbance of the solution was measured at wavelengths of \u03bb = 490 nm and \u03bb = 640 nm, using the Microplate Reader ThermoMultiskan Ascent 96 & 384 .Cells were seeded in 96-well plates and cultured in an atmosphere containing 5% CO3 per cm2. After attachment and spreading of the cells (8 h), RBITC-labelled capsules were added (10 capsules per cell) and incubated with the cells for 16 h. After that, the cells were washed three times with Hank\u2019s solution and were stained to show actin cytoskeleton  and the cell nucleus . Micrographs were taken on a Zeiss Axiovert 200  inverted microscope at a magnification of 63\u00d7 with oil immersion.Rhodamine B isothiocyanate (RBITC)-labelled capsules were used for intracellular visualisation. The cells were seeded in 35 mm Petri dishes with a central hole , at a density of 2 \u00d7 102\u2212x-loaded capsules were carried out using a Bruker Clinscan 7T MRI tomograph . For the measurements, the samples were diluted with an HEPES buffer solution to concentrations ranging from 0.1 mM to 1 mM, taking into account the efficiency of the loading of the nanoparticles and the concentration of nanoparticles per capsule. The SI dependencies on TI for each concentration were plotted and the T1 relaxation time was determined by the Mathcad approximation. 1/T1 (s\u22121) values were calculated from experimentally determined T1 relaxation times. T1 relaxivity values were calculated as the tangent of the inclination angle in the dependencies of the reverse T1 relaxation time on the Gd3+ concentration.MRI studies of CeGdO2O2 was generated under X-ray irradiation of the CeGdO2\u2212x NPs\u2019 suspensions. Irradiation was conducted using an X-ray therapeutic machine RTM-15  in a dose of 5 Gy, at a dose rate of 1 Gy/min, 200 kV voltage, 37.5 cm focal length, and 20 mA current. To evaluate the redox activity of the CeGdO2\u2212x NPs, the concentration of hydrogen peroxide after the X-ray exposure was measured by an enhanced chemiluminescence technique, using a luminol\u20134-iodophenol\u2013peroxidase system [2O2 content was determined using calibration chemiluminescence plots. The concentration of hydrogen peroxide used for the calibration was determined spectrophotometrically at 240 nm, using a molar absorption coefficient of 43.6 M\u22121\u00b7cm\u22121.He system . A TRIS t-test.Mean values and the standard deviation of the mean were calculated and the significance of differences between the groups was determined using the Student 2\u2212x nanoparticles, cerium chloride and gadolinium nitrate were used . Interestingly, the integration of CeGdO2\u2212x nanoparticles in the capsules provided them a luminescent property, which was revealed using a confocal microscope  cells . It was \u22121. The analysis of the relaxation rate of the synthesized LbL capsules loaded with CeGdO2\u2212x NPs demonstrated their lower relaxation rate (2.75 mM\u00b7s\u22121), which could be due to the aggregation of CeGdO2\u2212x NPs in the structure of the polyelectrolyte matrix and the low access level to water molecules (electron spins), or the possible partial loss of the nanoparticles during the multistage synthesis of the composite capsules, as previously shown [2\u2212x loaded microcapsules is significantly inferior to commercial preparations [The next task was to find out how the integration of the nanoparticles into the structure of the capsules affects the relaxation rate in MRI measurements . Previouly shown . At the arations . Given t2\u2212x NPs nanoparticles in their shell with an MRI contrasting property. The new microcapsules had a size of 3\u20134 \u00b5m. A comprehensive analysis of their physicochemical properties has confirmed the effective loading of nanoparticles into the structure of the capsules. Furthermore, the synthesized capsules effectively penetrated both normal and cancer cells, being localized in the cytoplasm after the internalization. The microcapsules were not toxic at concentrations below 100 capsules per cell. The combination of MRI imaging and the redox properties of the capsules opens up possibilities for their use as theranostic agents and drug carriers with an easy to trace localization.This paper has demonstrated the fabrication of biodegradable microcapsules with CeGdO"}
+{"text": "Acidovorax citrulli, the causative agent of bacterial fruit blotch, can be divided into two main groups based on factors such as pathogenicity and host species preference. PilA is an important structural and functional component of type IV pili (T4P). Previous studies have found significant differences in pilA DNA sequences between group I and group II strains of A. citrulli. In this study, we characterized pilA in the group I strain pslb65 and the group II strain Aac5. pilA mutants, complementation strains, and cross-complementation strains were generated, and their biological phenotypes were analyzed to identify functional differences between pilA in the two groups. pilA deletion mutants (pslb65-\u0394pilA and Aac5-\u0394pilA) showed significantly reduced pathogenicity compared with the wild-type (WT) strains; pslb65-\u0394pilA also completely lost twitching motility, whereas Aac5-\u0394pilA only partially lost motility. In King\u2019s B medium, there were no significant differences in biofilm formation between pslb65-\u0394pilA and WT pslb65, but Aac5-\u0394pilA showed significantly reduced biofilm formation compared to WT Aac5. In M9 minimal medium, both mutants showed significantly lower biofilm formation compared to the corresponding WT strains, although biofilm formation was recovered in the complementation strains. The biofilm formation capacity was somewhat recovered in the cross-complementation strains but remained significantly lower than in the WT strains. The interspecies competitive abilities of pslb65-\u0394pilA and Aac5-\u0394pilA were significantly lower than in the WT strains; Aac5-\u0394pilA was more strongly competitive than pslb65-\u0394pilA, and the complementation strains recovered competitiveness to WT levels. Furthermore, the cross-complementation strains showed stronger competitive abilities than the corresponding WT strains. The relative expression levels of genes related to T4P and the type VI secretion system were then assessed in the pilA mutants via quantitative PCR. The results showed significant differences in the relative expression levels of multiple genes in pslb65-\u0394pilA and Aac5-\u0394pilA compared to the corresponding WT stains. This indicated the presence of specific differences in pilA function between the two A. citrulli groups, but the regulatory mechanisms involved require further study. Acidovorax citrulli [Bacterial fruit blotch (BFB) is caused by citrulli  and is ocitrulli . Since icitrulli ,4.A. citrulli shows extensive intraspecies genetic diversity. The species is divided into at least two groups based on clear differences in pathogenicity [A. citrulli strains can infect cucurbitaceae crops such as melon and watermelon, due to the host preferences of different groups of strains, there are significant differences in pathogenicity between the two groups of strains to different hosts [genicity , host prgenicity ,7,8, andgenicity  between nt hosts ,6. Groupnt hosts ,11.A. citrulli pathogenesis has largely focused on the type III secretion system (T3SS) [A. citrulli virulence. These hair-like appendages are found on the surfaces of many bacteria and contribute to key bacterial activities and characteristics, such as surface adhesion, colonization, aggregation, biofilm formation, genetic material uptake, and virulence [Recent research into the molecular mechanisms associated with m (T3SS) ,14,15,16m (T3SS) ,18, the m (T3SS) , and quom (T3SS) . Type IVirulence ,22.Pseudomonas aeruginosa [Ralstonia solanacearum [Xylella fastidiosa [Xanthomonas spp. [Pseudomonas spp. [A. citrulli, T4P insertion mutants were characterized to determine the genetic function of pili. In the A. citrulli group II strain W1, insertion mutants for pilA showed reduced virulence, biofilm formation ability, and twitching motility capacity [A. citrulli group I strain M6; representative mutants were then further characterized [Genetic and functional characterizations of T4P were first conducted on the mammalian pathogen ruginosa . The rolnacearum , Xylellastidiosa , Xanthomnas spp. , and Psenas spp. . In A. ccapacity . In anotcterized .pilA gene sequences from plant pathogenic bacteria have revealed extensive genetic variability [pilA between A. citrulli strains. Based on differences in the pilA sequence, A. citrulli strains were divided into three types; the sequences of the three types of pilA genes can be found in A. citrulli group II strain AAC00-1 (GenBank accession number NC_008752.1), and the DNA sequences of those genes are identical to that of the group I strain M6 (GenBank accession number NZ_CP029373.1). We therefore hypothesized that differences in the pilA DNA sequences may lead to functional differences between the groups.PilA, one of the core components of the T4P structure and function, is a relatively small subunit (13\u201323 kDa), thousands of which combine to form T4P. Comparisons of iability . A previpilA deletion mutants, complementation lines, and cross-complementation lines from the group I strain pslb65 and the group II strain Aac5. Pathogenicity, twitching motility, biofilm formation ability, interspecies competition traits, and other phenotypes were assessed in these mutants. Quantitative PCR was also used to analyze the relative expression levels of T3SS-, T6SS-, and T4P-related genes in the two pilA deletion mutants to further explore the function of pilA in the two groups of A. citrulli strains.To test this hypothesis, here, we generated A. citrulli strain Aac5 was isolated from watermelon collected in Taiwan, China, and strain pslb65 was isolated from melon collected in Xinjiang, China. A. citrulli strains were grown in King\u2019s B (KB) broth or on KB plates at 28 \u00b0C. Escherichia coli strains were grown in Luria Bertani (LB) medium at 37 \u00b0C [The bacterial strains and plasmids used in this study are listed in at 37 \u00b0C . Liquid pilA and the amino acid sequences of PilA from A. citrulli strains pslb65 and Aac5 were aligned using Clustal Omega  and visualized with Jalview v2.11.2.4 [The DNA sequences of 2.11.2.4 .pilA deletion mutants were generated in the A. citrulli strains Aac5 and pslb65 using homologous double recombination [pilA were amplified from the wild-type (WT) pslb65 and Aac5 strains using the 65AL-F/R and 65AR-F/R primers (for pslb65) and the A5AL-F/R and A5AR-F/R primers (for Aac5)  to generate the pK18-pslb65pilA and pK18-Aac5pilA constructs in E. coli DH5\u03b1. pK18-pslb65pilA and pK18-Aac5pilA were then introduced to the A. citrulli strains pslb65 and Aac5, respectively, through triparental conjugation using pRK600 as a helper plasmid. Single-exchange colonies were selected on KB plates containing Amp and Km. Individual transformants were continuously sub-cultured in liquid KB medium containing Amp alone and then screened on KB supplemented with Amp and 10% sucrose. The pilA deletion mutants for pslb65 and Aac5, pslb65-\u0394pilA and Aac5-\u0394pilA, respectively, were confirmed via PCR using the 65-\u0394pilA-L/R, A5AL-L/A5AR-R, Km-F/R, and WFB1/2 primers  . The seq primers . InformapilA genes in pslb65 and Aac5 were amplified using the 65-\u0394pilA-L/R and HBA5L/R primers, respectively (pilA and pBBR-Aac5pilA constructs. pBBR-pslb65pilA was transferred into the pslb65-\u0394pilA and Aac5-\u0394pilA mutants to generate complementation (pslb65-\u0394pilAcomp1) and cross-complementation (Aac5-\u0394pilAcomp2) lines, respectively, using triparental conjugation. Similarly, pBBR-Aac5pilA was transferred into the pslb65-\u0394pilA and Aac5-\u0394pilA mutants to form cross-complementation (pslb65-\u0394pilAcomp2) and complementation (Aac5-\u0394pilAcomp1) lines, respectively. Successful transconjugants were confirmed with PCR.To generate complementation lines, the ectively . After cA. citrulli strain AAC00-1 and M6 genomes  using Primer Premier v5.0 (PREMIER Biosoft). All primers were synthesized by BGI Laboratories .All primers in this study were designed based on the pilA, pslb65-\u0394pilAcomp1, pslb65-\u0394pilAcomp2, Aac5, Aac5-\u0394pilA, Aac5-\u0394pilAcomp1, and Aac5-\u0394pilAcomp2) were measured in 3-week-old watermelon seedlings (Citrullus lanatus cv. \u2018Jingxin#3\u2019) and melon seedlings (Cucumis melo cv. \u2018TVF192\u2019). Briefly, A. citrulli strains were cultured overnight in KB broth to an OD600 of 0.3 (corresponding to ~3 \u00d7 108 colony-forming units (CFU)/mL). The leaves of the melon and watermelon seedlings were spray-inoculated with the bacterial suspension, with sterile water serving as the negative control. Each treatment had 15\u201320 melon or watermelon seedlings, and 200mL of bacterial suspension was evenly sprayed onto the front and back of the leaves of seedlings in each treatment. At 15 d after inoculation (DAI), the disease severity was determined by measuring the symptoms and calculating the disease index using previously described methods with slight modifications [The virulence levels of each strain  and three independent replicates of the experiment.All strains used in spray-inoculation experiments were also used in stem-inoculation experiments, which were performed as previously described  with sompilA deletion on HR induction, bacterial cell suspensions (~108 CFU/mL) were injected into the leaves of 3-week-old tobacco (Nicotiana tabacum var. samsun) plants as previously described [To evaluate the effects of escribed . Leaves A. citrulli strain using the method described by Bahar et al. [A. citrulli strains were grown on KB plates containing Amp at 28 \u00b0C for 72 h. Each strain was considered to have twitching motility if a thin, light halo was visible around the colonies via light microscopy. An IX83 microscope  was used. Nine plates were inoculated per strain in each replicate experiment, and there were three independent replicates.Twitching motility was measured in each r et al. . BrieflyA. citrulli strain at a concentration of 3 \u00d7 108 CFU/mL. The plates were incubated at 28 \u00b0C for 48 h without shaking. After incubation, 0.1% crystal violet was added to each well, and the plate was incubated for 30 min at room temperature. Each well was then gently washed three times with distilled water. To quantify biofilm formation, the stained biofilms were solubilized in 95% ethanol for 2 h, and then the OD575 value of each stained cell suspension was measured with an Evolution 300 UV/VIS spectrophotometer . There were three biological replicates for each A. citrulli strain in each experiment, and there were three independent replicates of this experiment.Biofilm formation was assessed as previously described . BrieflyE. coli DH5\u03b1 serving as the prey strain. The killer strains  were cultured in KB broth to an OD600 of 1.5, and the prey culture was grown in LB broth to an OD600 of 2.0. The killer and prey cultures were each centrifuged and resuspended in sterile water and then mixed at a 20:1 killer\u2013prey ratio. After co-culture, the mixed cultures were 10-fold serially diluted and spotted on LB plates containing 25 \u03bcg/mL Gm. After 3 h, the number of surviving E. coli DH5\u0251 was quantified in log10 (CFU/mL) [Interspecies competitions were conducted, with (CFU/mL) .pilA expression on T3SS genes, the WT A. citrulli strains pslb65 and Aac5 and the pilA mutants pslb65-\u0394pilA and Aac5-\u0394pilA were cultured in the T3SS-inducing broth XVM2 [pilA expression on T4P genes, the same strains were cultured in KB broth. To assess the effects of pilA expression on T6SS-related genes, the same strains were also cultured in KB broth to OD600 values of 1.0 and 1.5. Total RNA was extracted from each culture using Trizol reagent . cDNA fragments were synthesized using a HiScript II RT SuperMix kit . rpoB was used as the internal reference gene for expression normalization [\u2212\u0394\u0394Ct method [To assess the effects of oth XVM2 . To asselization . qRT-PCRt method . The prit-test (for sample pairs) or analysis of variance (ANOVA) and post hoc Duncan\u2019s multiple range test (for multiple samples). Differences were considered statistically significant at p < 0.05.Statistical analyses were conducted using SPSS version 22.0  and GraphPad Prism 7.0 software . Significant differences between treatment groups were assessed with Student\u2019s pilA gene of the group I strain pslb65 was 522 bp, and the pilA gene of the group II strain Aac5 was 507 bp. DNA sequence analysis revealed that the two pilA genes were highly similar from positions 1 to 107 bp, after which the sequences diverged significantly. The overall similarity between the two genes was only 61.64%  did not predict any typical protein domains in either of the two PilA proteins.The y 61.64% a. The aly 61.64% b. SMART pilA and Aac5-\u0394pilA), but there was no amplification when the primer pairs Km-F/Km-R, 65-\u0394pilA-L/65-\u0394pilA-R, or A5AL-L/A5AR-R were used   . These rpilA-pslb65 and \u2206pilA-Aac5 could cause the HR in a nonhost plant species  was statistically comparable to that inoculated with the WT strain. In melon, the average disease index of seedlings inoculated with pslb65-\u0394pilA was significantly lower than in those inoculated with WT pslb65. The average disease indices were significantly higher for both pslb65-\u0394pilAcomp1 and pslb65-\u0394pilAcomp2 compared to the knockout mutant but did not return to WT values . For plants inoculated with the complementation and cross-complementation lines pslb65-\u0394pilAcomp1 and pslb65-\u0394pilAcomp2, the average number of diseased seedlings increased again to 24.0 and 18.3, respectively, representing incidence rates of 80.0% and 61.0%, respectively. Seedlings inoculated with WT Aac5 averaged 23.6 diseased seedlings (incidence rate = 78.6%); this decreased to an average of 9.6 diseased seedlings (incidence rate = 32.0%) among those inoculated with the Aac5-\u0394pilA mutant. In seedlings inoculated with the complementation line (Aac5-\u0394pilAcomp1), there were an average of 22.3 diseased seedlings (incidence rate = 74.3%), which was comparable to the rate among those inoculated with the cross-complementation line Aac5-\u0394pilAcomp2 . These results demonstrated that the pilA mutants of both pslb65 and Aac5 exhibited low virulence in watermelon seedlings, and that the functional pilA genes of group I and group II strains had similar effects on watermelon seedling disease rates.In order to further reveal the role of differences in  strains . At 11 DpilA strain showed no outer halo when it was grown on KB plates, meaning it had completely lost the capacity for twitching motility. In comparison, the capacity for twitching motility was significantly reduced in the group II strain Aac5-\u2206pilA, but not completely lost. All four complementation lines  showed the restoration of twitching motility to WT levels . For both strains, both complementation lines restored biofilm formation to WT levels (pilA and Aac5-\u0394pilA showed significantly lower biofilm formation than the corresponding WT strains (p < 0.05); all four complementation strains somewhat restored biofilm formation, but only Aac5-\u0394pilAcomp1 reached levels comparable to the WT  and group II . The E. coli and A. citrulli colonies were then counted. E. coli was significantly more abundant (p < 0.05) when co-cultured with pslb65-\u2206pilA compared to WT pslb65. The complementation line (pslb65-\u0394pilAcomp1) completely recovered interspecific competitiveness with E. coli, whereas colony counts of the cross-complementation line (pslb65-\u0394pilAcomp2) were significantly lower than those of the WT or pslb65-\u0394pilAcomp1 after co-culture with E. coli. The results for Aac5 and derivative strains were similar. E. coli abundance increased significantly (p < 0.05) when it was co-cultured with Aac5-\u2206pilA compared with the WT; Aac5-\u0394pilAcomp1 completely recovered interspecific competitiveness with E. coli; and Aac5-\u0394pilAcomp2 competitiveness was significantly decreased compared to both the WT and Aac5-\u0394pilAcomp1 (p < 0.05) ( < 0.05) .hrpG, hrpX, hrpE, hrcJ, hrcQ, and hrcR) were measured in pslb65-\u0394pilA and Aac5-\u0394pilA to evaluate the effects of pilA on T3SS by comparing differences between the two groups of strains. In the group I strain pslb65, pilA deletion did not cause significant differences in the expression of six T3SS genes  were upregulated to varying degrees in pslb65-\u0394pilA compared to WT pslb65, while the expression levels of the other four genes showed no significant difference  were significantly downregulated to varying degrees in Aac5-\u0394pilA compared to WT Aac5 (p < 0.05), and the other five genes showed no significant differences -encoding genes (vgrGs). Because T6SS activation is affected by the concentration of a bacterial suspension, we tested the relative expression of T6SS genes in cultures at an OD600 value of 1.5.To evaluate the effects of pilA, six T6SS genes  were significantly upregulated; three T6SS genes  were significantly downregulated (p < 0.05), and the expression level of hcp showed no significant differences compared to the WT strain  were significantly upregulated; four T6SS genes  were significantly downregulated  were significantly upregulated; five genes  were significantly downregulated (p < 0.05)  were significantly upregulated; three genes  were significantly downregulated (For pslb65-\u0394 < 0.05) c. For Aaegulated d.A. citrulli exhibit differences in pathogenicity, host preference, and genome sequences [A. citrulli groups. In the present study, the sequence and function of pilA were analyzed in the two groups of strains. Significant differences in the sequences between the two groups were found to correspond to differences in biofilm formation ability, twitching motility, and interspecies competition.Previous studies have found that group I and group II strains of equences ,8,9. HowpilA deletion on biofilm formation during growth in KB and M9 media. In M9 medium, pilA deletion significantly reduced the capacity for biofilm formation in both strains, consistent with previous reports [pilA deletion differed between strains. In Aac5, the absence of pilA significantly reduced biofilm formation, but this was not the case for pslb65. Thus, for the group I strain pslb65, nutrient conditions determined the effects of pilA deletion on the capacity for biofilm formation. The relationship between nutrient metabolism and biofilm formation is largely unknown, but it has been suggested that carbon molecules may act as simple nutrients for biofilm-forming bacteria [pilA deletion mutants in the group I and the group II strain under consistent nutrient conditions may have been caused by group-specific differences in the regulatory pathways associated with pilA.Biofilm formation is a process that allows microorganisms to irreversibly adhere to and grow on a surface by producing extracellular polymers that promote adhesion and matrix formation; this enhances the ability of the bacteria to withstand harsh environmental conditions . The res reports ,29. Howebacteria . Alteratbacteria . These fpilA and pilO mutant strains of P. syringae pv tabaci 6605 reduced the HR in nonhost Arabidopsis leaves, and qPCR results showed that multiple T3SS-related genes of pilA and pilO deletion mutants were significantly downregulated in expression compared to the WT [pilA deletion mutant of A citrulli and the WT. The qPCR results showed that the expression of T3SS-related genes in pslb65-\u0394pilA did not significantly differ compared to the WT, and in Aac5-\u0394pilA strains, hrcJ and hrcR were significantly upregulated, whereas the other four genes showed no significant differences. This is different from the research results on P. syringae pv tabaci 6605. This indicates that in A citrulli, the effect of pilA on virulence may not be mediated through T3SS, but through alternative pathways. Vfr is a cAMP-binding protein and a transcriptional regulator that can regulate many virulence factors, such as quorum sensing, T4P biogenesis, and T3SS-related genes [P. aeruginosa Vfr in A. citrulli. Next, we will further investigate the regulatory relationship between vfr and T4P by constructing vfr mutants.Previous research found that the o the WT . Howevered genes . Taguchied genes . ThroughpilA was knocked out, consistent with previously published results for the group I strain M6 [pilA mutant. To understand the cause of the loss in motility, we identified seven genes that are predicted to have a close regulatory relationship with pilA in the group II strain AAC00-1 using the STRING website . We then measured the relative expression levels of those seven genes in pslb65-\u0394pilA and Aac5-\u0394pilA compared to the corresponding WT strains. Three genes  were upregulated to varying degrees in pslb65-\u0394pilA, while in Aac5-\u0394pilA, no genes were upregulated, and two genes (pilM and pilE) were significantly downregulated to varying degrees compared to WT Aac5. This indicates that there were certain differences in the regulation of T4P by the pilA gene between the two groups of A. citrulli.The group I strain pslb65 completely lost its capacity for twitching motility when train M6 . SurprisA. citrulli that contains 17 genes [A. citrulli strain Aac5 at a cell density of OD600 = 1.5 [E. coli showed no significant differences between the group I strain pslb65 and the group II strain Aac5; however, after pilA deletion, the interspecific competition ability of Aac5-\u0394pilA was significantly higher than that of pslb65-\u0394pilA, and complementation with endogenous pilA returned their competitive abilities to WT levels, whereas cross-complementation significantly enhanced the interspecific competition ability of both strains. To further analyze the effects of pilA deletion on T6SS functioning, we analyzed the relative expression of 17 T6SS genes and 12 vgrG genes in pslb65-\u0394pilA and Aac5-\u0394pilA at cell densities of OD600 = 1.5. Interestingly, the experimental results showed that the expression level of the hcp gene in Aac5-\u0394pilA was significantly downregulated (p < 0.0001), while there was no significant difference in pslb65-\u0394pilA. Aave_3347 (vgrG gene) also caught our attention, as its relative expression levels were significantly downregulated (p < 0.001) in both pslb65-\u0394pilA and Aac5-\u0394pilA. This may be one of the reasons for the decrease in interspecific competition with E. coli. The regulatory relationship between the pilA gene and Aave_3347 needs further research.The T6SS plays important roles in pathogenicity, biofilm formation, bacterial interactions, and other biological functions . There i17 genes . VgrG is00 = 1.5 . At thatNeisseria cinerea, a commensal of the human respiratory tract, can limit the growth of related pathogens. Custodio et al. (2020) believe that T4P affects T6SS-mediated antagonism by constructing and distributing bacteria in mixed microcolonies in this process; in other words, spatial segregation driven by type IV pili dictates prey survival against T6SS assault. In the process of bacterial interspecific competition, the pilA gene is more sensitive to attack by other bacterial T6SSs and can mount an earlier response [pilA and Aac5-\u0394pilA in the interspecific competition of E. coli is the loss of spatial isolation caused by the loss of T4P; in addition, key differences in the pilA-mediated regulation of T6SS and vgrG between pslb65 and Aac5 play a secondary role.T6SS activation in response . Based opilA in the A. citrulli group I strain pslb65 and in the group II strain Aac5. There were significant differences in phenotype between the two strains when pilA was deleted. Specifically, twitching motility was completely lost in pslb65-\u0394pilA, whereas Aac5-\u0394pilA only partially lost motility. Biofilm formation was significantly lower for Aac5-\u0394pilA than for Aac5, but there were no significant differences in biofilm formation between pslb65-\u0394pilA and the WT pslb65. The deletion of pilA also significantly reduced the interspecific competitiveness of pslb65-\u0394pilA and Aac5-\u0394pilA compared to the corresponding WT strains. qRT-PCR results showed that significantly more genes related to T4P and the T6SS were upregulated, and significantly fewer were downregulated in Aac5-\u0394pilA compared with pslb65-\u0394pilA. These results revealed critical differences in pilA function between group I and group II strains, indicating significant intraspecies divergence in T4P regulatory mechanisms.In the present study, we characterized the function of"}