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+{"text": "Applbaum demystifies some of the assumptions at work in the \"culture of marketing,\" toward the goal of explaining contemporary disease mongering. It is often said that leading drug companies now spend more on marketing than on research and development . While sThere is no doubt that drug company discoveries have profoundly improved upon our capacity to treat illness. But pharmaceutical marketing is more closely aligned with consumer marketing in other industries than with medicine, for which the consequences are not trivial. Once we view pharmaceutical industry activities in this light, we can disentangle industry's influence on contemporary medicine. Because we believe that we owe corporations our wealth and well-being, we tend not to question corporations' fundamental practices, and they become invisible to us. What follows is an attempt to demystify some of the assumptions at work in the \u201cculture of marketing,\u201d toward the goal of explaining contemporary disease mongering.There are three beliefs commonly associated with the \u201cfree market.\u201d The first is that human beings are creatures of limitless but insatiable needs, wants, and discomforts. The second is that the free market is a place where these needs might be satisfied through the exercise of free choice. The last of these beliefs is that the surest avenue to innovation in all industries is unfettered competition in the market.The anthropologist Marshall Sahlins theorizes that the belief in unlimited wants is unique in the West, and stems from the Christian notion of \u201cfallen man\u201d as sufferer. This results, says Sahlins, in a peculiar idea of the person \u201cas an imperfect creature of need and desire, whose whole earthly existence can be reduced to the pursuit of bodily pleasure and the avoidance of pain\u201d . A histoSahlins also points out that \u201cin the world's richest societies, the subjective experience of lack increases in proportion to the objective output of wealth\u201d . In otheIn a consumer society, when individuals make choices toward the satisfaction of their needs and wants, they experience this as constructing their own individuality and identity. This special consumer identity is what people refer to when they use the word lifestyle, though they may not realize the consumerist implications of the word. Marketing claims to provide a solution to the problem of unlimited needs and wants, while simultaneously enhancing free choice and the construction of lifestyle.In pharmaceuticals specifically, \u201clifestyle drug\u201d marketing techniques were honed in the 1980s and 1990s for cosmetic and sexual enhancements ,8. ThesAs a result of this sequence of events, industry opened the treatment of the inside of the body\u2014the final frontier\u2014to the same logic that governs all other marketing. Whether, in the antidepressant market, the \u201cdistribution channel captain,\u201d as marketers refer to the predominant competitor, ends up sailing the serotonin reuptake channel (the serotonin reuptake inhibitors) or the norepinephrine reuptake channel  may yet be determined by marketing rather than by medical jockeying.Harvard Business Review, \u201c it is about achieving customer-getting distinction by differentiating what you do and how you operate\u201d [It is an article of faith among free market devotees that breakthroughs spring not from paternalistic expert systems such as medicine but from industrial competition. As long as firms are committed to producing medications to treat diseases\u2014as they are classified by medical science\u2014this argument has some authority. But once a firm becomes principally driven by marketing\u2014the case for most companies in most industries since the 1980s\u2014then innovation comes to mean an elaboration of meaningless differences among a field of comparable \u201cme too\u201d products. \u201cIf marketing is seminally about anything,\u201d said Theodore Levitt, one of the towering figures of marketing and former editor of theAn assumption of boundless needs and wants is at the heart of marketing theory.New York Times article: \u201cFor a sizable group of people in their 20's and 30's, deciding on their own what drugs to take\u2014in particular, stimulants, antidepressants and other psychiatric medications\u2014is becoming the norm. Confident of their abilities and often skeptical of psychiatrist's expertise, they choose to rely on their own research and each other's experience in treating problems like depression\u2026.A medical degree, in their view, is useful but not essential\u201d [Since, in a consumer society, we see ourselves as individuals and as free agents when we exercise consumer choice, it is not difficult for pharmaceutical companies and other privatized health-care deliverers to convince us that it is empowering to think of ourselves not as patients but as consumers. This conversion from patient to consumer also paves the way for the erosion of the doctor's role as expert. A startling report of this was described in a recentential\u201d . This phPromoting consumer familiarity with drugs is one example of the very broad influence of the pharmaceutical industry. This influence extends to clinical trial administration, research publication, regulatory lobbying, physician and patient education, drug pricing, advertising and point-of-use promotion, pharmacy distribution, drug compliance, and the legal and ethical norms by which company practices themselves are to be evaluated. Actors traditionally found outside the \u201cdistribution channel\u201d of the market are now incorporated into it as active proponents of exchange. Physicians, academic opinion leaders, patient advocacy groups and other grass roots movements, nongovernmental organizations, public health bodies, and even ethics overseers, through one means or another, have one by one been enlisted as vehicles in the distribution chain. The inclusion of patients in the distribution chain fundamentally changes their role from recipients of medical care to active consumers of the latest pharmaceuticals, a role which surely helps to support industry profits.Because illness is one of the most tangible forms of suffering, the pharmaceutical industry, more than other industries, can link its marketing activities to ethical objectives. The result is a marriage of the profit-seeking scheme in which disease is regarded as \u201can opportunity\u201d to the ethical view that mankind's health hangs in the balance. Marketers and consumers in the West to some extent share a common vision of needs and the terms of their satisfaction. This apparent complicity helps even the most aggressive marketers trust that they are performing a public service. Pharmaceutical company managers that I speak to signal this when they characterize their engagement with the public as \u201cdoing good while doing well.\u201dThese managers also see nothing wrong with integrating doctors, patients, and other players into the drug distribution channel. On the contrary, they say, this is state-of-the-art management, making it professionally principled and tactically astute. Marketers also regard the incorporation of consumers into the channel as ethical because then people's needs can best be determined and satisfied, conferring upon them the power of self-determination through choice.But this choice is an illusion. For in our pursuit of a near-utopian promise of perfect health, we have, without realizing it, given corporate marketers free reign to take control of the true instruments of our freedom: objectivity in science, ethics and fairness in health care, and the privilege to endow medicine with the autonomy to fulfill its oath to work for the benefit of the sick."}
+{"text": "We thank A. D. Gowdy for his comments  on our aWe are not aware of data that convincingly demonstrate the impact of outreach visits by pharmaceutical representatives. Gowdy indicates that such information exists (\u201cProgress is tracked meticulously\u201d). We would very much like to see it!We have had informal discussions with executives from companies in Norway, and we have been struck by how they themselves question the effectiveness of their marketing strategies. At a recent conference in Denmark, the medical director of a major pharmaceutical company gave a talk on the impact of industry marketing on prescribing habits . He had The degree of interaction between the pharmaceutical industry and the medical profession is associated with differences in prescribing patterns . Thus, wEven more difficult to estimate is the cost-effectiveness of various marketing strategies. Considering that the pharmaceutical industry spends a five-digit amount ($US) per doctor per year on marketing alone , the indThe only study cited by Gowdy did indeed show promising results. However, changes in prescribing were compared between practices that chose to participate in the programme and practices that chose not to , and wheGowdy thinks our intervention sounds like \u201ca policing approach\u201d. This does not fit with our perception. The doctors were satisfied with the chance of meeting an industry-independent source of information and appreciated the opportunity to reflect on their own practice in light of the information and feedback that we provided them.Gowdy\u2019s use of the term \u201cevidence-based\u201d when describing the messages conveyed by pharmaceutical companies begs a brief comment. Several investigators have assessed the quality of advertisements and promotional material distributed by the pharmaceutical industry. They consistently conclude with a word of caution against basing clinical practice on claims made by pharmaceutical companies ."}
+{"text": "Big Pharma: The Rise of the Anti-Depressant Drug Industry and the Loss of a Generation. Since the mid-1990s, spending on drug promotion has grown steadily, reaching $21 billion in 2002. Berry explores the fallout of this expanded marketing blitz through the eyes of its masterminds, unwitting (and complicit) abettors, and victims through her portrayal of an advertising executive, a physician, and women and children who are prescribed heavily marketed antidepressants.Anyone who has experienced the assault of the pharmaceutical industry's marketing campaigns would appreciate Jennifer Berry's one-person play A primary target of the pharmaceutical industry, physicians receive not just advertising materials but office visits from drug representatives. Berry's physician, depicted as a pawn of the pharmaceutical industry, gratefully accepts the free drug samples, the free lunches, and the pharmaceutical industry\u2013sponsored trips to tropical islands. In fact, the pharmaceutical industry woos physicians with educational dinners, honoraria for participating in conference calls, consulting fees for participating in speakers' bureaus, research funding, and payments to write scientific publications. And physicians act as agents of the pharmaceutical industry in many ways, such as giving talks that favor a company's product, participating in clinical trials that increase physicians' exposure to a new drugs or new indications for old drugs, and publishing research articles that are financed and, in fact, written by pharmaceutical company employees .In addition to reaching physicians directly, the industry promotes consumer familiarity with particular drugs. In 2002, almost $3 billion of the industry's drug promotion budget was spent on direct-to-consumer advertising [Less obvious marketing strategies include using opinion leaders, and sponsoring education programs, scientific research, publications, and professional meetings. The pharmaceutical industry has also extended its influence to clinical trial administration, research design, regulatory lobbying, physician and patient education, drug pricing, pharmacy distribution, and drug compliance .There is a wide range of evidence suggesting that all of these marketing strategies influence physician prescribing and affect patient attitudes and behavior. Interactions with the pharmaceutical industry increase the likelihood of physicians prescribing inappropriately or making formulary requests for the company's product \u20139. Yet hEmpirical evidence regarding direct-to-consumer advertising shows that it acts as promotion rather than education, often includes inaccurate, misleading, or unbalanced information, frequently includes emotional appeals to anxiety about illness, underplays risks, omits mention of costs, and tends to promote the \u201cmedicalization\u201d of normal health and minor ailments \u201317. And The notion that the pharmaceutical industry creates diseases or \u201cdisorders\u201d when none exist surfaces as a major theme of the play. Berry's advertising executive proudly displays direct-to-consumer advertisements for antidepressants. The executive explains that she \u201ccan't just say \u2018buy drugs,\u2019\u201d so the advertisers develop a campaign that preys on women with societal problems, such as stress or homelessness. Clearly, pharmaceutical marketing practices impact definitions of social concern, health, and illness. For example, Moynihan and Cassels  and MoynAnd it is Berry's patient characters that have the most impact. The patients are portrayed as victims not only of a profit-seeking pharmaceutical industry, but of society in general. Berry describes the pharmaceutical industry's target market as women who are subjected to sexism, racism, and social inequalities. She describes how the pharmaceutical industry has transformed these societal problems into medical \u201cdisorders\u201d that can be treated with a pill\u2014in this case an antidepressant. Children who dare to be different or energetic are also labeled with a disorder. Berry's soliloquy poignantly describes how antidepressants transform vibrant individuals into quiet, passive people with a lack of affect. Her description of the side effects of antidepressants is completely accurate. Unfortunately, the play does give the impression that most women are not strong enough to help themselves. But, of course, this is what the pharmaceutical industry is counting on.My favorite part of the play is when Berry acts one half of a conversation with an elderly woman who lived through the Great Depression. She describes how everyone was sad, but, together, people survived. Berry contrasts this to our current era of the \u201canti-Depression,\u201d when unhappiness is considered a disease that must be eradicated.I have to admit that Berry's performance left me a bit depressed. I was depressed by the fact that the pharmaceutical industry manages to take advantage of societal problems to market their products and increase their profits. At the same time, the industry's tactics were exposed and the audience had a chance to think about how to channel unhappiness to confront our real problems and not just treat the symptom. After the play, I did have an urge to go home and be sad for a while...but not to pop any pills."}
+{"text": "By selecting for increasedlevels of specific cell wall components in the growth medium, we identified 13 genes thatare possibly implicated in different steps of cell wall assembly. Furthermore, 14 mutantsshowed a constitutive activation of the cell wall integrity pathway, suggesting that theyparticipate in the modulation of the pathway either directly acting as signaling componentsor by triggering the Slt2-dependent compensatory mechanism. In conclusion, our screeningapproach represents a comprehensive functional analysis on a genomic scale of geneproducts involved in various aspects of fungal cell wall formation.Using a hierarchical approach, 620 non-essential single-gene yeast deletants generated byEUROFAN I were systematically screened for cell-wall-related phenotypes. By analyzingfor altered sensitivity to the presence of Calcofluor white or SDS in the growth medium,altered sensitivity to sonication, or abnormal morphology, 145 (23%) mutants showing atleast one cell wall-related phenotype were selected. These were screened further to identifygenes potentially involved in either the biosynthesis, remodeling or coupling of cell wallmacromolecules or genes involved in the overall regulation of cell wall construction and toeliminate those genes with a more general, pleiotropic effect. Ninety percent of the mutantsselected from the primary tests showed additional cell wall-related phenotypes. Whenextrapolated to the entire yeast genome, these data indicate that over 1200 genes maydirectly or indirectly affect cell wall formation and its regulation. Twenty-one mutants withaltered levels of"}
+{"text": "Candida albicans. Hsf1 becomes hyperphosphorylated in response to heat shock and activates the transcription of genes with heat shock elements (HSEs) in their promoters, these genes contributing to thermal adaptation. However, the relevance of Hsf1 activation to C. albicans virulence is not clear as this pathogen is thought to be obligately associated with warm blooded animals, and this issue has not been tested because HSF1 is essential for viability in C. albicans. In this study, we demonstrate that the HSE regulon is active in C. albicans cells infecting the kidney. We also show the CE2 region of Hsf1 is required for activation and that the phosphorylation of specific residues in this domain contributes to Hsf1 activation. C. albicans HSF1 mutants that lack this CE2 region are viable. However, they are unable to activate HSE-containing genes in response to heat shock, and they are thermosensitive. Using this HSF1 CE2 deletion mutant we demonstrate that Hsf1 activation, and hence thermal adaptation, contributes significantly to the virulence of C. albicans.The evolutionarily conserved heat shock transcription factor Hsf1 plays a central role in thermal adaptation in the major fungal pathogen of humans,  These plasmids were integrated into C. albicans CAI4 selecting for the URA3 marker, and into C. albicans CLM62-1 using the NAT1 marker. Correct genomic integration was confirmed by PCR using oligos D1, D2, HSF-F and RPS1-GEN , and then 50\u00a0\u03bcl of chloroform and 20\u00a0\u03bcl of 1% SDS were added. Samples were equilibrated at 37\u00b0C for 10\u00a0min, and then reactions started by addition of 200\u00a0\u03bcl of pre-warmed OPNG (4\u00a0mg/ml). Samples were incubated until a yellow colour developed whereupon the reaction was stopped by addition of 0.4\u00a0ml of 1\u00a0M Na2CO3. \u03b2-galactosidase activities were measured in Miller Units.LacZ expression levels were assayed in quadruplicate on independent transformants as described previously . Briefly2.6Protein extracts were prepared and subjected to Western blotting using published protocols . Briefly2.7All animal experimentation conformed to the requirements of United Kingdom Home Office legislation and of the Ethical Review Committee of the University of Aberdeen.C. albicans strains SC5314, CE2t, CTMt and Basal-lacZ  from cultures grown overnight in NGY medium , prepared from overnight cultures in NGY medium, was used to intravenously infect six female BALB/c mice . Mice were monitored daily and culled when they showed signs of illness and/or when they had lost 20% body weight. Mice were humanely terminated and the kidneys aseptically removed. One kidney was used for fungal burden determination and the other was snap frozen in liquid nitrogen for subsequent RNA extraction and qRT-PCR analyses , a hydrophobic repeat region necessary for coiled-coil formation during Hsf1p trimerization (residues 326\u2013424), amino-terminal and carboxy-proximal transcriptional activation domains , a negative regulatory domain (CE2: residues 535\u2013550), and a carboxy-terminal modulator domain that alleviates the repression by CE2 in response to heat shock (CTM: residues 783\u2013833)  . C. albi domains .C. albicans Hsf1. Therefore we set out to create HSF1 truncation mutants in which the CTM domain alone, the AR2 and CTM domains together, and then all three carboxy-terminal domains (CE2-AR2-CTM) were selectively deleted. The approach was to create the truncations by integrating a URA3 cassette at the 3\u2032 end of the remaining wild type allele in the hsf1/HSF1 heterozygote, CLM61-1 . HSP104 mRNA levels were below detectable levels in untreated cells and were significantly induced in heat shocked cells. These data are entirely consistent with our previous observations  or glutamate codons (SDM-E) to generate non-phosphorylatable and phosphomimetic versions of Hsf1, respectively. Cells expressing these alleles were compared with cells expressing an equivalent wild type construct  to generate strains SN254-257  as a non-heat shock inducible control. lacZ mRNA levels increased when the growth temperature increased above 30\u00a0\u00b0C . Data from independent experiments confirmed the reproducibility of these observations, thereby reinforcing the idea that the Hsf1-HSE regulon tunes the expression of essential chaperones to the growth temperature of C. albicans.Our starting point involved the calibration of our qRT-PCR quantification methods using und SN2:  were grove 30\u00a0\u00b0C A, which ve 30\u00a0\u00b0C . The qRTve 30\u00a0\u00b0C A. Indeedy strong . In contperature A, and thlacZ,HSP90, HSP104 and ENO1 transcript levels in vivo, in C.\u00a0albicans cells from infected kidneys. Mice were sacrificed between days 3\u201310 after being injected intravenously with C. albicans SC5314 (HSF1/HSF1 control), SN1  or SN2 (HSF1/HSF1 cells containing HSE-lacZ) (10\u00a0CFU/g). We used the clinical isolate, SC5314, as the infection control in this experiment because the virulence of this strain has been shown to be equivalent to C. albicans CAI4 cells carrying CIp10  , once inng CIp10 , the vecng CIp10 . To main control B.lacZ mRNA was seen in C. albicans SC5134 cells, and minimal lacZ mRNA levels were observed in SN1 cells  from kidney infections in independent animals (lacZ mRNA levels were observed in SN2 cells (HSE-lacZ) infecting the kidney, indicating that the Hsf1-HSE regulon is activated in C. albicans during systemic infections. Also HSP90 and HSP104 were expressed during experimental infections with C. albicans SC5134 and SN1  it became possible to test whether activation of the Hsf1-HSE regulon is required for virulence in C. albicans.The above observations suggested that thermal homeostasis might contribute significantly to the physiological fitness of albicans  and therC. albicans SN127 (CE2t) and with the control strains SC5314 and SN128 C. albica Section  did not C. albicans.The CE2t mutant displayed significantly reduced fungal burdens and outcome scores compared with the control strains . Further4C. albicans. First, we conclude that the CE2 domain of the essential heat shock transcription factor, Hsf1, is required for its activation in response to heat shock. This CE2 domain lies between the trimerisation region and the activation domain AR2 of Hsf1  slightly increased thermotolerance (C. albicans thermotolerance than mutagenesis of S571, T575, S577 and T578 alone (The CE2 domain harbours four amino acid residues at which phosphorylation has been detected in olerance B. Howeveolerance A, and de78 alone B. We conC. albicans. Two key observations underpin this conclusion. Firstly, preventing the activation of Hsf1 severely attenuates the virulence of C. albicans (C. albicans, tuning the levels of essential chaperones to the temperature of the local microenvironment. This proposed function does not preclude a role for Hsf1 in protection against acute heat shocks in C. albicans. However, it is not clear when this pathogen might be exposed to the sudden and dramatic temperature up-shifts associated with the experimental heat shocks induced in vitro, given that C. albicans is thought to be obligately associated with warm blooded animals (Our second important conclusion is that activation of the Hsf1-HSE regulon contributes significantly to the virulence of albicans . Deletinalbicans . Secondlalbicans . Interesalbicans . Neverthalbicans . Taken t animals ."}
+{"text": "Candida albicans, has evolved robust stress responses that protect it against human immune defences and promote its pathogenicity. However, C. albicans is unlikely to be exposed to heat shock as it is obligatorily associated with warm-blooded animals. Therefore, we examined the role of the heat shock transcription factor (Hsf1) in this pathogen. We show that C. albicans expresses an evolutionarily conserved Hsf1 (orf19.4775) that is phosphorylated in response to heat shock, induces transcription via the heat shock element (HSE), contributes to the global transcriptional response to heat shock, and is essential for viability. Why has Hsf1 been conserved in this obligate animal saprophyte? We reasoned that Hsf1 might contribute to medically relevant stress responses. However, this is not the case, as an Hsf1-specific HSE-lacZ reporter is not activated by oxidative, osmotic, weak acid or pH stress. Rather, Hsf1 is required for the expression of essential chaperones in the absence of heat shock . Furthermore, Hsf1 regulates the expression of HSE-containing genes in response to growth temperature in C. albicans. Therefore, the main role of Hsf1 in this pathogen might be the homeostatic modulation of chaperone levels in response to growth temperature, rather than the activation of acute responses to sudden thermal transitions.All organisms have evolved mechanisms that protect them against environmental stress. The major fungal pathogen of humans,  Organisms exist in constantly changing and complex environments where they are subject to wide-ranging perturbations that are often perceived as stresses. For this reason organisms have evolved stress responses that promote their survival in these dynamic environments. In particular, the heat shock response protects cells against sudden changes in temperature by inducing the expression of heat shock proteins (HSPs) that protect proteins during thermal stress and facilitate the repair or degradation of damaged proteins . This reSaccharomyces cerevisiae the heat shock response is regulated by the heat shock transcription factor (Hsf1), which activates heat shock genes (HSPs) via canonical heat shock elements (HSEs) in their promoters. Hsf1 is essential for viability, binding to the HSE DNA sequence as a homotrimer even in the absence of stress , and life-threatening systemic infections in immunocompromised patients , wild mammals and marsupials  and birds   . These aprophyte .C. albicans have evolved to promote survival in animal hosts, and that these stress responses have diverged from those in benign model yeasts such as S. cerevisiae and Schizosaccharomyces pombe. For example, C. albicans is considerably more resistant to oxidative stresses than S. cerevisiae and S. pombe  they are particularly immunogenic during Candida infections, (ii) the levels of anti-HSP antibodies have been associated with increased survival in patients with systemic candidosis, and (iii) anti-Hsp90 antibodies have been reported to be immunoprotective against systemic candidosis  via HSEs in their promoters. However, neither Hsf1 nor HSE functionality has been characterized in C. albicans.Heat shock proteins have attracted special attention in C. albicans does have an HSF1 gene, that this gene is essential for viability, that Hsf1 activates transcription via the HSE, and that Hsf1 contributes significantly to the global transcriptional response to heat shock in C. albicans. Our examination of the cellular roles of Hsf1 in C. albicans has led us to suggest that although it has retained the capacity to act as an ON-switch in response to heat shock, the primary role of Hsf1 in the wild is to act as a thermostat that tunes the levels of essential chaperones to growth temperature. This would account for the strong conservation of \u2018heat shock\u2019 regulation in this obligatory animal saprophyte.In this study we show that C. albicans has a homologue of the S. cerevisiae and other metazoan HSF1 genes. Bidirectional blastn searches of the C. albicans genome (http://www.candidagenome.org/) revealed that orf19.4775 gene is the sequence orthologue of S. cerevisiae HSF1. The C. albicans orf19.4775 protein displays significant sequence similarity to S. cerevisiae Hsf1 (32.5% identity over the full lengths of these proteins). Furthermore this sequence similarity increases in their DNA-binding domains (71.9%), suggesting that orf19.4775 is C. albicans HSF1.Our first aim was to determine whether C. albicans orf19.4775 locus was initially named as CTA8 on the basis that an orf19.4775 cDNA clone was identified in a one-hybrid screen for C. albicans sequences capable of trans-activation in S. cerevisiae (http://www.candidagenome.org/). This reinforces the idea that orf19.4775 encodes a transcriptional activator. However, to our knowledge this locus has not been characterized further. We now refer to orf19.4775 as HSF1 because it is the sequence and functional orthologue of S. cerevisiae HSF1 (see below).The HSF1 is an essential gene in S. cerevisiae .revisiae . Given thsf1/tetp-HSF1 mutant was grown to mid-exponential phase and plated alongside HSF1/HSF1 (THE1) and hsf1/HSF1 (CLM61-1) controls on yeast-peptone-dextrose (YPD) medium containing or lacking 20 \u03bcg ml\u22121 doxycycline, which downregulates the tetp promoter. No significant growth was observed at 30\u00b0C for the hsf1/tetp-HSF1 cells on the doxycycline-containing plates, indicating that HSF1 is required for the growth of C. albicans  during the growth of C. albicans at normal temperatures, in addition to its predicted role(s) during stress responses.These strains were also examined in liquid culture. ycycline . The viainactive . Therefohsf1/tetp-HSF1 cells treated with doxycycline for 6 h. At this stage their growth had ceased, but their viability remained high in the absence of a heat shock (hsf1/tetp-HSF1 cells was reduced significantly compared with control cells not exposed to doxycycline (P < 0.001)  . FurtherS. cerevisiae in response to heat shock , but their expression increased further in response to the heat shock. Minimal HSP104 expression was observed under basal conditions, but this mRNA was strongly upregulated by the heat shock , the heterozygote (CLM61-1) and the conditional mutant (CLM62-1) grown at 30\u00b0C or subjected to a 45\u00b0C heat shock for 30 min . The HSPat shock . No indun viable . Furtherectively . TherefoS. cerevisiae and is thought to be essential in C. albicans  was subjected to a 30\u201345\u00b0C heat shock for 10 min, and this transcriptome compared to control cultures grown at 30\u00b0C. As expected, we found that HSP genes were strongly induced under these conditions . A total of 136 genes were reproducibly upregulated at least twofold by heat shock in wild-type C. albicans under our experimental conditions (Supporting information). This subset of heat shock-inducible genes was highly enriched in protein folding and refolding functions  upon the transcriptome of wild-type cells. To achieve this, the parental strain for our conditional unctions . Genes iC. albicans (Supporting information). These heat shock-inducible genes were highly enriched in ribosome biogenesis and RNA processing functions  and the different microarray platforms, there was reasonable overlap between this study and the current study with respect to the sets of C. albicans genes that were induced by heat shock . Recently a third study compared the transcriptional responses of C. albicans SC5314 and Candida dubliniensis CD36 to a 30\u201342\u00b0C heat shock  upon the at shock  . These heat shock-inducible genes included classical heat shock genes , as well as an array of chaperone-related functions , thereby reinforcing the view that protein folding is critical for heat shock adaptation in C. albicans. Stress-induced functions  and some transporters  were also included in this common set of heat shock-inducible genes.A subset of 46 C. albicans genes are dependent upon Hsf1 for their heat-shock regulation. To achieve this we took the set of heat shock-inducible genes in wild-type C. albicans cells. As described above, these genes were induced \u2265 2-fold by the 10 min 30\u201345\u00b0C heat shock in wild-type cells (THE1: HSF1/HSF1). Then to identify Hsf1-dependent genes, we compared the transcriptomes of doxycycline-treated and control hsf1/tetp-HSF1 cells (CLM62-1) following an equivalent heat shock. Given that THE1 and CLM62-1 are isogenic, we reasoned that strain differences in their transcriptomes would be minimal, but that genes identified in hsf1/tetp-HSF1 would also contain some that are affected by the doxycycline treatment used to achieve Hsf1 depletion. Therefore, control microarrays were performed to define which C. albicans genes were affected by doxycycline (Supporting information). The small number of genes whose expression was affected by doxycycline in THE1 cells was excluded from the list of Hsf1-dependent genes. To summarize, we defined Hsf1-dependent, heat shock-inducible genes in C. albicans as those that were: (i) upregulated \u2265 2-fold by heat shock in wild-type cells, but (ii) not significantly induced (< 2-fold regulation) by heat shock in hsf1/tetp-HSF1 cells after Hsf1 depletion, and (iii) not significantly affected (< 2-fold regulation) by doxycycline treatment in wild-type cells (Supporting information).Having defined the transcriptional response to heat shock in control cells, we then defined which C. albicans cells were found to be dependent upon Hsf1 for their induction  (hsf1/tetp-HSF1 cells). Nevertheless, Hsf1 had a large impact (\u2265 2-fold) upon most of these genes (73%). Furthermore, genes encoding Hsp104, Hsp90 and members of the Hsp70 family were among those that displayed the strongest dependence upon Hsf1 for their heat shock induction  . This linduction . This wanduction .C. albicans. The data also suggest that additional Hsf1-independent pathways contribute to the transcriptional response to heat shock. This is consistent with the situation in S. cerevisiae, where the general stress and cell wall integrity pathways also contribute to the regulation of the heat shock response  . Not surC. albicans genes was induced \u2265 2-fold by heat shock in doxycycline-treated hsf1/tetp-HSF1 cells, but not in wild-type cells subjected to heat shock (Supporting information). Similarly, 385 genes were downregulated \u2265 2-fold in response to heat shock in doxycycline-treated hsf1/tetp-HSF1 cells, but were not downregulated in wild-type cells under equivalent conditions (Supporting information). Most doxycycline-treated hsf1/tetp-HSF1 cells were viable at the time of transcript profiling  grown at 30\u00b0C in the presence or absence of doxycycline. Once again doxycycline-sensitive genes were excluded from the list of Hsf1-dependent genes (Supporting information). This revealed that Hsf1 contributes to the basal expression of 75 C. albicans genes even in the absence of heat shock. Significantly, this subset of Hsf1-dependent genes displayed significant enrichment in a single cellular process \u2013 protein folding 3). Six observations support this view. First, Hsf1 was required for the transcriptional activation of an HSE reporter in heat-shocked C. albicans cells . Of these 11 C. albicans HSP genes, all contain HSEs in their promoters.In this article we describe the identification of the ns cells . Second,ns cells . Third, in vitro  contain  the HSE . Sixth, S. cerevisiae . Furthermore, our microarray analyses extended these observations by defining Hsf1-dependent genes in C. albicans , possibly because the general stress response has diverged significantly in C. albicans . This is consistent with other well-documented examples of transcriptional rewiring in C. albicans that include Rfg1, a2, Gal4 and Msn2/4-like proteins . Following transformation into C. albicans THE1, this resulted in the deletion of codons 12\u2013746 of the 762 codon HSF1 open reading frame. This generated heterozygous \u0394hsf1/HSF1 mutants (e.g. CLM60-1)  . The Ura CLM62-1 . The genHSF1 coding region to create pACT1-FLAG-HSF1. pACT1-FLAG-GFP was made by inserting a double-stranded oligonucleotide encoding three copies of the FLAG epitope (Supporting information) into the HindIII site in pACT1-GFP, creating a new BamHI site  were cloned into the SalI site upstream of the basal ADH1 promoter in the CIp10-based URA3 plasmid, pLacBasal .Reporter constructs were based on the acZ gene . To creaACT1, HSP90 and HSP104 probes were specific for their corresponding mRNAs on Northern blots. However, Southern analysis of gene-specific PCR products showed that the HSP70 probe cross-reacted with transcripts from several HSP70 family members: HSP70, SSA2, SSB1 KAR2 and SSC1 (not shown).Published methods were used for RNA and DNA preparation, Southern blotting and Northern analyses  were treated in a 20 \u03bcl reaction mix with 1.5 \u03bcl of DNase I, 1.5 \u03bcl of RNase OUT, 2 \u03bcl of DNase I buffer (Invitrogen) at room temperature for 15 min. cDNA was prepared using Superscript II (Invitrogen) as per the manufacturer's protocol. Real-time RT-PCR SYBR green (Roche) assays were carried out as per the manufacturer's instructions using The LightCycler\u00ae 480 Real-Time PCR System (Roche).In some experiments real-time RT-PCR was used to measure the levels of the C. albicans strains THE1 (HSF1/HSF1) and CLM62-1 (hsf1/tetp-HSF1) was performed on exponential cells growing in YPD medium. Where appropriate, doxycycline was added to cultures for at least 6 h before heat shock. Heat stress was imposed by rapidly shifting cells from 30\u00b0C to 45\u00b0C and incubating for 10 min. Cells were frozen rapidly in liquid nitrogen, sheared mechanically using a microdismembrator  and RNA prepared by extraction with Trizol Reagent  as described previously  using published methods (Transcript profiling of tetp-HSF1 cells (compared with unstressed doxycycline-treated tetp-HSF1 cells). Finally, genes whose basal expression levels were dependent upon Hsf1 were defined as those whose transcript levels were: (i) affected at least twofold by the addition of doxycycline to tetp-HSF1 (CLM62-1) cells in the absence of heat stress (compared with untreated CLM62-1 cells in the absence of stress), and (ii) not affected in wild-type (THE1) cells under equivalent experimental conditions.Expression ratios were calculated by comparing stressed cells with the corresponding unstressed control, or by comparing doxycycline-treated cells with the corresponding untreated control. For example, heat shock-regulated genes were defined as those whose transcript levels were affected at least twofold by heat shock in wild-type (THE1) cells (compared with unstressed THE1 cells). Of these heat shock-regulated genes, those whose heat shock regulation was dependent upon Hsf1 were then defined as those whose transcript levels were no longer regulated at least twofold by heat shock in doxycycline-treated C. albicans genes were assigned using GO resources at CGD (http://www.candidagenome.org/cgi-bin/GO/goTermFinder), and on the basis of MIPS functional assignments for S. cerevisiae homologues (http://mips.gsf.de/proj/yeast/CYGD/db/index.html), as described previously , and then 50 \u03bcl of chloroform and 20 \u03bcl of 1% SDS were added. Samples were equilibrated at 37\u00b0C for 10 min, and then reactions started by addition of 200 \u03bcl of pre-warmed OPNG (4 mg ml\u22121). Samples were incubated until a yellow colour developed whereupon the reaction was stopped by addition of 0.4 ml of 1 M Na2CO3. \u03b2-Galactosidase activities were measured in Miller units.eviously . BrieflyTotal soluble protein was extracted and subjected to Western blotting using published protocols ("}
+{"text": "The interactions between stem cells and extracellular matrix (ECM) mediated by integrins play important roles in the processes that determine stem cell fate. However, the role of ECM/integrin interaction in the formation of embryoid bodies (EBs) during cardiogenesis from murine induced pluripotent stem cells (miPSCs) remains unclear.1 integrin were expressed and upregulated synergistically during the formation of miPSC-derived EBs, with a peak expression at day 3 of differentiation. The blockage of collagen/\u03b21 integrin interaction by \u03b21 integrin blocking antibody resulted in the production of defective EBs that were characterized by decreased size and the absence of a shell-like layer composed of primitive endoderm cells. The quantification of spontaneous beating activity, cardiac-specific gene expression and cardiac troponin T (cTnT) immunostaining showed that the cardiac differentiation of these defective miPSC-derived EBs was lower than that of control EBs.In the present study, collagen type I and \u03b21 integrin interaction is required for the growth and cardiac differentiation of miPSC-derived EBs and will be helpful in future engineering of the matrix microenvironment within EBs to efficiently direct the cardiac fate of pluripotent stem cells to promote cardiovascular regeneration.These findings indicate that collagen/\u03b2 Embryonic stem cells (ESCs) are characterized by unlimited proliferation capacity and multipotency and are one of the most promising stem cell populations for use in cardiovascular regenerative medicine. However, the use of ESCs is hampered by their embryonic origin and by the potential immune rejection of allogeneic cell grafts. These issues may potentially be circumvented by the use of induced pluripotent stem cells (iPSCs) that are reprogrammed from somatic cells by the ectopic expression of 4 transcription factors  ,2. The u1 integrin interaction in EB formation from miPSCs and the involvement of such interaction in the differentiation of miPSCs into cardiomyocytes.Lineage-specific differentiation within EBs is a response to a variety of environmental stimuli, including cell-cell adhesion and cell-matrix interactions. Recent strategies for engineering EB formation have focused primarily on controlling EB size, extracellular matrix (ECM) interactions and cell-cell adhesion . ProcessThe morphology of the undifferentiated miPSC colonies carrying the GFP transgene targeted to the Oct4 locus closely resembled that of the mESCs growing on either MEFs or gelatin-coated dishes under feeder-free conditions and expressing green fluorescent protein (GFP) Figure\u2009A. To ind1, \u03b12, \u03b21) was determined by semiquantitative RT-PCR in undifferentiated mESCs, miPSCs, and EBs. Undifferentiated mESCs and miPSCs expressed Itga1 and Itgb1 , whereas Col1A1, Col1A2, Col3A1 and Itga2 were not expressed . Phase-contract and fluorescent micrographs of miPSC-derived EBs formed after \u03b21 integrin blocking revealed a loose outer layer lacking a dense shell as observed in control EBs  to characterize the cardiac-specific protein. This immunostaining revealed organized sarcomeric myofilaments in the control group in contrast to less cTnT staining and unorganized myofilaments in the \u03b21 integrin blocking group , NK2 transcription factor related locus 5 (Nkx2.5), and myocyte enhancer factor 2C (Mef2c) were included as markers for cardiac mesoderm. \u03b1-myosin heavy chain (\u03b1-MHC) and myosin light chain 2 atrial and ventricular transcripts  were included as markers for cardiomyocytes. The semiquantitative RT-PCR and real-time PCR results both demonstrated that all of the above cardiac-specific markers were decreased significantly in the miPSC-derived EBs formed after \u03b21 integrin interaction plays an essential role in the formation and cardiac differentiation of iPSC-derived EBs, which closely resembles early embryonic development. To test this hypothesis, we examined the expression patterns of collagen and integrins during EB formation. Our findings, consistent with those of a previous study [1 and \u03b21 but not integrin \u03b12. During EB formation, collagen and \u03b21 integrin were upregulated synergistically and peaked on day 3 of differentiation, suggesting that the collagen/\u03b21 integrin interaction may be involved in EB formation.EB formation, which recapitulates various aspects of early embryonic development and displays a high degree of self-organization , has beeus study , showed 1 integrin interaction on the growth of miPSC-derived EBs. The size and shape of the EBs resulting from static suspension culture tend to be heterogeneous [1 integrin interaction on the cardiac differentiation of miPSCs.We used two common culture techniques, the hanging drop method and static suspension. Static suspension culture is performed by adding a suspension of ESCs or iPSCs to a bacteriological Petri dish and allowing the cells to aggregate spontaneously via cell-cell adhesion . This meogeneous , and thiogeneous -17. Suchogeneous . To excl1 integrin blocking antibody produced a greater decrease in EB size, indicating the essential role of collagen/\u03b21 integrin interaction in the control of EB size. The size of EBs formed by the hanging drop method was not affected by treatment with \u03b21 integrin blocking antibody. However, the EBs formed by this method following \u03b21 integrin blocking did not form a dense shell-like layer as is observed in normal EBs [1 integrin blocking, another recent study demonstrated that \u03b21 integrin blocking in differentiating EBs resulted in the detachment of this endoderm layer from the EB surface [The size analysis of EBs formed by static suspension culture showed that EB size was increased by treatment with ascorbic acid (a collagen stimulator) but decreased by treatment with CIS (a collagen inhibitor), indicating that collagen synthesis is potent regulator of EB growth. Treatment with \u03b2rmal EBs ,24. Durirmal EBs . Consist surface .1 integrin blocking during EB formation affects the cardiac lineage commitment of miPSCs. A study of spontaneous beating activity, the quantification of cardiac-specific gene expression and cTnT immunostaining all revealed decreased cardiac differentiation in miPSC-derived EBs formed after the blocking of collagen/\u03b21 integrin interaction. In addition, we also assessed some markers related to the structural/maturity characteristics of cardiomyocytes, such as cTnI, MLC2a, and MLC2v. cTnT staining showed that iPSCs-derived cardiomyocytes failed to form organized sarcomeric myofilaments with \u03b21 integrin blocking as that in the control group  feeder layer or on gelatin-coated dishes as described previously [The miPSCs used in this study , which carry the GFP transgene targeted to the Oct4 locus, were generated by retroviral transduction of adult fibroblasts from OG2 mice with pMX-based retroviral vectors encoding the transcription factors Oct4 and Sox2. Undifferentiated mESCs (CGR8) and miPSCs were cultured on a mitotically inactivated mouse embryonic fibroblast . For the hanging drop method, EBs were formed in hanging drops consisting of 500 cells in 20 \u03bcl of differentiation medium. For static suspension culture, dissociated cells were cultured to form spheroids (EBs) using a 3-dimensional culture system in the differentiation medium on uncoated Petri dishes . At day 3 of differentiation, the EBs were transferred onto ultra-low attachment 6-well plates  in suspension culture for 2 additional days. The differentiation medium was based on high-glucose glutamine-free DMEM and supplemented with 20% ES-qualified fetal bovine serum , 2 mM GlutaMAX, 0.1 mM nonessential amino acid stock, and 0.1 mM \u03b2-mercaptoethanol.1 integrin interaction in EB formation, ascorbic acid  or cis-4-hydroxy-D-proline  was administered prior to EB formation. In some experiments, 10 \u03bcg/ml \u03b21 integrin blocking antibody  was administered to block collagen/\u03b21 integrin interaction.To elucidate the role of collagen/\u03b21 integrin interaction affected the growth of EBs from mESCs and miPSCs. Images of EBs formed by the hanging drop method and by static suspension culture under various conditions were obtained on days 3 and 5. For size analysis, mean diameters were measured using the Image J software program and calculated from more than five sample EBs per field in 10 fields as described previously [Gross morphology was analyzed to determine whether the blocking of collagen/\u03b2eviously . When thDay 5 EBs were plated on 0.1% gelatin-coated 6-well culture plates at a density of\u2009\u2248\u200910 EBs per well. Starting at day 6 of differentiation, the differentiation medium described above was replaced every second day, and each EB outgrowth was examined daily for areas that displayed spontaneous beating.Whole EBs (day 3) and plated EBs (day 21) were stained to detect collagen and cardiomyocyte proteins, respectively. For Collagen I staining, whole iPSCs-derived EBs (day 3) were fixed in 4% paraformaldehyde for 40 min and embedded for frozen sectioning using OCT. Plated EBs (day 21) were fixed in 4% paraformaldehyde for 15 minutes at room temperature. All samples were permeabilized in 0.1% Triton X-100 for 10 minutes, and then blocked with 5% BSA for 30 minutes. The cells were incubated overnight with rabbit monoclonal anti-cardiac troponin T antibody  and rabbit monoclonal anti-collagen type I antibody . After incubation overnight, we identified that there were no Oct4-GFP residue in samples using a fluorescence microscope before incubation with the FITC-conjugated secondary antibody. The bound antibodies were visualized by incubation with the appropriate secondary antibody  for 40 minutes at 37\u00b0C. Then the cells were counterstained with DAPI  and analyzed using a fluorescence microscope.Total RNA was extracted from EBs using Trizol reagent (Invitrogen) according to the manufacturer\u2019s instructions. Following treatment with Recombinant DNAse I  to remove genomic DNA contamination, 400 ng/\u03bcl RNA was reverse transcribed using the PrimeScript\u2122 First Strand cDNA Synthesis Kit (Takara). The resulting cDNA was amplified by polymerase chain reaction (PCR) using standard methods. Primer sequences and PCR conditions are detailed in Additional file T method.SYBR Green real-time PCR studies were performed using SYBR Premix Ex Taq\u2122 II (Takara). All experiments were conducted in triplicate. Samples were subjected to the following cycle 40 times: 5 minutes at 95\u00b0C; 40 cycles of 30 seconds at 95\u00b0C, 30 seconds at 60\u00b0C, and 30 seconds at 72\u00b0C; a final step of 10 minutes at 72\u00b0C and 5 minutes at 4\u00b0C. The size of amplicons and the absence of nonspecific products were measured or confirmed using melting curves and gel electrophoresis. The transcripts for \u03b2-actin and GAPDH were used for internal normalization. Relative quantification was performed by the \u25b3\u25b3C2, and sputter-coated for 120 s at 2.0 KV using a Polaron SC7640 sputter coater. The samples were observed and photographed using a Philips XL20 scanning electron microscope.mESCs and miPSC-derived EBs (days 3 and 5) were fixed for 2 h in 2.5% glutaraldehyde diluted in sodium cacodylate buffer (Electron Microscopy Sciences) and post-fixed with 1% osmium tetroxide buffered with 0.1 M sodium cacodylate for 1 h. The fixed samples were dehydrated using a series of ethanol solutions of increasing concentration , critically point dried with COp values < 0.05 were considered statistically significant.The results are presented as the mean\u2009\u00b1\u2009SEM. The data were analyzed by Student\u2019s t-test or 1-way ANOVA with repeated-measures analysis. For all analyses, differences with ESC: embryonic stem cell; iPSC: induced pluripotent stem cell; EB: embryoid body; ECM: extracellular matrix; Vc: ascorbic acid; CIS: cis-4-hydroxy-D-proline.The authors declare that they have no competing interests related to the manuscript.QSZ conceived of the study, participated in its design and revised manuscript critically for important intellectual content. DZ and DBO performed most experiments in immunocytochemistry, scanning electron microscopic analysis, PCR, cell culture and cardiac differentiation of miPSCs. TW and LD analyzed the data. XL, XLH and XTL revised the paper. All authors read and approved the final manuscript.Expression of markers related to three germ layer within EBs. Semiquantitative RT-PCR measurement of markers related to the three germ layer  within 3d- and 5d-EBs derived from cells subject to integrin disruption and controls. Experiments were performed in triplicate, and the transcripts for GAPDH were used for internal normalization.Click here for fileLoss of pluripotency within EBs after integrin disruption. Semiquantitative RT-PCR (A) and quantitative PCR  measurement of pluripotent markers (OCT3/4 and Nanog) within 3d- and 5d-EBs derived from cells subject to integrin disruption and controls. Expression levels of each gene were normalized to GAPDH. Mean fold change relative to GAPDH and SD from triplicate experiments are shown.Click here for filePrimers and cycling conditions for RT-PCR.Click here for filePrimers for RT-PCR in supplementary figures.Click here for file"}
+{"text": "To investigate, using a Mendelian randomisation approach, whether heavier smoking is associated with a range of regional adiposity phenotypes, in particular those related to abdominal adiposity.CHRNA5-CHRNA3-CHRNB4 gene region) as a proxy for smoking heaviness, of the associations of smoking heaviness with a range of adiposity phenotypes.Mendelian randomisation meta-analyses using a genetic variant .Waist and hip circumferences, and waist-hip ratio.The data included up to 66\u2005809 never-smokers, 43\u2005009 former smokers and 38\u2005913 current daily cigarette smokers. Among current smokers, for each extra minor allele, the geometric mean was lower for waist circumference by \u22120.40% (95% CI \u22120.57% to \u22120.22%), with effects on hip circumference, waist-hip ratio and body mass index (BMI) being \u22120.31% (95% CI \u22120.42% to \u22120.19), \u22120.08% (\u22120.19% to 0.03%) and \u22120.74% (\u22120.96% to \u22120.51%), respectively. In contrast, among never-smokers, these effects were higher by 0.23% (0.09% to 0.36%), 0.17% (0.08% to 0.26%), 0.07% (\u22120.01% to 0.15%) and 0.35% (0.18% to 0.52%), respectively. When adjusting the three central adiposity measures for BMI, the effects among current smokers changed direction and were higher by 0.14% (0.05% to 0.22%) for waist circumference, 0.02% (\u22120.05% to 0.08%) for hip circumference and 0.10% (0.02% to 0.19%) for waist-hip ratio, for each extra minor allele.For a given BMI, a gene variant associated with increased cigarette consumption was associated with increased waist circumference. Smoking in an effort to control weight may lead to accumulation of central adiposity. This is a very large Mendelian randomisation study of the relationship between smoking and several anthropometric phenotypes relating to regional adiposity.Data included never, former and current smokers from a very wide spectrum of ages among 29 studies.By using a genetic variant associated with smoking heaviness as a proxy for smoking heaviness, bias from confounding is minimised and findings are not affected by reverse causality.Data for direct measures of fat, such as fat mass, and the biomarker leptin were available for only about one fifth of the participants whose weight, height, waist and hip were measured.Participants were exclusively of self-reported European ancestry, and were mostly recruited in European countries.Tobacco is the single most important cause of preventable death globally: one in two young people taking up lifelong cigarette smoking will die of causes related to it.3CHRNA5-CHRNA3-CHRNB4 gene region (rs16969968) codes for a functional amino acid change D398N in the nicotinic receptor \u03b15 subunit. The SNP rs16969968, which is in perfect linkage disequilibrium with SNP rs1051730 in European populations, is associated with smoking quantity among smokers.18A genetic variant in the chromosome 15 egion rs1969968 coregion rs969968 coWe previously used Mendelian randomisation methods to investigate the effect of smoking quantity on BMI.http://www.bris.ac.uk/expsych/research/brain/targ/research/collaborations/carta/): the 1958 Birth Cohort (1958BC), the Avon Longitudinal Study of Parents and Children , the British Regional Heart Study (BRHS), the British Women's Heart and Health Study (BWHHS), the Caerphilly Prospective Study (CaPS), the Christchurch Health and Development Study (CHDS), CoLaus, the Danish Monica study (Dan-MONICA), the Exeter Family Study of Child Health (EFSOCH), the English Longitudinal Study of Ageing (ELSA), the National FINRISK studies, GEMINAKAR, GS:SFHS , the Genomics of Overweight Young Adults (GOYA) females, GOYA males, the Helsinki Birth Cohort Study (HBCS), Health2006, Health2008, the Nord-Tr\u00f8ndelag Health Study (HUNT), Inter99, MIDSPAN, the Northern Finland Birth Cohorts (NFBC 1966 and NFBC 1986), the National Health and Nutrition Examination Survey (NHANES), the MRC National Survey of Health & Development (NSHD), the Netherlands Twin Register (NTR), the PROspective Study of Pravastatin in the Elderly at Risk (PROSPER) and Whitehall II. All studies received ethics approval from the local research ethics committees. Further details of these studies are provided in online supplementary material.We used data on individuals (\u226516\u2005years) of self-reported European ancestry from 29 studies from the CARTA consortium  and therefore represent the same genetic signal. Where studies had data available for both SNPs, we used the SNP that was genotyped in the largest number of individuals. Details of genotyping methods within each study are provided in online supplementary material.Within each study, individuals were genotyped for one of two single nucleotide polymorphisms (SNPs) in the Direct physical measurements included weight, height, waist and hip circumferences, arm circumference, triceps skinfold and subscapular skinfold thickness. Fat mass and fat-free mass were available from bioimpedance measures, while leptin and adiponectin were the two biochemical markers related to fat mass.2) and waist-hip ratio (waist/hip) were calculated.BMI . Individuals were classified as current, former, ever  or never cigarette smokers. Where information on pipe and cigar smoking was available, individuals reporting being current or former smokers of pipes or cigars but not cigarettes were excluded from all analyses.Smoking status was self-reported (either by questionnaire or interview) at the same time as regional adiposity measures for all studies, with the exception of 1958 BC (see online For studies with adolescent populations (ALSPAC children and NFBC 1986), analyses were restricted to current daily smokers who reported smoking at least one cigarette per day (current smokers) and individuals who had never tried smoking (never-smokers).http://www.R-project.org) software, following the same analysis plan. Analyses were restricted to individuals with full data on smoking status and rs1051730/rs16969968 genotype, and having data on at least one of the regional adiposity phenotypes.Analyses were conducted within each contributing study using Stata  and R  and sex, and adjusted for age. Apart from height, natural logarithmic transforms were taken of every anthropometric phenotype. An additive genetic model was assumed on log values, so that each effect size could be exponentiated to represent the percentage increase per minor (risk) allele. These analyses were presented separately for each smoking status category. All phenotypic measures were further adjusted for log(BMI) , thus assessing the effect of the particular adiposity measure after adjusting for this global weight measure. Log(weight) was adjusted for height instead of log(BMI). Since adjustment for ratio variables in anthropometric studies has been criticised,2; 95% CIs have been quoted for all effect sizes.Within each study, genotype frequencies were tested for deviation from the Hardy Weinberg Equilibrium (HWE) using a \u03c7Meta-analysis was also carried out of the relationship between reported daily cigarette consumption and rs1051730/rs16969968 genotype, among current smokers.Although analyses were carried out separately for males and females, the estimates were combined where no evidence for separate sex effects was seen. For NHANES, which has a survey design, Taylor series linearisation was implemented to estimate variances. For studies including related family members, appropriate methods were used to adjust SEs: in GEMINAKAR, twin pair identity was included as a cluster variable in the model; in MIDSPAN, linear mixed effects regression models fitted using restricted maximum likelihood were used to account for related individuals, while in NTR, only unrelated individuals were included. ALSPAC mothers and children were analysed as separate samples; as there are related individuals across these samples, sensitivity analyses were performed excluding each of these studies in turn.2 >50%), it was planned that both fixed and random effects analyses would be performed: however, as this never occurred, results for fixed effects analysis only are shown. Meta-regression analysis, using the \u2018metareg\u2019 command from Stata, was used to examine whether SNP effects varied by smoking status or by sex, or by a smoking by sex combination.Results from individual studies were meta-analysed in Stata (V.13) using the \u2018metan\u2019 command from Stata. Where there was evidence of heterogeneity between studies .The maximum sample size available, with genotype recorded, was 148\u2005731 for weight, height and BMI over 29 studies. The data on individuals with weight, height, smoking status and genotype recorded included 66\u2005809 never-smokers, 43\u2005009 former smokers and 38\u2005913 current smokers. Waist circumference was available in 28 studies (n=142\u2005381), and hip circumference and waist-hip ratio in 25 studies (n=139\u2005667). Measures of fat mass and fat-free mass were provided by 10 studies (n=28\u2005231), arm circumference by nine studies (n=72\u2005536), and skinfolds by five studies (n=7758). Finally, leptin and adiponectin were measured in nine studies . Overall, 47% of the combined study population was male. The median age within the contributing studies ranged from 16\u201374\u2005years. Descriptive statistics for each of the study populations are found in the Minor allele frequency for rs1051730/rs16969968 ranged between 0.31 and 0.36. There was no strong evidence for deviation from the Hardy-Weinberg Equilibrium in any of the studies .\u22125), non-significant in former smokers: \u22120.14%  and significantly inverse in current smokers: \u22120.74% . The full results for each contributing study are shown in online supplementary figure S1.\u22125): differences among smoking groups were highly significant , the minor allele of rs1051730-rs16969968 was not associated with waist circumference in either never-smokers: +0.01%  or former smokers +0.06% . However, in current smokers, the minor allele was associated with a 0.14%  higher waist circumference after adjustment for log(BMI). Very similar results were seen in all three smoking status categories after waist was adjusted for log(weight) and height instead of for log(BMI). Effects of genotype on waist circumference were shown to differ between smoking status categories before adjustment (p=3.85\u00d710\u22127) but only weakly after adjustment for log(BMI) (p=0.102), and after adjustment for log(weight) and height (p=0.018). Little heterogeneity of study results was evident . After restricting analysis to participants with BMI under 30\u2005kg/m2, we found that the percentage increases in waist circumference (after adjustment for log(BMI)) were 0.04% (95% CI \u22120.03% to 0.12%) for never-smokers, 0.03% (95% CI \u22120.06% to 0.13%) for ex-smokers and 0.12% (95% CI 0.02% to 0.21%) for current smokers: however, the test for difference in effects gave p=0.41.The waist circumference was higher per minor allele in never-smokers: +0.23% , non-significantly related in former smokers \u22120.07%  and lower in current smokers \u22120.40% . However, after adjustment for log(BMI), effects were not apparent in any of the three groups, and nor was the interaction of gene and smoking status.Results for the waist-hip ratio were similar to the BMI, waist and hip circumferences in direction but were smaller in magnitude: +0.07%, 0.00% and \u22120.08% increases in never-smokers, former smokers and current smokers, respectively . After adjustment for log(BMI), increases remained non-significant for never-smokers and former smokers (\u22120.01% and 0.04%) but increased significantly among current smokers (0.10%) (p=0.13 for differences among smoking groups).\u22125) and leptin (p=0.025), while the difference between smoking groups was only significant for arm circumference (p=3.29\u00d710\u22124). Both effects became non-significant after adjustment for log(BMI). Fat mass and fat-free mass, after adjustment by height, showed differences in effects by smoking group. These effects were more due to per-allele increases seen among never-smokers than decreases among current smokers.For several other phenotypes, per-allele decreases were observed in current smokers that exceeded those seen either in former or never-smokers (see online supplementary table S4). However, there was only statistical evidence for decreases among current smokers for arm circumference , but this effect was no longer apparent after adjusting for BMI (p=0.51).2=17%).The mean difference in daily cigarette consumption was 0.77 among current smokers .8Cross-sectional observational data from Switzerland has demonstrated that waist and hip circumferences were more strongly related to the number of cigarettes smoked per day than was BMI,Some observational studies have noted that low fat-free massOur findings resonate with observational studies which have shown associations between smoking and risk of diabetes,People who quit smoking appear to be at increased risk of acquiring diabetes in the short term but this was not explained by weight gain in a Japanese population.Mendelian randomisation studies have more potential than traditional observational epidemiological studies to establish causality for specific exposures,"}
+{"text": "CHRNA5-A3-B4 gene cluster associated with heaviness of smoking within smokers to confirm the causal effect of smoking in reducing body mass index (BMI) in a Mendelian randomisation analysis. While seeking to extend these findings in a larger sample we found that this SNP is associated with 0.74% lower body mass index (BMI) per minor allele in current smokers , but also unexpectedly found that it was associated with 0.35% higher BMI in never smokers . An interaction test confirmed that these estimates differed from each other (P\u200a=\u200a4.95\u00d710\u221213). This difference in effects suggests the variant influences BMI both via pathways unrelated to smoking, and via the weight-reducing effects of smoking. It would therefore be essentially undetectable in an unstratified genome-wide association study of BMI, given the opposite association with BMI in never and current smokers. This demonstrates that novel associations may be obscured by hidden population sub-structure. Stratification on well-characterized environmental factors known to impact on health outcomes may therefore reveal novel genetic associations.We previously used a single nucleotide polymorphism (SNP) in the  CHRNA5-A3-B4 gene cluster, which is known to influence smoking heaviness, is associated with lower body mass index (BMI) in current smokers, but higher BMI in never smokers. This difference in effects suggests that the variant influences BMI both via pathways other than smoking, and via the weight-reducing effects of smoking, in opposite directions. The overall effect on BMI would therefore be undetectable in an unstratified genome-wide association study, indicating that novel associations may be obscured by hidden population sub-structure.We found that a single nucleotide polymorphism in the  CHRNA5-A3\u2013B4 gene cluster associated with heaviness of smoking within smokers As obesity represents a substantial and growing threat to public health, efforts to identify the determinants of obesity are of considerable scientific and societal importance. Genome-wide association studies (GWAS) have identified numerous variants associated with body mass index (BMI) http://www.bris.ac.uk/expsych/research/brain/targ/research/collaborations/carta/). We used the same genetic variant, characterised by two SNPs (rs16969968 and rs1051730) which are in perfect linkage disequilibrium (LD) in samples of European ancestry, and therefore reflect the same genetic signal (hereafter rs16969968-rs1051730). This variant is associated with approximately 1% phenotypic variance in cigarettes per day and approximately 4% variance in cotinine levels  only affects the outcome through the exposure of interest) We sought to extend these findings in a larger sample drawn from the Causal Analysis Research in Tobacco and Alcohol (CARTA) consortium . We also confirmed the expected inverse association of rs16969968-rs1051730 with BMI in the 38,913 current smokers , consistent with a causal, weight-reducing effect of cigarette smoking on BMI. There was no evidence of association in the 43,009 former smokers . An interaction test indicated that these estimates differed from each other (P\u200a=\u200a4.95\u00d710\u221213). Similar associations were observed for weight . Between-study heterogeneity was low , and there was no evidence for effect modification by sex. Critically, when data were examined without stratification by smoking status no clear evidence of association with BMI was observed (P\u200a=\u200a0.22), indicating that a conventional GWAS would have failed to detect this signal.Our total sample size comprised 148,730 never smokers, former smokers and current smokers. In the 66,809 never smokers, we observed positive association of rs16969968-rs1051730 with BMI , indicatr weight  and wais2. This is smaller than the effect of rs9939609 in FTO (\u223c0.4 kg/m2) \u221212), consistent with previous studies The 0.35% per minor allele BMI increase in never smokers represents a change of approximately 0.09 kg/mCHRNA5-A3-B4 gene cluster was confirmed to be associated with heaviness of smoking, and downstream health outcomes including lung cancer and peripheral arterial disease Our results indicate that rs16969968-rs1051730 may be associated with BMI in never smokers, via pathways other than smoking, as well as with heaviness of smoking among current smokers. At this stage we can only speculate as to the mechanism through which rs16969968-rs1051730 may exert a positive effect on BMI in never smokers. In GWAS, the CHRNA3 gene and correlated with rs16969968-rs1051730 , shows association  with BMI (defined as <30 kg/m2 vs \u226530 kg/m2) (dbGaP Study Accession: pha003015.1). There is evidence from animal models that activation of hypothalamic \u03b13\u03b24 nAChRs leads to activation of pro-opiomelanocortin neurons, and subsequent activation of melanocortin 4 receptors, which have been shown to be critical for nicotine-induced decreases in food intake CHRNA3 and CHRNB4) may contribute to our finding. Clearly further work is required to explore this possibility. The use of more detailed body composition measures such as percent body fat and its distribution may also serve to refine the nature of the association.It is also notable that rs3743075, located within the P-value of 0.05, and even then would indicate an inaccurate effect size. This is essentially because the effect of rs16969968-rs1051730 on BMI that operates via pathways other than smoking is countered by the weight-reducing effect of smoking. Therefore, since there are roughly twice as many never smokers as current smokers on average across our sample, these two effects negate each other. On the other hand, a sample of approximately 160,000 never smokers would be required to detect the effect we observed with genome-wide significance. Assuming the proportions of never, former and current smokers in our sample, this would imply a total sample size of around 350,000. While this is larger than published GWAS of BMI Our results, if confirmed, have important implications for the design of future GWAS. The association we observed in never smokers would essentially be undetectable in an unstratified sample, since the effect size observed in the combined sample would require approximately 791,000 participants to detect even at an uncorrected larger than estimated with this variant. However, some caution must be exercised in conducting and interpreting the results of other Mendelian randomisation analyses using this variant because rs16969968-rs1051730 may influence outcomes through its effects on BMI, instead of or in addition to smoking heaviness. One possible solution is to use genetic variants for BMI as a method of reciprocal randomization to determine the direction of causation within inter-correlated networks of mechanistic pathways  The pleiotropic effect of rs16969968-rs1051730 , if shown to be robust via replication, has important implications for Mendelian randomisation studies assessing the causal effects of smoking. In this case, we can be reasonably confident that the BMI-reducing effect of the variant operates through smoking because the association with BMI in current smokers is in the opposite direction to the association in never smokers. Furthermore, if the effects on BMI that operate via pathways other than smoking and the effects that operate via the weight-reducing effects of smoking are independent, then the true causal estimate of the magnitude of effect of smoking in reducing BMI is likely to be A limitation to our analysis is that we were only able to control for potential population stratification indirectly in most samples, by restricting analyses to participants of self-reported European ancestry. We were not able to use other methods, such as adjustment for principal components, given that not all contributing studies hold the necessary genetic data. However, we note that the minor allele frequency of the rs16969968-rs1051730 differed only slightly across studies (between 0.30 and 0.36).Testing for gene-environment interaction in GWAS is not novel http://www.bris.ac.uk/expsych/research/brain/targ/research/collaborations/carta/): the 1958 Birth Cohort (1958 BC), the Avon Longitudinal Study of Parents and Children , the British Regional Heart Study (BRHS), the British Women's Heart and Health Study (BWHHS), the Caerphilly Prospective Study (CaPS), the Christchurch Health and Development Study (CHDS), the Cohorte Lausannoise (CoLaus) study, the Exeter Family Study of Child Health (EFSOCH), the English Longitudinal Study of Ageing (ELSA), FINRISK, the Danish GEMINAKAR twin study, Generation Scotland, the Genomics of Overweight Young Adults (GOYA) females, GOYA males, the Helsinki Birth Cohort Study (HBCS), Health2006, Health2008, the Nord-Tr\u00f8ndelag health study (HUNT), Inter99, the Northern Finland Birth Cohorts (NFBC 1966 and NFBC 1986), MIDSPAN, the Danish MONICA study, the National Health and Nutrition Examination Survey (NHANES), the MRC National Survey of Health & Development (NSHD), the Netherlands Twin Registry (NTR), the Prospective Study of Pravastatin in the Elderly at Risk (PROSPER) and Whitehall II. References to these individual studies are available on request. All studies received ethics approval from local research ethics committees  of European ancestry  from 29 studies in the Causal Analysis Research in Tobacco and Alcohol (CARTA) consortium  and therefore represent the same genetic signal. Where studies had data available for both SNPs, we used the SNP that was genotyped in the largest number of individuals.Within each study, individuals were genotyped for one of two single nucleotide polymorphisms (SNPs) in the 2.Height (m), weight (kg) and waist circumference (cm) were assessed within each study, directly measured for 99% of participants, and self-reported for GOYA females  and a sub-set of NTR (N\u200a=\u200a602). Body mass index (BMI) was calculated as weight/heightSmoking status was self-reported (either by questionnaire or interview). Individuals were classified as current, former, or never cigarette smokers. Where information on smoking frequency was available, current smokers were restricted to individuals who smoked regularly . Where information on pipe and cigar smoking was available, individuals reporting being current or former smokers of pipes or cigars but not cigarettes were excluded from all analyses. For studies with adolescent populations (ALSPAC children and NFBC 1986), analyses were restricted to current daily smokers who reported smoking at least one cigarette per day (current smokers) and individuals who had never tried smoking (never smokers). Descriptive characteristics of smoking frequency data are provided in Analyses were conducted within each contributing study using Stata and R software, following the same analysis plan. Analyses were restricted to individuals with full data on smoking status and rs16969968-rs1051730 genotype. Within each study, genotype frequencies were tested for deviation from Hardy Weinberg Equilibrium (HWE) using a chi-squared test. Mendelian randomisation analyses of the association between rs16969968-rs1051730 and BMI were performed using linear regression, stratified by smoking status  and sex, and adjusted for age. BMI was log transformed prior to analysis. An additive genetic model was assumed on log values, so that each effect size could be exponentiated to represent the percentage increase in BMI per minor (risk) allele.For NHANES, which has a survey design, Taylor series linearization was implemented to estimate variances. For studies including related family members appropriate methods were used to adjust standard errors: in GEMINAKAR, twin pair identity was included as a cluster variable in the model, in MIDSPAN linear mixed effects regression models fitted using restricted maximum likelihood were used to account for related individuals. ALSPAC mothers and children were analysed as separate samples; as there are related individuals across these samples, sensitivity analyses were performed excluding each of these studies in turn.2 values were all equal to or below 36% (indicating low to moderate heterogeneity), fixed effects meta-analyses were performed. The \u201cmetareg\u201d command was used to examine whether SNP effects varied by sex and estimates were combined as there was no evidence for effect modification by sex. Evidence for interaction between genotype and smoking status was assessed using the Cochran Q statistic. Data are available from the Institutional Data Access/Ethics Committees of the individual studies that contributed to this analysis, for researchers who meet the criteria for access to confidential data. Full details are provided in Results from individual studies were meta-analysed in Stata (version 13) using the \u201cmetan\u201d command. As Ihttp://biostats.usc.edu/Quanto.html). The following parameters were used: 80% power to detect associations, minor allele frequency of 0.33, mean and standard deviation for BMI of 25 kg/m2 and 3.8 kg/m2 respectively, alpha values of 0.05 and 5\u00d710\u22128.Sample size calculations were performed using Quanto software Click here for additional data file.Text S2Smoking heaviness in the CARTA studies.(DOCX)Click here for additional data file.Text S3Data access arrangements for individual contributing studies.(DOCX)Click here for additional data file."}
+{"text": "Given the speed of air travel, diseases even with a short viremia such as dengue can be easily exported to dengue na\u00efve areas within 24\u2009hours. We set out to estimate the risk of dengue virus introductions via travelers into Europe and number of secondary autochthonous cases as a result of the introduction. We applied mathematical modeling to estimate the number of dengue-viremic air passengers from 16 dengue-endemic countries to 27 European countries, taking into account the incidence of dengue in the exporting countries, travel volume and the probability of being viremic at the time of travel. Our models estimate a range from zero to 167 air passengers who are dengue-viremic at the time of travel from dengue endemic countries to each of the 27 receiving countries in one year. Germany receives the highest number of imported dengue-viremic air passengers followed by France and the United Kingdom. Our findings estimate 10 autochthonous secondary asymptomatic and symptomatic dengue infections, caused by the expected 124 infected travelers who arrived in Italy in 2012. The risk of onward transmission in Europe is reassuringly low, except where Aedes aegypti is present. The distribution of Aedes aegypti and Ae. albopictus mosquitoes - the vectors for dengue- is now the widest ever recorded; extensive in all continents, including North America and Europe, with over three billion people living in Aedes-infested countries or areas2. Although Aedes aegypti, remains a vector predominantly of the tropics and subtropics, the global spread of Ae. albopictus fuelled by global trade and travel3, and its resistance to colder weather2, put temperate areas such as Europe at risk for transmission of dengue and other Aedes transmitted viruses. Figure\u00a0Aedes albopictus in Europe. Some model studies predict that climate change will increase dengue epidemic potential in temperate regions, potentially flattening the differences between tropical and temperate zones6. Compared with the tropics, Europe shows pronounced seasonality which affects temperature-dependent proliferation of Aedes mosquitoes. Although vectorial capacity \u2013 the mosquito\u2019s capacity to transmit dengue virus to humans \u2013 is low in Europe according to modeling estimates, vectorial capacity is sufficient during the summer months for a dengue outbreak to occur in most of Southern Europe4. Indeed, small autochthonous dengue clusters occurred in Southern France and in Croatia in 20107, and a major dengue outbreak involving more than 2000 persons occurred in Madeira/Portugal in 20128, underpinning that the threat of dengue to Europe is real. The potential for dengue outbreaks to occur in Europe depends on two main factors: (1) the presence of Aedes vectors during the summer season when vectorial capacity is sufficient to sustain transmission and (2) the rate of dengue virus importations, that depends on the number of dengue viremic travelers entering Europe. Most of the modeling work has been focused on understanding the risk of local transmission and relatively little attention has been given to the extent of disease importations. As human movements resulting in global spread of infectious diseases including vector-borne diseases are increasing18 and the burden of diseases such as dengue increases, we can expect an exponential increase in arrivals of viremic passengers which may challenge traditional health infrastructures.Arboviral diseases are on the rise, with dengue viral infections taking the lead19 and increase of travel to tropical and subtropical countries19, diseases with short viremia such as dengue (around 7 days) can be easily exported to a dengue na\u00efve areas within 24\u2009hours, where mosquitoes may then be able to feed on viremic blood, and transmit the virus on to other humans. A sentinel surveillance study recently estimated that about 40% of travelers diagnosed with dengue are viremic at the time of arrival in Europe20. The first large dengue outbreak in Europe that occurred in Madeira in 2012 as a result of importation of the virus via incoming viremic air passengers most likely from Venezuela8 prompted us to study the extent of potentially dengue viremic travelers arriving in Europe as a whole in the same year and to estimate the probability of secondary transmission in Europe as a result of such importation. In the absence of good empiric data on importation of dengue via viremic travelers, mathematical models can provide an additional tool to estimate the number of dengue virus introductions21. The extent of dengue virus introduction is a function of travel volume and dengue incidence in the \u2018exporting\u2019 country23. We set out to model the estimated numbers of dengue-viremic air passengers from dengue-endemic countries to 27 European countries and the subsequent risk of autochthonous transmission as a result of such importation.Given the speed of air travel today21, and expanded it to further to include models on vectorial capacity5. To estimate the number of dengue viremic air passengers into Europe in one year we took into account air travel volume, the dengue monthly incidence in the country of origin, and the probability for the air passenger to be viremic at the time of travel.We applied a previously published mathematical model to estimate the risk of importation of infectious diseases via travelersAedes albopictus is currently present, and Madeira, where Aedes aegypti caused an important outbreak in 2012. Aedesalbopictus is a much less competent mosquito for dengue transmission than Aedes aegypti; Tables\u00a0We consider two types of countries, the \u2018exporting\u2019 country (where the infection is endemic) and those \u2018importing\u2019 or \u2018receiving\u2019 countries. We investigated the number of imported dengue-viremic travelers into 27 European countries based on the following variables: (1) the monthly dengue incidence in the exporting countries; (2) the monthly number of people leaving the airports of exporting countries and traveling to importing countries; (3) the expected monthly number of dengue-viremic travelers arriving at the importing countries. (4) the accumulated number of secondary cases in humans in the importing countries generated by the infected travelers from the exporting countries taking into account the vectorial capacity of Aedes mosquitoes in the importing countries over a one year period; and (5) the accumulated per capita risk of dengue infection in the population of the importing countries over one year caused by infected travelers arriving in that year. Items 4 and 5 will be exemplified by the cases of Italy, in which www.who.int). From the 85 countries that reported 1,597,220 dengue cases to WHO in 2012 we selected 16 countries which were responsible for 95% of all the cases. These countries, along with the number and relative contribution to the total number of dengue cases are shown in Table\u00a0The first step was to obtain information on dengue cases notified to the World Health Organization (WHO) from their website  time and c3 represents the \u201cvariance\u201d of the time distribution of cases. All parameters c1,i\u2009=\u20091, \u2026, 4 fitted to function (1), when used in the dynamical model described below reproduces the observed incidence of dengue for a given outbreak in a region preferably small as we will explain later. Parameter\u2009c4\u2009is given by:rect (t) is a rectangular function added to equation (\u03b8(t + ti) \u2212 \u03b8(t \u2212 ti). The rect (t) is zero for any t > |ti| and is equal to 1 for t < |ti|.The second step is to fit a continuous function to the number of actually reported dengue cases (incidence) multiplied by 4 to take into account the 4:1 asymptomatic:symptomatic ratio for dengue infectionsj (j\u2009=\u20091 \u2026 12) denoting the months of the year. We used previously developed and validated models (1) and (2)26 to the human components of the Ross-Macdonald model, in which \u03bcH and \u03b3H are the humans natural mortality and recovery from infection rates, respectively.The number of monthly reported dengue cases of all 16exporting countries, was used to estimate the monthly prevalence of dengue in each country. Therefore, all the quantities should have a superscript ci, i\u2009=\u20091, \u2026, 4 of equation  times the number of susceptible humans, denoted 27.The first term of the first equation in system of equations  (\\documeRemark: \u03bbj(t)is the dengue incidence:t, defined as the monthly individual risk of being infected and denoted The individual probability of being infected at time This risk is obtained as follows. From the second equation of model (3) for the infected humans we obtain:j, either among travellers who visited each of the selected countries or among local inhabitants. In addition, equation  travellers arrive at each month Note that both the humans\u2019 and mosquitoes\u2019 populations are assumed to be constant and the last terms of the susceptible humans and susceptible mosquitoes are included to mimic births as equal to deaths. This is based on the assumption that dengue does not have any significant impact in humans nor mosquitoes in terms of mortality.4. For the sake of clarity, let us exemplify how the parameters in model (7) enter the code.Let us take the biting rate, for instance, aj(t). As shown in4, it varies with temperature and hence with time, that is, it varies seasonally. Its value should be written as aj(t) = Aj[\u03b8(t + tj) \u2212 \u03b8(t \u2212 tj+1)], where Aj is the value of the biting rate obtained from4 for the month j. In other words, the biting rate assumes \u2018discrete\u2019 values in each month of the year.We did these calculations for Italy only where we used the average temperature month-by-month and applied the parameters from Table\u00a0The values of the pulse-like parameters are given inThe densities The above calculation is restricted to the autochthonous cases generated exclusively by infected travellers. If one is interested in the total number of dengue cases one should substitute the fourth and fifth equations of model (7) by:Figure\u00a0Aedes mosquitoes depends on the presence of such mosquitoes, and Fig.\u00a0Aedes albopictus distribution in Europe. From Fig.\u00a0Aedes albopictus. As the air passenger flight information was available only at a country level, we were only able to calculate the probability of onward transmission where the presence of Aedes albopictus is countrywide. Hence, we selected Italy to illustrate the method to calculate the number of secondary autochthonous cases in one year as a result of dengue virus importation via incoming air passengers from dengue endemic countries. Table\u00a0Aedes mosquitoes based on previous calculations on the seasonal variation of vectorial capacity in different European cities by Liu-Helmersson et al.4. Out model estimated that 10autochthonous (secondary) cases would occur as a result of 51 air passengers arriving in Italy at a time of being dengue viremic. These 10 persons include 2 (20%) symptomatic dengue cases, assuming a 4:1 ratio of asymptomatic to symptomatic infections.The risk of onward transmission via Aedes aegypti was responsible for a large dengue outbreak in 2012, as well as the expected number of secondary cases resulting from the introduction of imported cases from Venezuela and Brazil. Our models estimated the per capita probability to be 0.035 and the expected number of secondary cases to be 2205, which is well in agreement with the 2180 reported cases in the 2012 outbreak.We also validated our models against the well-documented dengue outbreak in Madeira with 2180 reported dengue cases. We estimated the risk of autochthonous transmission for the Madeira Islands where Aedes albopictus) and the Madeira Islands (Aedes aegypti). The parameters used for the simulations can be found in Table\u00a0Aedes albopictus) and S3 (Aedes aegypti) of the supplementary material.Table\u00a027, the extent of such a threat has not been quantified. This is the first attempt to quantify the actual number of dengue viremic air passengers from dengue endemic countries into Europe -an important parameter that determines the risk calculations of subsequent local dengue virus outbreaks. As not only clinically apparent viremic cases transmit dengue viruses28, we also included asymptomatic infections into our calculations at a published ratio of 4:1. Our estimated number of importations tally with the reported importations of dengue in Europe, although the numbers are slightly lower as we are focusing only on air passengers traveling to Europe still in the viremic phase (e.g. persons traveling at a time of viremia for the short period within 5 days after onset of symptoms)29. The majority of dengue cases reported in returning travelers to Europe are not viremic anymore at the time of arrival20.Although reported autochthonous cases in Europe in the recent past highlight the threat of dengue to EuropeAedes albopictus was recently introduced to Kent, United Kingdom but its distribution is very limited and not yet established30. In Germany, many more Aedes albopictus have been reported and established populations have been recorded in parts of Southern Germany31. However, risk for local transmission is limited under current climatic conditions. Both France and Italy receive a significant number of modeled dengue infected air passengers and both countries have significant presence of Aedes albopictus- indeed France experienced autochthonous dengue transmission in the year 2010 in Nice in the Southern part of its country7 and Italy experienced a chikungunya outbreak in 2007 in the Northern part of Italy32. We were limited to passenger flight information at a country level, so we restricted our analysis of the risk of onward transmission of dengue to Italy, a country where Aedes albopictus is distributed across the majority of the country.The highest number of dengue virus importations via air travelers were modeled to occur in Germany, France and the United Kingdom. 4. We translated these findings into per capita probability in Italy. The per capita probability was as low as 0.000000167, and we estimated 10 secondary infections which include around 2 symptomatic cases. For Madeira, we modeled the probability of an imported case to result in secondary cases to be 0.035 and the expected number of secondary cases to be 2205. The difference in the mosquitoes\u2019 species between Italy and Madeira is crucial in appreciating the risk difference between the two countries/areas. As Aedes albopictus is a much less competent mosquito for dengue transmission than Aedes aegypti, a large number of imported infections to Italy resulted in a small number of autochthonous cases, whereas a very small number of imported infections to Madeira resulted in a major outbreak.For Italy, we modeled the probability for an imported case to result in secondary cases by taking into account the monthly vectorial capacity where transmission is most suitable 33. Another limitation is the timing of imports. Here we were restricted to two countries as reference points for fitting monthly incidence curves. However, the highest uncertainties in our model were related to mosquito densities in Europe. There are no published data on the Aede smosquito abundance in relation to the host population which is a crucial parameter for modelling, nor is there sufficient information on biting rates, extrinsic incubation period, mosquito longevity under temperate climate conditions except for some pivotal temperature driven data derived from the work by Louis Lambrechts\u2019 team at the Institute Pasteur35.Our models have several limitations: From an empirical point of view, there is a need for accurate data on dengue incidence in the origin countries. We relied on notified dengue cases to WHO, which is probably a significant underestimate as recent modeling estimates showed at least 10 to 100 fold higher numbersAedes mosquitoes exist. This paper is hence intended mainly as a novel methodological proposal to estimate the risk of dengue introductions into Europe and subsequent probability and numbers of secondary  infections.We selected the year 2012 for our model parameters (dengue incidence in originating countries and air travel volume) in order to validate our model against the actual dengue outbreak that took place in Madeira in that year. Validating our model against the reported dengue outbreak in Madeira in 2012, we found that our model fits very well with the true number of cases reported: our model results estimated 2205 autochthonous infectious, which is well in agreement with the 2180 reported cases in the 2012 outbreak. Therefore, despite its limitations, the model proposed here can be very useful in the understanding of the risk of dengue virus importation into still unaffected areas, and calculate the probability of secondary cases in those areas where susceptible In conclusion, our estimates highlight that the risk is overall low which probably represents a good approximation of reality.Supplementary Material"}
+{"text": "Dendritic, antigen-presenting cells (DCs) determine not only whether lymphocytes produce different types of immune response but also tissue-homing profiles of lymphocytes they stimulate. For example, in health, mucosal DC stimulate T cells focused to home to the mucosa; DC/T-cell circuitry thus targets immune responses to specific tissue locations. Therapies being introduced for inflammatory bowel disease (IBD) include antibodies to gut-homing molecules such as \u03b14\u03b27 (Vedolizumab) used ostensibly to block gut-homing lymphocytes. However, such lymphocytes are dependent on the tissue specificity of DC that stimulated them.In health, blood DCs have the potential to home to multiple tissues including gut (\u03b14\u03b27+) and skin (CLA+). DCs have become gut-specific within the intestinal microenvironment stimulated partially by local retinoid to express \u03b14\u03b27  and/or CCR9  in the absence of skin-specific indicators. They spread veiled extensions, sample their environment, acquire/process antigens, produce cytokines and initiate innate immunity. Myeloid DC also traffic to draining lymph nodes where compartmentalization of adaptive immune responses is determined by DCs from the site of antigen exposure which dictate the homing profiles of lymphocytes they stimulate. In IBD, site and activity of disease are reflected in changes in homing/activation of gut DCs and T-cells they stimulate and also, in greater gut specificity and activation of blood DC. Homing potential of DC can be modulated toward mucosa or skin by vitamins A and D, respectively. Infliximab or interleukin-6 can divert homing profiles toward skin, perhaps predisposing to skin involvement in IBD. Probiotic bacteria or their products can also change homing profiles of gut DC toward skin homing and away from gut.In conclusion, development of gut focused inflammation and its treatment relies on changes in DC tissue specificity; therefore, removal or diversion of gut-homing DC as well as T-cells is likely to be critical in prevention of gut-focused inflammation in IBD. Dendritic cells (DCs) determine the tissue specificity of the lymphocytes they stimulate . Immune Chlamydia trachomatis. Elementary bodies of C. trachomatis bind to collagen, an association believed to underlie the development of arthritis occurring in the collagen rich environment of the joint. There was a prevailing view that cross-reactive stimulation of immunity to collagen occurred because of its association with chlamydia and caused accumulation and/or stimulation of collagen-specific T-cells within the joint. However, DCs in the joints, but not systemically, carried the chlamydial antigens in post-chlamydial arthritis. By contrast, T-cells present in the joint merely reflected the systemic increase in chlamydia-specific T-cells that followed the genital tract infection [An indication of acquired tissue specificity of human DC was first determined in early work on reactive arthritis following genital tract infection with nfection . In exacnfection ,4. This nfection  has beenThe knowledge of specific T-cell homing markers and their ligands ,6 was inSuch concepts of responsiveness and non-responsiveness in different circuits of DCs and T-cells, and the involvement of innate and adaptive arms of immune response, may inform understanding of the processes underlying the role of antigens within the gut such as food and the gut microbiota that could either promote or discourage inflammation in the gut. Direct evidence of the critical importance of site of sensitization in relation to the site of inflammation induced by gut antigens in humans in IBD was obtained from early studies of sensitivity to food antigens in Crohn's disease. In Crohn's disease, there is increased permeability of the gut mucosa predisposing to the breaching of this barrier by food antigens as well as by the microbiota. The induction of systemic sensitivity to such antigens was, therefore, studied in patients and controls by examining the development of recall or memory T-cell responses to a selection of antigens that are met in the gut. Antigens studied were extracts of cow's milk, cereals, cabbage group, citrus fruits, peanut group, yeast and bacteria. In Crohn's disease but not controls, the reactivity of peripheral lymphocytes to these antigenic extracts, particularly multiple sensitivities, was common  fig. 1b1b. DespiThe finding that the tissue source of the DC determined the migration pathways of lymphocytes they stimulate indicated that the DC themselves must have tissue specificity. The majority of blood T-cells, in health, express \u03b14\u03b27 in the absence of skin homing markers indicating that they have been \u2018educated\u2019 in the mucosa and have the capacity to home to the ligand mucosal vascular addressin cell-adhesion molecule 1 expressed by post-capillary endothelial cells in intestinal lamina propria. A smaller percentage of T-cells express skin homing markers without gut-homing potential. By contrast, around 80% of blood DCs in health have both gut and skin homing potential as indicated from expression of both \u03b14\u03b27 and CLA, respectively . Since tOnce the DCs reach the skin or gut, local environmental factors promote their tissue specificity; gut DC remain largely \u03b14\u03b27 positive but lose skin homing receptor for E-selection, CLA, although some colonic but not ileal DC may maintain or acquire some skin-homing potential ,17. SkinThere is increasing information describing the subsets and properties of human intestinal DC ,23,24,25A surprising finding is that blood DCs can act as a window on site and activity of disease in IBD; both the DCs and the T-cells in the blood in IBD can reflect the site of the disease within the gut and its activity. The DCs give a clearer picture of ongoing activity since expression of markers on activated DCs able to stimulate T-cells is not confounded by the persistence of homing markers such as \u03b14\u03b27 on pre-existing, long-lived, memory T-cells. The site of disease and its activity can be identified from changes in the migration markers and activation markers on blood DC. Thus, increases in CCR9 on blood DC concomitant with loss of skin homing potential is evident in many adult Crohn's patients and in a smaller proportion of pediatric Crohn's patients ,33; thisIn Crohn's disease with concomitant erythema nodosum, changes in the presence and acquisition of a skin homing profile in \u03b3\u03b4 T-cells was observed and resolved following successful treatment with steroids . Thus, dIn some patients, effective therapy is achieved by blocking gut-homing T-cells using natalizumab (anti-\u03b14) and particularly using vedolizumab (anti-\u03b14\u03b27); the latter avoids the potential dangers of interfering with brain tropic \u03b14\u03b21-positive cells, a possible risk with natalizumab . Such suLactobacillus casei was effective in changing the DC stimulatory potential of T-cells by promoting a homeostatic profile and making DC once again double positive for both skin and gut-homing markers; however, it failed to effect change in imprinting of CCR4 or of the gut-homing indicators CCR9 or \u03b27 [Lactobacillus plantarum which may have therapeutic value in IBD in part via its effects on homing profiles of DC and lymphocytes they stimulate. STp is a serine-threonine rich peptide which lacks cleavage sites and, therefore, resists breakdown by gut proteases. STp has anti-inflammatory effects on DC and reverses changes identified in gut DC studied ex vivo from ulcerative colitis patients. However, STp also changes the homing profiles of DC; through effects on the DC, STp instructs T-cells not only to reduce gut specificity but also increases their potential for migrating to the skin [However, other biological agents also alter the migration potential of DC and the lymphocytes they stimulate. The probiotic R9 or \u03b27 . An exciR9 or \u03b27 . We havethe skin ,37. Suchthe skin .Enteral nutrition in pediatric Crohn's is an effective treatment for a high proportion of patients and, in parallel with the therapeutic effects, reverses many of the defects and changes in blood DC in patients with colonic but not ileal disease. The latter non-responsive group with ileal disease can be identified by high CCR9 on blood DC. The enteral nutrition, when successful, normalizes the tissue-homing profile of the blood DC so that they once again show little sign of activation and have both gut and skin homing potential commensurate with that seen in health.The evidence for DC/T-cell immune circuits is now overwhelming and indicates that the normal immune system can be neatly compartmentalized into tissue-specific cell circuits; such a circuit will provide tissue-specific response to local foreign antigens or lack of inflammatory response to self-antigens and, probably, to self-associated, antigens for that tissue. Evidence for changes in these circuits is increasingly found in IBD which results in greater focus of immune activity toward the gut. Furthermore, reflections of this change of focus and activity can often be identified systemically as well as in the gut itself; indeed, the DC in the blood can provide a window suggesting both the location within the gut of ongoing immune activity and the level of activation which are indicated by DC homing markers and maturation/activation status respectively. The picture may be reflected in the circulating memory lymphocytes but is more difficult to gauge due to long-term lymphocyte survival and memory of past history within lymphocyte populations. Despite the growing evidence for the importance of the DC/lymphocyte axis in the development and control of immune responses, including those in the gut, therapeutic control of immune activity is still predicated on diverting or removing effector lymphocytes systemically or from migration to the gut. Tackling more directly the instigators of the immune localization, the DC, may provide a further step forward for treatment. Despite the focus and importance of effector T-cells, it is becoming evident that effectiveness of many treatments used for IBD may stem from changes in the DC which reduce the focus of immune activity from the gut and, generally, promote DC homing potential toward the skin. Further detailed analysis of the underlying and multiple mechanisms controlling the DC/lymphocyte circuitry will lead to more efficient methods of controlling immune activity; if localized tissue-specific, immune manipulation can be achieved it should remove the need for blanket immunosuppressive therapies and facilitate the development of tissue-specific control of immunity in IBD. When we can upgrade our cell-based postal/zip code system from general area codes to satnav precision, individualized tissue-specific treatments should become achievable.Stella Knight is a joint holder of a patent for the use of the bacterial peptide STp and is funded by the Biotechnology and Biological Sciences Research Council UK BB/J004529/1."}
+{"text": "Per1, Per2, Per3, Rev-erb\u03b1 and Dbp) expression level decreased and Bmal1 expression level increased. In both rat dams and their offspring, after continuous exposure to unfiltered air, we observed significant histologic evidence for both perivascular and peribronchial inflammation, increased tissue and systemic oxidative stress in the lungs. Our results suggest that chronic exposure to particulate matter can induce alterations of clock genes expression, which could be another important pathway for explaining the feedbacks of ambient particle exposure in addition to oxidative stress and inflammation.The symptoms of asthma, breathlessness, insomnia, etc. all have relevance to pulmonary rhythmic disturbances. Epidemiology and toxicology studies have demonstrated that exposure to ambient air particles can result in pulmonary dysfunction. However, there are no data directly supporting a link between air pollution and circadian rhythm disorder. In the present study, we found that breathing highly polluted air resulted in changes of the molecular clock genes expression in lung by transcriptome profiling analyses in a rodent model. Compared to those exposed to filtered air, in both pregnant and offspring rats in the unfiltered group, key clock genes ( In human and other mammals, circadian rhythms are intrinsic oscillations with a period near 24 h, which are synchronized by some environmental cues, such as light and food ,2,3. TheIt is well known that exposure to ambient air particulate matters (PM) can induce oxidative stress and excessive pulmonary inflammation . ChronicHence, we hypothesized that chronic exposure to PM can have an effect on the circadian functions in the lungs through regulating the circadian rhythm related genes by a certain pathway/mechanism. To address this hypothesis, we analyzed the expression levels of clock genes in the lung tissue of rats and tried to find an association between the circadian functions and the immune system.All animal experiments were conducted in compliance with the guidelines of ethical animal research. The study protocol was approved by the Medicine Animal Care and Use Committee at Peking University Health Science Center before commencement of experiments .3 in volume (1.2 \u00d7 0.8 \u00d7 1.2 m), were used in the present study. The chambers are divided into two layers. Each chamber could house a maximum of nine rat cages . Only two rats were housed in each cage . This ensures no overcrowding and that they can move freely in the cages. These chambers were placed side by side for whole body inhalation exposure of rats in a clean-level air-conditioned room. The ambient air is ducted to the chambers though the pipes directly from outside  by a suction blower and delivered to the chambers by the inlet duct on the top of the chambers. The only difference between the two chambers was the presence or absence of a high-efficiency particulate air (HEPA) filter placed in the inlet duct. The room was located in a heavy traffic area in Haidian District in Beijing. Particulate matter concentrations were measured using two instrument sets comprised of a single particle soot photometer and scanning mobility particle sizer and aerodynamic particle size, one for the chamber airstream and the other for ambient atmosphere. The removal efficiency of the HEPA filter in the chamber was 98.99% \u00b1 0.86% for particles larger than 2.5 mm in diameter and 70.61% \u00b1 19.34% for PM2.5. PM2.5 concentrations inside the chamber equipped with the HEPA filter were estimated using outdoor PM2.5 concentrations and the filter removal efficiency for PM2.5. The temperature inside these chambers was controlled at 24 \u00b1 1 \u00b0C.As in our previous paper , two ide2.5 concentrations were 73.5 \u00b1 61.3 \u03bcg/m3 in the unfiltered chamber and 19.8 \u00b1 9.0 \u03bcg/m3 in the filtered chamber. We further had the pups of rat dams prenatally exposed to unfiltered air stay in the unfiltered chamber, and the male pups prenatally exposed to filtered air stayed in the filtered chamber continuously until they were analyzed at 8 weeks of age.We started our experiments with pregnant Sprague Dawley rats. All the rats were kept in an animal care facility before the experiments commenced. We conducted the first set of experiments using 30 pregnant Sprague Dawley rats that were 12 weeks old. On gestation day 4, rats were randomized into two groups: 18 were placed in the unfiltered chamber, and the remaining 12 in the filtered chamber. Rats lived in these chambers naturally under a 12 h light/12 h dark cycle and were fed a normal chow diet. On gestation days 5\u201318, , PM2.5 concentrations were 64.6 \u00b1 72.7 \u03bcg/m3 and 16.7 \u00b1 9.6 \u03bcg/m3 in the unfiltered and the filtered chamber, respectively. After PM2.5 exposure, rats were sacrificed at ZT4 (where ZT0 is defined as the lights-on time and ZT12 as the lights-off time), and the blood and lungs were collected.To confirm some of the previous findings, we conducted a 2nd set of experiments using 16 pregnant SD rats at 12 weeks of age (10 in the unfiltered air group and six in the filtered air group). The experiments started at gestation day 1 and lasted for 19 days. Pups of these rats were not further studied. The exposure protocol was the same as the first set of experiment. The total exposure period was 19 days (12\u201331 March 2010), PMThe completely cut-down left lung were fixed in 10% formalin and embedded in paraffin for making hematoxylin-eosin stained (HE) slides, morphometric analysis and further immunohistochemistry using slides. The HE slides were analyzed by optical microscope with a digital camera. The right lung tissues were frozen in liquid nitrogen immediately for further RNA extraction. Other organs were collected, weighted and cryopreserved for further analysis.After euthanasia, the blood was collected in EDTA anticoagulant tubes , centrifuged at 3000 rpm for 10 min at 4 \u00b0C. The EDTA-plasma was separated and collected for malondialdehyde (MDA) and glutathione (GSH) assay kit analyses. Thirty g each of lung tissue were ground in 1 mL PBS on ice, centrifuged at 5000 rpm for 15 min. The clarifying middle layer was carefully aspirated for MDA and 8-isoprostane assay kit analyses. Plasma and lung oxidative stress biomarkers were measured using enzymatic colorimetric assays.2 ratio of data from the unfiltered air group versus the data from the filtered air group (log2 Ratio (unfiltered/filtered)). We sorted the lungs into four groups for RNA sequencing, the first time unfiltered mother  vs. first time filtered mother, the first unfiltered mother  vs. first time filtered mother, the second time unfiltered mother  vs. second time filtered mother, the unfiltered offspring (8 weeks) vs. filtered offspring (8 weeks).Second-generation RNA sequencing (RNA-Seq) was used to analyze the genome-wide gene expression profiling in the lungs. RNA-Seq was analyzed by a commercial company . We compared the transcriptomes in the lung of filtered group and unfiltered group by second-generation RNA sequencing. The gene expression level across all samples was evaluated by the bio-conductor package EDGER4. And the weakly expressed genes were filtered out when they had <1 read per million. Gene expression results are presented as the logThe real time PCR (qPCR) was used for verifying the significantly up or down regulated genes which were given out by the RNA-Seq analysis as described above. We tested all 9 rhythm genes by qPCR (t-test (two tailed) with equal or unequal variance depending on F-test results. p-value of less than 0.05 was considered statistically significant.SAS 9.1.3 for windows  was used to perform data analysis. The data were expressed as mean \u00b1 standard deviation (mean \u00b1 SD) in tabulate format. Pairwise comparison was conducted using Student\u2019s Both the lung sections of maternal and 8-week-old unfiltered air rats exhibited significant inflammation compared with the filtered air group. In pregnant rats, light microscopic examination in the unfiltered air group showed thickened alveolar septum, inflammatory cells in alveolar spaces and inflammation in the bronchus in comparison to filtered air exposed rats. The histologic sections at higher magnification show thickened alveolar septa and the presence of inflammatory cells in the airways of the unfiltered air groups compared to the lung tissues of rats in the filtered air chamber. Similar to that for the rat dams, lung histology showed both peribronchial and perivascular inflammation in these rats\u2019 offspring .As shown in Accordingly, there were significant reductions of GSH and significant increases of MDA in plasma in the maternal and 8-week-old rats in the unfiltered air group. Furthermore, we observed significantly higher concentrations of MDA and 8-isoprostane in lung tissues in the unfiltered air group compared to the filtered air group of 8-week-old pups.We overlapped all the RNA-Seq data of the four groups of lungs and picked out the same significantly expressed genes . By usinPer1, Per2, Per3 decreased and Cry1 and Bmal1 increased significantly after PM exposure in both the gestational mothers and their 8-week-old offspring, but the Cry2 expression level was decreased in the gestational mothers significantly, while it was only changed a little without meeting the requirement of |log2 ratio \u2265 0.5|  and cryptochrome (Cry1-2). The Bmal1/Clock transcriptional activator complex was upstream of the Period (Per1-3) and Cryptochrome (Cry1-2) genes, while Per1-3 and Cry1-2 repress Bmal1/Clock to form an autoregulatory transcriptional feedback loop [Per1, Per2, Per3 decreased and Bmal1 increased after PM exposure in both the gestational mother and the 8-week-old offspring. It is probably because that decreased Per1-3 resulted in the reduced repression of Bmal1. Meanwhile, the increased expression level of Bmal1 activated the expression of Cry1 in gestational mother. Moreover, the Bmal1/Clock complex can activate the Rev-erb\u03b1 expression and the over-expression of Rev-erb\u03b1 also represses Bmal1 transcription [Rev-erb\u03b1 promoted the increase of Bmal1. In addition, the Rora as a component of the mammalian circadian, was involved in normal Bmal1 expression [Rora and Casein kinase I in our RNA-seq data. Regarding the different expression of clock genes in the rat dams and pups, we can only make the ambiguous inference that the prenatal exposure to PM maybe not have an effect on the offspring, but this should be confirmed in the future.The mechanisms of the main circadian clock involve intracellular auto-regulatory transcriptional loops of specific genes called clock genes (CCGs), including and Cry2 . Rev-erb rhythms . General rhythms . There aack loop ,28. In tcription . In bothpression . Casein pression , but thePer1, Per2 and Bmal1 [ccl1/mcp-1 and cxcl1/kc) and cytokine release (CCL1/MCP-1and CXCL1/KC) [Circadian genes have also been determined to have a relationship with immunity and inflammation. An increased neutrophil response is associated with many inflammatory diseases presented in significant circadian rhythm cycle ,33. In tnd Bmal1 ,35,36. EXCL1/KC) . MoreoveXCL1/KC) . The nucXCL1/KC) . REV-ERBXCL1/KC) . In the In conclusion, our study showed that chronic exposure to particulate matter caused alterations of clock genes expression in rats\u2019 lung tissue, as well as increased lung inflammation. Previous studies demonstrated that the molecular clock genes could regulate the pulmonary inflammation and injurious responses. Our results highlighted the association between PM exposure and clock genes, which implicated that the alteration of clock genes expression may have a great effect on PM-mediated lung inflammation. It is well known that exposure to atmospheric particulates can cause cardiovascular disease. A better understanding of the relationship between clock genes and pulmonary inflammation caused by PM can help us to find ways for effective prevention and treatment. In the future, much more precise experiments need to be conducted, for example, what is the time point when circadian rhythm genes begin to change, whether the changes in circadian genes expression occur earlier and if the trend will be maintained, etc."}
+{"text": "Undernutrition is a serious health problem and highly prevalent in developing countries. There is no as such confirmatory test to measure undernutrition. The objective of the present study is to determine a new Composite Score using anthropometric measurements. Composite Score was then compared with other methods like body mass index (BMI) and mid-upper arm circumference (MUAC) classification, to test the significance of the method.Anthropometric data were collected from 780 adult Oraon  labourers of Alipurduar district of West Bengal, India, following standard instruments, and protocols. Nutritional status of the study participants were assessed by conventional methods, BMI and MUAC. Confirmatory factor analysis was carried out to reduce 12 anthropometric variables into a single Composite Score (C) and classification of nutritional status was done on the basis of the score. Furthermore, all the methods  were compared and discriminant function analysis was adopted to find out the percentage of correctly classified individuals by each of the three methods.The frequency of undernutrition was 45.9% according to BMI category, 56.7% according to MUAC category and 51.8% according to newly computed Composite Score. Further analysis showed that Composite Score has a higher strength of correct classification (98.7%), compared to BMI (95.9%) and MUAC (96.2%).Therefore, anthropometric measurements can be used to identify nutritional status in the population more correctly by calculating Composite Score of the measurements and it is a non-invasive and relatively correct way of identification. Human body needs a proper nutrition through well balanced diet to fulfill body requirements and to maintain basic body physiology. Improper nutrition leads to the consumption of excess calorie (over-nutrition) or insufficient supply of one or more essential nutrients (under-nutrition). Over-nutrition is a threat that increases body weight and causes several non-communicable diseases. On the other, undernutrition, caused due to the insufficient intake of energy and nutrients, is a serious health problem for the economically backward, developing countries like India . It causThe main causes of undernutrition can be broadly classified as biological, behavioral and sociological factors , 3. The Thus, undernutrition is a condition of poor nutritional status resulting from reduced food intake or impaired metabolism and evaluation of nutritional status is necessary to determine the severity of undernutrition. As there is no objective test to measure nutritional status, therefore numerous screening methods have been developed to determine the nutritional status of individual: (i) assessing clinical signs and symptoms, (ii) biochemical indicators (iii) dietary survey and (iv) anthropometric measurements. Assessment of clinical signs and symptoms need proper knowledge for evaluation, whereas biochemical indicators are relatively expensive and time consuming to perform in community level. On the other, dietary survey can give an idea of daily energy intake but there may have chances of misreporting and also need food consumption data of several days to obtain the estimate of usual diet.Anthropometry has a long tradition of assessing nutritional and health status of adults as this is an inexpensive, non-invasive method that provides detailed information on different components of body structure, especially muscular and fat components , 21. MorBMI (Body mass index) is generally considered as a good indicator and used for the assessment of chronic energy deficiency of adults, especially in developing countries \u201324. It iHowever, there are some difficulties associated with the sole use of BMI, for example the ratio of sitting height to standing height or cormic index can influence BMI . Cormic On the other, MUAC (Mid-upper arm circumference) is another important indicator for simple screening of adult nutritional status, specifically in developing countries , 32. TheIn view of the above, a more accurate and population specific method is necessary to assess the nutritional status of the population using anthropometric measurements. Present article documents the development of the method with specific statistical tools.Anthropometric data were collected as part of an ongoing bio-medical project on Oraon labourers of Alipurduar district of West Bengal, India. Data includes 780  adults from two occupational subgroups, one engaged in agriculture and other in tea garden, both having similar socio-economic status and living condition. The study was approved by the Ethical Committee of Indian Statistical Institute, Kolkata and was performed with the prior written consent from the participants. No statistical sampling was followed, because any kind of selection within the population would have raised suspicion in the minds of the people studied, regarding the purpose of the study. However, the participants were chosen without any conscious bias; actually the participants who could be persuaded to participate in the study and volunteered for participation in the study were included in the sample. Studies on the nutritional status of the indigenous population have an important significance in context of health planning. Thus, the present anthropometric measurements were collected from one of such indigenous group, Oraon in the Dooars foothill region of West Bengal.Anthropometric measurements were obtained following standard protocol and instrument . These wNutritional status of the study population was assessed in terms of the two conventional methods i.e. BMI (Body mass index)  and MUAC(BMI) has been calculated using the formula: The criteria used for the classification of the nutritional status have been described in Table As the mean of BMI and MUAC of the studied sample were 18.99 and 22.20 cm, so for the analytical purpose, nutritional status was classified into two categories; (a) Chronic energy deficiency or undernutrition and (b) normal.Initially, sixteen anthropometric measurements, which have previously been used for nutritional assessment, were used in the analyses. Among them twelve variables were found significant correlation with height, weight and MUAC. These were used for further statistical testing. Furthermore, by elimination method it was verified that the selected 12 variables can describe the highest variance of the newly computed variable. Thus these twelve variables created a single score variable that helped significantly for the assessment of nutritional status.Descriptive statistics of all the variables were calculated. Then First Order Confirmatory Factor Analysis (CFA) and Discriminant Function Analysis were performed. All of the statistical analysis was carried out using PASW, version 18.0  and STATA, version 13.0 .Confirmatory factor analysis (CFA) helps to test the hypothesis that a relationship between observed variables and their underlying latent construct exists. This analysis is primarily a theory driven statistical data reduction technique used to explain covariance among different observed random variables and thereby reducing large number of variables to parsimonious and meaningful groups of underlying unobserved variables named factors . The anax is a 12\u2009\u00d7\u20091 matrix of coefficients relating each variable to its latent factor, \u03be is the latent variable and \u03b5 is the measurement errors, which is a 12\u2009\u00d7\u20091 matrix.Where X is a column vector of 12 standardized anthropometric variables, \u039bThus latent variable was calculated from each linear equation for each individual and was termed as Composite Score. Later Composite Score was used as an alternative variable instead of the 12 anthropometric variables (observed variables) and a new classification was developed on the basis of this score. Negative Composite Scores were considered as undernutrition and positive Scores as normal nutritional status.Discriminant function analysis is primarily a multivariate test to observe the differences between groups. This is the reverse of MANOVA, where the independent variables are the continuous predictors and dependent variables are the groups .The analysis can be split into 2-steps- (a) testing significance of a set of discriminant functions, and, (b) classification. In the study, second step of the analysis was used for the classification of nutritional status in view of the equations created in the analysis. Computationally a canonical correlation analysis was performed and that determined the successive functions and canonical roots. Classification was then possible from the canonical functions. Individuals were classified in the groups in which they had the highest classification scores . This anTable Table Table Table For Composite ScoreEquations (1) and (2) have to be calculated for each case to get the undernutrition or normal value and the case will be classified for which computed value will be higher. For example, if eq. (2) gives the higher value for a particular case, then the case will be classified as normal.Table The present study tries to find out the most precise way of assessing nutritional status. Anthropometry has long been used as indicator of nutritional status because it is non-invasive and less expensive. Anthropometric measurements help in calculating both BMI and MUAC, which provide a simple and convenient value for assessing nutritional status. Nutritional assessments in rural population usually rely on BMI and MUAC, as it does not require much instruments, time and efficiency; but have independent limitations. For example, individuals with ectomorphic somatotype may be misclassified as undernourished with BMI classification. Again, human body has bilateral asymmetry; therefore, taking MUAC measurement on one side may provide erroneous assessment. On the other, variation of MUAC is very high depending on the physical activity and food intake of the individual. Moreover, both BMI and MUAC do not have population specific cut-off values. Therefore, there are every chances of misclassifying nutritional status of individuals.The effort of the present study was to develop a better and rigorous tool that can easily identify nutritional status. A number of anthropometric measurements were used to construct a new method for assessing nutritional status. All the anthropometric traits were tested for its association to height, weight and MUAC, however, found 12 anthropometric traits that have precise and good association with height, weight and MUAC. Those 12 anthropometric traits represented into one Composite Score by confirmatory factor analysis (Fig. A separate equation for undernutrition and normal was obtained from discriminant function analysis Table  for BMI,The assessment of nutritional status by anthropometry was previously done primarily on the basis of BMI or MUAC , 20, 41.Moreover, the cut-offs of all the nutritional assessment tools with anthropometric traits have same values, irrespective of ethnicity. Adult individuals of different ethnic backgrounds display differences in body shape and cormic index. Studies found a higher percentage of body fat at lower BMI in case of Asians . So, it But in calculating the Composite Score, the values of anthropometric data are being used of the study population, so the classification category is more specific for that study group. Finally, it is believed that the newly developed method will be useful in identifying nutritional status of the individuals of a particular study group more correctly.The present study tried to assess the nutritional status of a particular ethnic group with selected anthropometric measurements by computing Composite Score. There are few limitations also in calculating such Composite Score; it needs some statistical computation which may not always be feasible in the field situation. Moreover, the method is applicable to identify only the under-nourished and normal individuals, not the obese individuals. In the present data, as there were no obese individuals, so this method has been applied successfully. Further study in different population with more sample size is needed to classify the nutritional status for three categories following the present Composite Score method.The adverse effect of undernutrition on health, development and economic productivity is well established. It needs proper and accurate identification to get the idea of severity to address the issue. The newly computed Composite Score can predict the nutritional status more accurately than BMI and MUAC. Moreover, it will give population based cut-off values which will lower the probability of mis-classification. Lastly, it is expected that scientific ventures will continue to develop such scores with the data of other population groups and it will provide a comprehensive understanding over this newly developed method."}
+{"text": "The comparison of estimated step lengths to reference measurements showed no significant difference (p > 0.05) and acceptable agreement . Step length estimations further improved  after smoothing the original estimated step lengths with a third order polynomial function (R2 = 0.94 \u00b1 0.04). In conclusion, during maximal sprint acceleration, acceptable estimates of stride and step length were obtained from IMU-derived step times and 30-m (split) and 60-m sprint times. During sprint acceleration, step length, step rate, ground contact, and airtime are key variables for coaches to guide the training process and technical development of their athletes. In the field, three of these variables are easily obtained with inertial measurement units (IMUs), but, unfortunately, valid estimates of step length with IMUs currently are limited to low speeds (<50% max). A simple method is proposed here to derive step length during maximal sprint acceleration, using IMUs on both feet and two timing gates only. Mono-exponential velocity-time functions are fitted to the 30-m (split) and 60-m times, which in combination with IMU-derived step durations yield estimates of step length. To validate this approach, sixteen well-trained athletes with IMUs on the insteps of both feet executed two 60-m maximal sprints, starting from a three-point position. As a reference, step lengths were determined from video data. The reference step lengths combined with IMU-derived step durations yielded a time series of step velocity that confirmed the appropriateness of a mono-exponential increase of step velocity (R In many sports, sprint acceleration is essential for good performance. Sprint speed is the product of step rate and step length . TherefoOptoelectronic systems are the gold standard for motion analysis in sports and rehabilitation ,14 and cThe present study focuses on step length during maximal sprint running. In a recent study, step length (and joint kinematics) during sprinting was recorded with an optoelectronic set up consisting of 21 cameras . Fortuna\u22121) and constant velocities ). Predicted 60-m time (7.59 \u00b1 0.49 s) of the feet also closely matched the corrected 60-m gate time  \u22120.0025 s, LOA [\u22120.0056 0.00058]. Thus, fitting a mono-exponential velocity-time function solely based upon the measured 30-m (split) and 60-m times (\u22120.045 s) introduced systematic errors of only a few milliseconds. Thus, during maximal sprint acceleration, mono-exponential velocity-time functions constructed with the 30-m (split) and 60-m (finish) times as input accurately describe the increase of step velocity during individual sprint accelerations.The predicted 30-m split time of the feet obtained with the timing gate based exponential velocity-time functions was 4.28 \u00b1 0.25 s. It closely matched the corrected gate time at 30-m  and similar to the measured video steps and strides  and similar to measured reference step-length . Based on the interpolation of these steps, the feet, on average, crossed the 30-m and 60-m lines 0.045 s earlier than the measured gate times. Thus, the tip of one of the feet crossed the 30-m and 60-m lines slightly ahead of the chest, while the chest usually contacted the mechanical timing rods. At the 30-m line, the average velocity was about 8.5 m\u00b7s\u22121 for the current athletes, which means that the tip of one of the feet was ~0.38 m (=8.5 m\u00b7s\u22121 \u00d7 0.045 s) ahead of the chest at ground contact. This distance is in line with the touchdown distance (about 0.32 m) reported for physical education students running at 8.04 m\u00b7s\u22121 [Horizontal velocity increases mono-exponentially during sprint acceleration  [\u22121 in their analysis and showed that the error of stride length estimations increased above 5 m\u00b7s\u22121 for the different algorithms that they evaluated [\u22121 already after two or three steps, and attained maximal velocities ranging from 8.0 to 10.5 m\u00b7s\u22121. Despite the inclusion of the acceleration phase and these high speeds, the errors in step length estimation were similar compared to the studies of Brahms et al. [To the best of our knowledge, our estimations of step length are considerably more accurate than previous predictions at high running speeds. Deriving accurate estimates of step length during maximal sprint acceleration from the IMU-signals using sensor fusion techniques ,24 is no\u22121) . Zrenner\u22121)  and the valuated . The aths et al.  and Zrens et al. . TherefoThe runners\u2019 chest did not always contact first with the rods of our mechanical timing gates. Occasionally the arm or even the hand contacted the rod, which introduced some variation in the sprint times, but this also is the case with single beam optical timing gates.The manual digitization of videos introduced a random error of a few cm in our reference step lengths. Unfortunately, we did not have 40 m of a more accurate optical measurement system at our disposal that others used to obtain step length ,7.Our unsmoothed estimates of step length were subject to some error  sprints are commonly used to evaluate sprint performance in many sports. In principle, the velocity-time function of sprinters can also be derived with a series of more than two  timing gates or directly with radar or laser equipment. Although the latter devices measure instantaneous velocity rather well, the data need filtering and the accuracy depends on properly aiming these devices at the sprinters lower back . In pracThe athletes in the present study did not use starting blocks, because block-starts require a technique that was not mastered to the same extent by all the athletes. Football, rugby, and soccer players also do not use starting blocks, which facilitates the generalization of the present results to other sports. An additional strength of the present study is that the performance of the present athletes covered quite a broad range, although it unfortunately only included three women. Thus, our results are representative of athletes with different sprint capacities. The foregoing considerations suggest that the proposed method of step length estimation most likely applies to sportsmen and women in many other sports.Speed is the product of step rate and step length. IMUs attached to the shoes of athletes provide valuable information about ground contact time, airtime and step rate, which together with step length are key performance determining variables in sprinting ,3,5,6,7.In conclusion, 30-m and 60-m times in combination with IMU-derived step timing suffice to obtain good estimates of stride and step length during maximal sprint acceleration. Step length estimates complement other important sprint performance related variables  that can be accurately measured with IMUs and are used by coaches to monitor the training process and technical development of their athletes."}
+{"text": "Pseudoaneurysm is a vascular injury typically caused by rupture of arteries with extravasation of blood. The involvement of this entity in facial arteries after firearm aggression is extremely rare, and they need treatment as early as possible, thus avoiding irreversible damage to patients.A 40-year-old male victim of gunshot attack with an entrance orifice in the right posterior cervical region with ascending trajectory, lodging in the ipsilateral zygomatic-orbitary complex, which was submitted to removal of the bone fragments and the bullet. In the intraoperative period, the patient developed profuse hemorrhage and, after complementary examinations, he was diagnosed with pseudoaneurysm of the internal maxillary artery, which was treated by selective endovascular embolization.The patient was hemodynamically stable, with no complaints and was discharged after 48 hours, without postoperative bleeding recurrences. He had no more complications after 8 months of follow-up.The main forms of treatment and diagnosis of vascular lesions are reviewed, and embolization is demonstrated as a technically safe procedure with few complications. Key words:Gunshot wound, pseudoaneurysm, maxillary artery, therapeutic embolization. Pseudoaneurysm (PA) is a vascular injury typically caused by rupture of the arteries with extravasation of blood. The compressed perivascular tissue forms the wall of the aneurysmal sac and develops in the mass with active pulsation. Pseudoaneurysm differs from true aneurysm in that the latter involves abnormal expansion of the arteries due to the weakening of vessel walls with blood accumulating between the arterial wall layer .The traumatic involvement of PAs in the internal maxillary arteries is rare because of the anatomical location and protection of the surrounding tissues, although the other branches of the external carotid artery may be more commonly involved in complications and concomitant vascular lesions. Although uncommon, traumatic PAs of the internal maxillary artery are well-recognized complications in facial trauma. The penetrating and blunt trauma is the most common cause .The internal maxillary artery arises behind the neck of the condylar process of the mandible and is well protected by the maxilla. A traumatic injury can result in deep bleeding and shock due to its deep location of difficult control only by direct compression . There aThe objective of this study is to report an unusual case of pseudoaneurysm of the internal maxillary artery in a victim of a gunshot wound treated by catheter embolization using the micro springs technique, as well as to make a review on the subject.A 40-year-old male patient came to the hospital emergency after a gunshot lesion in the cervical region. He was conscious, hemodynamically stable, and without signs of active bleeding or cervical spine injuries. Physical examination showed significant edema in the region of the mandibular angle, trismus, restriction of mandibular movements, absence of rhinorrhea or epistaxis, and soft tissue injury compatible with the bullet entrance orifice in the right posterior cervical region without clinical signs of exit bullet orifice.Computed tomography showed a comminuted fracture of the coronary and mandibular right ascending branches associated with ipsilateral zygomatic-orbital fracture Fig.  and the After physical and imaging evaluation, vascular surgery and neurosurgery teams opted for conservative treatment. However, the maxillofacial surgery team indicated surgical removal of the bone fragments due to the restrictions of the mandibular movements and removal of the fragments of the projectile due to discomfort and superficialisation in the genic region.On the third day after trauma, under general anesthesia, removal of the bone fragments was initiated by intraoral access in the ascending ramus of the mandible, which evolved intraoperatively with an intense arterial bleeding, incompatible with the surgical procedure. Local compression maneuvers were performed using compresses, attempts to pinch with instruments after local exploration and the use of hemostatics, but they were not enough to contain the bleeding. After failure, it was decided to submit the patient to angiography of the external carotid artery.The examination was performed by percutaneous puncture of the right femoral artery and selective catheterization of the external carotid artery and internal maxillary artery, which verified the presence of an PA Fig.  with indThe patient was transferred and remained under observation for 12 hours in the intensive care unit. A new angiography was performed after 24 hours for control, confirming the complete resolution of the PA. The patient was submitted to a second surgery 72 hours after the hemorrhagic episode when a large part of the bone fragments were removed by intraoral access. Also the palpable and superficial portion of the projectile located in the genic region by infraorbital access was removed.The patient was hemodynamically stable, with no complaints and was discharged after 48 hours, without postoperative bleeding recurrences. He had no more complications after 8 months of follow-up.Pseudoaneurysms (PAs), or traumatic aneurysms, are different from true aneurysms because they involve rupture of the internal layers of an artery after blunting, penetrating or even surgical trauma. They may also occur in interruptions of all three vascular layers if the original hematoma is buffered by the surrounding soft tissues. In a penetrating trauma, as reported in this case, the usual mechanism is high velocity maxillofacial trauma, in which fragments of bone or projectile lead to arterial laceration ,6. The pThe rarity of occurrence of PAs after firearm injuries is evidenced by the few cases described in the literature. This demonstrates the lack of data on this topic -9. A seaAfter analysis of the literature, it was observed that all the patients described were male, with ages varying from 15 to 41. This data was aligned with the case described here.Several factors may contribute to the development of PA. The most common causes are accidents or iatrogenic injury and infection associated with the arterial wall . Most ofMost of the PAs of the internal maxillary artery are consequences of penetrating and forceful trauma, and may occur as complications in temporo-mandibular joint surgery, orthognathic surgery, mandibular condyle fractures, as well as the consequence of excessive effort by trumpeters and in the disease of Von Willebrand .The branches of the internal maxillary artery are also vulnerable to lesions at different levels of their anatomical location, either by trauma or surgical intervention. An injury to an arterial wall with maintenance of blood flow produces a hematoma at the boundaries of the surrounding soft tissues. The size of the hematoma is limited by the balance between the pressure inside the vessel and the pressure within the hematoma itself. In a few days, this hematoma begins to liquefy in the center, while producing a new layer of connective tissue along the outer wall. With the progression of central liquefaction, The PA evolves as it pulsates and expands progressively. This may cause pressure on the surrounding tissue, rupture causing severe hemorrhage or embolic thrombi release .The diagnosis of PA is strongly suggested by the patient\u2019s history and physical examination. Patients typically report a pulsatile mass in an area that has recently been traumatized ,14. In oAngiography is a radiological technique that describes the pathway that feeds the lesion and locates the exact site of bleeding, thus allowing precise treatment . In factEmbolization has been widely used in the treatment of vascular lesions and bleeding in the head and neck. This procedure was first described by Dawbom who injected paraffin and Vaseline to block blood flow in a facial sarcoma in 1904. The goal of embolization in cases of PA is not to block blood vessels near the lesion as this would serve to stimulate the growth of a collateral network of blood supply, but rather to deposit a substance in the aneurysm network . This isThe most commonly used permanent embolization materials for embolization of vascular lesions in the head and neck are polyvinyl alcohol (PVA), micro springs and liquid adhesives. Polyvinyl alcohol has been widely used for the treatment of idiopathic epistaxis or epistaxis caused by preoperative tumor embolization. The disadvantage of PVA is recurrent PA bleeding after embolization. Sometimes it can be difficult to place the particles in the affected artery due to blockage of the lumen of the microcatheter by the PVA itself. Micro springs, such as platinum springs or removable Guglielmi springs, have been successfully used in the treatment of intracranial aneurysms and arteriovenous fistulas ,11. The The great majority of authors adhered to the non-surgical treatment of vascular lesions of the internal maxillary artery. Yin  reportedSurgical exploration and hemostasis are usually performed as a last resort if embolization procedures are not satisfactory. Percutaneous catheterization embolization is a known, safe, fast, and effective technique for the treatment of these PAs. With this technique, it is possible to reach damaged vessels that are difficult to approach surgically. The technique allows immediate verification and efficacy of treatment by post-embolization angiography.Excision of an AP is usually the last option in cases where embolization has been ineffective. The contraindications are similar to those of compression therapy. The PAs with signs of local infection are better treated surgically, but this depends on the location of the aneurysm and is easily accessible through open removal .If left untreated, pseudoaneurysm tends to become fatal due to frequent bleeding or thromboembolism. Clinical signs of PA of the internal maxillary artery often arise several weeks or months after trauma. Unlike most cases reported in the literature, this patient presented an almost immediate post-traumatic vascular malformation in his infra-temporal fossa, which was confirmed by angiographic studies.The treatment of pseudoaneurysm aims to control the proximal and distal arterial blood flow of damaged vessels. Endovascular treatment is the best method because of the anatomical location of the PA of the internal maxillary artery . Using tPseudoaneurysms of the head and neck region are rare and there are few reports in the literature about gunshot wounds. It was observed that the use of embolization by catheterization is a less invasive, efficient and safe technique in the treatment of these complications, thus avoiding invasive methods of surgical interventions in this anatomical space."}
+{"text": "Antheraea pernyi. Cecropin DH possesses effective antibacterial activity, particularly against Gram-negative bacteria, with very low cytotoxicity against mammalian cells. Interactions between cecropin DH and the highly anionic lipopolysaccharide (LPS) component of the Gram-negative bacterial outer membrane indicate that it is capable of dissociating LPS micelles and disrupting LPS aggregates into smaller assemblies, which may play a vital role in its antimicrobial activity. Using LPS-stimulated mouse macrophage RAW264.7 cells, we found that cecropin DH exerted higher potential anti-inflammatory activity than cecropin B, as demonstrated by the inhibition of pro-inflammatory cytokines nitric oxide production and secretion of tumor necrosis factor-\u03b1. In conclusion, cecropin DH has potential as a therapeutic agent for both antibacterial and anti-inflammatory applications.Cecropins, originally found in insects, are a group of cationic antimicrobial peptides. Most cecropins have an amphipathic N-terminal segment and a largely hydrophobic C-terminal segment, and normally form a helix-hinge-helix structure. In this study, we developed the novel 32-residue cecropin-like peptide cecropin DH by deleting the hinge region (Alanine-Glycine-Proline) of cecropin B isolated from Chinese oak silk moth,  In recent decades, antibiotic resistance has emerged as a major threat to global healthcare and food security , and newFor Gram-negative bacteria, lipopolysaccharide (LPS) is the major barrier protecting against host defense molecules such as AMPs. LPS, also known as endotoxin, forms a highly anionic layer at the outer leaflet of the outer surface membrane of Gram-negative bacteria, contributing greatly to structural integrity and preventing entry of host molecules . LPS canIn insects, cecropins constitute a large family of cationic AMPs that are active against Gram-positive and Gram-negative bacteria, as well as fungi, with low toxicity toward eukaryotic cells such as erythrocytes . Most ceAntheraea pernyi  from cecropin B (KWKIFKKIEKVGRNIRNGIIKAGPAVAVLGEAKAL) that was isolated from Chinese oak silk moth, a pernyi . The antCecropin DH and its parent peptide cecropin B were synthesized by GL Biochem Ltd.  using the solid-phase synthesis technique. And the success of peptide synthesis was identified via electrospray ionization mass spectrometry. The purity of the synthetic peptides used to evaluate biological activity was higher than 95% confirmed with analytical reverse-phase high-performance liquid chromatography.Escherichia coli ATCC 25922, E. coli DH5\u03b1, Pseudomonas aeruginosa ATCC 27853, Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 25923 and Micrococcus luteus NCIMB 8166 were determined using the modified classical microtiter broth dilution method  and compared with those of cecropin B and melittin purchased from . Briefly, bacteria were incubated in Mueller Hinton Broth (MHB) medium overnight at 37 \u00b0C (30 \u00b0C for B. subtilis) with shaking and diluted into fresh MHB at a cell density of 5 \u00d7 105 colony-forming units (CFUs)/mL. A 100 \u03bcL volume of cell suspension was dispensed in each well of a sterile 96-well plate from column 1 to column 11, and 100 \u03bcL of MHB was dispensed in column 12 for sterility control and to blank the plate scanner. A 11 \u03bcL sample of each of the peptides (10\u00d7 stocks) was dissolved separately in 0.01% acetic acid and 0.2% bovine serum albumin, and added to each well (columns 1\u201310) to a final concentration of 100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78, 0.39 and 0.195 \u03bcM, respectively. Column 11 without peptides served as positive controls (untreated bacteria). The plate was incubated at 37 \u00b0C (30 \u00b0C for B. subtilis) for 18\u201324 h in an incubator, and the absorbance was recorded at 590 nm. Three replicates were generated for each test sample. In the present study, the MIC was taken to be the lowest concentration of peptides that prevented visible turbidity. Then 10 \u03bcl 10\u22126 dilution of the first three wells that showed no visible growth of above overnight cultures, were plated onto Mueller Hinton Agar (MHA) plates and incubated at 37 \u00b0C (30 \u00b0C for B. subtilis) for 18 h. The lowest concentration of peptides that prevent any residual colony formation is the minimal bactericidal concentration.The minimum inhibitory concentrations (MICs) of cecropin DH against 2 or MgCl2. MIC values against E. coli ATCC 25922 were measured as described above in the presence of 100 mM NaCl, 2 mM CaCl2 or 1 mM MgCl2.Next, the salt sensitivity tests for cecropin DH were performed at fixed concentrations of NaCl, CaClE. coli ATCC 25922 were also determined. Exponentially growing E. coli cells were resuspended in MHB to a density of 4 \u00d7 106 CFUs/mL. Bacterial suspensions were treated at 1\u00d7 and 4\u00d7 MIC with peptide. At various time periods , 50 \u03bcL samples were removed and diluted appropriately in MHB, and CFUs were determined by spotting 100 \u03bcL of serially diluted samples onto MHB agar plates and incubating for 18 h at 37 \u00b0C.The in vitro bacteria killing kinetics of cecropin DH against The hemolytic activities of cecropin DH and cecropin B peptides were determined as the amount of hemoglobin released by the lysis of mouse red blood cells (mRBCs) . Fresh m2 atmosphere. Cells were pre-seeded on a 96-well plate at a density of \u223c6 \u00d7 104 to 1 \u00d7 105 cells/mL and cultured overnight. Increasing concentrations of the peptides (0.195\u2013100 \u03bcM) in DMEM medium were added, and wells containing cells without peptides served as controls. After incubating plates for 1 day, 10 \u03bcL of CCK-8 solution was added to each well and plates were incubated for an additional 2.5 h at 37 \u00b0C. The absorbance at 450 nm was measured using a microplate autoreader .The cytotoxicity of cecropin DH and cecropin B peptides against RAW264.7 mouse macrophage cells  was determined using a CCK-8 cell proliferation and cytotoxicity assay kit . RAW264.7 cells were cultured in Dulbecco\u2019s modified Eagle\u2019s medium  supplemented with antibiotics  and 10% fetal bovine serum  at 37 \u00b0C in a humidified chamber under a 5% COE. coli 055:B5 purchased from  was determined using the fluorescent dye BODIPY-TR-cadaverine  displacement assay  and F100 is the fluorescence upon addition of 10 \u03bcg/mL polymyxin B purchased from  to the LPS-probe mixture .The binding affinity of cecropin DH to the lipid A portion of bacterial LPS from nt assay , in whicE. coli 055:B5 purchased from  were analyzed by exciting FITC-LPS (0.5 \u03bcM) at 480 nm in 20 mM TRIS and 100 mM NaCl (pH 7.4) in the absence and presence of different concentrations of peptide . Fluorescence emission spectra at 515 nm were recorded using a SpectraMax i3x microplate autoreader .Interactions between cecropin DH and fluorescein isothiocyanate (FITC)-conjugated LPS from To obtain information on the particle size distribution of LPS micelles in the absence and presence of cecropin DH, static light scattering (SLS) measurements were performed on a Malvern Zetasizer \u03bcV instrument . Measurements were made for LPS micelles at a final concentration of 1 mg/mL mixed with 0.25 mM peptides. LPS and peptide were dissolved in TRIS buffer , and all solutions were filtered and degassed prior to use. Data were acquired at 298 K using disposable cuvettes. Scattering data were collected at 90\u00b0 and analyzed using the software supplied with the instrument.Transmission electron microscopy (TEM) was performed to observe the influence of peptides on the morphology of LPS micelles. A total of 50 \u03bcL solutions containing 0.25 mM cecropin DH and 1 mg/mL LPS micelles in Milli-Q water were incubated at room temperature overnight. Untreated LPS micelles were used as controls. After incubation, a drop of the mixture was deposited onto a carbon-coated grid, negatively stained with phosphotungstic acid and visualized using a Tecnai G2 Spirit BioTWIN 120 kV TEM instrument .E. coli ATCC 25922 bacterial cells in 10 mM sodium phosphate buffer (pH 7.4). The peptide concentration was 0.2 mg/mL. The CD spectra of cecropin DH in sodium phosphate buffer, in LPS or E. coli cells environment were obtained by subtracting the peptide control spectrum. Spectra were collected in quartz cuvettes with a 0.5 mm path length and averaged across three consecutive scans. A 1 nm data pitch, 0.4 s time-per-point and 1 nm bandwidth were generally used. In addition, E. coli bacterial cell cultures for CD experiments were grown in MHB medium at 37 \u00b0C overnight, harvested  and resuspended in 10 mM sodium phosphate buffer (pH 7.4) at OD600 values of 4.0, 2.0, 1.0 and 0.5. Peptide solution was added to the resuspended E. coli cells to give a final OD600 of 2.0, 1.0, 0.5 and 0.25, and samples were incubated at room temperature for 4 h (Circular dichroism (CD) experiments were performed at room temperature with a Chirascan qCD spectropolarimeter . Spectra were recorded at wavelengths ranging from 190 to 240 nm. CD spectroscopy was used to investigate the secondary structure of cecropin DH in the absence and presence of 2 mg/mL LPS and  for 4 h .2O buffer at pH 5.8. STD experiments were recorded on a Bruker 500 MHz spectrometer at a temperature of 298 K with 1,024 scans. Selective saturation of LPS resonances was performed by selective irradiation at frequency at \u22122.0 ppm (on-resonance) or 40 ppm for the reference spectrum (off-resonance). A cascade of 44 selective Gaussian-shaped pulses was applied for a total saturation time of 2 s. The low power Gaussian pulses had a duration of 45 ms and inter-pulse delays of 450 \u03bcs. The difference spectrum, which contains signals arising from the saturation transfer, was obtained by subtracting the off-resonance spectrum from the on-resonance spectrum by phase cycling.For saturation transfer difference (STD) experiments, the nuclear magnetic resonance (NMR) sample contained 1 mM cecropin DH, 0.3 mg/mL LPS, in 10 mM sodium phosphate D31P nuclear magnetic resonance spectra of LPS were recorded on a Bruker 850 MHz spectrometer at 298 K. Interactions of peptides with LPS were examined by recording a series of one-dimensional 31P NMR spectra of LPS, whereby 4 mg/mL LPS in 20 mM TRIS (pH 7.4) and 100 mM NaCl was titrated with various peptide concentrations .5 cells/well) and cultured overnight. Cells were then stimulated with or without 200 ng/mL LPS (negative controls) in the presence or absence of peptides (10 \u03bcM) for 3 h. Total RNA was prepared using an RNAprep Kit , and equal amounts of total RNA were reverse-transcribed into cDNA using a RevertAid first-strand cDNA synthesis kit  according to the manufacturer\u2019s protocol. Products were amplified from cDNAs by PCR using the following specific primers: interleukin-1\u03b2 (IL-1\u03b2) ; interleukin 6 (IL-6) ; macrophage inflammatory protein (MIP)-1 ; MIP-2 ; TNF-\u03b1 ; inducible nitric-oxide synthase (iNOS) ; glyceraldehyde-3-phosphate, used as an internal control . Thermal cycling consisted of an initial denaturation step of 5 min at 94 \u00b0C, followed by 35 cycles of denaturation at 94 \u00b0C for 1 min, annealing at 55 \u00b0C for 1.5 min and extension at 72 \u00b0C for 1 min, and a final extension step of 10 min at 72 \u00b0C.RAW264.7 murine macrophage cells were plated in six-well plates (5 \u00d7 105) cultured in DMEM containing 10% fetal bovine serum, 100 U/mL penicillin and 100 \u03bcg/mL streptomycin were plated and adhered to a 96-well culture plate. After stimulating with LPS (200 ng/mL) in the presence or absence of peptides (10 \u03bcM) for 24 h, culture medium was collected to determine nitrite levels using Griess reagent  according to the manufacturer\u2019s instructions. The absorbance at 540 nm was measured using a SpectraMax i3x microplate reader  and converted to nitrite concentration by referring to a standard curve generated using NaNO2.RAW264.7 murine macrophage cells (1.5 \u00d7 104 cells/well) and incubated overnight. Cells were pretreated with peptides (10 \u03bcM) and incubated at 37 \u00b0C for 1 h before the addition of 20 ng/mL LPS. After incubation for another 6 h, supernatants were harvested for TNF-\u03b1 and IL-6 analyses using commercially available mouse TNF-\u03b1 and IL-6 enzyme-linked immunosorbent assay (ELISA) kits  according to the manufacturer\u2019s protocols. The absorbance at 450 nm was measured using a SpectraMax i3x microplate reader .RAW264.7 murine macrophage cells were seeded in 96-well plates . Differences with E. coli ATCC25922, E. coli DH5\u03b1 and P. aeruginosa) and three Gram-positive  bacterial strains . In the STD-NMR experiments, selective saturation pulses were applied at LPS resonances, and magnetic saturation was subsequently transferred to LPS-bound peptides. Saturation difference spectra were generated from the intensity difference between LPS-bound peptide and free peptide signals. 1H spectrum of free peptides and the STD spectrum of peptides bound to LPS. A relatively strong STD effect of cecropin DH was observed for the aromatic ring protons of Trp2 and Phe5 (6\u20138 ppm), as well as a number of aliphatic side chain proton resonances (0\u20135 ppm) (Further experiments were performed to investigate the interactions of cecropin DH and LPS. STD-NMR spectra were recorded on 1 mM peptide samples with 0.3 mg/mL LPS (in 10 mM sodium phosphate D0\u20135 ppm) , implyinE. coli cells, \u03b1-helical structure also formed, and the \u03b1-helical content was dependent on cell concentration. This is due to the fact that more LPS molecules are available for peptide binding with the increment of cell concentration. Fourier-transform infrared (FTIR) spectra subsequently provided additional evidence; in the presence of LPS, the appearance of a peak at 1,655 cm\u22121 in the amide I region indicates an \u03b1-helical conformation for cecropin DH . The 31P NMR spectra of LPS micelles exhibit a strong peak close to \u22121.30 ppm and two weak peaks at approximately 0.8 and 1.78 ppm, corresponding to the diphosphate and monophosphate groups of LPS, respectively . It was proposed that the antibacterial activity in cecropin-like model peptides requires the flexible region between the N-terminal amphipathic \u03b1-helix and the C-terminal hydrophobic \u03b1-helix  can be released from the cell wall and elicit strong innate immune responses in animals . Once LP5L7, may lead to a loosening of the bacterial outer membrane and the formation of transient \u201ccracks\u201d that allow the passage of a variety of molecules. In this study, cecropin DH exerted strong activity against Gram-negative organisms, probably due to its ability to breach the LPS-mediated barrier by interacting with LPS, and using large structural perturbations of LPS aggregates.Lipopolysaccharide covers >70% of the outer leaflet of Gram-negative bacteria and forms a permeability barrier that must be overcome by broad-spectrum AMPs. A variety of biophysical techniques have been employed to investigate the interactions between AMPs and LPS/mimetic membranes to understand the mechanisms of membrane recognition and perturbation by AMPs . Herein,E. coli bacterial cells , 30% trifluoroethanol (TFE)/water and 15 mM dodecylphosphatidylcholine (DPC).CD spectra of cecropin DH (0.2 mg/mL) in HClick here for additional data file.10.7717/peerj.5369/supp-2Supplemental Information 2\u22121), 2 mg/mL LPS in D2O alone or with peptide (0.5 mM) were lyophilized and spectra were collected. For the negatively charged phosphate region measurements , similar procedures were performed as for those in amide I region, except H2O was selected as the solvent instead of D2O.(A) Amide I region of LPS. (B) Antisymmetric phosphate region of LPS. For amide I region measurements (1,750\u20131,550 cmClick here for additional data file.10.7717/peerj.5369/supp-3Supplemental Information 3Click here for additional data file."}
+{"text": "N-Palmitoylethanolamide (PEA) is a non-endocannabinoid lipid mediator belonging to the class of the N-acylethanolamine phospolipids and was firstly isolated from soy lecithin, egg yolk, and peanut meal. Either preclinical or clinical studies indicate that PEA is potentially useful in a wide range of therapeutic areas, including eczema, pain, and neurodegeneration. PEA-containing products are already licensed for use in humans as a nutraceutical, a food supplement, or a food for medical purposes, depending on the country. PEA is especially used in humans for its analgesic and anti-inflammatory properties and has demonstrated high safety and tolerability. Several preclinical in vitro and in vivo studies have proven that PEA can induce its biological effects by acting on several molecular targets in both central and peripheral nervous systems. These multiple mechanisms of action clearly differentiate PEA from classic anti-inflammatory drugs and are attributed to the compound that has quite unique anti(neuro)inflammatory properties. According to this view, preclinical studies indicate that PEA, especially in micronized or ultramicronized forms , could be a potential therapeutic agent for the effective treatment of different pathologies characterized by neurodegeneration, (neuro)inflammation, and pain. In particular, the potential neuroprotective effects of PEA have been demonstrated in several experimental models of Alzheimer\u2019s disease. Interestingly, a single-photon emission computed tomography (SPECT) case study reported that a mild cognitive impairment (MCI) patient, treated for 9\u2009months with ultramicronized-PEA/luteolin, presented an improvement of cognitive performances. In the present review, we summarized the current preclinical and clinical evidence of PEA as a possible therapeutic agent in Alzheimer\u2019s disease. The possible PEA neuroprotective mechanism(s) of action is also described. N-palmitoylethanolamide (PEA) has attracted much attention because it exerts a local anti-injury function through a down-modulation of mast cells and protects neurons from excitotoxicity through several mechanisms (Neuroinflammation and synaptic dysfunction in Alzheimer\u2019s disease (AD) have been originally considered as epiphenomena with inflammation and altered neurotransmission occurring when damaged neurons provoke glia activation and changes in neuron biology. However, the growth of knowledge about the molecular mechanisms underlying AD converted this earlier view and points to a causal role of these events in the pathology . Specifichanisms .N-acylethanolamine (NAE) phospolipids, which also includes the first endocannabinoid to be discovered, N-arachidonoyl-ethanolamine  and the anorectic mediator N-oleoyl-ethanolamine (OEA). PEA was firstly isolated from soy lecithin, egg yolk, and peanut meal  are already licensed for use in humans  as a nutraceutical, a food supplement, or a food for medical purposes, depending on the country. PEA is especially used in humans for its analgesic and anti-inflammatory properties , oleoyl-ethanolamide , and linoleoylethanolamide (C18:2 N-acylethanolamine). These compounds are much more abundant than the endocannabinoid anandamide in several animal tissues and endowed with important biological actions. The biosynthesis and metabolism of PEA have been deeply described elsewhere  are endogenously synthesized lipids exerting interesting anti-inflammatory properties . PEA  able to interact with specific DNA sequences in the promoter regions of selective genes, thus leading to complex anti-inflammatory responses  of 3.1 \u00b1 0.4\u202f\u03bcM inflammatory effects of PEA. PPAR-\u03b1 is, in fact, known for its protective role against (neuro)inflammation, and PPAR-\u03b1 ligands are recognized as possible anti-inflammatory compounds . When acesponses . In vitr\u00b1 0.4\u202f\u03bcM . Numerou\u00b1 0.4\u202f\u03bcM , thus su+ in a cellular model of Parkinson\u2019s disease, but an agonist of GPR55 did not enhance neuroprotection in GPR55-expressing cells  estimated to be > 5  was able to penetrate through the blood\u2013brain barrier (BBB), but only in small amounts with a brain bioavailability corresponding to 0.95% of the oral dose  administration, hrocytes . Moreoveral dose . It has ral dose . The pooral dose .Cmax = 420 \u00b1 132\u2005nM); PEA plasma levels returned to the baseline ones \u223c2\u2005h after the compound administration. The formulation of PEA as micronized or ultramicronized particles  has been more recently proposed as a strategy to possibly increase PEA bioavailability, also in the CNS, without affecting the pharmacological efficacy of the compound (t) 1, 2, and 3\u2009h; the administration of m\u2010PEA and \u00b5m\u2010PEA leads to similar peak concentration values of PEA in serum  measured in the blood samples taken 1\u2009h after the compound administration, with PEA concentrations returning to basal values at t = 2\u2009h. 13C]4-PEA or a na\u00efve [13C]4-PEA (30\u2009mg/kg of body weight) formulation to healthy and carrageenan-injected rats, that ultramicronization increases the ability of PEA to reach peripheral and central tissues under either healthy or local inflammatory conditions. In particular, the plasma concentrations of [13C]4-PEA were measured at 5, 15, 30, and 60\u2009min after the oral administration of the compound in ultramicronized and na\u00efve formulations to healthy rats. Rats receiving \u00b5m-[13C]4-PEA showed higher mean plasma levels of the compound than rats receiving na\u00efve [13C]4-PEA. In rats receiving \u00b5m\u2010[13C]4-PEA, the peak concentration of [13C]4-PEA (5.4 \u00b1 1.87\u2009pmol/ml) was found after 5\u2009min, and it was five times higher than the concentration measured in rats administered with the na\u00efve formulation (1.1 \u00b1 0.35\u2009pmol/ml), in which no significant peak plasma concentrations were found.In addition, a PEA suspension in corn oil administered to rats by gastric gavage (100\u2005mg/kg of body weight) led to an about 20-fold increase in basal PEA plasma levels . The higcompound . It has compound . Subsequcompound . Anothercompound . In thisCollectively, the above findings suggest that micronized or ultramicronized formulations of PEA maximize the compound bioavailability and efficacy, although further studies are necessary to undoubtedly confirm this hypothesis. Other strategies have been proposed to improve PEA bioavailability. For instance, PEA ester derivatives, prepared by conjugating PEA with various amino acids, have been synthetized as PEA prodrug and allowed to modulate the kinetics of PEA release in plasma and stability in liver homogenate . Two derin vitro and in vivo experimental models of AD.Several preclinical and some clinical indications support the view of PEA as a therapeutic tool with high potential for the effective treatment of different pathologies characterized by neurodegeneration and neuroinflammation . In this\u22127\u2009M) to mitigate A\u03b2 -induced astrogliosis in primary cultures of rat astrocytes. The results indicate that PEA treatment attenuated A\u03b2-induced astrocyte activation, as proven by its effects in reducing astrocyte hypertrophied cell bodies and thickened processes, along with the expression of glial fibrillary acidic protein (GFAP) and S100 calcium-binding protein B (S100B), two specific markers of astrocyte activity also linked to AD pathogenesis. Furthermore, PEA was able to blunt A\u03b2-induced neuroinflammation by significantly diminishing either the altered expression of pro-inflammatory molecules, such as cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), or the enhanced release of prostaglandin PGE2, nitric oxide, IL-1\u03b2, and TNF-\u03b1. Interestingly, the PPAR-\u03b1 antagonist MK886 was able to partly blunt the PEA-induced effects against A\u03b2-induced astrogliosis and neuroinflammation, thus suggesting a significant, but not exclusive, involvement of the PPAR-\u03b1 in mediating the above-mentioned PEA actions. Concerning the possible intracellular signaling involved in PEA-induced effects, it has also been demonstrated that PEA critically diminished the A\u03b2-induced activation of p38 and Jun N-terminal kinase (JNK), as well as the subsequent activation of nuclear transcription factors, such as nuclear factor kappaB (NF-kB) and activator protein 1 (AP-1)  . Later, 1 (AP-1) . In part1 (AP-1) . Finally1 (AP-1) . In line1 (AP-1) .in vitro toxic effects of A\u03b2. For instance, in a very elegant study, it has been demonstrated that in wild-type (WT) mice, the addition of several acylethanolamides (including PEA) partially reverted A\u03b2-induced inflammation. However, the genetic deletion of FAAH  in astrocytes induced an increased sensitivity to the pro-inflammatory A\u03b2-induced action, and this effect involved PPAR-\u03b1, PPAR-\u03b3, and TRPV1 receptors, but not CB1 or CB2 receptors  and closely mimic many aspects of AD in humans. In fact, these animals are characterized by age-dependent build-up of both plaques and tangles in the cerebral cortex, hippocampus, and amygdala regions, along with early synaptic dysfunction and cognitive decline, thus constituting a widely used and validated AD model. The results indicated that A\u03b21-42 fragment  treatment induced a reduction of cell viability and an increase in glutamate levels in cultured cortical neurons and astrocytes from non-Tg mice, but not in those from the genetic model of AD. The A\u03b2-induced effects in non-Tg cell cultures were counteracted by a pretreatment with PEA (0.1\u202f\u03bcM). Based on these findings, it has been hypothesized that exogenous A\u03b2 treatment failed to induce deleterious effects in 3xTg-AD mice-derived cortical neurons, as these cells at 8\u2009days in vitro were already exposed to a quite high concentration of endogenous peptide fragment. In fact, A\u03b2 levels were observed in these cell cultures after 6\u2009days in vitro , but it promoted neuron viability and counteracted reactive astrogliosis in mature 3xTg-AD primary astrocytes.Moving from the above data, we evaluated the protective role of PEA against A\u03b2-induced toxicity on cell viability and glutamatergic transmission in primary cultures of cerebral cortex neurons and astrocytes from the triple-transgenic murine model of AD (3xTg-AD) and their WT littermates , there was a reduction of neuronal viability, an increase in the number of neuronal apoptotic nuclei, a decrease in the number of MAP-2-positive neurons, and an increase in the number of neurite aggregations/100\u2009\u03bcm as compared with control  astrocyte\u2013neuron co-cultures. Taken together, these data indicate that astrocytes contribute to A\u03b2-induced neurotoxicity and neuroinflammation. Interestingly, these effects were not observed when neurons were cultured in the presence of astrocytes pre-exposed to PEA (0.1\u202f\u03bcM), applied 1\u200ah before and maintained during A\u03b2 treatment. Thus, it has been concluded that PEA, by blunting A\u03b2-induced astrocyte activation, improved neuronal survival in mouse astrocyte\u2013neuron co-cultures.In a further study, we evaluated whether astrocytes could participate in regulating the A\u03b2-induced neuronal damage, by using primary mouse astrocyte cell cultures and mixed astrocyte\u2013neuron cultures . The res1-42  induced a reduction of cell viability and neuroinflammatory responses. These effects were counteracted by the pre-treatment with co-ultraPEALut  for 2\u2009h . Under these experimental conditions, the pre-treatment with co-ultraPEALut significantly reduced iNOS and GFAP expression, restored neuronal iNOS and brain-derived neurotrophic factor (BDNF), and reduced the apoptosis , which demonstrated antioxidant properties. Previous studies, in fact, indicated that the association of these two molecules, in a fixed ratio of 10:1 in mass, induced a strong neuroprotective activity . The exp for 2\u2009h . Similarpoptosis . In linepoptosis .in vitro results prompted the development of in vivo studies aimed at evaluating the neuroprotective properties of PEA in animal models of AD. Firstly, 25\u201335 peptide (9\u2009nmol). To this purpose, PEA was administered once a day , starting 3\u2009h after A\u03b225\u201335, for 1 or 2\u2009weeks, while water-maze, water-maze working memory, and novel object recognition tests were used to assess cognitive performances. The authors demonstrated that, depending on the dose, PEA reduced (10\u2009mg/kg of body weight) or prevented (30\u2009mg/kg of body weight) the cognitive impairments induced by A\u03b225\u201335 peptide injection. In line with previous in vitro findings, the beneficial effects of PEA appear mediated by PPAR-\u03b1, as the compound failed to rescue memory deficits induced by A\u03b225\u201335 peptide injection in PPAR-\u03b1 null mice, and GW7647 (a synthetic PPAR-\u03b1 agonist) mirrored the effects of PEA. These encouraging behavioral results were corroborated by the evidence that in the same animals used for cognitive tests, PEA reduced brain lipid peroxidation, protein nitrosylation, iNOS induction, and caspase3 activation  was intraperitoneally administered once a day for seven consecutive days, starting from the day of A\u03b21\u201342 injection, it was able to partially or completely antagonize A\u03b21\u201342-induced toxic effects. Again, the effects of PEA were prevented by the treatment with GW6471 (2\u2009mg/kg), thus demonstrating the involvement of PPAR-\u03b1. Moreover, the authors demonstrated the PPAR-\u03b1-dependent ability of PEA to restore the alteration in the Wnt signaling pathway caused by A\u03b21\u201342 hippocampal infusion. This is relevant since Wnt signaling pathway plays different roles in the development of neuronal circuits and also in the adult brain, where it regulates synaptic transmission and plasticity and has been also implicated in various diseases including neurodegenerative diseases  of \u00b5m\u2010PEA in 3xTg-AD mice, at two different stages of the pathology, were evaluated by administering the compound via a subcutaneous delivery system to groups of 3-month-old and 9-month-old animals . In fact, the bioavailability and apparent volume of distribution have not been clearly evaluated; and blood PEA levels, at least in animals, do not accurately reflect levels in the CNS  slowed down disease progression and disability .in vitro or in vivo data  are actually used for certain medical indications, especially inflammatory pain. Moreover, as an endogenous compound, PEA has a safely profile at pharmacological doses. Relevant PEA-induced side effects were not seen in humans at oral doses up to 1,800\u2009mg/day. Finally, PEA has proven efficacious in humans in a number of clinical settings, and none of the clinical trials with PEA to date have reported treatment-related adverse events  to LF (grant # 20151001) and from the University of Ferrara (FAR 2018).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": "Drosophila. Under crowded conditions, fly larvae form coordinated digging groups (clusters), where individuals are linked together by sensory cues and group membership requires prior experience. However, fitness benefits of Drosophila larval clustering remain unknown. We demonstrate that animals raised in crowded conditions on food partially processed by other larvae experience a developmental delay presumably due to the decreased nutritional value of the substrate. Intriguingly, same conditions promote the formation of cooperative foraging clusters which further extends larval stage compared to non-clustering animals. Remarkably, this developmental retardation also results in a relative increase in wing size, serving an indicator of adult fitness. Thus, we find that the clustering-induced developmental delay is accompanied by fitness benefits. Therefore, cooperative foraging, while delaying development, may have evolved to give Drosophila larvae benefits when presented with competition for limited food resources.Cooperative behavior can confer advantages to animals. This is especially true for cooperative foraging which provides fitness benefits through more efficient acquisition and consumption of food. While examples of group foraging have been widely described, the principles governing formation of such aggregations and rules that determine group membership remain poorly understood. Here, we take advantage of an experimental model system featuring cooperative foraging behavior in The online version of this article (10.1007/s00359-020-01434-6) contains supplementary material, which is available to authorized users. Group foraging is a major component of cooperative animal behavior Allee . It can Examples of cooperative foraging providing fitness benefits have been documented among a broad range of animal taxa. Group hunting strategies that increase success rate of prey localization and acquisition were described in carnivorous mammals Clark , birds , and fisDrosophila melanogaster. Interestingly, although behavioral and developmental aspects of larval solitary foraging behavior were addressed a long time ago , and w\u2212; Sp/CyO; TM2/TM6B-Tb flies were kindly provided by Dr. Susan Doyle, University of Virginia.Wild-type Canton S (CS) flies were donated by Ed Lewis , blind GMR-hidDrosophila melanogaster stocks were raised in food vials  containing standard Caltech food mixture  at 22\u00a0\u00b0C, 30% humidity under standard 12/12\u00a0h light\u2013dark cycle (unless further specified in experiments involving dark rearing) at\u2009~\u20091000\u00a0lx light intensity. For egg production,\u2009~\u200950 adult flies 3\u20134\u00a0days old were transferred into egg cups and kept in the same conditions. Eggs were laid on 35-mm Petri dishes (egg plates) containing standard agar-molasses food and yeast. For collection of larvae at designated developmental stages, egg plates were changed every 6\u00a0h and subsequently kept (unless further specified) in the same light/temperature/humidity conditions. Depending on the experimental purposes, larvae were collected from egg plates in the middle of L2 stage , or middle of L3F2 stage (110 hAEL).All Techniques of \u201cpre-processed\u201d vial production and transplantation of 200 L2 (60 hAEL) larvae were adapted from our previous studies  starting immediately after transplantation. Videos were recorded on an iPhone 5 at full resolution and 1 frame/60\u2033 using \u201cLapseit\u201d software for iOS and subsequently analyzed using iMovie and ImageJ (32-bit version for Windows). Percentage of frames with a clearly observed larval cluster  was calculated for each 12-h light period during 3 consecutive days of recordings for each individual vial (days 3\u20135 after transplantation for L2 larvae and days 1\u20133 after transplantation for L3F2 larvae) of a designated genotype/condition (each of these measurements represents a single data point on the corresponding graphs). Values were subsequently averaged and represent mean values for each genotype/condition.Newly formed pupae were counted on each food vial/plate of a designated genotype/condition twice a day . Eclosed flies were counted  and collected using CO2 anesthesia twice a day , and females were subsequently frozen at \u2009\u2212\u200920\u00a0\u00b0C in 1.5-mL plastic tubes for subsequent wing size measurements. For pupariation and eclosion measurements, percentage of animals that reached a designated developmental stage was calculated relative to the total number of pupariated/eclosed animals counted by the final day of observations . Survival values were estimated as ratios of eclosed flies to the total number of originally transplanted larvae. In the case of mixed populations, only wild-type CS flies were counted.In all experiments involving light deprivation, dark-reared larvae were kept in a completely dark room for a designated time period in a food plate/vial and were additionally covered with a layer of aluminum foil. Dark rearing began immediately after larval transplantation at a designated stage and until the eclosion of all adult flies. Daily eclosion and pupariation rate measurements were performed in a room with dim red lights for each vial individually to minimize the time of possible light exposure.Wing size (which serves as an estimate of body size) of previously collected and frozen females was measured using a technique adapted from Gilchrist and Partridge  distanca. A singp\u2009<\u20090.05; **p\u2009<\u20090.01; ***p\u2009<\u20090.001. Linear regression analysis was used for Fig.\u00a0Unless otherwise stated, all data are presented as mean values and error bars represent 95% confidence intervals. Statistical significance was calculated by one-way ANOVA using Tukey\u2019s method. When comparing two groups of normally distributed data, Student\u2019s two-tailed unpaired T-test was used. *Processed food and crowded conditions increase the duration of larval development  with either fresh or processed food and compared their pupariation and eclosion rates Fig. e and S1cTo examine the role of cooperative foraging in larval development, we took advantage of approaches shown in Fig.\u00a0To provide more evidence in support of this hypothesis, we used cluster \u201cdisruptors\u201d as another tool to manipulate clustering frequency Fig.\u00a0a, as our. Importantly, clustering frequency was significantly reduced in L3F2 transplants . Therefore, our study indicates that cooperative foraging in fruit fly larvae can be beneficial in some but not all conditions Fig.\u00a0e. This rLewontin , althougLewontin . This hiDrosophila are governed by insulin-like hormones and TOR signaling in prothoracic gland , as well as antagonistic actions and complex interplay of ecdysone and juvenile hormone Supplementary file2 (TIF 1492 kb)Below is the link to the electronic supplementary material."}
+{"text": "Individuals presenting with respiratory symptoms (ILI) were tested for influenza A and B virus (Alere BinaxNOW). Subsequently, we tested influenza-negative nasopharyngeal (NP) swabs for SARS-CoV-2. We extracted total nucleic acid using the bioM\u00e9rieux eMAG automated platform and performed real-time RT-PCR (qRT-PCR) assays specific for SARS-CoV-2 N and E genes . Out of et al.  directly on specimen RNA, thereby avoiding cell culture passage and potentially associated mutations. This generated an NGS dataset of 20.7 to 22.7 million paired-end reads per sample. We mapped quality-filtered reads to a reference SARS-CoV-2 genome (MN908947) using BBMap (version 39.64) to generate three full-length genomes: AZ-ASU2922 (376x coverage), AZ-ASU2923 (50x) and AZ-ASU2936 (879x) (Geneious prime version 2020.0.5). We aligned a total of 222 SARS-CoV-2 genome sequences comprising at least 5 representative sequences from phylogenetic lineages defined by Rambaut et al. . The ASUet al. .Similar to SARS-CoV, the SARS-CoV-2 genome encodes multiple open reading frames in the 3\u2019 region. We found that the SARS-CoV-2 AZ-ASU2923 genome has an 81 base-pair deletion in the ORF7a gene resulting in a 27 amino-acid in-frame deletion . The SARCollectively, although global NGS efforts indicate that SARS-CoV-2 genomes are relatively stable, dynamic mutations can be selected in symptomatic individuals.1"}
+{"text": "The photoperiod marks a varied set of behaviors in plants, including bulbing. Bulbing is controlled by inner signals, which can be stimulated or subdued by the ecological environment. It had been broadly stated that phytohormones control the plant development, and they are considered to play a significant part in the bulb formation. The past decade has witnessed significant progress in understanding and advancement about the photoperiodic initiation of bulbing in plants. A noticeable query is to what degree the mechanisms discovered in bulb crops are also shared by other species and what other qualities are also dependent on photoperiod. The FLOWERING LOCUS T (FT) protein has a role in flowering; however, the FT genes were afterward reported to play further functions in other biological developments . This is predominantly applicable in photoperiodic regulation, where the FT genes seem to have experienced significant development at the practical level and play a novel part in the switch of bulb formation in Alliums. The neofunctionalization of FT homologs in the photoperiodic environments detects these proteins as a new class of primary signaling mechanisms that control the growth and organogenesis in these agronomic-related species. In the present review, we report the underlying mechanisms regulating the photoperiodic-mediated bulb enlargement in Allium species. Therefore, the present review aims to systematically review the published literature on the bulbing mechanism of Allium crops in response to photoperiod. We also provide evidence showing that the bulbing transitions are controlled by phytohormones signaling and FT-like paralogues that respond to independent environmental cues (photoperiod), and we also show that an autorelay mechanism involving FT modulates the expression of the bulbing-control gene. Although a large number of studies have been conducted, several limitations and research gaps have been identified that need to be addressed in future studies. It has been reported that the degree of bulb growth over long photoperiods increases [The photoperiod is demarcated as the portion of light and dark hours in a diurnal cycle of 24 h . The conncreases . Under sncreases . Photopencreases .Allium, proposing the turnover of all controllers of bulb advance in varied taxonomy [The FLOWERING LOCUS T (FT) gene family in Arabidopsis encrypts the transportable flowering indicator formed in the leaves that is responsible for generating bulb development in the subversive stolon, thereby providing a vital innovation about the way bulbing is controlled. Further research in onion exhibited that diverse FT genes similarly control bulb enlargement, stressing the evolutionary-focused maintenance of FT proteins as key producers of the bulb alteration. This too influences the offshoot meristems at the axillary buds and vascular cambium etches for this progressive changeover. FT-like genes switch onion bulb development; however, unlike Arabidopsis FT, it is not a long-distance indicator. These fallouts and the preceding information indicate that FT-like genes activate bulb development in taxonomy . Furthertaxonomy , recommePlants respond to different seasons to initiate evolving strategies at specific times of year. The photoperiod controls several evolving developments in plants. Genomic studies have revealed the fundamental responses to alterations in photoperiod and the generation of a vigorous cyclic response. Current advances in plant genome analysis have established the variation in these controlling structures in several crops. Plants are visible to a sturdy rhythm of day and night due to the rotation of the earth around its own axis. This rhythm varies throughout the year, and the more distant an area is from the Equator, the sturdier these variations are, therefore demanding the adaptation of life procedures to this altering rhythm. The adjustment sequence and thus the synchronization of frequent biological and progressive developments with the ecological sequences is understood by an inner circadian clock. Throughout evolution, a quantity of plant classes has achieved the skill of distinguishing their leaves, stems, or roots into storage organs (bulb), as observed in garlic and onion. The formation of these bulbs is attracted throughout drought and freezing conditions that pacify plant feasibility, acting as a mechanism for asexual propagation that delivers a survival strategy to the plant. As such, these bulbs are continuously dormant in soil throughout divergent cold and dry times, after which they are sown in the following term and produce a healthy plant. Early growing of the new shoot is liable on the metabolic properties that have accumulated in these bulbs, generally in the formula of starch or soluble sugars, which makes them an outstanding caloric supplementation to the human nutritional requirements. An iterative breeding collection for bigger bulbs and form to diverse latitudes could improve the recent cultured genotypes, which is of high financial significance and tactical in equilibria of food safety. Therefore, understanding the mechanisms recycled by the plant to signal the distinction of these bulbs is a vital area to see the nutritious stresses of the increasing world population, and it is also an ultimate query in developing environmental skills.Allium sativum [A bulb is a vegetative growing point or an unexpected flowering shoot. The base is from a reduced stem, and plant growth occurs from this basal plate. Root development forms from the underside of the base and new stems and leaves form from the upper side. A long photoperiod is crucial for bulb initiation and growth in  sativum ,15,16,17 sativum . At larg sativum . In onioAllium sativum have attracted limited investigation reflection, apart from the research of Kamenetsky and Rabinowitch [Allium species including garlic have been shown to be influenced by day length, temperature, and carbohydrates [Up to the present time, the result of photoperiod on bulb development has been researched for some Alliums. The bulb enlargement of onion and its subsequent development were influenced by the photoperiod, and bulbing was encouraged by long days. Furthermore, in some cultivars, bulbing only occurred once double thresholds of a least thermal period of 600-degree days and a photoperiod of 13.75 h were achieved ,21,22,23inowitch , Mathew inowitch , and Atiinowitch . Findinginowitch . Far-redinowitch . Furtherinowitch ,28,29. Rinowitch . A lowerinowitch ,32. Enviinowitch . In onioinowitch ,39,40,41hydrates ,43. Earlhydrates , and in hydrates . Equallyhydrates ,46.Phytohormones are considered the most important endogenous substance for modulating physiological and molecular responses, and they are a critical requirement for plant survival as sessile organisms. Phytohormones act either at their site of synthesis or elsewhere in plants following their transport. The examination of the paraphernalia of photoperiod on bulb growth will offer vision into the mechanism of bulbing and environmental tools (adaptable photoperiods) for developing parameters. The directive of bulbing is comprised of intricate evolutions determined by an intricate system of signaling pathways. In order to improve propagation ability and bulb production through horticultural practices, regulating environmental conditions (photoperiod) and phytohormones are the most effective ways, which have a key role in the development of bulbs ,15,47,483 application could increase shoot weight in carrot [3 increased bulb weight in onion under deficit irrigation [s caused a decline in tuber production in potato [3 treatment improved the number of tubers per plant [3 also encouraged tillering in welsh onions [Jatropha curcas [Endogenous gibberellins were found at high levels in the storage leaf during clove sprouting, and they also inhibited dormancy induction in whole tulip bulbs ,52,53. Ln carrot , seed yin carrot , and frun carrot  and plumn carrot . Earlierrigation . On the n potato ; fruit pn potato , grape [n potato , and pean potato ; and roon potato . In potaer plant . The apph onions , encourah onions , and proa curcas . GA was a curcas ,73,74  are a growth-encouraging phytohormone [s might significantly increase plant weight in cauliflower and sweet peppers [3 and high frequency of application GA3 inhibit lettuce growth [3 decreased root, tuber, fruit weight, and yield in other crops as well [3 had a positive and negative effect on the organ nutritive eminence of potatoes, sweet peppers, and sweet cherries [3 can significantly induce lateral bud formation and increase the clove number per bulb, which improves the reproduction efficiency in garlic. However, the physiological and metabolic changes of garlic plants under GA3 treatment are unknown [3 treatment [3 on garlic plant architecture and bulb structure. Here, they investigated the effect of both soaking seed cloves in GA3 solution and injecting plants with GA3 on plant growth and bulb development in garlic. They detected that soaking seed cloves in GA3 solution induced secondary plant  development and expressively increased the incidence rate of secondary plants, clove numbers per bulb, and bulb weight. Clove number per bulb and bulb weight were sharply increased by the application of GA3. Exogenous GA3 induced the axillary bud formation of garlic via the changes of soluble sugar content and soluble protein content in the stem [3) induced axillary bud formation and promoted the growth of lateral branches in tomato [Jatropha curcas [3 not only intensely increased clove number per bulb but also changed bulb morphology [3 is considered a new means for improving propagation competence and garlic yield. These results highlight the status of exogenous GA3 as a start of axillary bud development [3 solution and injecting plants with GA3 caused a substantial increase in the incidence rate of secondary plants, clove numbers per bulb, and bulb weight, which can be used for improving propagation efficiency in horticultural practice.Gibberellins are imperative phytohormones that control many evolving developments in plants . Accordin of GA3 . Gibbereohormone ,77,78, a peppers ,79,80. Ie growth  and decre growth . The app as well ,64,67. Pcherries ,82,83. G unknown . Lately,reatment ,85,86,87reatment  also stuthe stem . Exogenon tomato , potato n tomato , cherry n tomato , Jatropha curcas , and wela curcas . The apprphology . Currentelopment . Accordielopment , soakingAllium fistulosum L.), which improved expressively and reduced subsequently after the flower bud variation. According to the outcomes of Wu et al. [Allium cepa L.), where the level of ABA was higher during the dormancy period and decreased when dormancy broke. Furthermore, later research established that exogenous ABA delays sprouting in Allium wakegi plants [Abscisic acid (ABA) normally plays a vital part in plant resistance to biotic or abiotic stresses. It was expected that ABA plays an important role in the initial phase of plant bolting ,93. Su  describeu et al. , ABA alsu et al. . Abscisiu et al. . In Dutcu et al. . ABA weru et al. . ABA wasu et al. . A furthu et al. . The rolu et al. ,101,102.i plants . ABA\u2019s ri plants . Accordii plants , signifiSolanum tuberosum L.) tuber growth. Endogenous auxin levels were found to be high just prior to and during stolon swelling, after which auxin levels steadily decreased [Indole acetic acid (IAA) exhibits inconsistent results, decreasing with rising flower bud variation degree and then growing expressively throughout the bolting procedure of welsh onion . It is necreased . Moreoveecreased  detectedecreased  and ARF ecreased  have beeecreased . Arf6 execreased   was reported to have an enhancing effect on plant bolting . Accordied roots ,124,125 Jasmonic acid (JA) and connected amalgams are extensively dispersed amongst higher plants and play significant parts in the directive of plant growth ,127. JasSalicylic acid (SA) also has a significant role in garlic bulb development, and MeJA probably boost the endogenous SA content of garlic plant, therefore refining bulbing. Cytokinins have also been shown to have a part in tuberization by persuading local cell propagation throughout the initial tuberization start. Tuber development can be tempted in stem node carvings cultured in media with a high absorption of sucrose in the occurrence of these hormones. There was a decrease in the number of tubers per plant in transgenic lines ectopically articulating the cytokinin oxidase (CKX) inactivating enzyme . It is sCultivars grown at diverse latitudes required a least day length for bulbing, and cultivars are classified on this into short-day (SD), intermediate, and long-day (LD) categories. The short-day cultivars procedure bulbs at low latitudes wherever the day length is close to 12 h, whereas intermediate ones grow bulbs at mid-latitudes wherever the day length lies between 12 and 16 h, and long-day cultivars initiate bulbing at high latitudes wherever the day length close or above 16 h . BulbingArabidopsis thaliana [Oryza sativa), maize (Zea mays), wheat (Triticum aestivum), and legumes such as soybean (Glycine max) and barril medic (Medicago truncatula) [Allium development [Allium bulb enlargement is reliant on day length, and it is therefore similar to the day-length retort of flowering [A difference among garlic cultivars in bulbing and responses to environmental indicators is predictable and is furthermost probably alike to what mutual happens in other Allium crops ,145,146.thaliana  and the ncatula) ,153,154.elopment . Allium lowering . In contlowering ,141.Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as transfer RNA (tRNA) or small nuclear RNA (snRNA) genes, the product is a functional RNA ,161,162.Bulbing is an alterable development, and plants grown under inductive environments promote bulb formation, but if they are moved to non-inductive condition, they quickly return to vegetative growth ,184. BulArabidopsis flowering and onion bulbing are both photoperiodically-controlled developmental events  that areArabidopsis thaliana [The FLOWERING LOCUS T (FT) gene was first recognized in thaliana ,203 and thaliana . FT playthaliana . Moreovethaliana ,180. Forthaliana . These rthaliana . For insthaliana ,205,206 thaliana ,208. Oththaliana . Describthaliana   gene plays a central role in integrating flowering signals in Arabidopsis, because its expression is regulated antagonistically by the photoperiod and vernalization pathways. FT encodes a mobile signaling protein involved in regulating flowering, as well as other aspects of plant development such as bulb formation ,209. FT rization ,163. AcFrization . Manoharrization  examinedrization . The LM rization . Additiorization . Manoharrization  also antrization . Tuberizrization , signifyrization . FT generization , tomato rization , sugar brization , and wherization .Genes encrypting FT-like proteins show a main part in monitoring both onion bulb enlargement and flowering. A model of the roles of FT-like genes in the periodic switch of bulb crops growth specified that in juvenile plants and those grown under a non-inductive photoperiod, AcFT4 prevents the up-regulation of AcFT1. Once the plants are mature and the day length holds a risky length, AcFT4 is down-regulated and AcFT1 is up-regulated, leading to the initiation of bulb enlargement. Constant with this prototypical, constitutive expression of AcFT4, AcFT1 up-regulation and bulb enlargement are both prevented. In sugar beet, two FTs with different expression profiles and antagonistic purposes control flowering are involved ,221. RasMolecular genetic analysis in a wide collection of plants has exposed that sequence variations in mechanisms of the circadian clock ,225,226 Substantial progress has been made in understanding the mechanisms regulating the initiation of bulb enlargement in plants. Several genes have been identified to control bulb crops, and alterations of their expression levels confirms the regulation of bulbing. Critical detections have been the outcome that clock mechanisms and a CO-FT component connected to that testified in crops such as Arabidopsis are concerned in the day-length switch of bulb enlargement, while as high gibberellins persuade the enlargement of these tissues in types where this development has been evolutionarily stifled. Bulbs can be tempted in axillary buds of stem node models, and it is being established that phytohormones play an imperative role in axillary meristem instigation and obligation for bulb growth and enlargement. Florigen FT proteins control the axillary shoot branching, and it will be vital to evaluate if extra FT family affiliates are similarly concerned in the bulbing modification. It will be interesting to examine if bulb initiation by high-temperature frameworks demonstrate leading fundamentals with the photoperiod regimes. More importantly, bulb growth and development in geographical regions with low temperatures that pacified the cultivation of these bulb crops might be interesting. Furthermore, the main emphasis of future research should be on the identification of genes and gene products controlling the bulbing parameters under different photoperiodic environments. Identification of both host as well as photoperiodic specific protein factors regulating the synergetic association and the key cellular and metabolic pathways under different photoperiodic conditions can be hot areas for future research. Understanding the photoperiodic-induced modulations in the bulbing mechanisms and the cross-talk mechanism triggered to regulate the bulbing performance can help improve crop productivity. Taken together, the photoperiod must be explored at all levels to further investigate their role in nature as an environmental cue for managing and improving horticultural production."}
+{"text": "Trichinella spiralis muscle stage larvae (mL1) produce excretory-secreted products (ESPs), a complex mixture of protein, which are believed to be important for establishing or maintaining an infection niche within skeletal muscle and the intestine. Studies of both whole ESPs and individual cloned proteins have shown that some ESPs are potent immunogens capable of eliciting protective immune responses. Here we describe two novel proteins, Secreted from Muscle stage Larvae SML-4 and SML-5 which are 15 kDa and 12 kDa respectively. The genes encoding these proteins are highly conserved within the Trichinellids, are constituents of mL1 ESP and localized in the parasite stichosome. While SML-5 is only expressed in mL1 and early stages of adult nematode development, SML-4 is a tyvosylated glycoprotein also produced by adult nematodes, indicating it may have a function in the enteral phase of the infection. Vaccination with these proteins resulted in an impaired establishment of adult stages and consequently a reduction in the burden of mL1 in BALB/c mice. This suggests that both proteins may be important for establishment of parasite infection of the intestine and are prophylactic vaccine candidates. Author summaryTrichinella spiralis. These worms cause infection when L1 (newborn) larvae migrate and invade skeletal muscle remodeling muscle cells into long-lived nurse cells which serve as reservoirs for the parasite. The establishment of nurse cells allows T. spiralis to better survive within the host because factors, such as blood vessel formation, bring required components for survival to the worms. We previously found three novel proteins termed Secreted from Muscle stage Larvae (SML)-1, -2, and -3 by transcriptomic analysis. In this study, we further expand our previous findings by identifying two novel low molecular weight proteins SML-4 and SML-5, which can elicit protective responses against challenge infections with T. spiralis.Trichinellosis, also known as trichinosis, is a disease caused by consuming raw or undercooked meat from animals infected with muscle stage larvae of  Trichinella spiralis is a parasitic nematode that can infect a wide variety of mammalian hosts, including humans. During T. spiralis\u2019s life cycle this parasite occupies two intracellular niches within its host, skeletal muscle  and intestinal epithelial cells . In these environments the parasite remodels the cells or tissues it inhabits. Skeletal muscle fibers are remodeled by newborn larvae (NBL) into a novel structure called a \u201cnurse cell,\u201d which serves as a long-term home as they develop into mL1[T. spiralis and other Trichinellids remain important human and agricultural pathogens with millions of people worldwide chronically infected, and are classified as a neglected tropical disease [ into mL1. The nem into mL1\u20138, espec disease ,10.Trichinella have helped define the processes and cells underlying the development of protective immunity to enteral and muscle stages of the parasite. Time course experiments have shown that parasite ESPs are rapidly recognized by the immune systems of rodents and both protein and glycan antigens can elicit protective immune response [T. spiralis ESPs along with identification of the key determinants that influence their effectiveness in vaccination trials will provide a platform for building and refining general strategies for vaccination against Trichinella and other medically or agriculturally important nematode pathogens [Infection of laboratory mice with response ,12. In aresponse ,14. Efforesponse . To dateresponse ,18,19. Hathogens .T. spiralis transcriptome that identified three novel proteins secreted by mL1 [T. spiralis challenge infections. Based on these initial proof of principle results and parallel studies in other parasitic nematodes, we suggest that these two novel T. spiralis ESP proteins are key vaccine antigens worth exploring in vaccine development programs.We have previously published the results of an informatics-based screen of the d by mL1 . Here, wThe study was approved by the Imperial College Ethical Review Committee and performed under license from the UK Home Office (PPL: 70/6073) and the University of Utah Institutional Animal Care Use Committee (Protocol Number 17\u201304002). All procedures were carried out in accordance with institutional guidelines for the care and use of animals.ad libitum. mL1 of T. spiralis (T1 ISS930) were recovered from chronically infected Sprague\u2013Dawley rats (2 months post-infection or more). Larvae were isolated and ESPs collected after culturing in serum-free RPMI-1640 for 72\u201380 hours as described in Gounaris et al. [Mice and rats were purchased from Harlan  or Jackson Laboratories . Food and water were available s et al. . Adult ns et al. . NBL wern-octyl \u03b2-D-glucopyranoside and SigmaFast Protease Inhibitor cocktail). Parasite soluble protein extracts were then extracted after 30 minutes incubation on ice, followed by brief sonication  and centrifugation at 15,000 x g for 15 minutes; SXT were stored at -20\u00b0C. The protein content of concentrated ESPs and SXTs was determined by the BCA (Pierce) or Bradford (BioRad) microplate assay.Soluble whole worm extracts (SXT) were prepared by disruption of nematodes first in a custom-made Bessman tissue pulverizer and protein extraction buffer  datasets were locally searched for open reading frames predicted to encode secreted proteins [The informatic procedures used to identify SML-4 and SML-5 have been previously published in Guiliano et al. . Brieflyproteins . cDNA clde novo gene prediction was performed using Artemis and BLAST sequence alignments [T. spiralis life cycle stages was analyzed by reverse transcriptase (RT)-PCR. T. spiralis nematodes were flash frozen in Trizol (Invitrogen) and disrupted using a custom made Bessman tissue pulverizer and RNA isolated according to the manufacturer\u2019s protocols. Isolated RNA was treated with RNase-free DNAse and then further purified using RNAeasy mini column purification (Qiagen). Reverse transcription was performed using 2\u03bcg of total RNA, Superscript II and oligo (dT) (Invitrogen) according to the manufacturer\u2019s protocols. PCR was performed with gene specific SML primers , standard reaction conditions and PCR cycle parameters  and Taq polymerase (NEB). PCR reactions were resolved on 2% agarose gels, visualized with ethidium bromide and documented. T. spiralis alpha tubulin was used as an internal positive control , and reactions with no reverse transcriptase were used as negative controls.For each candidate protein, a corresponding full-length cDNA sequence was identified in GenBank: SML-4, ps46d08.y1 and SML-5, ps03c05 (GenBank accession numbers BG521264.1 and BG520988.1). cDNA clones were obtained from Washington University  using the nematode cDNA/EST clone provision service, and the sequences confirmed using vector and gene-specific primers. The verified sequences were deposited in GenBank . SML-4 and SML-5 gene family members from other Trichinellids were identified within the assembled Trichinellid genome sequences deposited in Genbank using BLAST . When reignments . Multiplignments ,34. For NdeI and XhoI sites of the Escherichia coli expression vector pET-29b (Novagen). Proteins were expressed using either standard IPTG induction or via culture in Overnight Express media . Insoluble recombinant proteins were purified under denaturing conditions using nickel affinity chromatography and His-Bind Quick 900 cartridges  or Ni-Sepharose FF using an FPLC system. Denaturing agents were then removed by dialysis or on-column refolding performed on an FPLC using Sephacryl S200-HR resin and a redox-refolding buffer. Proteins were buffer exchanged into PBS, concentrated and then quantified by the BCA or Bradford assay. SDS-PAGE and Coomassie blue staining was used to assess the purity of recombinant proteins. Endotoxin was removed from protein samples using Pierce High Capacity Endotoxin Removal Columns (Thermo Scientific). Following endotoxin removal, the Pierce Chromogenic Endotoxin Quant Kit (Thermo Scientific) was used to determine the success of endotoxin removal.Open reading frames corresponding to predicted mature proteins of SML-4 (amino acids 23\u2013140) and SML-5 (amino acids 21\u2013109) were fused to a poly-histidine epitope tag (His-tag) by cloning into the Polyclonal antisera to SML-4 and SML-5 for use in Western blots, IHC and immunogold transmission electron microscopy (TEM) was produced in BALB/c mice. Antigens were emulsified in 1:1 ratio of Freund\u2019s incomplete adjuvant (FICA) to a final concentration of 1mg/mL by light sonication. Mice were given a primary immunization 50\u03bcg of antigen and FICA via intraperitoneal injection. After four weeks animals were boosted twice, three weeks apart, with each boost containing 25\u03bcg of recombinant SML-4 or 5 in FICA. Antibody responses to SMLs were quantified by ELISA and Western blotting prior to final bleed and collection of serum.For vaccination against SMLs BALB/c mice were given a primary immunization of 100\u03bcg of recombinant SML-4 or SML-5 absorbed onto 1mg Imject Alum (Sigma-Aldrich) via intraperitoneal injection. Animals were rested for three to four weeks and then boosted twice with 25\u03bcg of recombinant SML-4 or SML-5 in alum allowing a one-to-three-week interval between boosts. Unvaccinated animals were given intraperitoneal injections of PBS in alum following to the same schedule as vaccinated animals.E. coli lysate (Promega). Antibody binding was visualized using standard procedures with horseradish peroxidase (HRP)-conjugated anti-mouse IgG secondary antibody (BioRad) and chemiluminescent detection .Protein samples were resolved by 12 or 15% SDS-PAGE. For Western blot analysis, 0.5\u03bcg of recombinant or 10\u03bcg of ESP or soluble crude extract (SXT) protein samples were resolved by SDS-PAGE, transferred onto polyvinylidene fluoride (PVDF) membranes, and blocked with 5% skimmed milk powder/PBS/0.1% Tween-20 for 1 hour. Blots were then incubated at 4\u00b0C overnight with anti-SML-4 or anti-SML-5 murine polyclonal antisera diluted (1:200) in blocking solution and pre-absorbed with -1 of enzyme added and the reaction incubated for 12 hours at 37\u00b0C. PNGase F reactions were resolved by SDS-PAGE and visualized by Western blotting using SML-4 or SML-5 specific antisera.Deglycosylation of ESP using N-Glycosidase F  was performed according to the manufacturer\u2019s protocol. Briefly, 10\u03bcg of crude protein extract was denatured, 50 U mlE. coli-expressed recombinant SML-4 and SML-5 was examined by Western blot using pooled sera collected from chronically infected BALB/c mice. Antibody binding was detected using goat anti-mouse IgG-HRP .The antigenicity of the T. spiralis were collected from rats 40 days post-infection. Tissue or isolated mL1 were fixed in 10% neutral buffered formalin overnight and then embedded in paraffin according to standard protocols. Paraffin sections were de-waxed with Histoclear, rehydrated, and antigen retrieval was performed by microwaving sections in a solution of citrate-based antigen unmasking solution (Vector Labs) for 15 minutes. Sections were incubated in 2% H2O2 to remove endogenous peroxidase activity and then blocked in PBS/5% normal goat serum/5% BSA for 1 hour at room temperature. Endogenous biotin and streptavidin binding sites were blocked using a commercial blocking kit (Vector Labs). Sections were incubated overnight at 4\u00b0C with murine anti-SML-4 or anti-SML-5 (diluted 1:100\u20131:200) in blocking solution, washed thoroughly with wash buffer, incubated with biotin-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Labs) for 1 hour, washed again and detected with Streptavidin-HRP complex and DAB substrate . Sections were counterstained with Mayer\u2019s hematoxylin, mounted with DPX mounting media (BDH), and visualized by light microscopy.Muscles infected with T. spiralis were fixed for 1 hour in 0.25% glutaraldehyde and 1% sucrose in 0.1M phosphate buffer pH 7.4 and then processed for immunoelectron microscopy as previously described [T. spiralis. For immuno-localization of the native parasite protein corresponding to recombinant SML-4 and SML-5, thin sections (70 nm) of embedded worms were incubated with anti-SML-4 and anti-SML-5 murine polyclonal antisera (1:20 dilution), followed by incubation with 15 nm gold particles coated with goat anti-mouse IgG . Pre-immune serum was used as the control.For immunogold TEM, adult escribed ,36. mL1 ELISAs for recombinant SML-4 or SML-5 proteins were performed using 96 well plates (Nunc Polysorp) coated with 50\u03bcL of 0.05 M carbonate buffer containing 5\u03bcg/mL of recombinant SML-4 or SML-5. The plates were incubated at 4\u00b0C overnight. Plates were blocked with 1% BSA or 5% non-fat milk and sera samples were loaded at a dilution of 1:200 or 1:1000. After an overnight incubation, 50\u03bcL of anti-mouse detection antibody conjugated to horseradish peroxidase was added according to the manufacturer\u2019s instructions . Bound antibodies were detected using 100\u03bcL/well of 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) Super AquaBlue ELISA substrate (Invitrogen) and readings were taken at a wavelength of 405 nm.6 splenocytes per well were stimulated in 96 well round bottom plates for 72 hours with 20 \u03bcg endotoxin-free recombinant SML-4 or SML-5 protein. Concanavalin A  was added at a concentration of 1\u03bcg/mL as a positive control stimulus, and media alone served as a negative control. Supernatants were analyzed by Luminex  according to the manufacturer\u2019s instructions. After 72 hours, splenocytes were transferred to a fresh 96 well flat-bottomed plate containing bound \u03b1-CD3  and cells were restimulated for 6 hours along with soluble \u03b1-CD28  with the addition of Brefeldin A (Biolegend) in the last 4 hours of stimulation. After blocking with addition of 10 \u03bcg/mL CD16/32 , cells were then surface stained for CD4 , CD44  and zombie live/dead . After fixation and permeabilization using Fix/Perm buffer (BD) cells were intracellularly stained for IFN-\u03b3  and IL-4 . Cells were read on an LSRFortessa X-20 flow cytometer (BD) and data analyzed using FlowJo software.Spleens were removed from vaccinated animals one week after the second boost, and splenocytes isolated aseptically using standard methods. 1 X 10Groups of 8\u201310 female BALB/c mice 4\u20136 weeks old were purchased from Harlan  or Jackson Laboratories  and immunized as described above. Pre-vaccination control sera were collected prior to vaccination. Control animals were immunized with PBS and alum.T. spiralis (high dose) mL1. Forty days post-infection serum was collected from infected animals and the number of encysted mL1 recovered from each animal quantified after digestion of mouse carcasses to assess the effect of vaccination on expulsion of adult worms. This was further verified by enumerating the recovery of adult worms from the intestines of BALB/c mice infected with 500 T. spiralis (high dose) mL1 at days 2, 6, 9, and 12 post-infection. Briefly, the intestine was opened lengthwise and rinsed with PBS and adults were collected by sedimentation using a Baermann funnel before being counted.Immunogenicity of the vaccines was checked by ELISA one week after the second boost or by western blot before mice were challenged with either 250 (moderate dose) or 500 T. spiralis mL1 and adult worm recovery were analyzed using ANOVA or General Linear Modeling to remove the effects of experimental variation (Minitab Inc). Dunnett\u2019s pairwise comparison post-test was used to determine differences between experimental groups. Where duplicate experiments were carried out, experimental consistency was evaluated by inclusion of a vaccination-by-experiment interaction term in the model and was not found to be statistically significant. Residual variation was analyzed for normality (Anderson Darling test) and homogeneity of variance (Levene\u2019s test) in each case. The mL1 and IgG2a data were logarithmically transformed, and the adult recoveries (n+1) were square root transformed to meet the requirements of parametric testing.All experiments, with the exception of adult recoveries over 4 different time points and high-dose challenge , were unet al. [T. spiralis mL1 were also identified and include gp45, gp43, Ts53, prosaposin, and GM2A [The candidate secreted proteins abundantly represented in the EST datasets isolated in the informatics-based analysis previously described by Guiliano et al.  are showet al. . A numbeand GM2A .T. spiralis genome [Trichinella T6 genome indicates that SML-4 is present as a multicopy gene family with three genes identified in the Trichinella T6 genomic contigs submitted to Genbank. Similarly, analysis of SML-5 indicates it is present as a multicopy gene family in Trichinella nativa. The predicted polypeptide of SML-4 contains two potential N-glycosylation sites  and excretory-secreted proteins (ESP). Murine antisera generated against SML-4 detected two protein species migrating at 16 and 18kDa  , but the SML-5 vaccinated animals did not show a statistically significant difference in mL1 recovery as compared with control animals . This indicates that SML-4 is a more effective vaccine candidate when compared to SML-5.BALB/c mice vaccinated with SML-4 or SML-5 protein were challenged with a moderate (250) or high (500) dose of infective mL1. At forty days post-infection, the number of encysted mL1 collected from vaccinated mice challenged with a moderate dose was significantly lower compared to the control group  . There w2,75 = 26.83, p = 0.000; Dunnett\u2019s post-test, p<0.05 for both antigens). This protective effect was apparent from day 2 post-infection  showing a 67% and 48% reduction in adult recoveries for SML-4 or SML-5, respectively . The efficacy of the single antigen vaccine is slightly lower than vaccinations with whole ESP which can reach 70\u201390% reductions in adult recovery but similar if not better than many previously published vaccines based on single recombinant protein proteins [To determine if the reduced larval burden after vaccination with SML-4 or SML-5 is a result of reduced establishment of adult worms in the intestine, the adult worm recovery from groups of vaccinated mice that were challenged with a moderate dose of infective mL1 was quantified at 2, 6, 9, and 12 days post-infection . Vaccinaproteins ,21,23\u201325T. spiralis has facilitated the identification of numerous new ESP proteins. Building on the previously published analysis of SML-1, -2 and, -3 [Analysis of ESTs from different lifecycle stages of the parasitic nematode While it is difficult to speculate what the functions of these two proteins might be, their differential expression and localization suggests they may have roles linked to the development of mL1 into adults and/or the parasite\u2019s transition into the enteral environment. The presence of SML-4 throughout this phase of the parasite\u2019s lifecycle hints that its function is requisite for successful adaptation and persistence within the enteral niche. This contrasts with the restricted expression of SML-5 in the early stages of intestinal invasion, which could indicate that it might have a role more specific to the initial phases of infection or developmental processes, like molting, which occurs during the first two days of infection. While both SML-4 and SML-5 are constituents of mL1 ESP, protein localization studies do not indicate they are secreted into the nurse cell. However, given the specific limitations of these techniques, such as loss of antigenicity after fixation and low protein concentrations outside the nematode, we cannot exclude the possibility they are secreted into the nurse cell and participate in its development or maintenance.Recombinant SML-4 and SML-5 can act as potent immunogens when administered to BALB/c mice with alum. Surprisingly, assessment of the cellular and humoral responses to these proteins indicates that despite being administered with alum, which promotes the development of Th2 responses, both proteins are capable of eliciting Th1 (SML-4) or mixed Th1/Th2 (SML-5) responses and mixed IgG Th1/Th2 associated isotype production. Preparations of recombinant SML proteins were routinely tested for LPS contamination prior to use  and low levels of residual endotoxin cannot fully explain the development of the Th1 or mixed Th1/Th2 responses we observe after immunization with these proteins. Potentially, these recombinant proteins have inherent polarizing Th1 properties which are skewing the development of balanced Th1/Th2 away from Th2 known to be activated in alum formulated vaccines. Such a potential bias has been observed with other nematode derived proteins  and may Trichinella antigens.Vaccination of BALB/c mice with these proteins prior to challenge infection has shown that both proteins are able to induce high levels of immune responses eliciting protective immunity. Assessment of infection time courses show that parasite attrition happens at early time points post-infection, indicating protective immune responses may be targeting the parasite during this critical point of adaptation and establishment in the enteral niche. In general vaccination generates adaptive immune responses against the target antigen(s) that can neutralize pathogens and pathogen-derived products. Although the majority of vaccinations are parenterally administered, immunity can extend to specific organs of the body. Given that SML-4 and SML-5 are secreted proteins from mL1, and that mL1 emerge into the small intestine which would have mucosal antibodies that recognize SML-4 and SML-5 from vaccination as measured in T. spiralis transcriptome continues to yield novel proteins whose biological or immunological properties may be important in mediating evasive host-parasite interactions and/or the development of protective immune responses. Moving forward, while the biological functions of SML-4 and SML-5 remain unknown, we have established their efficacy as novel vaccinogens. Further work refining the antigenicity and mechanisms of action of these novel proteins will provide critical clues to design anti-Trichinosis vaccines with increased protective efficacy.Analysis of the S1 FigT. nativa , Trichinella , T. pseudospiralis , T. papuae  and T. zimbabwensis  genomes. The location of the conserved intron site is indicated with a red triangle and potential N-glycosylation sites with the blue hexagons. (B) A rooted cladogram showing the consensus of the tree found in the maximum parsimony analysis of the SML-4 protein sequence alignment. Nodes which are supported bootstrap values of > 60% are shown and the bootstrap values are placed at the base of the node.(A) A multiple protein sequence alignment yielded by ClustalX analysis of the 16 SML-4 homologues found in sequenced genomes of Trichinellid species contained in Genbank. The Genbank accession number of the protein is given along with the species/isolate identifier. In five instances a manual genome prediction (MGP) was required to refine gene prediction models obtained from searching contigs isolated from the (TIF)Click here for additional data file.S2 Fig(A) A multiple protein sequence alignment yielded by ClustalX analysis of the 16 SML-5 homologues found in sequenced genomes of Trichinellid species contained in Genbank. The Genbank accession number of the protein is given along with the species/isolate identifier. The location of the conserved intron site is indicated with a red triangle. (B) A rooted cladogram showing the consensus of the tree found in the maximum parsimony analysis of the SML-5 protein sequence alignment. Nodes which are supported bootstrap values of > 60% are shown and the bootstrap values are placed at the base of the node.(TIF)Click here for additional data file.S3 FigFlow cytometry of activated CD4+ cells following recall responses in BALB/c mice vaccinated with SML-4 (A) or SML-5 (B) protein in alum adjuvant. Cell stimulations  are indicated within respective flow plots. Figures are representative of two independent experiments with n = 5 per group.(TIF)Click here for additional data file.S1 TableT. spiralis mL1 EST dataset with their names and Genbank accession numbers. The columns next to the identifier show the number of ESTs for each transcript that were identified in the T. spiralis mL1 and adult EST datasets. Homologies to sequences contained within Genbank, the NEMBASE4 cluster (accessed 08/11/19) and number of ESTs derived from these clusters are shown [This table shows the top 11 candidates identified in the informatics screen of the re shown . No NBL (DOCX)Click here for additional data file."}
+{"text": "HL skills and perceived needs for support were identified through thematic analysis. Women appeared to be highly engaged in the decision-making process. They mentioned searching and selecting general information about pregnancy and labor, constructing their preferences based on their own pre-existing knowledge and experiences and by discussions with partners and significant others. However, women with low basic skills and primigravida perceived difficulties in finding reliable information, understanding probabilistic information, constructing preferences based on benefit/harm information and preparing for consultations. Women also emphasized dealing with uncertainties, changing circumstances of pregnancy and labor, and emotions. Maternity care professionals could further support clients by guiding them towards reliable information. To facilitate participation in decision-making, preparing women for consultations  and supporting them in a timely manner to understand benefit/harm information seem important.Shared decision-making requires adequate functional health literacy (HL) skills from clients to understand information, as well as interactive and critical HL skills to obtain, appraise and apply information about available options. This study aimed to explore women\u2019s HL skills and needs for support regarding shared decision-making in maternity care. In-depth interviews were held among women in Dutch maternity care who scored low ( Clients  in maternity care are expected to participate in decisions about available options during pregnancy and labor . For manA typical decision-making process requires women to be able to access, understand and apply benefit/harm information to make informed decisions, as well as to communicate effectively and exchange information with maternity care professionals, such as midwives, obstetricians and maternity care nurses . These sMaternity care professionals usually play an important role in helping women to find and interpret information of available options. They also typically support pregnant women to make their own decisions during pregnancy, birth and postnatal . As cliePrevious research showed that SDM positively influences satisfaction, adherence and well-being among patients making longer-term decisions and/or having chronic diseases . SDM praWhat health literacy skills do women in maternity care use for (shared) decision-making?What needs for support in shared decision-making do clients in maternity care perceive?There has been little attention for SDM practiced in the maternity care setting and the specific needs and skills in decision-making of clients. Therefore, this qualitative study aimed to explore skills and needs for support regarding SDM in maternity care, by building on the perspective of clients. The research questions were:In this qualitative study, we conducted individual face-to-face interviews with clients in maternity care from March to July 2019 in the Netherlands. A qualitative design was chosen to explore the perspectives of clients regarding skills they use in the decision-making process and their needs for support in SDM. According to Dutch law, this study was waived from requiring medical ethical approval (W18_307). We guaranteed the anonymity of the participants and ensured that written informed consent was obtained from all participants prior to conducting the interviews.The research population involved (pregnant) women with varying reading, understanding and calculating skills in a health context or \u2018basic skills\u2019 to achieve a representative sample of clients. Women with a gestational age of 25 weeks onwards and women who had given birth in the past four months were included. From 25 weeks of pregnancy, women would have had enough experiences to discuss decisions they have or had to make during their pregnancy, e.g., on pain relief during labor or place and mode of labor. To discuss the decisions that women make after birth , we chose to include women until four months after birth.Participants were recruited from primary and secondary care practices in representative areas in the Netherlands for a mixed demographic using various strategies. Childbirth classes and pregnancy courses were visited by the first author (L.M.) to recruit pregnant women personally. Participants were also personally recruited in the waiting rooms of midwifery practices and child health clinics. Maternity nurses and obstetricians were asked to inform eligible women about our study and sent their contact details to the research team after receiving the consent of their client. The interviewer (L.M.) contacted the client for further information about the study and to make an appointment. We also used an online approach through a Dutch client organization of pregnant women and mothers (Geboorte Beweging) and social media.The qualitative interviews took on average 0.5\u20131 h each (time to fill in the questionnaire and to take the basic HL screening tests is excluded in the interview time) and took place at a location the participant preferred, such as her home. A translator was present during one interview. Clients\u2019 partners were allowed to be present during interviews, but did not participate. We estimated women\u2019s basic understanding and reading skills with the Short Assessment of Health Literacy-Dutch (SAHL-D) and calculating skills with the Newest Vital Sign-Dutch (NVS-D) (see below). We used two measures to assess self-reported HL of a non-Dutch speaking participant, because it was not possible to measure basic reading, understanding and calculating skills ,14. BasiMcCaffery\u2019s framework  was usedThe following variables were assessed orally before the interview: educational level, ethnic background and relationship status. Pregnancy-related outcomes assessed before the interview included: parity, gestational age or the number of weeks postpartum.Measures that are often applied to assess performance-based basic literacy and numeracy skills are the SAHL-D and NVS-D. The SAHL-D assesses both word recognition and comprehension in the health domain. It contains single words that refer to medical specialties, tests, treatments and symptoms. Participants have to select the correct meaning of each word, using a multiple-choice format. For the SAHL-D, individuals with <9 out of 13 correct responses were considered as having low reading and understanding skills . The NVSWe used two measures to assess self-reported HL of a non-Dutch speaking participant. The set of brief screening questions (SBSQ) in Dutch is a self-reported measure focusing on completing medical forms and reading and understanding medical information. An average score of \u22642 indicates inadequate HL . The PerAll interviews were audio-recorded, transcribed literally by a professional organization and analyzed using thematic analysis . The traAll transcripts were coded by the first coder (L.M.). A second coder (M.E.D.) randomly and independently coded three transcripts in the first stage of coding. M.E.D. and L.M. agreed on the majority of the coding, and the remaining disagreements between authors were solved in consensus sessions. This was an iterative process. The new emergent codes were mapped into themes and subthemes. The decision-making stages (themes) and HL skills (subthemes) were categorized using the framework of decision-making stages and skills as a conceptual framework. Women\u2019s needs for support were explored by analyzing how they used the identified skills and by exploring the difficulties they experienced when using these. Furthermore, we also assessed their perceived needs. We compared the emerging (sub)themes with the framework and assessed which HL skills were mentioned in the framework and not in the interviews, and vice versa. Next, themes and interpretations were discussed between researchers . The final coding scheme was applied to the entire dataset. The analysis was performed using MAXQDA 18.1.1. .In total, 51 women intended to participate and 22 were excluded because of no response or withdrawal. Six women scored high on SAHL-D and/or NVS-D and were excluded when convenience sampling shifted to purposive sampling. We included 23 clients, who were on average 33 years old. Ten participants scored low on either SAHL-D or NVS-D . The parDuring the interviews, women mentioned the following decisions: which pregnancy courses to follow, diabetes test, induction of labor, maternal pertussis vaccination and options for breech birth . In addition, the following decisions were discussed presented in the timeline: place of birth  (discussed 14 times), prenatal screening for Down syndrome (first trimester combined test (FCT) or Non-Invasive Prenatal Testing (NIPT)) (discussed 11 times), pain relief during labor (discussed 11 times), infant feeding (bottle or breastfeeding) (discussed 9 times), healthcare providers (choice between midwifery and maternity care practices) (discussed 7 times) and twenty-weeks ultrasound (discussed 3 times). Women explained that they collected general information about pregnancy from various sources see , some woWomen often stated that they used multiple sources of information to be reassured, to affirm their decision, to develop an overview of options available and/or to prepare for consultations with their professional. The skill \u2018using multiple sources\u2019 was brought forward by low basic skilled women as well as by women with higher basic skills. However, women with lower basic skills stated that they tended to use multiple sources when they were in doubt about their own decision and asked other peers and professionals for reassurance.Women reported that they had appraised information on websites of hospitals, the National Public Health Institute or websites that professionals recommended as trustworthy. They mentioned that an excessive amount of online information is available and that they had to select trustworthy information. Women with higher basic skills stressed that they avoided online fora and social media for information, because they might get worried by reading negative experiences of peers and they wanted to avoid invalid information. Several low basic skilled women stated that they use social media. They reported social media groups for pregnant women to be trustworthy, because it was information provided by women themselves, who had experienced pregnancy and labor.Some women stated that they did not want to gather a lot of information and decided to stop looking for more information. Women with higher basic skills stressed that they wanted to avoid an overload of information, whereas other women with higher and lower reading, understanding and calculating skills indicated that they wanted to receive more information from their professional, for instance about pain relief options. Non-active information seekers were mostly multigravidas, who often mentioned they felt experienced to make a decision. Women with low and higher basic skills both stated that they were often not able to understand medical terminology used by professionals or online. However, women with higher basic skills stated to be able to derive the meaning of medical and pregnancy-related terms from the context, and this was facilitated by repetition in the course of pregnancy. Low basic skilled women reported to be able to derive meaning from context, but also that medical terminology, for instance about the NIPT test, remained abstract for them quote 2, . Women expected their professional to recommend websites with reliable information , had received from their maternity care professional or from peers (both women with low and higher basic skills). Low basic skilled women mentioned that the harms related to pain relief (2 clients), breech birth (1 client) and induction of labor (2 clients) were unclear, which made it difficult for them to decide. High basic skilled women did not mention difficulties in understanding harms and benefits, except for one woman who mentioned that the side effects of a maternal pertussis vaccination were unclear.One woman with low basic skills and two women with higher basic skills seemed to estimate the likelihood of harms  occurring by using heuristics to estimate the risks, for example by estimating how many clients or children they knew who had experienced a certain event. Both high and low basic skilled women explained that they found it important to know probabilities about harms, but also stressed that they had experienced difficulties in interpreting them. One low basic skilled woman explained that risk information provided on the National Public Health Institute\u2019s website was abstract and therefore without meaning for her quote 3, .Both low and higher basic skilled women mentioned having compared options in two ways. Some looked up the advantages and disadvantages of all available options and discussed the information with their partner and/or professional, others compared the information they gathered from various sources themselves. Information about benefits and harms was in general perceived as important by women with low and higher basic skills to be able to make decisions. Women expressed that they needed more benefit/harm information, for example about caesarean section when the baby is in a breech presentation. After seeing the decision aids during the interview, three women explicitly expressed understanding benefits and harms better when it is presented in an overview. Most women preferred to receive benefit/harm information from their professional rather than online, because they trust their health professional\u2019s expertise. To understand the likelihood of harms occurring, some women preferred to read stories of other women, whereas others preferred factual information , articulated their choice to their professional, and discussed their preferences in their conversation with the professional. Women indicated that during their regular check-ups and postnatal care, they had asked questions about what they read to verify if that was correct. One participant, for example, explained that she had difficulties with breastfeeding and asked several care professionals for help quote 8, . Not all women expressed that they were involved in the decision-making process. However, women with low and higher basic skills both expressed that, in hindsight, they found it important to be involved, and to create a sense of control. In addition, they emphasized this because they did not want to regret the decisions made . Most women were aware of the costs and the need for insurance related to certain options, such as receiving care of lactation consultants.Women with low and higher basic skills both mentioned being motivated to be involved in decisions, because of the perceived importance of taking responsibility for their child. It was emphasized that this sense of responsibility made them motivated to gain control over their pregnancy and make well-informed decisions. Responsibility towards partners was also mentioned, i.e., making decisions not only for her child but also her partner\u2019s child  skills and needs for support regarding participation in decision-making of clients in Dutch maternity care. Our findings showed that the HL skills discussed by women largely corresponded with the skills described in the framework of decision-making stages and HL skills developed by McCaffery et al. . AdditioWomen appeared to collect information about pregnancy and labor themselves from an early stage onward. Women with low and higher basic skills both searched for information online, however they appraised online sources in different ways. Women with higher basic skills explained to use websites of professional institutes  and hospitals, whereas women with low basic skills seemed to look for experiences of peers (other pregnant women) online. This information might be easier to understand compared to information from professional institutes that often contains more medical jargon and statistical information. In our study, women with low basic skills explained that they considered anecdotal information, provided by women who went through pregnancy and labor, as reliable. However, the quality of these sources remains questionable, especially when it is used as the only information source. A previous systematic review also showed that low HL individuals use evaluation criteria that do not correspond to established quality criteria . It is aMcCaffery\u2019s framework for decision-making stages and HL skills largely follows a rational decision-making model , based oWe found that women experienced difficulties coping with uncertainty and changing  circumstances, which in turn led to emotional reactions. Women experienced uncertainty related to the unpredictability of pregnancy and/or labor as well as uncertainty related to the unpredictability related to harms occurring. Another study also showed that especially first-time mothers experience uncertainty, e.g., about physical and emotional reactions during labor and birth . MaterniPrimiparous women also dealt with their own uncertainties being in limbo, which was also shown by Soltani et al . Their sA strength of our research is that we managed to represent the general population by including clients with various reading, understanding and calculating levels. However, the mean SAHL-D and NVS-D scores among our low basic skilled group (6 for SAHL-D and 2 for NVS-D) show that we were not able to include the group with lowest basic skills. The group with lowest basic skills might experience more difficulties in applying HL skills to make decisions and may need a different type of support than perceived by our study population.Maternity care professionals could further support women in participating in important decisions during pregnancy and labor. Guiding them towards reliable and easy to understand information is an important first step. Furthermore, preparing women explicitly for consultations , supporting them in understanding and discussing benefit/harm information seem important to facilitate participation in decision-making. To support women in coping with uncertainty and changing circumstances, a narrative can be adopted that emphasizes the ability to change preferences over time in advance of labor. For example, peers\u2019 coping strategies according to changing circumstances could be presented in decision aids. However, narratives should be carefully designed, because it might also bias decision-making.Future research should gain insight into strategies to provide affective meaning to probabilistic information, consequently, to support comprehension of women by meeting their current decision-making style. Our study did not include individuals with the lowest HL levels. Therefore, future research should provide insight into the perceived needs of this group, since they may need a different type of support in (shared) decision-making.Women with various reading, understanding and calculating levels appeared to be highly engaged in the decision-making process. They mentioned to search and select general information about pregnancy and labor, and to construct their preferences based on their own pre-existing knowledge and experiences, and by discussions with partners and significant others. However, not all women were able to find reliable information, understand probabilistic information, construct preferences based on benefit/harm information and to cope with changing circumstances and uncertainties. Especially low basic skilled women and primigravidas perceived needs for support to apply these skills."}
+{"text": "Imax) of AlGaN double channel HEMTs reaches 473\u2009mA/mm with gate voltage of 0\u2009V. Moreover, the superior breakdown performance of the AlGaN double channel HEMTs is also demonstrated. These results not only show the great application potential of AlGaN double channel HEMTs in microwave power electronics but also develop a new thinking for the studies of group III nitride-based electronic devices.In this work, AlGaN double channel heterostructure is proposed and grown by metal organic chemical vapor deposition (MOCVD), and high-performance AlGaN double channel high electron mobility transistors (HEMTs) are fabricated and investigated. The implementation of double channel feature effectively improves the transport properties of AlGaN channel heterostructures. On one hand, the total two dimensional electron gas (2DEG) density is promoted due to the double potential wells along the vertical direction and the enhanced carrier confinement. On the other hand, the average 2DEG density in each channel is reduced, and the mobility is elevated resulted from the suppression of carrier-carrier scattering effect. As a result, the maximum drain current density ( Vb) and improving the device reliability at high temperatures. Therefore, in view of the larger bandgap and superior thermal stability of AlGaN over GaN, AlGaN channel devices have been proposed as promising candidate to further improve the performance limits of nitride HEMTs in high-voltage and high-temperature applications [Group III nitride-based high electron mobility transistors (HEMTs) have been identified as the most promising candidate for next-generation microwave power electronics owing to their fast-switching speed and low-switching loss \u20135. Latelications \u201315.0.53Ga0.47N/Al0.38Ga0.62N HEMTs with the gate-drain distances of 10\u2009\u03bcm [0.40Ga0.60N/Al0.15Ga0.85N HEMTs to 1700\u2009V [2/V\u00b7s, and the records of maximum drain current and Ion/Ioff ratio were reported for the fabricated AlGaN channel HEMTs [VT) of 1.0\u2009V [T of 28.4\u2009GHz and fMAX of 18.5\u2009GHz were determined from small signal S-parameter measurements [Nanjo et al. demonstrated the remarkable breakdown voltage enhancement of AlGaN channel HEMTs, and the obtained maximum breakdown voltages were 1650\u2009V in the Alo 1700\u2009V . Xiao etel HEMTs . Whereafurements . These rHowever, the limitations of the previously reported AlGaN channel devices are equally obvious. On one hand, on account of the ternary alloy disordered scattering effect, the two dimensional electron gas (2DEG) mobility in AlGaN channel is much lower than that in GaN channel. As a result, the current drive capacity of AlGaN channel devices is much lower than that of the traditional GaN channel devices. On the other hand, in order to induce the same amount of 2DEG in AlGaN channel, the AlN component in AlGaN barrier layer should be higher than that of conventional GaN channel heterostructures, which will increase the difficulties in material growth process. These contradictions seriously inhibit the widespread application of AlGaN channel devices, and the optimizations of heterostructure layout are urgently needed.Double channel technique is an intriguing approach to promote the channel carrier density of nitride-based heterostructures without any adverse impact on the electron mobility, and the current conduction capability of the devices will be improved \u201318. Howe0.10Ga0.90N channel layers for the AlGaN double channel heterostructure, corresponding to the double 2DEG channels. In addition, it can be observed that the 2DEG density in upper channel is higher than that in lower channel, which can be explained from two aspects. On one hand, the lower AlGaN barrier acts as back barrier of the upper channel, which is beneficial to promote the 2DEG confinement of upper channel. On the other hand, the main supplying source of the channel 2DEG in nitride heterostructures is the donor-like surface states [The cross-section schematic of the AlGaN double channel heterostructure is shown in Fig. e states , which aFigure 0.10Ga0.90 interfaces. In addition, the carrier distribution properties can be extracted from C-V curve and the result is illustrated in Fig. 19 and 2.5 \u00d7 1019\u2009cm\u22123, which is in accordance with the calculated result as shown in Fig. 13\u2009cm\u22122 and 1130\u2009cm2/V\u2219s by the Hall effect measurement.Capacitance-voltage (C-V) measurement with mercury-probe configuration was performed to investigate the double channel characteristics of the heterostructure. As shown in the inset of Fig. 2 atmosphere. Then, the mesa isolation was performed by Cl2/BCl3 inductively coupled plasma etching to a depth of 200\u2009nm, and 100-nm-thick SiN passivation layer was formed by plasma-enhanced chemical vapor deposition. Afterwards, a gate area with a length (LG) of 0.8\u2009\u03bcm was defined by photolithography after etching the top SiN with CF4 plasma, and then a Ni/Au schottky gate electrode was deposited. The gate-source (LGS) and gate-drain (LGD) distances are 0.8 and 1\u2009\u03bcm, respectively. For comparison purposes, two additional HEMTs samples based on the conventional AlGaN single channel and GaN double channel heterostructures were also fabricated, and the cross-section schematics are shown in Fig. The standard HEMTs fabrication process was carried on the AlGaN double channel heterostructure. The device fabrication process started with ohmic contact formed with Ti/Al/Ni/Au multilayer metal stack deposited by electron beam evaporation, followed by a rapid thermal anneal at 850\u2009\u00b0C for 30\u2009s in NGS and VDS were swept from 0~\u2212 10\u2009V and 0~10\u2009V. The maximum drain current density (Imax) and differential on-resistance (Ron) of the AlGaN single channel sample are 265.3\u2009mA/mm and 27.1\u2009\u03a9\u2219mm, respectively. These results are in accordance with the previous reports, suggesting the deficiency of AlGaN channel HEMTs in current drive capacity. For the AlGaN double channel HEMTs, the Imax dramatically increases to 473\u2009mA/mm, which is 1.8 times higher than that of AlGaN single channel HEMTs. We attribute the improvement in Imax to the superior transport properties of the AlGaN double channel heterostructure. On one hand, double channel structure possesses two potential wells along the vertical direction, and the carrier storage capability of the AlGaN conduction channel is promoted. On the other hand, although the total channel carrier density is increased, the average electron density in each channel is reduced. As a result, the carrier-carrier scattering effect is suppressed and the channel electron mobility is elevated. However, it can be observed that the Ron of AlGaN double channel HEMTs is 12.5\u2009\u03a9\u2219mm, still larger than that of GaN double channel HEMTs. This phenomenon is related to the large contact barrier height of the AlGaN barrier layers, for which the AlN composition is as high as 31%. Due to the self-heating effect resulted from the high power dissipation, the negative differential resistance effect can be observed for the GaN double channel HEMTs when VGS >\u2212 4\u2009V and VDS> 6\u2009V. Nevertheless, for the AlGaN channel HEMTs (both single channel and double channel), this negative differential resistance effect is significantly suppressed, manifesting the superior performance of AlGaN channel HEMTs in elevated temperature conditions.The typical output characteristics of the HEMTs are illustrated in Fig. VDS of 10\u2009V. The AlGaN single channel HEMTs exhibit a threshold voltage (VT) of \u2212 3.8\u2009V, together with an inferior peak extrinsic transconductance  of 80.5 mS/mm in the vicinity of VGS = \u2212 1.5\u2009V. For the AlGaN double channel and GaN double channel HEMTs, the VT remarkably decreases to \u2212 9.2 and \u2212 11.2\u2009V, which is resulted from the high channel carrier density and the relatively long distance from the gate electrode to the lower 2DEG channel. The high VT may result in high power loss of the devices at off state, and this issue can be improved by further optimizing the growth parameters of double channel structures, such as properly reducing the thickness of barrier and upper channel layers. Specially, double-hump characteristics can be observed of the transconductance curves of AlGaN double channel and GaN double channel HEMTs. For the AlGaN double channel HEMTs, two peak values of 97.9 and 42.5 mS/mm can be extracted at VG = \u2212 1.0 and \u2212 6.0\u2009V. The sub-peak value reaches 43% of the Gm,max, indicating the decent gate-control ability and linearity of the AlGaN double channel HEMTs. Moreover, based on our previous research achievement [Figure ievement , the resVb is defined by the criteria of leakage current reaching 5\u2009\u03bcA/mm. It can be observed that all the three samples present hard breakdown characteristics, and the breakdown performance of AlGaN channel HEMTs is obviously better than that of the GaN channel HEMTs. The Vb of the AlGaN double channel HEMTs is 143.5\u2009V, more than two times higher than that of the GaN double channel HEMTs. Taking the LGD = 1\u2009\u03bcm into consideration, the Vb,standard (defined by Vb/LGD) is as high as 143.5\u2009V/\u03bcm for the AlGaN double channel HEMTs. The Imax and Vb,standard results of the AlGaN double channel HEMTs in this work are benchmarked against the GaN channel and AlGaN channel HEMTs reported by other groups in Fig. Imax. Noticeably, the Imax of the AlGaN double channel in this work is comparable to most results of the GaN channel HEMTs. Moreover, it is necessary to note that the Imax value in this work is obtained at VGS = 0\u2009V, which is conservative and can be further improved by applied positive gate voltage. Therefore, these results convincingly demonstrate the enormous potential of AlGaN double channel HEMTs in microwave power device applications.The off-state breakdown characteristics of the HEMTs based on different heterostructures are measured and shown in Fig. In summary, AlGaN double channel heterostructure is proposed to fabricate high performance HEMTs. The superior transport properties of AlGaN double channel heterostructure is revealed, leading to the improved current drive capability of the HEMTs. In addition, the excellent breakdown performance of the AlGaN double channel HEMTs is demonstrated. The results in this work show the great potential of AlGaN double channel devices in microwave power applications in the future."}
+{"text": "Preeclampsia (PET) is a multisystem inflammatory disorder that represents a leading cause of feto-maternal morbidity and mortality, complicating 2\u20135% of all pregnancies. PET incurs an increased risk of venous thromboembolism, which is one of the leading causes of death in pregnancy and in the postpartum period. This prothrombotic phenotype is attributable to the maternal phase of PET, which is characterized by a systemic inflammatory response and coagulation activation. Research continues to be undertaken in terms of preventative measures, however, currently revolves around pharmacological low dose aspirin initiated in the first trimester of pregnancy for those with risk factors. Treatment involves antenatal corticosteroids for fetal lung development in preterm birth, parenteral magnesium sulfate for fetal neuroprotection and maternal seizure prophylaxis, and timely birth of the fetus and placenta being the only definitive treatment of PET. Patients with a venous thromboembolism (VTE) risk deemed to be >1\u20133% are treated with pharmacological thromboprophylaxis in the form of low molecular weight heparin. Completing each woman\u2019s VTE risk assessment is crucial, particularly in the setting of PET, as there is also a proven associated competing hemorrhagic risk. Preeclampsia (PET) complicates 2\u20135% of all pregnancies and represents a leading cause of feto-maternal morbidity and mortality worldwide \u20133. PET iRisk factors for PET include history of PET, chronic hypertension, pregestational diabetes mellitus, multiple pregnancy, obesity, and antiphospholipid syndrome , 12. WomPreeclampsia pathophysiology is considered to occur in two stages in the first trimester and 2nd/3rd trimester .Reduced oxygen tension and persistent hypoxia appear to play an important role  which plays a role in vascular remodeling  remains a leading cause of death in pregnancy and in the postpartum period . During Under normal physiological circumstances, pregnancy is characterized by the development of a hypercoagulable state, characterized by an increase in procoagulant factor activity and a down-regulation of endogenous anticoagulant and fibrinolytic pathways. It is postulated that this hypercoagulable state develops to limit the risk of major bleeding associated with labor and birth , 62. AltThis baseline pregnancy-associated elevated thromboembolic risk is increased in the presence of additional VTE risk factors. These risk factors may pre-date pregnancy, arise during pregnancy or occur peripartum, highlighting the crucial importance of performing a VTE risk assessment at several times during pregnancy and at labor and birth. A Norwegian register-based case-control study including 600,000 pregnancies reported a four-fold increased risk of VTE in patients with PET in the postpartum period, however, no association was identified between VTE and PET in antepartum period . These rMechanisms which may underly this prothrombotic phenotype can be attributed to the maternal phase of PET which is characterized by a systemic inflammatory response accompanied by coagulation activation . The incUnder normal physiological conditions, the endothelium includes an intact, negatively charged, and non-adhesive glycosaminoglycan layer which acts to inhibit thrombin generation and the adhesion of platelets and leucocytes . This enAside from endothelial dysfunction, relative to normal pregnancy, PET is characterized by alterations in circulating platelet-derived microparticle (MP) and extracellular vesicle (EV) profiles, which may contribute to the PET-associated VTE risk, although a proven mechanistic association has not yet been defined .Preeclampsia prevention and treatment continues to be investigated in ongoing studies. Simpler approaches have explored hygienic and dietetic strategies. Measures including bed rest , sodium Currently, PET prevention centers around low dose aspirin. In 2019, a Cochrane meta-analysis of 77 trials  determined that the risk of pre-eclampsia was 18% lower with low dose aspirin  . In the Optimal timing of initiation and dose remain uncertain . Most evDetermining which women should be started on aspirin prophylactically is very challenging. Current evidence shows that no single test predicts pre-eclampsia with sufficient accuracy to be clinically useful , and thuPreeclampsia without severe features is managed expectantly until 37 weeks, in the presence of severe features in those <34 weeks it may be managed expectantly with birth indicated at any time with deterioration of fetal and maternal status. The pharmacological management of mild to moderate hypertension (systolic <160 and diastolic <110) is not currently recommended by the ACOG, as it does not appear to attenuate disease progression and may increase the risk of fetal growth restriction. As this mild to moderate hypertension may be associated with a 4% risk of stroke, its treatment is still subject to debate . TreatmeDespite the fact that pre-eclampsia complicates a significant number of pregnancies and is the leading cause of morbidity and mortality in pregnancy, therapeutic strategies remain poorly characterized . The eleThe authors of a 2014 Cochrane review concluded that \u201cthere is insufficient evidence on which to base recommendations for thromboprophylaxis during pregnancy (and that) large scale, high-quality randomized trials of currently used interventions are warranted\u201d . HoweverConsequently, to date, guideline recommendations are mainly based on expert opinion rather than high-quality evidence , 105\u2013107Both PET and VTE remain a leading cause of maternal morbidity and mortality, complicating a significant number of pregnancies , 54. UndTR-B and OE wrote the sections of the manuscript. FN revised the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor declared a shared affiliation with one of the authors, TR-B, at the time of review.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": "Zn (5.0 mg/L) and Se (0.1 mg/L) increased the migrated number of neutrophils (p < 0.01) to an extremely significant degree. These findings provided a theoretical and experimental basis for mammary gland immunity in dairy cows. Thus, we suggest that adding moderate amounts of different trace elements can improve the immune function of dairy cows.Neutrophils represent the first line of mammary gland defense against invading pathogens by transmigration across the mammary epithelial cell barrier. The effect of trace elements on the migration of bovine neutrophils is not clear. In this study, we investigated the effect of copper , zinc  and selenium  on the migration of bovine neutrophils by using a Transwell assay. The results showed that Cu, Zn and Se promoted the number of neutrophils in the trans-mammary epithelium. With the increased concentration of Cu at 1.5 mg/L, the number of neutrophils in the trans-mammary epithelium was increased significantly ( Neutrophils are the main leukocyte cells and play an important role in the non-specific immune system of dairy cows. In the healthy mammary gland, neutrophils have a low concentration of cell population from 5 to 20%). When pathogens invade the bovine mammary gland, neutrophils are recruited to the mammary gland and execute important functions including phagocytosis, respiratory bursts, and the formation of an extracellular trapping network (NET) to kill pathogens ,2,3,4. T to 20%. C. albicans, but their ability to kill the ingested C. albicans was decreased [4 to the diet of calves can increase Zn levels in the liver and serum, enhance macrophages\u2019 random migration and change the proportion of neutrophils in peripheral blood [Several researchers have found an association between trace elements in bovine nutrition and the ability to resist infections ,13,14. Secreased . Jones eecreased ,20. Percecreased . The traecreased . Bednareal blood . Releaseal blood . Supplemal blood . Ndiwenial blood .These studies suggest that the nutritional status of trace elements in dairy cows has an impact on the function of neutrophils. However, how trace elements Cu, Zn and Se affect the migration of bovine neutrophils is unknown. Therefore, the objective of this work is to investigate the effect of Cu, Zn and Se on the migration of neutrophils and provide useful information for the further study of trace elements on the function of neutrophils.\u00ae . Then, 4 mL Separate A, 2 mL Separate C, and 2 mL sodium citrate anticoagulated whole blood were successively added into a 15 mL centrifuge tube, forming a liquid gradient. The mixture was then centrifuged at room temperature in an Eppendorf high-speed centrifuge at 900\u00d7 g for 30 min. After the centrifugation step, neutrophils were found in the lower gradient phase as a white ring. Neutrophils were extracted into a new 15 mL centrifuge tube and erythrocytes were lysed with Red Blood Cell Lysis Buffer. Then, the suspensions were centrifuged  and washed three times; neutrophils were resuspended in Roswell Park Memorial Institute (RPMI) 1640 medium  containing 10% fetal bovine serum .The test animals were selected from a sizeable intensive dairy farm in Daqing, Heilongjiang Province, China. Three healthy 2-year-old cows of similar body condition and at mid-lactation were randomly selected. All cows were healthy and did not exhibit any clinical signs of mastitis. Whole blood was collected from the cows via the middle tail vein. Neutrophils were isolated and purified by using the bovine peripheral blood neutrophil purification kitSeparated neutrophils (2 \u03bcL) were used to make a Giemsa-stained smear, which was checked for purity by a Nikon Eclipse TS100 inverted fluorescence microscope . The obtained cell suspensions were stained with Trypan blue, and their concentration and viability were measured using Countess II Life Technologies Cell Counter .2/95% air). Cell migration assays were performed by using Transwell chambers . Using sterile forceps, the permeable supports were inserted into a sterile 25 mm-deep tissue culture dish. Each Transwell permeable support was coated with 0.012 g/L Collagen I . Then, 50 \u03bcL of the MAC-T cells suspension (1 \u00d7 105 cells) was added to each permeable support. The lid was carefully placed on the dish at 37 \u00b0C with 5% CO2 for about 6 h. The permeable supports were placed into a 24-well plate containing 0.2 mL DMEM/F12 per well. Then, 0.1 mL DMEM/F12 was added to the upper reservoir of each permeable support and incubated at 37 \u00b0C with 5% CO2. The medium was replaced every 2 days. After 3 days, the permeable supports were seeded with MAC-T, and the confluence of the cells was assessed  was obtained from Jilin University . MAC-T cells were cultured in Dulbecco\u2019s Modified Eagle Medium: Nutrient Mixture F-12  medium supplemented with 10% FBS and 1% penicillin/streptomycin  at 37 \u00b0C in a humidified atmosphere  was diluted in HBSS  at a concentration of 1.0, 2.0, and 3.0 mg/L. ZnSO4\u00b77H2O  was diluted in HBSS at a concentration of 2.0, 10 and 20 mg/L. Na2SeO3  was diluted in HBSS at a concentration of 0.2, 2.0 and 4.0 mg/L. Then, 990 \u03bcL RPMI 1640 and 10 \u03bcL fMLP were added into the lower chamber of Transwell chambers, and 100 \u03bcL of cell suspension (2 \u00d7 105 Cells) was added into the upper chamber. Three chambers were selected and 100 \u03bcL HBSS was added to each upper chamber as the blank control group.A stock solution of N-Formyl-methionyl-leucyl-phenylalanine  was prepared at 10 \u03bcmol/L in DMSO . CuSO2 and incubated at 37 \u00b0C for 2 h  test were used for parameter estimation and hypothesis testing. Significant differences were established at Neutrophils were isolated as per the manufacturer\u2019s instructions, as described previously. The morphology of neutrophils is presented in p > 0.05). However, when the concentration of Cu was 1.5 mg/mL, the number of neutrophils in the trans-mammary epithelium was increased significantly (p < 0.05).The results of Cu on the migration of neutrophils are shown in p < 0.01), when the concentration of Zn was 5.0 mg/mL.The results of Zn on the migration of neutrophils are shown in p < 0.01).The results of Se on the migration of neutrophils are shown in p > 0.05). With the increased concentration of Cu at 1.5 mg/L, the number of neutrophils in the trans-mammary epithelium was increased significantly (p < 0.05). This result might be because the Cu could promote the deformability of neutrophils. However, the normal physiological concentration of Cu is 0.7~0.9 mg/L in bovine serum and Cu excess may be even more harmful and can result in serious toxicity [Neutrophils represent the first line of mammary gland defense against invading pathogens by transmigration across the mammary epithelial cell barrier . Admittetoxicity .p < 0.01). One possible explanation is that Zn acts as a chemoattractant and enhances chemotaxis toward fMLP in rat neutrophils [Zn is an essential ion for animal nutrition and development and plays an important role in cell functions, such as protein synthesis and enzymatic regulation, but excess Zn induces drastic toxicity symptoms. Past studies have repeatedly shown that immune cells are affected by free intracellular Zn and impaired cell migration in clinical and experimentally induced Zn deficiency ,31. In btrophils . Howeverp < 0.01), which is characterized by an increase in Se concentration (from 0.1 to 2.0 mg/L). We hypothesize that Se may increase the intracellular calcium flux of neutrophils, which enhanced neutrophils\u2019 random motility.Se is one of the essential trace elements, with various important biological functions in mammals. It is a necessary component of numerous enzymes that are involved in redox reactions and this antioxidative function causes Se to protect immune cells from oxidative stress caused by respiratory burst. In bovine serum, the normal physiological concentration of Se is over 0.08 mg/L . Innate In summary, our results indicated that trace elements Cu, Zn and Se could promote the trans-mammary epithelium migration of neutrophils in vitro. These findings provide a theoretical and experimental basis for mammary gland immunity in dairy cows. Thus, we suggest that adding moderate amounts of different trace elements may improve the immune function of dairy cows."}
+{"text": "PSB-CB-27 shifted the concentration-response curves (CRCs) of PSB-MZ-1415, PSB-MZ-1440, NAGly and Abn-CBD to the right; O-1918 caused rightward shifts of the CRCs of PSB-MZ-1415 and NAGly. Endothelium removal diminished the potency and the maximum effect of PSB-MZ-1415. The potency of PSB-MZ-1415 or NAGly was reduced in male patients, smokers and patients with hypercholesterolemia. In conclusion, the novel GPR18 agonists, PSB-MZ-1415 and PSB-MZ-1440, relax hPAs and the effect is inhibited by the new GPR18 antagonist PSB-CB-27. GPR18, which appears to exhibit lower activity in hPAs from male, smoking or hypercholesterolemic patients, may become a new target for the treatment of pulmonary arterial hypertension. GPR18 receptor protein was detected in the heart and vasculature and appears to play a functional role in the cardiovascular system. We investigated the effects of the new GPR18 agonists PSB-MZ-1415 and PSB-MZ-1440 and the new GPR18 antagonist PSB-CB-27 on isolated human pulmonary arteries (hPAs) and compared their effects with the previously proposed, but unconfirmed, GPR18 ligands NAGly, Abn-CBD (agonists) and O-1918 (antagonist). GPR18 expression in hPAs was shown at the mRNA level. PSB-MZ-1415, PSB-MZ-1440, NAGly and Abn-CBD fully relaxed endothelium-intact hPAs precontracted with the thromboxane A In the mouse, GPR18 is located in the central nervous system, the gastrointestinal tract, in the lymphoid system thiazin-3(5H)-one . The three ligands were synthetized at the Department of Technology and Biotechnology of Drugs, Faculty of Pharmacy, Jagiellonian University, Krak\u00f3w, Poland, and identified and characterized in the Department of Pharmaceutical & Medicinal Chemistry, University of Bonn, Germany . The final concentration of DMSO was <1.0% v/v.U46619 ((5Z)-7-{-6-[-3-hydroxyoct-1-en-1-yl]-2-oxabicyclo[2.2.1]-heptan-5-yl}hept-5-enoic acid), O-1918 -3-methyl-6-prop-1-en-2-ylcyclohex-2-en-1-yl]benzene), NAGly, and Abn-CBD were purchased from Tocris Bioscience . Stock solutions of these substances were made to 10 mM in ethanol; their final concentrations were prepared by dilutions with deionized water, which led to final concentrations of ethanol of <0.7% Results are given as the mean \u00b1 SEM of n (number of patients). The relaxation elicited by the GPR18 agonists  or the appropriate solvent was expressed as a percentage of the precontraction induced by U46619.X is the logarithm of the molar concentration of the agonist and Y is the percent response. The curves were used to determine potency  of the agonist that produced 50 or 25% of the maximal effect (Rmax)) [GraphPad Prism 5.0 software  was used to plot the mean data as sigmoidal concentration-response curves (CRCs): (Rmax)) .25 values. The antagonistic potency (apparent pA2) of PSB-CB-27 or O-1918 against GPR18 agonists was calculated from the equation: apparent pA2 = log (CR \u2212 1) \u2212 log[B], where [B] is the molar concentration of the antagonist and CR is the concentration ratio of the EC25 values of GPR18 agonists in the presence and absence of an antagonist.Antagonist- or endothelium removal-induced rightward shifts of the CRCs relative to the control curve were calculated on the basis of the ECt-test for unpaired data was used when two groups (one treatment and one control group) were compared. Data were analyzed using GraphPad Prism version 5.00 for Windows, GraphPad Software . Differences were considered to be statistically significant at p < 0.05.When two or more treatment groups were compared to the same control, a one-way analysis of variance (ANOVA) followed by Dunnett\u2019s test was used. Post hoc tests were run only if F achieved the necessary level of statistical significance and there was no significant variance inhomogeneity. Student\u2019s gpr18 mRNA is shown. GPR18, the activity of which is reduced by endothelial denudation and is lower in hPAs from male, hypercholesterolemic, or smoking patients, may become a new target for the treatment of pulmonary arterial hypertension.In conclusion, the following facts lend further support to the role of GPR18 in isolated human pulmonary arteries: (i) Two new agonists, PSB-MZ-1415 and PSB-MZ-1440, relax hPAs, (ii) the vasodilatory effect of these agonists is inhibited by the new GPR18 antagonist PSB-CB-27, and (iii) expression of"}
+{"text": "With the author(s)\u2019 decision to opt for Open Choice the copyright of the article changed on 21 September 2021 to \u00a9 The Author(s) 2021 and the article is forthwith distributed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article\u2019s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article\u2019s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit The Original articlehas been corrected."}
+{"text": "Students from disadvantaged backgrounds continue to be underrepresented in medical education. Widening participation (WP) or outreach schemes seek to increase diversity. Drawing on previously unexplored data from a scheme called Realising Opportunities in England, this study aimed to investigate which high\u2010achieving socioeconomically disadvantaged students in a national WP scheme went on to study medicine at university.This retrospective longitudinal study analyzed data of 2665 16\u2010year\u2010olds on a WP scheme in England between 2010 and 2014. Descriptive statistics and logistic regression analyses investigated any differences between those that went on to study medicine and those that did not. Eligibility for studying medicine, student's neighborhood, gender, ethnicity, parent's higher education experience, exam attainment, interest in medicine, and their subject of choice for university at age 16 were considered.Of the 1850 students who were tracked to a university destination, only 55 (3%) studied medicine. Participants with high exam results, female, Asian, and from neighborhoods of higher university entry were most likely to study medicine. In the multivariate model, only prior attainment and intention to study medicine predicted studying medicine. Three hundred and forty Realising Opportunities participants expressed interest in studying medicine at age 16, but 80 (24%) were found to have unrealistic aspirations based on their prior academic attainment.Attainment and intention were key factors for predicting medicine enrolment among these outreach scheme participants. Some students interested in studying medicine had insufficient academic attainment to compete for medical school places. Efforts to equalize attainment and provide guidance for career choice are crucial factors for students from disadvantaged backgrounds. This study draws on data from the scheme to discover which high\u2010achieving socioeconomically disadvantaged students went on to study medicine at university with the aim of discovering factors that could inform recruitment of students from WP backgrounds.22.1This study is a longitudinal retrospective analysis of routinely collected data from \u201cRealising Opportunities\u201d (RO),Data access was through the RO partnership, within their data protection and legal compliance parameters. Key considerations were that participants had explicitly opted for their data to be used for research purposes and that data were anonymized. Ethical approval was from the University of Exeter.2.2Realising opportunities participant's data were analyzed from 2010 to 2014, with the exception of year 2011 as consent from participants had not been obtained. RO data on participants were linked to advance level exams at age 18, higher education destination, and university course through the Higher Education Access Tracker (HEAT), which draws information from databases including the Higher Education Statistics Agency (HESA).The data set included 2670 participants. All those participants (817), without a university destination, were included in the data when looking at the sociodemographic distribution of the sample but removed when analyzing the participant's Higher Education destination.We do have a significant amount of missing data in the analysis. This should be considered our first finding in itself: It is surprising for an outreach scheme of the scale and intent as RO to have such low tracking record. We found that data on university destination were more likely to be missing for those who were White (550 or 63% of those not tracked as compared to 980 or 53% of those tracked) and with lower GCSES (450 or 51% of those not tracked had 0 to 3 GCSEs compared to 690 or 37% of those tracked). Conversely, those tracked are more likely to be Asian and also were not in receipt of discretionary payments. The lower attainment of the non\u2010tracked group would align with a hypothesis that these students have not entered higher education at all, giving the explanation for no tracked destination being found but the data themselves are inconclusive.The information known about each participant on entry to the WP program was gender, ethnicity, if parents had higher education experience, their preferred choice of university course aged 16, their age 16 exam results and subjects, and if they received a bursary . The POLAR dataThe preferred course at university was derived from RO's question to the participants on joining the scheme: \u201cHave you already considered what subject you may want to study at university?\u201d If more than one area was mentioned only the first one was retained unless it was medicine then this was retained.All the nonnumerical information was categorized into groups called \u201cvalues\u201d and each value was assigned a numerical code. Missing data were coded as a separate value. The subject participants interested in studying were divided into six values.Ethnicity was converted into five groups: White, Asian, Black, and other, and missing.A\u00a0=\u00a05 points, to E\u00a0=\u00a01 point, and their top three grades were only used.The grades achieved in the exams at age 16 were given a score that counted all the participants' A and A* grades (the top grades). Exams taken at age 18 were converted to a numerical number with The data were analyzed on IBM SPSS Statistics Data Editor V25. Descriptive statistics were generated to discover if there were any common characteristics among those that studied medicine. In addition, multivariate regression was carried out\u2014standard assumptions of independence of predictors held. Data were not analyzed in years of entry to the scheme as the number of students who went on to study medicine in the whole population was low. Also, there was no hypothesis considering changes in medical aspirations over time and nothing in the data or policy context to expect annual variations.2.3In building the multivariate regression in SPSS, we expect from the bivariate association that the exams at age 16 are the strongest predictor of studying medicine and that advanced exams taken at age 18 might also play a role. In terms of social background, ethnicity, POLAR quintile, and discretionary payments are expected to be significant. However, when including background and academic factors simultaneously such background effects might disappear.We based our regression analysis\u2014predicting studying medicine at university\u2014on the 1850 cases who matched to their university destination  and who also matched a complete set of age 16 exam results resulting in 1840 cases for the analysis.Each factor was individually entered to test significance as a univariate predictor of studying medicine in a binary logistic regression model with medicine at university coded as 1  grades for different groups Table\u00a0. The numGiven the wide\u2010reaching importance of exams at age 16 (GCSE results), Figure\u00a03.1\u03c72\u00a0=\u2009132.6, P\u2009<\u2009.01); number of A and A*s , and A\u2010level score , although, perhaps surprising, eligibility for studying medicine was significant but added comparatively less explanatory power . The multivariate investigation showed that the most parsimonious model with the fewest predictors of studying medicine is a model of knowing GCSE scores and the intention to study medicine : Adding other factors and the associated degrees of freedom is not associated with an improvement to model fit, or significance. This is true for eligibility for medicine and A\u2010level scores, although A\u2010level scores are the only additional significant predictor after knowing GCSE scores, and intention to study medicine, although the number of unknowns regarding A\u2010level reduces the sample size, and thus model fit statistics . None of the social factors: ethnicity, gender, POLAR, parents having HE experience, and eligibility for discretionary payments are significant when added to the base model or increase the model fit. We tested an interaction effect between gender and GCSE scores and gender and intention for medicine, as men seemed underrepresented, neither main nor interaction term was significant.For background factors, the chi\u2010squares varied from 0.3 for parental higher education to 21.0 for ethnicity. The most powerful univariate predictors were intention to study medicine (4Which disadvantaged students study medicine? In this study, these students were more likely to be Asian, and less likely to be White, they were living in an area with high university attendance and more likely to receive discretionary payments, were high attaining in exams, and had an expressed interest to study medicine from age 16.The RO study participants were mostly from areas with low rates of higher education entry and all from underperforming state secondary schools but in line with the national average, 3% went on to study medicine.More females than males entered medical school despite equal numbers being interested in studying medicine and similarly high exam grades at age 18. University statistics for students entering medical school in 2017 found the female\u2010to\u2010male ratio 1.4:1 and the present study 2.6:1.The concern from this database is the lack of White students who were interested in medicine\u2014they represented 57% of the population but they accounted for only 19% of the population interested in studying medicine in contrast to the Asian students of whom represented 28% of the population but almost a half of the students wanted to pursue medicine as a career. It has been found that Asian parents emphasize the duty that students have to succeed and this success is limited to a narrow field of options that include medicine.These high aspirations would need to be appropriate to the individual students as some students in this study appeared to have unrealistic aspirations when considering medicine as a career at age 16. The UK medical schools vary on the weighting they place on exams at age 16. Most medical schools specify five GCSEs with a minimum of C in English and Maths, but some require six GCSE passes at A or above to include English, Mathematics, and two Science subjects.4.1The strength of this longitudinal study was the ability to track students from joining a WP scheme, to their entry to university via HEAT data, and that the key findings resonate with previous research. RO ensured that participants were only eligible to join the scheme if they fulfilled two or more WP criteria, and they only accepted participants from targeted state schools. A unique feature of this study was the recording, at age 16, of the subject that participants were interested in studying at university. This enabled correlations to be made between this interest and the subject they studied at university. Data were also used from five cohorts of participants increasing the sample size and strengthening the reliability of the data. However, as with all research, there were some limitations. The data were not collected specifically for this study and this may have affected the accuracy of the recorded data needed for this research question. The destination of the students who did not have a university destination recorded was not recorded; however, if a university was not matched using the HEAT data, it is likely that they did not enter university immediately after school. This may be a heterogeneous group, comprising participants who did not go to university, those who studied abroad, and those who took time out before going to university, and those for whom the linkage procedure failed. The exam grades were converted into a numeric score without reference to the subjects studied. Some medical schools stipulate which subjects students study for their exams at age 18. Therefore, students might have a high numerical score but with qualifications in the wrong subjects for medicine. Furthermore, a range of factors influence studying medicine, including parental and community attitudes and individual personalities and values. Our study is silent on these factors. There were missing data in all the variables, which should be considered when assessing the outcomes.5The 2018 report by the Medical Schools CouncilsThe reasons for the low proportion of WP men entering medicine require further research, to see if barriers can be overcome. If the barrier to success is the medical school interview or the application process, then career advisers and widening participation schemes could target resources in these areas. If the barrier is their perceptions of the career itself then this could be a priority for focus in a widening participation program or through the media. In addition, understanding the reasons for some academically able WP student choosing to go to university but not to study medicine would provide valuable insights into factors influencing student's career choices.Further evaluation is required to understand which, of the multiple strands of the Realising Opportunities WP Scheme, might help participants to successfully apply to medical school, or whether it is the combination of the strands that is beneficial. To increase the WP proportion in medicine, further qualitative research is needed to understand if there are surmountable barriers preventing this group from considering medicine as a career. If all these barriers can be successfully targeted then a more diverse medical school population could develop.The authors declare no conflicts of interest regarding this study.Conceptualization: Carolyn Murray, Anna Mountford\u2010Zimdars, Karen MattickData Curation: Carolyn MurrayFormal Analysis: Carolyn Murray, Anna Mountford\u2010ZimdarsFunding Acquisition: Not applicableInvestigation: Carolyn Murray, Anna Mountford\u2010ZimdarsMethodology: Carolyn Murray, Anna Mountford\u2010Zimdars, Karen MattickProject Administration: Carolyn MurrayResources: Carolyn MurraySoftware: Carolyn Murray, Anna Mountford\u2010ZimdarsSupervision: Anna Mountford\u2010Zimdars, Karen MattickValidation: Carolyn Murray, Anna Mountford\u2010ZimdarsVisualization: Not applicableWriting\u2014Original Draft Preparation: Carolyn MurrayWriting\u2014Review and Editing: Carolyn Murray, Anna Mountford\u2010Zimdars, Karen Mattick\u2003\u2003All authors have read and approved the final version of the manuscript.\u2003\u2003Carolyn Murray confirms that she had full access to all of the data in this study and takes complete responsibility for the integrity of the data and the accuracy of the data analysis.Carolyn Murray affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned have been explained."}
+{"text": "Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) disease (COVID-19) has been linked to thrombotic complications and endothelial dysfunction. We assessed the prognostic implications of endothelial activation through measurement of endothelin-I precursor peptide (proET-1), the stable precursor protein of Endothelin-1, in a well-defined cohort of patients hospitalized with COVID-19.We measured proET-1 in 74 consecutively admitted adult patients with confirmed COVID-19 and compared its prognostic accuracy to that of patients with community-acquired pneumonia (n\u2009=\u2009876) and viral bronchitis (n\u2009=\u2009371) from a previous study by means of logistic regression analysis. The primary endpoint was all-cause 30-day mortality.Overall, median admission proET-1 levels were lower in COVID-19 patients compared to those with pneumonia and exacerbated bronchitis, respectively . Although COVID-19 non-survivors had 1.5-fold higher admission proET-1 levels compared to survivors (81.8\u00a0pmol/l [IQR: 76 to 118] vs. 53.6 [IQR: 37 to 69]), no significant association of proET-1 levels and mortality was found in a regression model adjusted for age, gender, creatinine level, diastolic blood pressure as well as cancer and coronary artery disease . In patients with pneumonia  and exacerbated bronchitis  we found significant associations of proET-1 and mortality.Compared to other types of pulmonary infection, COVID-19 shows only a mild activation of the endothelium as assessed through measurement of proET-1. Therefore, the high mortality associated with COVID-19 may not be attributed to endothelial dysfunction by the surrogate marker proET-1. Coronavirus disease 2019 (COVID-19), a novel human pathogenic severe acute respiratory syndrome coronavirus counts meanwhile worldwide more than 60 million confirmed cases and more than 1.4 million deaths . Most ofAnother target of SARS-CoV-2 infections is the endothelium, which also expresses ACE2 , 11, leaEndothelin-1 (ET-1), mainly synthesized by activated endothelial cells which are stimulated by bacterial endotoxin and various inflammatory cytokines , is a potent vasoconstrictor agent . FurtherThis prospective observational study included all consecutively hospitalized adult patients (\u2265\u200918\u00a0years) with confirmed COVID-19 at the Cantonal Hospital Aarau, a tertiary care medical center in Switzerland, between February 26, 2020 and April 30, 2020.Baseline characteristics of the analyzed COVID-19 patients have been published elsewhere . In brieClinical data, including socio-demographics, comorbidities as well as pre-existing medical prescription and COVID-19-specific inpatient medication were assessed until hospital discharge or death and extracted from the electronic health records. Experimental antiviral treatment was recorded if given and included hydroxychloroquine  and sometimes tocilizumab. Comorbidities were also assessed via chart review and based on the International Statistical Classification of Diseases and Related Health Problems codes (ICD10). Additionally, patient outcomes including admission to the intensive care unit (ICU), length of hospital stay (LOS) as well as length of ICU stay were collected via chart review. 30-day mortality was collected by abstraction of hospital records, or where necessary by systematic telephone interviews. Laboratory test results were available according to clinical routine. ProET-1 was batch-tested at the end of the study and was therefore not available to the treating team during the index hospitalization.We used patients with confirmed community-acquired pneumonia or acute and chronic exacerbated bronchitis included in a previous prospective study as a control group , 23. TheThe primary endpoint of this investigation was all-cause 30-day mortality. For the COVID-19 patients and the control group, we assessed vital status 30\u00a0days after admission by abstraction of hospital records and/or systematic telephone interviews.Plasma and serum samples on admission and during follow-up were collected in BD Vacutainer\u00ae Heparin and SST tubes. Routine left-over samples were stored at \u221280\u00a0\u00b0C until analysis. Results from routine laboratory tests were recorded. C-terminal proendothelin-1 (CT-proET-1) was assessed in batch using a commercially available automated fluorescent sandwich immunoassay , as described in detail elsewhere , 26. The0 T1 (day 3/ day 4)T2 (day 5/ day 6)T3 (day 7/ day 8)TFor the COVID-19 affected patients various time points during hospitalization were analyzed, depending on the available data:For the control group, blood samples for later marker measurement were collected upon admission, i.e., within the first 24\u00a0h upon hospital admission. ProET-1 levels were batch-measured in plasma with sandwich immunoassays (Kryptor\u00ae Thermo Scientific Biomarkers) .p-value of\u2009<\u20090.05 was considered significant. Statistical analysis was performed using Stata 15.1 .Discrete variables are expressed as frequency (percentage) and continuous variables as medians with interquartile ranges (IQR) or mean with standard deviation (SD). In addition to descriptive statistics, we investigated the association of proET-1 levels at different time points with the primary endpoint by means of multivariable logistic regression analysis with reporting of odds ratios (OR) and corresponding 95% confidence intervals (CI) and p-values as a measure of association. As predefined, three types of regression models were calculated, namely an unadjusted model, a model adjusted for age and gender and a model adjusted for age, gender, creatinine level, diastolic blood pressure and comorbidities. The confounders were based on previous reports published by Bhandaria et al.  as well Overall, we included 103 patients with confirmed COVID-19 . Four patients were excluded from the analysis . Further 25 cases had to be excluded because of missing aliquots for biomarker analysis (n\u2009=\u20099), or due to missing blood sampling within 24\u00a0h from admission (n\u2009=\u200916). Thus, the final analysis encompassed 74 COVID-19 patients. In addition, 1247 control patients from a previously conducted study were used with a diagnosis of pneumonia (n\u2009=\u2009876) or acute or chronic exacerbated bronchitis (n\u2009=\u2009371). Figure\u00a02 with (32\u00a0mmHg (IQR 31 to 35\u00a0mmHg) and without initial O2-supply (31\u00a0mmHg (IQR 29 to 33\u00a0mmHg) at admission compared to the control patients (p\u2009<\u20090.01). Regarding 30-day outcomes, COVID-19 patients had higher rates of all-cause mortality and admission to ICU. Length of stay was also longer in COVID-19 patients.Table Median proET-1 was lower in COVID-19 patients compared to pneumonia and exacerbated bronchitis patients, respectively . Still, among all patient groups, non-survivors had higher proET-1 levels at admission compared to survivors Table . In the The association of proET-1 levels at measured time points with the primary endpoint is shown in Table Prognostic accuracy of proET-1 to predict 30-day mortality was analyzed for specific proET-1 cut-offs Table . Based oThis prospective study evaluated endothelial activation through measurement of initial proET-1 in patients with confirmed COVID-19, as well as patients with other types of respiratory infections. Although we expected an increase in proET-1 in COVID-19 patients compared to other types of respiratory infections, our results showed quite the opposite. In fact, our results indicate a less pronounced activation of proET-1 in COVID-19 compared to other respiratory tract infections with non-significant associations with mortality in multivariable analyses. Although proET-1 provided some prognostic information regarding mortality, discrimination analysis based on C-statistic showed only a moderate prognostic accuracy. These results suggest that in COVID-19 patients proET-1 is not a powerful marker for calculating morbidity and mortality in contrast to its validity in other respiratory infections.Several pro-inflammatory cytokines and prognostic markers from different pathways have been investigated in patients with COVID-19 , 31. YetThere is a strong rationale for looking at proET-1 in COVID-19 patients based on results of previous research in other types of infections. More specifically, the prognostic relevance of proET-1 was analyzed in several prior studies focusing on critical-ill patients with and without sepsis , patientIn our study population, COVID-19 patients were mainly transferred to ICU due to the need for non-invasive and/or invasive ventilation support. In contrast, reason for ICU admission in patients with other respiratory infection is often due to shock and need for vasopressor support. Other than in septic patients, where the need of pressure support and rising levels of the vasoconstrictor ET-1 are crucial, the reason for severe disease and death in patients with COVID-19 may rather be related to the infection-mediated endothelial injury and endothelialitis. This in turn may trigger excessive thrombin production, inhibit fibrinolysis, and activate complement pathways, which consequently initiate thrombo-inflammation and can lead to micro-thromboembolism and microvascular dysfunctions .Compared to prior studies , the optThis study has several limitations. The results of this analysis have to be interpreted in the context of the study design: First, due to the single center design of this study the number of analyzed COVID-19 cases was small and external validation is needed. Second, clinical data were limited and not all evaluated laboratory parameters and characteristics were available for all patients, resulting in few missing data. Third, due to incomplete data, left atrial size, which represent a known confounder of proET-1 levels, was not considered in the adjusted regression models.In conclusion, as compared to other types of pulmonary infection, COVID-19 causes increases of proET-1 concentrations to a lesser extent, which might be explained by either only a mild activation of the endothelium or by the reduced possibility to produce the hormone from damaged endothelium. Thus, we could not find any evidence that the high mortality associated with COVID-19 can be estimated by the endothelial function marker proET-1. Based on our results, the use of proET-1 for prognostic risk stratification in patients with COVID-19 is not recommended."}
+{"text": "Advanced bipolar devices  have a lower blade temperature than ultrasonically activated devices  during activation, potentially enabling accurate lymph node dissection with less risk of postoperative pancreatic fistula (POPF) due to pancreatic thermal injury in laparoscopic gastrectomy. Therefore, we compared the efficacy and safety of ABD and USAD in laparoscopic gastrectomy for gastric cancer patients.A retrospective cohort study was conducted on patients who underwent laparoscopic distal gastrectomy (LDG) between August 2008 and September 2020. A total of 371 patients were enrolled, and short-term surgical outcomes, including the incidence of ISGPF grades B and C POPF, were compared between ABD and USAD. The risk factors for POPF in LDG were investigated by univariate and multivariate analyses.p\u2009<\u20090.001), the morbidity rate was lower , the length of postoperative\u00a0hospitalization was shorter , and the lymph node retrieval rate was higher  with an ABD than with a USAD. There were no mortalities in either group. A multivariate analysis showed that a USAD was the only independent risk factor with a considerably high odds ratio for the occurrence of POPF .A propensity score-matching algorithm was used to select 120 patients for each group. The POPF rate was significantly lower (0.8 vs. 9.2%, An ABD may improve the safety of laparoscopic gastrectomy for gastric cancer patients. According to Globocan, gastric cancer is the fifth-most common malignancy and the third leading cause of cancer-related death worldwide  . DespiteHowever, radical lymphadenectomy recommended in the Japanese guidelines involves the removal of the peripancreatic lymph nodes, which may be accompanied by postoperative pancreatic complications. Although postoperative pancreatic fistula (POPF) is aseptic in the majority of cases, infection of POPF is a significant risk factor for further complications, especially bleeding and mortality . RecentlAdvanced bipolar devices  and ultrasonically activated devices  are the most widely used energy devices in such surgeries \u201318. AlthWe previously reported that the grasping range and repetition of dissection are essential factors that affect the blade temperature of these devices . In partA retrospective cohort study was conducted on patients who underwent laparoscopic distal gastrectomy (LDG) with lymph node dissection to evaluate the short-term surgical outcomes of using an ABD versus a USAD. The inclusion criteria were patients who underwent LDG for gastric cancer at University of Occupational and Environmental Health, Kitakyushu, Japan, between August 2008 and September 2020 Fig.\u00a0.Fig. 1CoThe primary endpoint was grades B and C POPF according to the International Study Group for Pancreatic Fistula (ISGPF) classification . The secData on patient characteristics and surgical outcomes were obtained from medical charts. Clinical TNM classification and stage were determined according to the 15th version of the Japanese Classification of Gastric Carcinoma . The extAll procedures were performed according to the ethical standards of the respective committees on human experimentation  and complied with the 1964 Declaration of Helsinki and later versions concerning Animal Rights. The Ethics Committee of University of Occupational and Environmental Health, Kitakyushu, Japan, approved this study.LDG was performed by 10 surgeons, including 3 who were board-certified by the Japanese Society of Endoscopic Surgery. Surgical procedures were standardized for LDG and have been described in detail elsewhere \u201327. LDG Most of the surgical procedures using ABD were similar to those using USAD, except for the clipless sealing procedure of certain small-caliber vessels. Intracorporal Billroth I reconstruction with functional end-to-end anastomosis or Roux-en-Y reconstruction was performed following distal gastrectomy , 29. A pPOPF was defined as localized fluid accumulation on computed tomography (CT) and elevated amylase levels in the drain with accompanying abdominal pain and a fever . We consU test, chi-square test, or Fisher\u2019s exact test, as appropriate. Propensity scores were calculated for all patients based on 11 crucial preoperative clinical variables that might affect the short-term outcome of gastrectomy: Endoscopic Surgical Skill Qualification System for laparoscopic distal gastrectomy (ESSQSG); age; sex; BMI; clinical T, N, and M, tumor size; neoadjuvant chemotherapy; extent of lymphadenectomy; and type of reconstruction [Comparisons were made using the Mann\u2013Whitney truction . SubsequP\u2009<\u20090.01 in univariate analyses, and odds ratios (ORs) with 95% confidence intervals (CIs) were estimated for each variable. The R release 3.6.3 software program was used for all calculations [P\u2009<\u20095% level.Furthermore, the risk factors for POPF were investigated in the propensity score-matched (PSM) cohort by univariate and multivariate analyses. The model included the sex, age, BMI, ESSQSG, cStage, extent of lymphadenectomy, and type of energy device used as possible confounding factors. The multivariate logistic regression models included variables with ulations . SignifiA total of 796 consecutive patients underwent surgery for gastric cancer, of whom 425 met the exclusion criteria Fig. . Thus, tP < 0.01), a lower rate of ESSQSG (P < 0.01), and a higher rate of Roux-en-Y reconstruction (P < 0.01) than the ABD group. After matching, all covariates were well balanced between the ABD and USAD groups.The background characteristics for all patients and matched patients are shown in Table P <0.001). The overall postoperative complication rate for ISGPF grade B was not significantly different between the USAD and ABD groups , but the USAD group had more blood loss  and fewer harvested lymph nodes  thanthe ABD group. In the matched analysis, POPF was significantly more frequent in the USAD group than in the ABD group . The USAD group had fewer harvested lymph nodes  than the ABD group, as well as longer postoperative hospitalization  and higher overall postoperative morbidity rates . There was no marked difference in the types of postoperative complications between the USAD and ABD groups, except for POPF. No 90-day mortality was observed in either group.Energy devices and surgical outcomes, including details concerning postoperative complications before and after matching, are shown in Table P <0.001), higher BMI , higher cStage , greater extent of lymph node dissection , and the type of energy device used for dissection  but not with the sex (P = 0.358) or surgeon qualification (P = 0.338). A multivariate analysis indicated that the type of energy device was the only independent risk factor with a considerably high OR for the occurrence of grades B and C POPF .To identify risk factors for POPF, a risk factor analysis was performed. The results of univariate and multivariate analyses of clinical factors are summarized in Table When considering the safety and feasibility of LG, it is vital to assess the risks of POPF formation. PF after gastrectomy is thought to be caused by intraoperative injury to the pancreas. Possible causes of pancreatic injury during LG include thermal injury from energy devices, compression from forceps, accidental injury from anatomical variations in the shape and location of the pancreas, and pancreatic steatosis , 33\u201338. ABD create a vessel seal by applying bipolar electrosurgical radiofrequency energy to vessels interposed between the jaws of the device . In contThere have been many reports comparing the outcomes of procedures using ABD with those of conventional surgery for various surgical procedures , 40, 41.Several limitations associated with the present study warrant mention. First, this study employed a retrospective, single-center, non-randomized design. We compared the short-term outcomes of ABD and USAD after matching two groups using propensity scores to reduce any bias in the comparison. However, there may have been unknown confounders, including the potential for selection biases, so the overall results should be interpreted cautiously. Second, the superiority of an ABD with regard to oncological outcomes is not yet conclusive, as long-term surveillance has not been conducted. Third, other factors, such as advances in individual surgical techniques, might have influenced the improvement in surgical outcomes. Because LDG with the ABD was a new procedure for some surgeons at the start of the study, the ABD group included cases in which the surgeon was using the device for the first time. Thus, even in single-center trials, it can be very difficult to ensure that all doctors have the same level of experience and proficiency. However, this study is the largest series comparing the short-term surgical results of an ABD and a USAD for LDG and is the first to demonstrate the safety of an ABD over a USAD in reducing POPF and mortality following LDG.In conclusion, the present study suggested that the ABD may be a safe and beneficial energy device for LDG, as the ABD was significantly more advantageous than the USAD in terms of the POPF and morbidity rates, number of lymph nodes retrieved, and postoperative hospital stay. To safely perform LG with lymph node dissection in patients with gastric cancer, the mechanism underlying the heat conductance of energy devices needs to be better considered, which may reduce the incidence of POPF. However, no matter what kind of energy device is used, surgeons need to be careful to avoid damaging normal tissues during surgery and ensure patient safety. Further studies involving multiple centers, a larger population, and a randomized controlled trial with long-term surveillance are needed to establish solid evidence concerning the safety of ABD."}
+{"text": "Our research found that a combinational approach blending kappa-carrageenan and silk fibroin could enhance the biological properties of the nanostructured scaffold. kappa-carrageenan could also enhance the osteogenic potential and bioactivity properties of silk fibroin nanofibers, while genipin crosslinking preserved the mechanical strength of hybrid nanofibrous mats, indicating that the electrospun hybrid scaffolds could be a potential candidate for bone regeneration applications.Bone tissue engineering has recently been considered as a potential alternative approach to treating patients with bone disorders/defects caused by tumors, trauma, and infection. Scaffolds play a crucial role in the field because they can serve as a template that can provide optimal structural and functional support for cells. In this study, we prepared a series of electrospun silk fibroin/kappa-carrageenan (k-CG)  was prepared using electrospinning and genipin (GP) as a crosslinker. The presence of k-CG in SF nanofibers was analyzed and confirmed using Fourier transform infrared spectroscopy (FTIR). In addition, X-ray diffraction (XRD) analysis confirmed that GP could cause SF conformation to shift from random coils or \u03b1-helices to \u03b2-sheets and thereby facilitate a more crystalline and stable structure. The ultimate tensile strength (UTS) and Young\u2019s modulus of the SF mats were enhanced after crosslinking with GP from 3.91 \u00b1 0.2 MPa to 8.50 \u00b1 0.3 MPa and from 9.17 \u00b1 0.3 MPa to 31.2 \u00b1 1.2 MP, respectively. Notably, while the mean fiber diameter, wettability, and biodegradation rate of the SF nanofibers increased with increasing k-CG content, a decreasing effect was determined in terms of UTS and Young\u2019s modulus. Additionally, better cell viability and proliferation were observed on hybrid scaffolds with the highest k-CG content. Osteogenic differentiation was determined from alkaline phosphatase (ALP) activity and Alizarin Red staining and expression of osteogenic marker genes. To this end, we noticed that k-CG enhanced ALP activity, calcium deposition, and expression of osteogenic genes on the hybrid scaffolds. Overall, hybridization of SF and k-CG can introduce a promising scaffold for bone regeneration; however, more biological evaluations are required.In this study, a novel nanofibrous hybrid scaffold based on silk fibroin (SF) and different weight ratios of  Over the last few decades, bone tissue engineering (BTE) has been considered as a potential alternative approach to treating patients with bone disorders/defects caused by tumors, trauma, and infection ,2,3,4. SBombyx mori silk fibroin (SF) is one of the most commonly used because of its desirable attributes such as biocompatibility, biodegradability, nontoxicity, low immunogenicity, and good mechanical strength  were obtained from Sigma-Aldrich (USA), absolute ethanol 99.8%, GP, and isopropanol were provided from Merck Company (Germany). In addition, silk cocoons from Bombyx mori were purchased from the Golestan Silkworm Research Institute (Iran). ALP\u2019s kit was obtained from Pars Azmun (Iran). Furthermore, fetal bovine serum (FBS) and other components of cell culture were purchased from Vasegh Company (Iran).Bombyx mori was boiled in 0.02 M Na2CO3 for one hour, then rinsed thoroughly with deionized water three times; finally, it was dried at 37 \u00b0C for 24 h. Moreover, degummed SFs were dissolved in 9.3 M LiBr at 80 \u00b0C for 8 h. In order to purify SF and eliminate the LiBr from the aqueous solution, dialysis was performed in cellulose tubing against distilled water for three days, changing the water every two hours. Then, the aqueous SF solution was centrifuged to remove impurities, and finally, it was lyophilized until a dried pure SF sponge was obtained.Purification of SF was performed according to previous studies . To be sv/v) HFIP/CHCl3 solution and stirred for about two hours using a KA\u00ae-RCT basic magnetic stirrer. Following that, different amounts of k-CG powder  were added to the SF solution, and all of the mixing solutions were placed on a shaker plate for 4 h before electrospinning to make a homogenous solution at room temperature. Then, the viscosities of different solutions were measured using a Brookfield viscometer (Model LVDVE 230). Pure SF solution was also considered as the control group, and all selected solutions were poured into a 10 mL syringe with a needle diameter of 0.5 mm for the electrospinning setup . The solution flow rate was fixed to 0.3 mL/h, and droplets of the injected solutions were subjected to a high voltage of 20 kV. Finally, at a distance of 12 cm from the syringe tip, nanofibers were gathered on the aluminum foil surface, then crosslinked with 1% (w/v) GP. For this purpose, GP powder was first dissolved in distilled water and agitated for 30 min until a homogenous solution was achieved. Then, the electrospun fibers were immersed in the GP solution for 24 h. The resulting scaffolds are called SF-CG1-GP, SF-CG3-GP, and SF-CG5-GP as GP-crosslinked nanofibers containing k-CG with a concentration of 1, 3, and 5 mg/mL, respectively. Further, noncrosslinked and crosslinked pure SF nanofibers were called SF and SF-GP, respectively.To obtain 12 wt% SF solutions, the freeze-dried SF sponge was first dissolved in an 8/2 , as well as the porosity measurements. The results were expressed as \u201cmean \u00b1 standard deviation\u201d.To observe the fiber morphology, high-resolution FE-SEM (TESCAN MIRA3) with a solid-state secondary ion detector (accelerating voltage: 30 keV) was employed. Prior to imaging, the dried samples were cut into round discs (diameter: 5 mm), mounted on an aluminum stub, and sputtered with gold (8\u201310 nm) for 3 min. The average diameter and fiber distribution were determined by measuring 100 fibers at random from each SEM image and analyzing these images using ImageJ2) with an initial weight of For swelling capacity measurements, a certain amount of sample (size of 1 \u00d7 1 cm2). Then, the images of the droplets were shot with a 2\u00d7 lens using a DFK 23U618 USB 3.0 color industrial camera. The angles around the image were analyzed, and the average was used to determine the eventual contact angle value.The wetting test was conducted using a contact angle measuring device . First, 4 \u00b5L of deionized water was dropped onto the surface of each mat . The instrument was purged with nitrogen gas to eliminate the spectral contributions of atmospheric water. Data were collected at a spectral range of 400 cmThe XRD pattern of the samples was evaluated using an automated X-ray diffractometer . The samples\u2019 scans were performed from diffraction angles of 2\u03b8 = 0\u201340\u00b0 with a CuKa source (l = 0.154 nm) at a scan rate of 0.015 \u00b0/s.v/v ninhydrin solution at 100 \u00b0C for 15 min; afterward, the solution\u2019s optical absorbance was measured using a spectrophotometer  at 570 nm. Glycine solutions of different known concentrations were utilized as standards. Finally, the amount of free amino groups in the mats was proportional to the optical absorbance of the solution. A standard curve of glycine concentration versus absorbance was employed to calculate the concentration of free NH2 groups in the sample. The degree of crosslinking was calculated using Equation (2).2 remaining in noncrosslinked and crosslinked samples, respectively.A ninhydrin assay was conducted to measure the degree of crosslinking; the percentage of free amino groups in GP-crosslinked nanofibrous samples was compared to noncrosslinked nanofibers. For this purpose, the samples were first heated with a 2 wt% 2 and an average thickness of 0.2 mm based on the method proposed by previous researchers [For tensile testing, nanofiber mats were prepared in rectangular pieces with dimensions of 10 \u00d7 40 mmearchers ,27,28. NTo calculate the biodegradation rate of nanofibrous mats, samples of the same size as those used in swelling tests were separately soaked in 20 mL of PBS (pH: 7.4) containing protease XIV (5 U/mL). At the designated time points of 1, 3, 7, 14, \u2026, 28 days, the samples were removed from PBS, rinsed with deionized water, dried in an oven for 12 h at 37 \u00b0C, and weighed. Finally, the mass loss percentage was calculated using Equation (3).The initial and final masses of the nanofibrous mats are represented by 2, 37 \u00b0C) as reported earlier [4 cells/well. After one day of cell culture, samples were washed 3 times with PBS solution and fixed in GTA (4% v/v) for 2 h, then dehydrated with ethanol. Finally, the completely dried samples were covered with a thin gold layer for SEM examination.To study the cell behavior, all nanofibrous mats were clipped into disc-like samples (diameter of 12 mm and height of 0.2 mm) and transmitted into Petri dishes. The samples were sterilized through immersion in 70% ethanol solution for 1 h; then, they were rinsed three times, each for 15 min, in PBS and air-dried in a sterile environment. Human osteoblast-like cells, MG 63, were obtained from the Materials and Energy Research Center . The cells were cultured in Dulbecco\u2019s modified Eagle\u2019s medium (DMEM) supplemented with 10% FBS and 1% penicillin under standard conditions  and propidium iodide . Cells cultured on the membrane\u2019s surface for 1 and 3 days were followed by removing the culture medium and PBS washing. Then, loaded cell samples were incubated in a mixture of 5 \u03bcM acridine orange and 5 \u03bcM propidium iodide, in 10 mL culture medium, to assess the presence of both live (green) and dead (red) cells at the same time. After the incubation period , cell membrane scaffolds were observed using a fluorescence microscope .4 per well for 24 h. Then, the culture medium was replaced with 10 \u03bcL of 12 mM MTT solution and placed in an incubator for 4 h. In the next step, 50 \u03bcL of dimethyl sulfoxide (DMSO) was supplied to each well to dissolve the formed formazan crystals. The deposited formazan crystals were dissolved in each well with 50 \u03bcL of DMSO, after 10 min of keeping it at 37 \u00b0C, the absorbance was measured at 570 nm with an ELISA reader (Stat Fax-2100).To assess the eventual cytotoxicity, an indirect 3--2, 5-diphenyltetrazolium-bromide (MTT) bioassay was employed. For this purpose, all samples were extracted in culture medium for 3, 7, and 14 days of incubation at 37 \u00b0C. Different extraction solutions were placed in contact with MG 63, with a density of 1 \u00d7 104) obtained from the Materials and Energy Research Center , were cultured on different samples. Finally, after 3 and 7 days of cell culturing, the cells were lysed using 200 \u03bcL of radioimmunoprecipitation lysis buffer system  and 2 \u03bcL of phenyl methyl sulfonyl fluoride (PMSF). ALP activity was calculated based on the ALP kit  protocol and was finally normalized corresponding to cell number.The ALP production of MC3T3-E1 cells (osteoblast precursor cell line) seeded on the mats was assessed based on earlier reports ,30. BrieAlizarin Red staining was used to determine the calcium concentration of the cell membrane scaffold. For this purpose, MC3T3-E1 cells cultured on different samples after 14 and 21 days in osteogenic medium  were fixed with 4% paraformaldehyde in 20 min, then washed with PBS. A 0.5 g amount of Alizarin Red-S was dissolved in 25 mL of deionized water, and the pH was adjusted to 4.2 using 0.5% ammonium hydroxide. Each well received one milliliter of Alizarin Red-S solution, which was then incubated for one hour at room temperature. Subsequently, the samples were properly rinsed with deionized water, then dried at ambient temperature before being examined under an inverted microscope . To quantify the calcium concentration, each stained sample was moved into a fresh 24-well plate and treated with 1 mL of 10% cetylpyridinium chloride solution for one hour to desorb calcium ions. The solution\u2019s absorbance was measured at 540 nm in an ELISA reader (Stat Fax-2100), and the results were normalized by cell number.5 cells per sample (disc-like sample with a diameter of 5 mm and thickness of 2 mm). After 24 h of attachment, the culture medium was replaced with 1 mL of osteogenic medium . The osteogenic medium was changed every 2 days until the end of the experiment. After 7 days of cell culture on the scaffolds, the total RNA was extracted using the Qiagen RNeasy Mini kit  according to the manufacturer\u2019s instructions, then converted to complementary DNA (cDNA) with first-strand cDNA using the TaKaRa RNA PCR kit, Shiga, Japan. The differentiation of MC3T3-E1 was monitored by measuring mRNA expression levels of Col I and Runx2. All the fold changes in gene expression were normalized to GAPDH. All results were quantified using the 2\u2212 (\u0394\u0394CT) method. The genes and the related specific primer sequences for Col I and Runx2 were designed and illustrated in The expression of some osteogenic genes, including collagen type I (Col I) and run-related transcription factor 2 (Runx2), was performed using the real-time PCR technique. The MC3T3-E1 cells were seeded at a density of 5 \u00d7 10p < 0.05 was used in the statistical calculations.All of the experiments were carried out at least three times, and the findings were averaged and given as mean \u00b1 SD. A statistical significance threshold of k-CG hybrid nanofibers was observed with SEM. The average fiber diameter, porosity percentages, and viscosity of the electrospun solution are presented in The morphology of SF and SF/k-CG also increased the swelling capacity to 160 \u00b1 1.5%, 170 \u00b1 1.8%, and 190 \u00b1 1.98% for SF-CG1-GP, SF-CG3-GP, and SF-CG5-GP, respectively. We believe this to be related to the hydrophilic nature of k-CG due to its abundant hydroxyl groups [k-CG content. SF-CG5-GP nanofibers had the lowest water contact angle (\u03b8 = 46.4 \u00b1 3.7\u00b0), while these values were 57.1 \u00b1 1.1, 62.5 \u00b1 1.3, 83.8 \u00b1 0.1, and 83.2 \u00b1 0.2 for F-CG3-GP, F-CG1-GP, SF-GP, and SF respectively. It was therefore concluded that k-CG, due to its hydrophilic nature, results in more suitable wettability. It has been reported that surfaces with a water contact angle in the range of 40\u201370\u00b0 are optimal for osteoblast attachment and proliferation [k-CG in nanofibrous mats can effectively moderate the hydrophobic properties of SF-based nanofibers, which are mostly generated by SF amino acid domains such as glycine-alanine and glycine-serine [Scaffold wettability plays a significant role in tissue regeneration, as a high water swelling ratio is conducive to transferring nutrition and wastes . Hence, feration ,35. The e-serine .\u22121 for amide v, 1235 cm\u22121 for amide III, 1533 cm\u22121 for amide II, and 1673 cm\u22121 for amide I in the SF structure. After GP crosslinking, the bands corresponding to amide III, amide II, and amide I slightly shifted to 1260, 1524, and 1640 cm\u22121, respectively. This could be due to the change in conformation from random coils or \u03b1-helices to \u03b2-sheets reminiscent of a more stable SF structure [\u22121 appeared as well [\u22121, which could be associated with the stretching vibration peak of tertiary amine C-N as a result of the emerging covalent bond in the SF-GP sample, which was due to the covalent crosslinking between GP and amino groups in SF [FTIR analysis was performed to examine the functional groups contained in each membrane scaffold, and the results are presented in tructure . Some ch as well . Moreoveps in SF . Genipinps in SF ,38.\u22121 are related to the sulfate group (stretching S-O) and galactopyranose rings (C-C vibrations) of k-CG, respectively. With increasing k-CG content, sharper sulfate-related peaks were observed. Additionally, we found a peak at around 2910 cm\u22121 attributed to C-H bonds in both SF and k-CG. Peaks at around 3400 cm\u22121 are attributed to hydrogen bonds (OH groups) that can occur between nanofiber components [In hybrid nanofibrous scaffolds , the bands that appear around 1265 and 1050 cmmponents ; in the k-CG content. The peak is characteristic of the \u03b2-sheet crystalline structure, and it is suggested that after GP crosslinking, the amorphous SF (Silk I) is partially transformed into a more rigid and stable \u03b2-sheet structure (Silk II). This finding proves that GP could be acting as a crystallization-inducing agent. The other hybrid scaffolds exhibit a similar pattern to crosslinked SF nanofibers, thereby indicating that the presence of k-CG in the nanofiber composition does not damage their crystalline structure. This result is consistent with the FTIR results, demonstrating that the crosslinked nanofibers had a higher percentage of \u03b2-sheets due to the addition of GP, which caused SF molecules to be structurally rearranged. These findings were also consistent with the results of a previous study by Wang et al. reporting that the uncrosslinked SF film showed arc-shaped scattering at around 20\u00b0, corresponding to the amorphous silk (Silk I), while after GP crosslinking, there appeared an obvious characteristic peak at around 20.7\u00b0 and 24.3\u00b0, illustrating the content of \u03b2-sheet (Silk II) conformation [As shown in ormation .The crosslinking degrees of different fabricated mats were assessed using the ninhydrin assay. The obtained values were 65.2 \u00b1 1%, 55 \u00b1 0.5%, 49.5 \u00b1 2.5%, and 42.3 \u00b1 1.1%, for SF-GP, SF-CG1-GP, SF-CG3-GP, and SF-CG5-GP, respectively. Since GP reacts with the SF amino acids spontaneously, the degree of crosslinking increases with increasing SF content. The maximum percentage (65.2 \u00b1 1%) is related to SF-GP, which contains the highest concentration of amino groups in its structure.k-CG content, the UTS was reduced to 7.31 \u00b1 0.2 MPa in SF-CG1-GP, 5.20 \u00b1 0.2 MPa in SF-CG3-GP, and 4.04 \u00b1 0.09 MPa in SF-CG5-GP. These results are in agreement with the SEM analysis, which established a correlation between increasing k-CG content and a larger fiber diameter. Several studies have already proved that the reduction of fiber diameters enhanced tensile strength because of the sharper orientation of the polymeric chain along the fiber axis [The results of the mechanical studies are shown in ber axis ,43. The ber axis ,44. Prevber axis ,45. Anotber axis ,46. AccoA similar trend was also obtained for Young\u2019s modulus, as shown in k-CG and the amorphous structure, which can be susceptible regions for enzymatic attack [The characterization of the degradation behavior of nanofibers is shown in c attack ; this fik-CG content. In comparison to SF nanofibrous mat, where the cell phenotype is rounded, on hybrid samples, cells were shown to be elongated and adopted a spindle-shaped morphology, showing that k-CG has a favorable influence on cell attachment to the matrix. In fact, based on the findings of many other studies, hydrophilic and nanorough surfaces such as k-CG-containing materials promote the adsorption of a wide variety of glycoproteins such as vitronectin and fibronectin, consequently promoting the adhesion and spreading of osteoblast or osteoblast-like cells [The morphology of osteoblast-like cells MG 63 after 24 h of cells seeding on the surface of different mats is illustrated in ke cells ,47.The distributions and viability of MG 63 cells in electrospun nanofibers were visualized using confocal microscopy following live/dead cell staining. As shown in k-CG exhibited better cell viability than the pure SF nanofibers. More cellular proliferation was observed on the sample with the highest k-CG content (SF-CG5-GP). On Days 7 and 14, significant differences  in cell number were observed between SF-CG5-GP and SF nanofibers. Improved cell proliferation can be associated with the presence of k-CG, which improves the surface hydrophilicity of hybrid fibers, resulting in greater absorption of bioactive proteins [k-CG and its negatively charged functional groups, similar to native ECM characteristics, could provide a more suitable matrix for adhesion and proliferation of osteoblastic cells [Cytotoxicity of nanofibers against the MG 63 cell was determined by an MTT bioassay and is presented in proteins . It has ic cells ,49.2+ and thereby increasing ECM mineralization. The ALP enzyme is a transcription factor that activates the cell differentiation factors, as well as playing a role in message transmission and osteoblast differentiation [k-CG were significantly higher than that of pure SF, with the rate increasing over time. The highest amount of ALP expression was observed on SF-CG5-GP on Day 7. This could be attributed to the presence of sulfonate groups in k-CG. A similar trend was observed by Felgueiras et al., who found that grafting of sulfonate groups on Ti alloys could improve osteoblast differentiation [ALP is a biomarker of osteogenesis that appears in the early stage of osteogenic differentiation and is linked to osteoblast cell mineralization activity ,51. Calcntiation . ALP actntiation .p < 0.01). This is in accordance with the previously suggested action of CG on promoting osteogenic differentiation [3H) in CG causes Ca2+ to be taken up, followed by phosphate ions, resulting in calcification via nucleation and growth. In mineralized ECM, Alizarin Red-S can bind to Ca2+, leading to bright red stains [ntiation . In factd stains .k-CG hybrid nanofibrous scaffolds was evaluated through a quantitative analysis of relative mRNA expression levels of the main osteogenic genes, including Col I and Runx2. Col I is the most abundant protein found in the organic phase of the bone ECM. Collagenous matrix formation is also important for the development of bone cell phenotype and is thought to be a cell differentiation modulator. The relative gene expression of Col I from seeded MC3T3-E1 under osteogenic conditions is presented in k-CG enhances osteogenic differentiation of MC3T3-E1 cells. Runx2 is also an important transcription factor that activates the osteoblast differentiation marker genes in all osteogenic differentiation processes. Both Col I and Runx2 genes exhibited low expression levels in the cells on the pure SF and SF-GP scaffolds while showing the highest expression on the SF-CG5-GP nanofibers. From the literature, the bioactive sulfonate groups of k-CG were reported to be associated with an increase in the expression of osteogenic marker genes. It was reported that k-CG could promote MC3T3-E1 preosteoblast adhesion and spreading, metabolic activity, proliferation, and osteogenic differentiation [The osteogenic ability of pure SF, SF-GP, and SF/ntiation .k-CG nanofibrous membranes with the aim of mimicking bone ECM structure and composition and improving the biological properties of SF-based nanofibers. It was concluded that a combinational approach blending k-CG and SF could enhance the biological properties of the nanostructured scaffold. k-CG could also enhance the osteogenic potential and bioactivity properties of SF nanofibers. while GP crosslinking preserved the mechanical strength of hybrid nanofibrous mats. The incorporation of k-CG into the SF nanofibrous scaffolds in electrospun nanofibers was confirmed by FTIR, XRD, and contact angle measurements. Wettability studies indicated that the presence of k-CG in nanofibrous mats could effectively moderate the hydrophobic properties of SF-based nanofibers, which in turn enhanced cell viability and proliferation. On the other hand, degradation studies proved that the GP crosslinking process could stabilize SF nanofibrous scaffolds and make them more stable in biological solutions. Furthermore, GP crosslinking increased the mechanical characteristics of the scaffolds, which is another crucial aspect of bone tissue engineering. From the regulation of ALP activity, mineral deposition, and osteogenic gene expression, k-CG was found to guide MC3T3-E1 toward the osteogenic lineage and induce the mineralization and formation of bone tissue in vitro. In particular, SF-CG5-GP displayed the highest effects on MC3T3-E1 in terms of ALP activity, calcium deposition, and Col I and Runx2 gene expression, indicating that it could be a potential candidate for bone regeneration applications, though more in vitro and in vivo studies are inevitable.In this study, we prepared a series of electrospun SF/"}
+{"text": "Monitoring was carried out through the control of physico-chemical and microbiological parameters, such as temperature, pH, humidity, Rottegrade, Solvita tests, the presence of Salmonella sp. and Escherichia coli, total coliform, and Enterococcus sp. concentrations. After carrying out the tests, it can be affirmed that the three types of installations used worked correctly in terms of the monitoring of physico-chemical parameters, giving rise to a compost of sufficient stability and maturity to be applied on agricultural soil. In all cases the bacterial concentrations in the final compost were lower than those detected in the mixture of initial components for its preparation, thus complying with the requirements established in RD 506/2013 and RD 999/2017RD on fertilizer products. However, it cannot be affirmed that one of the three types of installation used produces a greater bacterial inactivation than the others. When composting with different types of facilities, it is of interest to optimize the irrigation and aeration system in order to have a better control of the process and to study the possible temperature gradients in the piles to ensure good sanitization without the risk of bacterial proliferation a posteriori. Finally, the different initial mixtures of OFMSW and SM used in this study did not have a significant influence on the functioning of the composting process or on the microbiological quality during the process. The irrigation water can provide a bacterial contribution that can lead to increases in concentration during the composting process. This study is part of the Life-NADAPTA project (LIFE16 IPC/ES/000001), an integrated strategy for adaptation to Climate Change in Navarra, where NILSA participates in water action and collaborates in agricultural action, which includes among its objectives the development of new soil amendments from different organic waste.The aim of this work was to carry out a pilot experiment to monitor OFMSW  composting processes using different types of installations . To carry out the process, pruning waste was used as structuring material (SM), in a 1:1 and 1:2,  Municipal waste is made up of different components, with its organic fraction being predominant. This fraction represents a percentage by weight of 44\u201349% of waste  and is aIn Spain, around 18 million tons of municipal waste was collected in 2018, 17% of which was composted. In the same year, one million tons of biodegradable waste was collected separately, 65% of which was composted [The organic fraction of waste can be treated by biological processes, both aerobic (composting) and anaerobic, in order to hygienize it and stabilize the organic matter. In the case of composting, woody plant residues (pruning) are fundamental since their function is that of structuring materials  .The composting process can be carried out in different types of facilities: open systems such as windrows or piles, either aerated static or turned; and closed systems such as reactors or composters . In all The resulting material, compost, is a stable material that can be used as an agricultural amendment as it brings many benefits to soils: it provides organic matter and biofertility, improves soil structure and water retention, decreases erosion, etc. .The use of compost in agricultural soils is regulated in Spain by Royal Decree 506/2013 on fertilizer products . The lawIn the Autonomous Region of Navarra, where this study was carried out, the \u201cWaste Plan of Navarra 2017\u20132027\u201d was drawn up, in accordance with current legislation on waste, whose objectives include \u201cMoving towards high quality selective collection, maximizing waste recovery and recycling and guaranteeing the co-responsibility of the waste generator  and taking into account the principle of gender equality, through the usual management routes and through emerging processing routes, especially for domestic bio-waste and industrial waste\u201d.Navarra de Infraestructuras Locales S.A. (NILSA), a company which specializes in sanitation and water treatment and manages the Waste Consortium of the Autonomous Region of Navarra, in collaboration with the Water and Environmental Health group, a reference research group recognized by the Government of Aragon and belonging to the University Institute for Research in Environmental Sciences of Aragon, is developing a project (to which this study belongs) to promote the correct management of bio-waste in Navarra, encouraging its recycling through composting processes, from a safe environmental and health perspective. Its objective is the application of composting processes in the treatment and recovery of organic waste, with the aim of promoting its use as an agricultural fertilizer, while minimizing the associated environmental and health risks. The project also aims to establish guidelines for action that facilitate the adoption of good agricultural practices for the application of the different fertilizers obtained and that encourage coordination of the strategy for the management of organic waste by the public sector with the needs of the agricultural sector.Specifically, the aim of this work is to study the influence on the physico-chemical and microbiological characteristics of the compost obtained by the type of installation used in the composting process at pilot scale  and of the characteristics and mixing ratio of the initial waste used .The waste used in this study came from a waste treatment plant in Navarra, which processes the organic fraction of municipal solid waste (OFMSW). Pruning waste was used as a structuring material (SM) for composting and the water used to provide moisture for the process was taken from the water level.OFMSW composting was carried out in three types of pilot facility with the operating conditions described below:The reactor has two compartments of approximately 125 L capacity with polyurethane insulation walls and a longitudinal axis connected to the aeration system. Both aeration and turn-over can be carried out automatically by means of a microprocessor. Water was added manually. The temperature was controlled automatically by means of PT100 sensors. A picture of the automatic reactor can be seen in 3 capacity were used. Air was supplied through 3 pipes installed in the floor of the piles and water through a drip system until the piles reached a humidity of 40%. Covers were placed over the piles to help maintain humidity. A PT100 sensor was used to automatically monitor the temperature of the piles and the ambient temperature. Two aerated static piles of 4\u20136 m3. Aeration was carried out manually by mechanical turning of the piles with a shovel and water was supplied manually through a water hose until the pile reached a humidity of 40%. The piles were covered with layers to help maintain humidity. A PT100 sensor was used to automatically monitor the temperature of the piles and the ambient temperature. The turned piles had a capacity of 4\u20136 mv/v), in function of the available pruning material and the relation C/N of the mixture. This is summarized in OFMSW composting was carried out in the facilities described above, with different initial ratios between the OFMSW and SM  was carried out according to the \u2018Test Methods for the Examination of Composting and Compost\u2019 . DiffereThe solid samples were pre-treated to analyze the microbiological and physico-chemical parameters. This pre-treatment was based on that described by Carter (1993) . For thev/v), and the compost samples throughout the four months of the composting process. All of these samples were analyzed in the three facilities used in this study .The samples analyzed in this part of the study were: the initial OFMSW, the initial SM, the mixtures of initial OFMSW and SM at different ratios  , which eof waste ,20, clasThe samples analyzed in this part of the study were the same as those used in the analysis of the physico-chemical parameters and, in addition, samples of the water used to provide humidity during the composting process.Escherichia coli, Enterococcus sp., Clostridium perfringens, total mesophiles and Salmonella sp. Among these bacteria, those selected for monitoring microbiological parameters during the composting process and in the irrigation water were Salmonella sp. and Escherichia coli, as they are the microorganisms for which maximum concentrations in the compost are established by current legislation, and total coliforms and Enterococcus sp., due to their staining characteristics  and since they are parameters commonly used in the microbiological control of environmental matrices.The initial microbiological characterization of the materials used for composting, OFMSW and SM, was carried out through the analysis of six bacteria: total coliforms, The microbiological parameters analyzed in the samples, the culture media used, and the standard methods of analysis are shown in To reliably determine the concentration of bacteria in the samples, serial dilutions were performed. These dilutions were carried out by dissolving 1 mL of sample in 9 mL of 0.9% NaCl. All samples were analyzed using the plate count method. After surface seeding or using the membrane filtration method, the samples were subjected to the appropriate incubation period for each bacterium (time and temperature), resulting in plates with colored colonies that could be counted as colony forming units (CFU). The microbiological concentration of solid samples is expressed as CFU per gram of dry matter  and the microbiological concentration of water samples is expressed as CFU per 100 mL.Lepidium sativum L. seeds was analyzed on three different bases: distilled water, unamended soil, and commercial peat.To determine the fertilizing capacity of the compost obtained, the germination index (GI), representative of the phytotoxicity of the compost , was anaThe initial physico-chemical properties of OFMSW and SM used in the composting are shown in As can be seen in terms of the physicochemical properties of OFMSW and SM, the main differences lie in humidity, which is higher in OFMSW, total solids, which are higher in SM, and nitrogen and phosphorus, which have higher values in OFMSW. The rest of the physico-chemical parameters present similar values in both components. As for heavy metals, although the values detected in OFMSW and SM are different, at no time are the maximum concentrations established in RD 506/2013 (modified by RD 999/2017) for heavy metals in compost exceeded. The two initial mixtures tested in this study result in C/N ratios between 5.2 and 5.5.Escherichia coli and Enterococcus sp., with the exception of total coliforms, whose concentration is similar in both components. In addition, Salmonella sp. was not detected in either of them.As shown in 2 to 3.1 \u00d7 105 CFU 100 mL\u22121 of total coliforms, 5.0 \u00d7 101 to 1.4 \u00d7 105 CFU 100 mL\u22121 of Escherichia coli and 5.0 \u00d7 101 to 1.1 \u00d7 104 CFU 100 mL\u22121 of Enterococcus sp. were found, concentrations quite variable depending on the time of analysis during the process.With regard to the microbiological analysis of the irrigation water, used to provide humidity during the composting process, concentrations of 6.2 \u00d7 10v/v) is described below.The control of the composting processes was carried out by monitoring temperature, pH and humidity. The evolution of these parameters in the three pilot scale composting facilities  and with the two initial ratios between OFMSW and SM (OFMSW:SM of 1:1 and 1:2 (a)Automatic reactorAs can be seen in In general, slightly higher humidity values are observed for the initial 1:2 mixture during the process, probably due to the fact that although SM has a much lower humidity than OFMSW initially see , a highe(b)Aerated static pileAs can be seen in (c)Turned pileAs shown in The humidity values are between 20 and 50% during the whole process in both piles, and low values are observed in spite of the multiple additions of water during the process see . The greComparing the results obtained in terms of the monitoring of the physico-chemical parameters of the composting process using the three types of facilities, it is observed that the highest temperatures are reached in the turned pile, followed by the aerated static pile and the automatic reactor, although in no case is the temperature corresponding to \u201cmicrobial suicide\u201d  exceeded\u00ae test indicate that the compost obtained in the three types of facility and with different initial mixtures of OFMSW and SM is highly stabilized and sufficiently mature at the end of the process (grade V according to Rottegrade and maturity index 6 according to Solvita\u00ae). Moreover, the C/N ratio of the final compost obtained from the three facilities has values of 12.5\u201313.5, complying with the requirements established in the current legislation.Finally, the Rottegrade and the SolvitaEscherichia coli and Enterococcus sp.) in the compost during the composting process using the three types of installation  with both OFMSW/SM ratios. Salmonella sp. was not detected at any time in any of the facilities used.8 CFU g\u22121) with the three types of pilot composting facilities, the concentrations are lower at the end of the process, complying with the requirements established in RD 506/2013 on fertilizer products (modified by RD 999/2017). However, the overall bacterial reductions are different depending on the facility used: in the case of the automatic reactor, these concentrations are reduced to 102\u2013103 CFU g\u22121 for all bacteria; using the aerated static pile, the final concentrations vary between 103 CFU g\u22121 for Escherichia coli and 106 CFU g\u22121 for total coliforms; and using the turned pile, the final concentrations are between 101 CFU g\u22121 for Escherichia coli and 105 CFU g\u22121 for total coliforms, with the concentration of Enterococcus sp. being around 103\u2013104 CFU g\u22121 in both piles.As can be seen, starting from the same initial bacterial concentrations  or 1:2 (v/v).The results for the automatic reactor show a progressive decrease in the concentrations of the three bacteria analyzed, being more pronounced during the first days, probably due to the fact that the first days of the process is when there is a greater increase in temperature in the reactor see . FifteenEscherichia coli decreases progressively throughout the process, with a reduction in concentration of about 5 logarithmic units at the end of the process with respect to the initial concentration of the mixture, this being the bacterium whose concentration is most reduced. In the case of Enterococcus sp., concentrations of the order of 104 CFU g\u22121 are reached at the end of the process, which means a reduction of four logarithmic units. Some increases in concentration can be attributed to the bacterial contribution through the irrigation water, since during the process maximum concentrations of 104\u2013105 CFU 100 mL\u22121 are detected for the three bacteria. In addition, if during the thermophilic phase there has not been a homogeneous temperature throughout the pile, bacteria may remain in the compost and proliferate over time. Again, as with the automatic reactor, no significant differences in bacterial evolution are found in the cases of composting with an initial mixture of OFMSW and SM of 1:1 (v/v) or 1:2 (v/v).The aerated static pile results show different behavior depending on the bacteria. The concentration of total coliforms remains practically constant throughout the process; only variations of approximately 1 logarithmic unit are observed. The concentration of Escherichia coli decrease, especially at the beginning of the composting process, coinciding with the period in which a higher temperature increase is observed in the piles . With regard to Enterococcus sp., as in the previous case, a progressive decrease is observed during the process, achieving at the end a reduction of five logarithmic units in its concentration. As in the case of the aerated static pile, some increases in the concentration of the bacteria analyzed throughout the process may be due to the bacterial contribution of the irrigation water or to the non-homogeneity of the temperature in the pile. No significant differences in bacterial evolution are found in the cases of composting with an initial mixture of OFMSW and SM of 1:1 (v/v) or 1:2 (v/v), as was the case using the other two pilot facilities.Finally, the results obtained using the turned pile show a similar behavior as in the case of using the aerated static pile, although the final bacterial concentrations are lower in all cases. The concentrations of total coliforms and iles see . SpecifiEscherichia coli occurs in the turned pile. The latter may be due to the fact that Escherichia coli is a thermotolerant bacterium and it is in the turned pile where higher temperature values are reached throughout the composting process, although without exceeding the temperature of \u201cmicrobial suicide\u201d, conditions in which an inhibition of the biological treatment would occur. In the case of Enterococcus sp., the reductions obtained are similar in the three installations. Therefore, it cannot be stated that there is one type of facility among those used that produces a greater bacterial inactivation in general. It is important to have a good control of the temperature and to detect unfavorable points that could cause bacterial proliferation.A comparison of the three facilities shows that the automatic reactor produces the highest total coliform removal, while the greatest reduction of With regard to the fertilizing capacity of the compost obtained, in all cases high values of the germination index were obtained, proving that the compost is stable for use as an agricultural amendment . According to Zucconi et al. (1981), germination rates below 50% show a strong presence of phytotoxic substances and, therefore, a compost not stable for use.Escherichia coli, which is a thermotolerant bacterium. In all cases the bacterial concentrations in the final compost were lower than those detected in the initial components, complying with the requirements established in RD 506/2013 on fertilizer products (modified by RD 999/2017). However, it cannot be affirmed that there is a type of installation among those used that produces a greater bacterial inactivation in general. When composting with different types of facility, it is of interest to optimize the irrigation and aeration system to have a better control of the process and to study the possible temperature gradients in the piles to ensure good sanitization without the risk of bacterial proliferation posteriori. Finally, the different initial mixtures of OFMSW and SM used in this study did not have a significant influence on the functioning of the composting process or on the microbiological quality throughout the process. It should be pointed out that the irrigation water can provide a bacterial contribution that leads to increases in concentration during the composting process.The composting process of OFMSW has been analyzed at pilot scale in three types of facility, automatic reactor, aerated static pile, and turned pile. All three performed correctly in terms of the monitoring of the physico-chemical parameters, resulting in a compost of sufficient stability and maturity to be applied on agricultural soil. Although the maximum temperature during the composting process was reached using the turned pile, this does not imply that this installation produces the greatest elimination of bacteria in general, but it does eliminate"}
+{"text": "The vast structural and chemical diversity of metal\u2212organic frameworks (MOFs) provides the exciting possibility of material\u2019s design with tailored properties for gas separation, storage and catalysis. However, after more than twenty years after first reports introducing MOFs, the discovery and control of their synthesis remains extremely challenging due to the lack of understanding of mechanisms of their nucleation and growth. Progress in deciphering crystallization pathways depends on the possibility to follow conversion of initial reagents to products at the molecular level, which is a particular challenge under solvothermal conditions. The present work introduces a detailed molecular-level mechanism of the formation of MIL-53(Al), unraveled by combining in situ time-resolved high-resolution mass-spectrometry, magic angle spinning nuclear magnetic resonance spectroscopy and X-ray diffraction. In contrast to the general belief, the crystallization of MIL-53 occurs via a solid-solid transformation mechanism, associated with the spontaneous release of monomeric aluminum. The role of DMF hydrolysis products, formate and dimethylamine, is established. Our study emphasizes the complexity of MOF crystallization chemistry, which requires case-by-case investigation using a combination of advanced in situ methods for following the induction period, the nucleation and growth across the time domain. The discovery and control of the synthesis of metal\u2013organic frameworks remains challenging due to the lack of understanding of their nucleation and growth. Here, the authors report a detailed molecular-level mechanism of the formation of MIL-53(Al). Their topology can be changed by varying the connectivity of the organic linker and/or the inorganic unit. The perceptible advantage of MOFs over materials, such as zeolites and other porous oxides, is their larger surface area, larger pore diameter, and chemical versatility that result from their composition and crystalline structure. However, it took almost twenty years to extrapolate this peculiarities to areas such as catalysis6 and gas separation and storage12. Equally, MOFs can function as support for metal or oxide nanoparticles, finding application in photovoltaics and electrocatalysis14.Metal-organic frameworks (MOFs) are solid materials comprising multi-functionalized organic molecules bound to metal ions or clusters through coordinating moieties, such as carboxylates, imidazoles, amines, sulfates, and phosphates, to form microporous and mesoporous crystalline compounds possessing one-, two-, and three-dimensional structures15. However, even after several decades of research, the field of understanding of the synthesis of MOFs remains a gray area due to the absence of insight to the mechanisms of their nucleation and crystallization20. The lack of this knowledge hinders the rational design of MOF for target applications23. Unraveling the synthesis mechanism of solid compounds is of enormous interest to both academia and industry due to the possibility to engineer and then synthesize materials with specific properties24. Needless to say, studying the crystallization processes of MOFs is a complex endeavor due to the heterogeneity of the synthesis medium, comprising a solvent, inorganic precursors, organic linkers, modulators, templates, etc. and high sensitivity to various physical and chemical parameters, such as temperature, pressure, pH, the composition of the reaction mixture, and aging time28.There are numerous examples showing the engineering of novel MOFs at the molecular level29 are among the most accepted mechanisms established, for instance, for the family of aluminum trimesates of MIL-96, MIL-100, and MIL-110 topology30. According to this model, the crystallization of MOF starts with the formation of SBU, comprising metal clusters or ions, bound to the organic linker. Next, they aggregate and nucleate, forming the seeds for further crystallization. The transport of SBUs or/and molecular precursors from the liquid phase to the seeds finalizes the crystallization of the material, which can subsequently undergo Ostwald ripening, resulting in larger crystals. Simultaneously, more elementary mechanisms, such as growing from oligomers or pre-formed clusters, have also been suggested in the case of UiO-6632. In contrast, the evolution of amorphous nanoparticles, observed during the crystallization of MIL-89(Fe) and developing into ordered structures, constitutes an alternative to the liquid transport crystallization model34. Crucially, none of these models explicitly explains the frequent observation of intermediate phases, such as MOF-235 or EHU-30, formed at the early stages of the solvothermal synthesis36. This issue is highlighted in the recent reviews, urging the researchers to develop and use in situ methods for monitoring the synthesis22.Nucleation and growth from soluble, pre-formed, secondary building units (SBUs) or related molecular structures37. Equally, a very limited number of reports is devoted to the application of NMR38, Raman39, X-ray absorption spectroscopy33, or mass spectrometry40, which makes the overall area of the understanding synthesis extremely underexplored. Moreover, no single technique can interrogate the many species that exist during MOF synthesis on timescales from seconds to days, but each chemical reaction and process during framework construction affects the product that forms and its properties. NMR spectroscopy can provide the information on the kinetics of nucleation, being, however, intrinsically blind to the induction period due to the low resolution and signal broadening. In contrast, high-resolution mass spectrometry is ideal for the direct establishing the structure of soluble building units and pre-nucleation species. X-ray diffraction (XRD) is of natural choice to follow the formation of the crystalline phase of target MOF or its by-products. A combination of various techniques becomes essential to explore the synthesis in the time domain from induction period through nucleation to final precipitation, hence enabling the refinement of the current crystallization models for different synthetic protocols.There are indeed quite a number of research papers devoted to the in situ analysis of the formation of MOFs under solvothermal conditions, mostly using X-ray scattering techniques, which are intrinsically sensitive to the observation of crystalline phases, and, in some cases, to the amorphous nuclei40. The formation of this thermodynamically stable phase is frequently observed during the synthesis of other MOFs, such as MIL-101, MIL-68, and MOF-23541. In this work, we focused our attention on studying MIL-53(Al) crystallization mechanism using in situ electrospray ionization coupled to high-resolution mass spectrometry (ESI-HRMS), magic angle spinning NMR and X-ray diffraction. To do so, we have developed the methodology, which can become a compelling approach when studying the mechanism of the synthesis of (in)organic functional materials. The combination of physical-chemical tools, data processing approach, and discovered mechanistic concepts pave a road towards understanding the synthesis mechanism and engineering properties of metal-organic frameworks, with broad implications in fields ranging from adsorbents and catalysts to photovoltaics and semiconductors.Aluminum-based MOFs are especially important due to their high thermal stability (>773\u2009K) and relatively low synthesis cost. MIL-53, one of the most studied MOFs, is of particular interest due to its breathing behavior upon adsorption of guest molecules2BDC) dissolved in N,N-dimethylformamide (DMF). Figure\u00a0m/z, coordinated to the ligands NO3\u2212, BDC2\u2212, HDBC\u2212, DMF, OH\u2212, OCH3\u2212, and HCOO\u2212. The first four originate from the initial synthetic mixture, while the latter are coming either from the hydrolysis of aluminum nitrate nonahydrate and N,N-dimethylformamide or coordination of methanol, which is used as a solvent for ESI-HRMS analysis. The structure of the aluminum-containing species does not vary significantly in negative and positive modes\u2014the main difference is associated with the coordination of one or two DMF molecules to each positively charged specie, which it is not the case for anions. At a longer reaction time, the formation of dimethylammonium nitrate salts ((CH3)2NH2)x(NO3)x-1+ was observed in positive mode. Comparison of the time-resolved responses of the normalized intensities of each identified MS peak enabled the grouping of the peaks in the classes with very similar temporal behavior. Three main groups comprise the following compounds:(i)monomeric Al, which has only one aluminum atom in the structure, charge-balanced with NO3\u2212, OH\u2212, OCH3\u2212, and DMF. None of the aluminum coordinated to terephthalic acid species follows a similar behavior.(ii)Al - BDC adducts, where the mono- and multinuclear aluminum species coordinated to terephthalic acid were found together with other charge-balancing anions, such as NO3\u2212, OH\u2212, OCH3\u2212, and DMF.(iii)Al - BDC - HCOO adducts, which are structurally similar to the previous group, but also possessing formate anions in the structure. Their temporal behavior is significantly different from the other two classes and therefore they were placed in a separate group.The chemical processes and reactions leading to the formation of the final MOF start immediately after the mixing of the initial reagents aluminum nitrate and terephthalic acid . The formate-containing group Al - BDC - HCOO slowly progresses during the 0\u2013150\u2009min reaction time and stabilizes. Strikingly, the temporal behavior of monomeric Al shows gradual decrease from the beginning of the reaction until the start of crystallization, followed by the rapid increase of the signal and its stabilization. In contrast, the consumption of cationic monomeric Al takes place rapidly, with almost no effect of the crystallization on its concentration. The difference in temporal behavior of anions and cations, however, can not be directly correlated and might originate from the different structure and/or adducts formed. The intensity of the Al - BDC - HCOO peaks passes through the maximum at 150\u2009min, while Al - BDC group shows consumption of these species during the entire reaction time.Following this classification, we compared the time-resolved response of the sum of normalized intensities of these main classes Fig.\u00a0. In the 39, numerous Al- and terephthalic acid-containing species can be identified during the induction period, including those due to the gradual hydrolysis of DMF, namely, dimethylamine and formate anions, with the latter participating in the coordination to aluminum. The species containing formate anions progress slowly, which is seemingly associated with the low hydrolysis rate of DMF. Most of the species containing terephthalic acid are consumed at a longer reaction time, showing its depletion from the solution and transfer to the solid phase. The monomeric aluminum anions show unexpected behavior implying spontaneous increase of the concentration after beginning of MIL-53 crystallization. Even though it can only be interpreted semi-quantitatively, HRMS suggests the presence of such monomeric aluminum anions.In line with previous reports40 establishing the structure of molecular precursors and secondary\u00a0building units of MIL-53 in solution, MAS NMR can follow38 the evolution of both liquid and solid phase. Figure\u00a013C and 27Al MAS NMR spectra acquired during the synthesis at 343\u2009K. After heating the synthetic mixture to the reaction temperature, several sharp overlapping signals are visible in 27Al MAS NMR spectrum , the strongest transition appears at \u2212150\u2009ppm. The time-resolved spectra show the signals around 0\u2009ppm gradually decreasing, and that at \u2212150\u2009ppm developing after some induction period of about 200\u2009min. This points to the consumption of the aluminum species from the liquid phase and the start of formation of MIL-53.While HRMS enablesum Figs.\u00a0, S5\u2013S7. 13C MAS NMR acquired in two modes\u2014direct excitation without proton decoupling and with proton cross-polarization and high-power proton decoupling (CP-MAS), respectively. The latter pulse sequence allows observation of the solid phase, while the former is dominated by the species in solution .Figure\u00a0on Figs.\u00a0\u2013S13. Theon Figs.\u00a0. Asymmet2BDC with aluminum ions. The reaction of deprotonated and protonated carboxyl groups with aluminum is finished simultaneously.Figure\u00a01H-13C CP-MAS data show the presence of an induction period prior the observation of any signals coming from the solid MIL-53 nuclei within the studied temperature range 39. In contrast to the previous report, no other phases such as MOF-235 were found in the course of the synthesis35. The obtained background corrected data were used to calculate the excess of crystallinity. Comparing these data with those of 27Al MAS NMR confirms the compatibility of NMR and XRD: the progression of the MOF phase signals coincides at all temperatures studied 2NH2)x(NO3)x-1]+[NO3]\u2212.The nucleation process starts after achieving the maximal concentration of deprotonated terephthalate in the mixture Fig.\u00a0. The forI\u2009=\u20091/2 nuclei such as 1H and 13C. For the linker concentration, typical for MOF synthesis (about 0.1\u2009M), the time resolution higher than 5\u2009min is achievable with high signal-to-noise ratio. 1H-13C CP-MAS NMR spectroscopy is ideal for monitoring nucleation phase, both kinetics and chemical composition of nuclei. Remarkably, by introducing magic angle spinning, no effect on the synthesis kinetics can be observed: at the spinning rates employed, the local heating due to the viscous friction and centrifugation effect are negligible. However, NMR suffers from low resolution for quadrupolar nuclei such as 27Al and here very little structural information can be gained, while keeping sufficient sensitivity of the analysis of consumption/accumulation kinetics of precursors and MOF. In this respect, we expect the boost in application of X-ray absorption spectroscopy for monitoring the evolution of the metal core. Finally, proton NMR was shown to be surprisingly informative in terms of following hydrolysis of the solvent and monitoring pH, even without adding the specific amines, typically used as in situ NMR pH meters.Our study on a practical example highlights the capabilities of various time-resolved physical-chemical methods for monitoring the synthesis at different stages and emphasizes the need for a combined approach. It identifies a roadmap towards understanding the chemical processes that occur during the quite underexplored field of synthesis. In particular, NMR spectroscopy shows a great potential for following the fate of molecular precursors, notably organic linkers comprising NMR-active spin Equally, we appreciate that NMR is completely blind to the induction phase. We show however that ESI-HRMS can fill this gap by providing structural information about secondary\u00a0building units and other molecular adducts formed right after the preparation of synthetic mixture. On the one hand, mass spectrometry possesses an almost infinite resolution, dictated by the equipment, and on the other, the deciphering of the spectrum in the region of large masses represents a challenge. However, we show that the isotope labeling allows unambiguous assignment of most observed peaks. Moreover, smart design of the experiments allow extracting (semi)quantitative information, which can be used for kinetic analysis.Finally, we confirm that the scattering techniques will always go hand in hand with other physical chemical methods, enabling the cross validation of the results and placing them into the context of currently available literature data. Their unique temporal resolution achievable at synchrotrons can not be compared with NMR or HRMS so far, making this data unique in terms of precise kinetic analysis and modeling crystallization.Critical parts of the mechanism of crystallization of one of the most important aluminum-containing MOFs is revealed. In contrast to what is generally assumed, the crystallization of MIL-53 begins via the aggregation and transformation of pre-formed nuclei rather than via secondary\u00a0building units and molecular precursors. We show that formate species formed during the hydrolysis of N,N-dimethylformamide partially incorporate in both nuclei and crystals, possibly acting as modulators for the morphology and/or creating defects in the structure. The revealed complexity of the synthesis indicates that unnecessarily simplification and generalization of the mechanisms for the families of MOFs can mislead understanding of their crystallization. In contrast, case studies are highly demanding to explain the experimental observations for each particular MOF. The combination of multiple advanced spectroscopic methods with kinetic measurements is essential to establish any mechanism of nucleation and growth. Our discovery will lead to the creation of a direction in investigation of the synthesis of solids, rejuvenating and boosting activity in this complex field and ultimately enabling the rational design of novel materials.13C2-labeled terephthalic acid, d7-DMF, d4-methanol , and 15N-aluminum nitrate  were used.All synthetic mixtures were prepared using aluminum nitrate nonahydrate and terephthalic acid by dissolution in N,N-dimethylformamide . The final concentrations were 0.66\u2009M and 0.33\u2009M, respectively. For ESI-HRMS and MAS NMR experiments, the m/z. Prior to the analysis, the samples where diluted with MeOH . To prevent the cross-contamination of the samples, single-use 1\u2009ml plastic syringes were utilized. The flow was set to 5\u2009\u00b5l/min. The following mass spectrometer parameters were used: gas temperature: 423\u2009K, drying gas flow: 11\u2009l/min, nebulizer pressure: 10\u2009psi, sheath gas temperature: 473\u2009K, sheath gas flow: 4.5\u2009l/min. The capillary voltage was set to 3000\u2009V. These conditions were selected to avoid severe fragmentation of ions45. Mass spectra acquisition time was 1\u2009min. The data analysis was performed using Agilent MassHunter Workstation Qualitative Analysis, version 10.0. The obtained mass spectra were averaged over the acquisition time, and detected peaks and corresponding intensities were extracted as CSV files for further evaluation.The direct injection analysis of the MOF reaction mixture in the negative and positive ion modes was performed using Agilent 6550 iFunnel Q-TOF high-resolution mass spectrometer, controlled by Agilent MassHunter Workstation Data Acquisition version 10.1. The device was calibrated in positive and negative modes using Agilent ESI-L tuning mix for the mass range 100\u20133000\u2009The synthesis of MIL-53 was carried out in a sealed glass insert of 5.59\u2009mm outer diameter and ~10\u2009mm length, which was securely placed in the 7-mm zirconia rotor for subsequent in situ NMR analysis of the reaction products. The samples (~150\u2009mg) were placed into the glass tube then kept in liquid nitrogen and flame-sealed.1H, 13C, and 27Al MAS NMR spectra were recorded on an AVANCE III HD 400WB (Bruker Biospin) spectrometer (9.4\u2009T). A double-resonance 7\u2009mm MAS probe (Bruker Biospin) was employed for detection, the MAS frequency being between 2.8 and 3.6\u2009kHz. VTU unit was used to control the temperature in the stator; the calibration was performed using 80% ethylene glycol in d6-DMSO mixture. Direct polarization without high-power decoupling (NODEC), 1H-13C cross-polarization (CP-MAS) and single-pulse 1H and 27Al were the main pulse sequences. For 13C NODEC, 128 scans were acquired with 4\u2009s recycling delay. For 1H direct excitation, 4 scans were acquired with 5\u2009s recycling delay. For 27Al direct excitation, 2048 scans were acquired with 0.25\u2009s recycling delay. In the case of CP-MAS experiments, the CP-RAMP modification with TPPM proton decoupling was used implying accumulation of 128 scans with recycling delay of 3\u2009s and contact time of 5.0\u2009ms. The chemical shifts were calibrated on the 13C and 27Al spectrum of adamantane and aluminum nitrate as a secondary external reference (38.5 and 0\u2009ppm), respectively. The resulting free induction decays (FIDs) were zero filled and apodizated by multiplying with a decaying exponential function with line broadening (LB). After Fourier transform, the signal phasing was manually adjusted and the signal intensity was normalized to the number of scans. To process the data, the TopSpin 3.6 and Mestrenova 10.0 software were applied.The synthesis of MIL-53 was carried out in a sealed glass capillaries of 2.0\u2009mm outer diameter, 10\u2009\u00b5m thickness and ~60\u2009mm length, which was securely placed into the home made cell, enabling continuous heating and spinning of the capillary. Manual goniometer head was used to align the capillary in the beam. The samples (~50\u2009mg) were placed into the glass tube then kept in liquid nitrogen and flame-sealed.46. Pilatus 6M detector installed at 50\u2009cm from the capillary was used. After focusing and alignment of the beam, the spinning of the capillary was started together with the heating to the desired temperature with 10\u2009K/min rate. After achieving the desired temperature, the time-resolved series of XRD data were acquired every 1\u2009min until complete crystallization of MIL-53. The data treatment was performed using Medved and Mathematica software47.The time-resolved measurements were performed at Material Science beamline at Swiss Light Source, Switzerland, using 25.2\u2009keV radiationSupplementary InfoPeer Review File"}
+{"text": "Recently proposed methods in intrusion detection are iterating on machine learning methods as a potential solution. These novel methods are validated on one or more datasets from a sparse collection of academic intrusion detection datasets. Their recognition as improvements to the state-of-the-art is largely dependent on whether they can demonstrate a reliable increase in classification metrics compared to similar works validated on the same datasets. Whether these increases are meaningful outside of the training/testing datasets is rarely asked and never investigated. This work aims to demonstrate that strong general performance does not typically follow from strong classification on the current intrusion detection datasets. Binary classification models from a range of algorithmic families are trained on the attack classes of CSE-CIC-IDS2018, a state-of-the-art intrusion detection dataset. After establishing baselines for each class at various points of data access, the same trained models are tasked with classifying samples from the corresponding attack classes in CIC-IDS2017, CIC-DoS2017 and CIC-DDoS2019. Contrary to what the baseline results would suggest, the models have rarely learned a generally applicable representation of their attack class. Stability and predictability of generalized model performance are central issues for all methods on all attack classes. Focusing only on the three best-in-class models in terms of interdataset generalization, reveals that for network-centric attack classes , general representations can be learned with flat losses in classification performance  below 5%. Other attack classes vary in generalized performance from stark losses in recall (\u221235%) with intact precision (98+%) for botnets to total degradation of precision and moderate recall loss for Web attack and infiltration models. The core conclusion of this article is a warning to researchers in the field. Expecting results of proposed methods on the test sets of state-of-the-art intrusion detection datasets to translate to generalized performance is likely a serious overestimation. Four proposals to reduce this overestimation are set out as future work directions. Every day, a larger and more varied collection of devices is connected to the Internet. This connectivity exposes the same devices to the malicious actors who are active on the Internet, continually looking for exploitable targets. The discovery, classification and tracking of malicious activity against any host or network thus becomes increasingly important. Creating this type of defense has been an active research field since at least 1985 . At the Because network connectivity kept increasing, a second branch that places communication between devices at its center was added. Network intrusion detection systems (NIDS) aim to model attacks from the information present at various abstraction levels of network traffic. At the highest level of resolution, deep packet inspection decides whether traffic is malicious or not based on the information encapsulated in the data portion of the network packets. This type of IDS is becoming more obsolete because many connections use encryption at least on the payload . Packet-At each resolution, a subdivision can be made into roughly two categories. Either the system operates based on a set of rules or it has a more abstract representation of legitimate behavior and reports the anomalous events. Traditionally, rule-based systems rely on a signature database with condensed, unique representations of known attacks. Packets or flows are matched against the signatures and rejected if they match an attack signature. Precision is the main advantage of this approach. The signature\u2019s metadata immediately conveys the attack class, the specific attack and potentially even which strain of the attack. The main downside is the fragility of these systems. Attackers try to fool rule-based IDS by applying the smallest possible alteration that leads to a mismatch in the signature engine ,5. DefenAn IDS that relies on anomaly detection tries to model normality and reports the anomalous events. Under modern conditions this definition only includes unsupervised learning methods, which is overly restrictive. This is a historical artifact because early anomaly detection IDSs relied on thresholds  derived from the networks on which they operated. Many recent methods are based on supervised machine learning algorithms (ML), and the authors still classify their proposals within the anomaly detection IDS category. ML-based IDS promises to deliver models that have solid, general representations of just benign traffic  or of benign and malicious traffic (supervised classification). If successful, ML-based IDSs would overcome the core deficiency of rule-based IDS and supersede them.Unfortunately, ML-based IDS has been studied for well over twenty years, and adoption into open-source or proprietary network defense software is almost nonexistent. We posit that this is in part due to the way in which ML-based IDSs are evaluated and adopted by the research community. Well-adopted literature ,12,13,14This work is the first, to the authors\u2019 knowledge, large-scale investigation into the interdataset generalizability of ML methods in intrusion detection. A varied set of well-established ML methods which have been part of many published works is tested for their ability to use their learned representations to recognize new samples from the same attack classes without retraining.Binary classification models have been trained for every attack class present in CSE-CIC-IDS2018 . Previous work  has showThose positive signals give the impression that the models are capable of learning a robust, general representation of their attack class, but this article demonstrates that claim is false. The general performance across datasets is erratic and the resistance to data reduction does not carry through even though the samples are closely related. Despite the unpredictability, a subset of models for the network-centric attack classes  do maintain their discriminating power on the novel samples.The remainder of this article is split into 6 other sections. The related work in Because the result section is in-depth and verbose, the discussion  takes a The related work demonstrates the need for research into generalization from different perspectives. It starts by noting the attention that is given to robust, generalized performance in the computer vision (CV) and natural language processing (NLP) domains. Subsequently, the lack of experimental research into generalization in network intrusion detection is highlighted with some examples.How well learning methods generalize beyond the test sets of benchmark datasets has been getting attention in the fields that are pushing the supremacy of machine learning in pattern recognition. In computer vision research, object detection models trained on CIFAR-10 and ImageNet were tasked with evaluating images from reproduced CIFAR and ImageNet data generation experiments. The results showed a significant loss of generalized performance  . The fraML-NIDS eagerly adopts new techniques and model architectures from CV and NLP research but without validating general performance ,21,22.This can be explained, in part, by the sparse dataset landscape. The lack of ample, qualitative data in network intrusion detection research has been criticized frequently in the literature . CentraMentions of generalization in intrusion detection are either limited to theoretical considerations during algorithm choice  or mentialways find schemes that yield marginally better ROC curves than anything else has for a specific given setting. Such results however do not contribute to the progress of the field without any semantic understanding of the gain.\u201d The authors argue in favor of an evaluation methodology that takes more into account than metric gains on benchmark datasets. At various points in the text, they remind the reader of the contrast between academic intrusion detection research which works toward a silver bullet solution and the collection of highly specialized tools that protect current networks.The potential for machine learning IDSs to overpromise and underdeliver in terms of general ability has been pointed out by proponents of rule-based systems. Perhaps foremost in a paper co-authored by one of the original inventors of Bro, currently Zeek IDS, which uses deep packet inspection to detect specific threats. Sommer et al.  wrote inGates et al.  challengThe methodology section is split in two parts. The sparsity of the dataset landscape in intrusion detection has been a continuous issue exemplified by the positions in the literature of KDD99, published in 1999, and NSL-KDD, a KDD99 refresh from 2009. These datasets have become fixtures with novel publications still using them as the central dataset(s), despite damning but valid criticism ,32.The one-off nature of these dataset creation experiments as well as their proprietary methods of data collection and preprocessing have barred the addition of new samples that reflect more modern threats. The tendency of singular efforts that culminate in one published dataset progressed throughout the past decade with datasets such as UTwente2009 , CTU-13 https://github.com/ahlashkari/CICFlowMeter (accessed on 28 December 2022) Github. Sharing the feature extraction tooling has at least opened the door to other researchers to evaluate novel samples from new dataset generation experiments in proposed models, validated on the CIC\u2019s datasets. A strong case can be made for open sourcing other parts of dataset creation methodology. Because this level of access is presently not available, this work focuses on working with the four recent CIC datasets .CIC-IDS2017  includesCIC-DoS2017  is a oneCSE-CIC-IDS2018  is a dirCIC-DDoS2019  focuses The intradataset framework is directly taken from the previous work that pushed the efficient use of training data and learning difficulty for 12 learning algorithms on four intrusion detection datasets  . The intThe CIC-IDS2018 collection of pretrained models includes twelve supervised classifiers. Three classical algorithmic families are represented. The seven tree-based learners include multiple improvements to the base decision tree model. All but plain decision trees are ensemble methods that include bagging, some of which add boosting mechanisms and/or rely on randomization for efficiency and robustness reasons. The other five methods fall into two simple neighbor-based algorithms, two support vector machines and logistic regression. The inner workings of these algorithms are described in detail in the following references:  Decision trees with bagging (bag)Adaboost (ada)Gradient-boosted trees (gradboost)Regularized gradient boosting (xgboost)Random forest (rforest)Randomized decision trees (extratree)Neighbor methodsK-nearest-neighbors (knn)Nearest-centroid (ncentroid)Other methodsLinear kernel SVM (linsvc)RBF-kernel SVM (rbfsvc)Logistic regression (binlr)As alluded to earlier, when classifying within the included datasets, all models showed great resistance against the two major forms of data reduction. Vertical data reduction, i.e., setting increasingly stringent constraints on the amount of samples made available to train/validate with the complement of samples for testing, could be pushed to extreme splits such as 0.66%/0.33%/99% without incurring significant losses. There was some variation between models and attack classes but stable convergence of performance on intradataset test sets always occurred before using 10% of the data for training/validation. Any sampling happened in a stratified  manner. To increase the learning difficulty even further an opposite approach to the standard feature selection procedure was adopted. The top 20 features, derived from averaging the feature importances of the aggregate of random forests for CSE-CIC-2018, is listed in The following subsections summarize and highlight the important results of this analysis. First, the base recognition of the models trained on the attack classes in CSE-CIC-IDS2018 is established https://gitlab.ilabt.imec.be/lpdhooge/reduced-unseen-testing (accessed on 1 February 2023). With transparency and reproducibility in mind, the same repository also contains the experiment code.The visualizations in this article are in support of the most salient results. The total amount of available, interactive graphs however is much larger . All raw result files  and plotting code is publicly available at To establish a baseline, the newly trained models were re-exposed to all data of the subset on which they were trained. For models trained and validated on larger portions of the data, this means that more samples that had been seen during training will be re-evaluated. This might introduce some skew in the results. For models trained and validated on little data, i.e., <5% of the samples, this is less of an issue.This subsection will contain new pieces of information as well because CSE-CIC-IDS has multiple instances where two subsets of traffic from the same class have been collected. Therefore, it is possible to test generalization within CSE-CIC-IDS2018 between models trained to classify the same attack class. The DoS (subset 1 and 2), DDoS (3 and 4), Web attack (5 and 6) and infiltration (7 and 8) attack classes each have two subsets. For the Web attack and infiltration classes the documentation remains vague on what the differences between the two subsets are, so a substantial amount of overlap should be assumed. This overlap is more clearly stated for the DoS and DDoS classes, where some tools to generate attacks are used in both subsets and some are different.Tree-based models as well as knn, linsvc, binlr and rbfsvc reach perfect classification scores. Both relationships training volume\u2014classification performance and top-feature removal classification performance behave as expected with an upward trend when increasing training volume and a flat reduction in classification scores when disallowing the models to use the most discriminative features.Layer-7 denial of service (DoS) attacks are extremely well-classified by all models except nearest-centroids. F1-scores above 99.5% even for models trained and validated on just 0.1% (associated 99.9% test) of the data are the norm even when removing the 25% most discriminative features prior to training. Unfortunately, when feeding the benign and malicious samples of the second subset of DoS traffic to models trained on the first subset and vice versa, these perfect scores do not remain. By and large the models revert to balanced accuracies of 50%, which indicates models that are not better than chance at recognizing the attacks. The new F1-scores mostly stay below 10%, showing that the classification of attacks is lackluster. The confusion matrices for the individual models confirm this, showing that most of the errors are type II . Learning with regard to training volume is nonexistent. There is one glaring exception to this crash in classification performance. The linear SVMs trained on the normalized features in subset 2 of DoS traffic generalize as expected to the samples in DoS subset 1 . F1-scorDistributed denial of service (DDoS) attack samples (subsets 3 and 4) are as well classified as Layer-7 DoS samples . The results of exposing the pretrained models to the other subset containing DDoS samples paints a different picture of the classification performance. While it is possible to achieve models that have generalized perfectly to the class, this happens rarely . The strWeb attack traffic is harder to classify, as evidenced by the lower overall classification scores for all algorithms. The tree-based methods converge to 95% classification metrics but take more training data than they did to accurately classify the previous attack classes. They are now also significantly weakened when removing the most discriminative features. Methods such as nearest centroids, binlr, linsvc and rbfsvc have such poor precision scores that they are not even usable. Subsequently, pretrained models based on these methods are not expected to do well when evaluating the samples of the other subset. The experimental results confirm this with similar classification profiles . The tree-based methods fare much better, but for this behavior to emerge the models had to be trained on nonscaled features and none of the most discriminative features should have been removed from the data prior to training. Those parameters lead to F1-scores that are consistently in the 80\u201385% range . The behavior of these models when removing top features is as expected, showing a flat drop in classification performance (up to \u221235%), but stability is retained. For tree-based methods this stability is peculiar, but it is an essential property if they are to be included in real IDSs. This result does show that it is possible to train stable tree-based methods, however further inquiry is needed to specify what the training conditions should be. As stated in the introduction to this section, the Web attack class has two separate subsets of traffic, but a substantial overlap in the attack methods should be assumed as per the documentation of CSE-CIC-IDS2018.Recognition of infiltration attacks is poor for all methods on the first subset (7) but substantially better for subset two (8). The documentation of CSE-CIC-IDS2018 does not state how the infiltration attacks were executed in both subsets, but based on the classification scores, it is safe to assume that the methodology differs. In concrete numbers, classification metrics on subset 7 typically only have F1-scores below 50%. F1-scores on subset 8 reach between 85\u201395% for the tree-based methods, with the other methods trailing significantly behind at around 50% (F1). The models with decent performance are also stable with regard to training volume, showing an upwards trend when allowed more data to train on. Furthermore, this stability extends to models that were trained without access to the most discriminative features. As expected, the models pretrained on subset 7 that all performed poorly overall have poor performance when classifying the samples of subset 8. The other half with models pretrained on subset 8, evaluating the samples from subset 7, reach a conclusion that is much more interesting. That conclusion is a performance regression to low levels, indicating that the results obtained on the subset itself are misleading. A learnable pattern does exist in the data, but it is not descriptive of the difference between regular traffic and infiltration attacks . F1-scorThe final class in CSE-CIC-IDS2018 contains botnet traffic from two different botnets .Subset 0 of CIC-IDS2017 contains attacks generated by the Patator tool against an FTP and an SSH endpoint. Patator is designed as a multipurpose brute-forcer, a general framework to execute brute force attacks against an array of services  DoS attacks in a single subset, whereas CSE-CIC-IDS2018 split this into two hence two pretrained models have been tested. The obvious result is the loss of perfect classification and the introduction of dramatic performance swings. The fork in which metrics such as the F1-score lie has a top end above 90% and a bottom as low as 0%. The relation between training volume and performance is not predictable, further complicating the matter. The models pretrained on DoS subset 2 of CSE-CIC-IDS2018 are consistently worse than those trained on subset 1, which is surprising given that CIC-IDS2017 is actually biased toward the attacks contained in DoS subset 2 of CSE-CIC-IDS2018. The models that exhibited decent performance required either minmax- or no feature scaling prior to training. Removal of top features cripples these methods further to the point of irrelevancy.Curiously, the simplest method, nearest centroids, is quite capable of distinguishing between DoS and normal traffic  with class separability at 82%. Methods such as adaboost, gradient-boosted trees and XGBoost , hold thThe final class that is represented in both CIC-IDS2017 and CSE-CIC-IDS2018 is distributed denial of service. Both datasets rely largely on the same tooling to generate samples of this attack class. When exposing the same pretrained models to this DDoS data, a similar conclusion can be drawn. Good to great generalization is possible, with tree-based methods as well as the logistic regression and SVMs. Even the nearest-centroids classifier performs reasonably , despite summarizing each of the two classes into one label each. This shows that the feature values that are computed for the flows do differ significantly for DDoS versus benign traffic. Models with more complex means of separation classify the DDoS samples better. Here, the tree-based methods manage peak F1-scores of 90%  and maintain these when removing the most discriminative features. Only two downsides remain: the occasional downward performance spikes and the lack of lockstep performance metrics between the models pretrained on subset 1 and subset 2 of 2018 DDoS traffic . These sSummarizing interdataset general performance from models pretrained on the attack classes of CSE-CIC-IDS2018 to samples from the same attack classes in CIC-IDS2017 is straightforward. True network attacks  can be recognized with minor regressions in absolute performance. Botnet traffic sits at an intermediate level, and models for Web and infiltration attacks that just use the network for transport have really poor performance. Tree-based learners typically outperform versus the other learners on the classification metrics.Absolute performance, however, is not the entire picture. Reliability is much weaker in the interdataset evaluation than it was for the exact same models during intradataset evaluation. As expected, the fragility is more pronounced for tree-based learners than it is for distance-based learners or regression models. Other properties that were lost in the interdataset results versus the intradataset results are the clear relation between training volume and model performance, the resistance against deliberate data quality reduction and the indifference to feature scaling.In short, even with tight overfitting control during training and hyperparameter search, the excellent classification scores on the extensive test sets of CSE-CIC-IDS2018 cannot be trusted as one-to-one proxies for general model performance. Advances in model training and preprocessing may solve the reliability problem for network-centric attack classes, but the other attack classes will require other solutions first.Letting the models pretrained on the DoS subset of CSE-CIC-IDS2018 classify the samples from the specific CIC-DoS2017 data set yields no satisfactory results. The overwhelming majority of pretrained models do not reach F1-scores above 40%. This is in spite of the fact that the malicious samples in the data set come exclusively from low-volume layer 7 DoS attacks. Combining the results described in The most recent dataset iteration published by the CIC focuses on DDoS attacks. It contains both reflection-based DDoS attacks as well as exploitation-based ones. As a rationale for the creation of this dataset around a specific attack type, the authors cite the small scope of attacks, covered under earlier taxonomies. In concrete terms, CIC-DDoS2019 contains 12 different DDoS attacks against a diverse collection of target machines . The raw packet captures as well as the labeled CSVs amount to a very large sample collection . In order to cut down on processing time, the files have been reduced to reach a balance between benign and malicious samples. Since the benign traffic far outstripped the attacks in volume, the reduction matches a sampling of normal traffic equal to the malicious count for every subtype of DDoS attack . The disThe traffic in this category is generated by abusing genuine commands that exist in the implementations of several protocols. Attackers try to find a request that is small in size but has a large response, thereby achieving amplification. The secondary component to these attacks is executing them from a network that allows IP spoofing, redirecting the responses to an arbitrary IP. In lab settings, IP spoofing is not even required as ARP poisoning can redirect all traffic on a subnet to a specific host.DNSDNS amplification is a typical DDoS attack whereby spoofed clients send small requests such as \u201ddig ANY [domain] @[resolver] + edns = 0 + notcp + bufsize = 4096\u201c that lead to large responses (50\u00d7) sent over UDP. The requests are sent to open DNS-resolvers that respond to spurious requests. Some of the tree-based methods have very high performance with up to perfect recall, matched with 60\u201380% precision, but the volatility is staggering . MoreoveLDAPThe subsets labeled 1 and 11 of CIC-DDoS2019 contain LDAP DDoS amplification attacks. The ingredients for this form of attack are similar. LDAP servers will typically communicate over TCP, but they offer a UDP endpoint as well. Well-configured LDAP servers should not respond to information requests by systems that have no authority to ask, but unfortunately, misconfigured LDAP servers exist that serve the open Internet. Services like Shodan scan the Internet looking for all kinds of vulnerable machines and subsequently expose this data through an open and a paid API. The tested algorithms show that they are capable of separating the novel malicious samples from the benign traffic with which they have been mixed and that separation can be done with a certain amount of stability  that informs clients of the capabilities of an MSSQL cluster is vulnerable by design to be abused for DDoS purposes. It works on top of UDP and the functionality is to serve information to interested clients so attackers can blend in with legitimate users. Subsets labeled 2 and 12 from CIC-DDoS2019 contain samples of this attack class. Overall recognition here is once again done best by meta-estimators based on decision trees. A random forest trained on minmax-scaled features has the expected pattern with regard to training volume and maintains a stable profile even when removing the 15 most discriminative features from the data prior to training . These mNetBIOSThe NetBIOS amplification attack abuses the NBSTAT query within the protocol that gives a status about an endpoint that responds to NetBIOS queries. Although the amplification factor is low at 3.8\u00d7 , this atNTPThe monlist command in implementations of the network time protocol (NTP) used for clock synchronization between hosts is perhaps the best-known amplification DDoS attack. Like the others, it works over UDP, but unlike the others NTP reflection attacks have an amplification factor of more than 550. The command is a diagnostic tool that gives a list of 600 clients that recently made use of the NTP server. Subset 4 of CICDDoS2019 contains the NTP amplification attack samples. By now, the pretrained models that have shown good stable performance in the previous sections only have to continue exhibiting this behavior to solidify their position. This does appear to be the case with all tree-based methods scoring high . Stability is strong for methods such as the bagging classifier, random forests and the gradient-boosted trees. There are some discrepancies between the models pretrained on DDoS subset 1 of IDS2018 compared to the models pretrained on subset 2 of the same dataset. That does weaken the results somewhat.SNMPSNMP amplification abuses the GET BULK request type of the protocol which bundles smaller amounts of information that would otherwise have to be retrieved through several GET NEXT calls. Like the other attacks it is wrongly configured SNMP servers that respond to illegitimate use of a legitimate feature that causes the problem. Classification of this subset by the models pretrained on IDS2018 DDoS loses the roughly equal performance by models trained on either of the subsets. Several methods also perform worse in absolute terms on this specific attack that had been strong performers for the other variants . Good classifiers maintain large stable sections with perfect recall and 60\u201370% precision. The single constant so far have been nearest-centroid models, which score with identical performance numbers on all subsets  is a part of the Universal Plug and Play (UPnP) protocols, allowing seamless discovery and access of services on a network by interested clients. The devices themselves can announce their presence and capabilities on the network with passive listeners, but an active version exists where a newly connected device can query the network for services to which it would like to connect. An M-SEARCH query looking to obtain responses from all service endpoints can easily reach more than 20\u00d7 bandwidth amplification. SSDP amplification attacks were recognized fairly well both by tree-based models trained on standardized features as well as minmax-scaled features. This is rare, because most tree-based models built on normalized features have terrible performance across the board. It should be noted that the recall of models on standardized features is about half that of models trained on minmax-scaled features . PerformTFTPThe trivial file transfer protocol (TFTP) is a lightweight version of FTP that works over UDP. It has primarily seen adoption in network-booted environments. It was never intended to be exposed to the Internet, but as with all of these reflection attacks, misconfigured TFTP and firewalls allow misuse of the protocol. Recognition by the pretrained models follows the pattern of SSDP with mostly tree-based models that perform well and with some stability even retaining lots of performance if the features were normalized prior to training. This indicates that it is no fluke and while models built on minmax-scaled features still outperform, it might be possible to abstract over this choice altogether with the right training scheme. One other algorithm that has the interesting property of stable metrics that are tightly clustered and stable with regard to training volume and feature reduction is the RBF-kernel SVM. All metrics are densely packed around 55%, which is not high enough to be immediately usable, but the tightly clustered metrics are interesting . They haPortmapThe portmapper service  provides a linking of program number, program version and listening port. The client can ask for this information and then pick a preferred level to interact with the available service(s) through remote procedure calls (RPC). This access should be restricted to authorized users, but because there is no authentication/authorization by default, attackers can simply ask for a listing of the entire table and send it to a spoofed victim because UDP is available as the transport layer. Results on this class are almost equal to those on the previous classes. Decent patches of performance exist, but the variability is large and no overall recommendations can be made that will guarantee stable, high performance. There is almost no agreement between the pretrained models on subset 1 of DDoS from IDS2018 versus those pretrained on subset 2. This issue has been intermittently showed up for the other attack classes as well. Good performance in numbers for this class ranges from perfect recall paired with 60% precision, which can be fairly stable, to models with recall\u2013precision pairs above 99.5% without stability.The attacks described in the previous UDP-floodUDP-floods simply overwhelm a server by sending packets to random UDP ports, not even interested in a particular service. The OS on the receiving end has to process the packet and potentially send an ICMP packet back. If there is a firewall between the target and the attacker that will take the first hit, but it too has to handle the traffic somehow. Even if it manages to ignore the majority of malicious packets, the link itself can get filled up with the packets. Despite their simplicity, these attacks can have the same practical outcome as the reflection-based DDoS attacks. Basic UDP floods are well-recognized, with some degree of consistency between the models and relative stability. This applies most to the tree-based meta-estimators. Raw performance numbers are perfect recall with 60\u201392% precision. Minmax scaling had to be applied to the inputs before training for these results. Models trained with normalized features can still be decent, up to perfect recall and 89% precision, but hampered by stability issues. These conclusions apply to both subsets with UDP-flood traffic (7 & 16).SYN-floodSYN floods work by an attacker creating lots of half-open TCP connections  but never actually using them. Because the number of connections always has an upper bound, it is only a matter of resources to tie up all available connections. The result pattern for SYN-flood attacks is strange. On the one hand, the models pretrained on DDoS subset 1 of CSE-CIC-IDS2018 do not perform well at all, except for some cases where high classification metrics are posted, but only at minute training volumes (<1%). Models trained on DDoS subset 2 of IDS2018 score much better, but even the tree-based models no longer hold perfectly stable recall. The observation that some of the best models were trained on little data, flows to lesser or greater extent through all attack types. It is possible that for clear network attacks, limited exposure is enough to achieve robustness, rather than the typical assumption that more data is better .UDP-lagThe UDP-lag attack is a niche network attack whereby a client abuses an existing connection between itself and the server by introducing lag at the client side. Typically, clients do not immediately disconnect from the service as that would hurt the user experience but instead can continue local interaction with the service and the network will catch up when the link improves. This may create advantages for the client because the server is not in control and might have to deal with a state generated on the client side that would have been impossible if the connection had stayed intact. This attack is classified under DDoS because one way of introducing it is by DDoSing your own machine. The clearest case of requiring models to train on a limited amount of samples to have a good general representation is this DDoS subclass and the nearest neighbors algorithm. Even though it is so simple, F1-scores above 90% happen with a large degree of consistency, not just with regard to feature removal but also between the two sets of pretrained models and the two sets of UDP-lag traffic of CIC-DDoS2019. The only caveat is a serious performance regression when increasing the training volume (most visible with standardization). These performance regressions have been observed for the logistic regressions and SVMs for this and other attack types of this data set, but the slope of the decline is much less steep, because these models did not achieve good metrics to begin with. One final illustration that summarizes many of the observations in all the previous subsections is shown in quadrant When tasking CIC-IDS2018\u2019s pretrained DDoS models with classifying CIC-DDoS2019, the interdataset generalization of these models can be summed up as follows. Classification results can be near-perfect and highly volatile (for tree-based learners) or significantly less accurate, but more often stable for SVMs and simple distance-based learners. Consistency when classifying is possible, but it is not the norm. Nevertheless, the existence of consistent sets of models indicates that it is possible to train them. Further investigation is required to compare this sparse set of stable, well-generalizing models to their failing counterparts. As a network-centric attack class, models with pockets of good generalization were expected and confirmed, but they exhibit the same flaws observed from generalization on the other datasets. The inconsistent loss of key intradataset generalization properties such as high discriminating power, stability with regard to data reduction and invariability to scaling seriously hamstrings dependable, effective use of these methods.The intermediate conclusions  interspeThe tables show the numeric confirmation of the statement from the intermediate conclusions that it is possible to train models that generalize well to novel samples, generated by unseen attacks from the same class, executed in a different network environment. Structuring the entire result section around these results would have been misleading because the top 3 models obscure the loss of stability and invariance to data reduction and feature scaling.Finally, the generalized results of 2018\u2019s DDoS models to every specific attack represented in CIC-DDoS2019 are listed in Generalizability will need improvement to start adopting learning IDS models. The major issue to resolve is consistency. In our opinion, four options are available. On the pure data science track, it is possible that models trained on an intelligent selection of features might suffer less from instability. Second, models with a higher capacity might achieve better abstracted versions of the attack classes. A third solution could lie in the expansion of the quality and variety of academic data available for model building. By doing so the inconsistency issue may be overcome because the models are trained on much more comprehensive samples of the attack classes. A fourth option would be to compare the sets of best-in-class general models for each attack class and compare their internals to those of the unsuccessful models trained for the same attack class. This would provide more insight into the factors that drive general performance. The future work and hypotheses This work exposes supervised ML intrusion detection models with excellent recognition on CSE-CIC-IDS2018 to the novel samples of CIC-IDS2017, CIC-DoS2017 and CIC-DDoS2019. Those datasets share the included attack types but have been generated with mostly different individual attacks and in different network environments. Based on the results obtained by these models within CSE-CIC-IDS2018, it was assumed that the pretrained models would generalize well and successfully classify the other datasets\u2019 samples. This assumption has been standard but untested in ML-based intrusion detection research, in part because of the low availability of compatible, labeled datasets. As a consequence, new proposed IDS systems are recognized as an improvement in the state-of-the-art if they succeed at reaching higher classification scores, with training, validation and testing limited to samples of one or two datasets without crossover testing. This work shows experimentally that the assumption that good intradataset generalization will yield good interdataset generalization  is faulty.Models for attacks that rely heavily on network interaction tend to suffer the smallest performance regression in interdataset testing. Recognition of the malicious samples for the brute force, DoS and DDoS attack classes decreases by 5\u201315 points , rendering the models useless. Interdataset recognition of infiltration traffic is nonexistent as generalized precision never exceeds 1%. The full tables with the performance numbers of the three best generalizing models for each attack class and each dataset are part of the discussion .Whereas data reduction (both features and samples) and feature scaling had little to no impact on the excellent intradataset classification , in intIn short, it is impossible to tell in advance if a model will have a good general representation of the attack class. It is possible, but not the norm and metrics from the standard intradataset generalization overestimate real classification ability. Unless a solution is found that guarantees a model\u2019s general performance, having to train and test many models and cherry-picking those with good intra- and interdataset performance, is a costly endeavor and a procedure that would still only be viable for the network-centered attack classes.Ultimately, this work urges researchers to consider adopting a more rigorous validation approach that includes testing with unseen samples by default. Novel methods should be recognized as improvements to the state-of-the-art primarily by how well they continue to function in the most realistic test scenario available.Several routes remain open for further investigation. First, the models in this work were pretrained as part of an earlier publication which investigated how far data could be reduced/damaged prior to training and still yield models with high discriminating power . The pooA second route for future investigation is the inclusion of newer ML-based intrusion detection models. Many publications have adopted techniques from the deep learning research field and applied them to the IDS pattern recognition problem ,53,54,55If neither classical models trained with optimal features or deep neural networks provide an answer for the generalization problem, then solutions three to five should be considered. Solution three consists of setting up new data generation experiments for intrusion detection. The emphasis should be on generation of samples from a large variety of modern attacks in variable, but controlled mirrors of realistic network environments. Variety and interoperability should be the main goals. In this regard, the LUFlow project  is interSolution four includes comparing the rare instances of sets of models of the same algorithm that generalize well to their poorly generalizing counterparts. The focus should be on examining what makes these models different and if they can be adapted or trained in such a way that they are more likely to exhibit the desired behavior.Solution five tailors IDS to a specific protocol. Rather than trying to build models that can work to detect misuse of any service on the network, specialized models could integrate knowledge of how specific protocols should operate. Within wireless intrusion detection, this approach has shown promising results ."}
+{"text": "The calculated absorption spectra lie between 248 nm and 496 nm, leading to optical activity of the 2D multilayer CH3NH3PbI3 systems in the visible and ultraviolet regions. The strength of the optical absorption increases with an increase in thickness. Overall results from this theoretical study suggest that this 2D multilayer CH3NH3PbI3 is a good candidate for photovoltaic and optoelectronic device applications.Two-dimensional (2D) hybrid halide perovskites have been scrutinized as candidate materials for solar cells because of their tunable structural and compositional properties. Results based on density functional theory demonstrate its thickness-dependent stability. We have observed that the bandgap decreases from the mono- to quad-layer because of the transformation from 2D towards 3D. Due to the transformation, the carrier mobility is lowered with the corresponding smaller effective mass. On the other hand, the multilayer structures have good optical properties with an absorption coefficient of about 10 3NH3PbI3 and its multilayer halide perovskites. These systems have a high formation energy. The optical properties absorption spectra lie between 248 nm to 496 nm with an absorption coefficient of about 105 cm\u22121.We have studied 2D CH To overcome this situation many researchers and scientists are working on finding alternative effective, clean and sustainable sources of energy. In the past two decades, researcher have been working on solar energy, because it is a clean and cost-free source of energy. The task is how to efficiently harvest the solar energy. Many research groups are working on finding new technologies and materials for harvesting solar energy more efficiently.1,2 The flexible structural and compositional properties of hybrid perovskite materials make them preferable candidates for solar cell applications,3,4 light-emitting diodes,5,6 field-effect transistors,7 lasers, non-volatile memory devices8\u201310 and photodetectors,11etc. The use of hybrid halide perovskites with the structural formula ABX3, where A is an organic molecule like CH3NH3, NH2CH etc., B is a cation such as Ge, Pb, Sn etc. and X represents anions such as Cl, Br, I, etc. has been explored. In 2009, hybrid halide perovskites as light absorbers were reported for the first time, with a power conversion efficiency (PCE) of 3.8%,12 which was useful to increase research in the photovoltaics field13,18 due to thhe tunable bandgap, charge carrier transport properties, and intense photoluminescence.14\u201316,19\u201324 Within a few years, the PCE of hybrid halide perovskites reached 22%, in comparison with traditional silicon-based solar cells.14,25\u201328In recent years, low cost and high performance hybrid perovskites have attracted much attention from the scientific community.29\u201331 The use of 2D forms of these structures is an efficient way to increase the stability, transport charge mobility, and flexibility.32\u201335 Therefore, it is important to study perovskite formation to understand the interaction between the inorganic PbI3 and organic CH3NH3 compound.Despite good efficiency, three-dimensional hybrid perovskite shows low stability and device degradation when interacting with oxygen and moisture.et al. were first to describe the single layer and multilayer thick single crystal of 2D (C4H9NH3)2PbBr4 perovskites, with a conformation of 2D perovskites separated by layers of PbBr4\u2212. They showed the electronic properties of the 2D materials were different from the 3D bulk structure.36 As the absorber, 2D perovskites (BA)2PbI4 fabricated by Tsai et al. reached an efficiency of up to 12%, with greater stability and high efficiency.37 Also, the light\u2013matter interactions, photoluminescence and photoresponse of 2D perovskites have been reported, and these 2D perovskites can be suitable for optoelectronic device applications.38,39 Experimentally Liu et al. have reported the 2D (MA)2PbI4, thin film as efficient in solar absorber applications and the electronic and optical properties.40 The halide perovskite\u2019s structural and compositional properties modulation has been achieved in 0D, 1D, 2D, and 3D.36 It has been found that the 2D layered BA2GeI4 and BA2SnI4 are potential candidates for light-emitting devices.41,42 Also, Zhang et al.43 found that the bandgap, Rashba spin splitting, and effective mass vary with the thickness of 2D perovskites 2PbI4) suggesting the 2D materials as good candidates for optoelectronic devices.In experimental studies, Dou 3NH3PbI3 vary with thickness has rarely been studied. In this study, the transport charge mobility and optical absorption of multilayered CH3NH3PbI3 are systematically investigated at the density functional theory level. First, structural properties of monolayer and multilayer CH3NH3PbI3 are discussed. Then, with the electronic and charge carrier properties, the absorption properties of the multilayer hybrid are uncovered.Up to now, many studies have been done on 2D hybrid perovskites. But how the effective transport and optical properties of the 2D hybrid perovskite CH244\u201346 The exchange\u2013correlation functional (GGA-PBE) was used.47,48 It is well known that GGA-PBE cannot describe the weak interaction between CH3NH3 and PbI3 molecules. Therefore we applied Grimme DFT-D2,48 nonlocal density functional vdW (DFT-D2) implemented in VASP in the study. We also included SOC and HSE49 to get clear images of the electronic and transport properties of the 2D multilayer CH3NH3PbI3. In our previous work on the 2D single layer CH3NH3PbI3 perovskite, we have observed the underestimation and overestimation of the bandgaps using SOC and HSE calculations as shown in Fig S2 and S3.\u202049,52 The kinetic energy cut-off was set to 500 eV. To avoid successive interaction, a vacuum space of 20 \u00c5 was applied along the Z-direction. For the bilayer, trilayer and quadlayer, the distance between two successive layers was set as 3 \u00c5, to check the interaction between layers and the effect of N layers on the cell phase. As we increase the number of layers, the Z direction increases up to \u223c20 \u00c5, \u223c30 \u00c5, and \u223c45 \u00c5 for the bilayer, trilayer and quadlayer, respectively.Density functional theory (DFT) was employed with VASP implemented with the PAW method.\u22121 with energy convergence about 10\u22124 eV for multilayer CH3NH3PbI3. The lattices agree well with experimental and theoretical studies.50,53Structural optimization was performed using a (5 \u00d7 5 \u00d7 1) grid, and a (15 \u00d7 15 \u00d7 1) grid was used for the electronic properties. All atomic structures were optimized up to a force of 0.001 eV \u00c554The formula for the perovskite crystal structure, with effective ionic radii for the ions at the A, B, and X sites, is shown in 53,55,58,593NH3PbI3 can be calculate using the Bardeen and Shockley40,54 method which was used in a previous report:52The dielectric function optical absorption coefficient can be calculated using the following equation.33.13NH3PbI3 are shown in a = 6.33 \u00c5 and b = 6.21 \u00c5.50\u201353,60 The calculated bond length PbI is in the range of 3.09\u20133.27 \u00c5 for multilayer CH3NH3PbI3.The equilibrium configurations of multilayer CHEform = (Elayer \u2212 nEPb \u2212 nEI \u2212 nE3NH3CH)] where Eform is formation energy, Elayer is total energy of the layer, EPb is total energy of the Pb atoms, EI is the total energy of the iodine atoms, E3NH3CH is the total energy of the CH3NH3 molecules. The formation energies of the multilayer perovskites are negative and increase with the corresponding increase in number of layers of CH3NH3PbI3, as shown in 38\u201340The calculated formation energy is [3.23NH3PbI3 monolayer is 1.63 eV; in the bilayer case it is 1.81 eV; for the trilayer 1.68 eV; and for the quadlayer, it is 1.55 eV as shown in 3NH3PbI3 systems also have direct band gaps. The variation in the direct band gap occurred when we shifted from the monolayer to bilayer to trilayer to quadlayer due to an increase in localized states in the CBM and VBM near the Fermi level. Which appears as denser bands for the VBM compared to the CBM. In the case of the bilayer, the energy of the bottom of the near Fermi conduction band decreases and the energy of the top of the valence band increases, as shown in Fig. S5.61 Moreover, The calculated band gap for the perovskite CH3.33NH3PbI3 hybrid halide perovskites as shown in 3NH3PbI3. As we can observe, with tensile and compressive strain, the total energy of the system decreases with respect to the unstrained systems. From Fig. S4Further we have explored the strain dependent electronic properties of the multilayer CH3.43NH3PbI3, we determined the carrier mobility via calculations of the deformation potential by applying compressive and tensile strain up to 2%. as displayed in Fig. S4 where \u03b5(k) is the wave vector k\u2019s eigenvalue along the transport direction. This is an effective way to moderate transport properties. The charge carrier has some limitations in the bulk hybrid halide perovskite.To evaluate the charge carrier efficiency in multilayer CH40,54 of CH3NH3PbI3 with multilayer 2D CH3NH3PbI3 perovskite. In the comparison of the 2D single layer system with the 2D multilayer system, the average directional dependent effective mass of the electrons is decreased, from 3.92 to 0.72, while in the case of holes, the effective mass decreases from 1.51 to 0.79 as shown in 63 2D CH3NH3PbI3 has flexible structural properties, which affect the transformation from monolayer to bilayer and carrier mobilities are decreased. In the case of 2D multilayer hybrid halide perovskites, from bilayer to quadlayer, carrier mobilities increase from 3.00 cm2 V\u22121 s\u22121 to 24.00 cm2 V\u22121 s\u22121, which is consistent with previously reported 3D bulk halide perovskites, where 0.4 cm2 V\u22121 s\u22121 to 25 cm2 V\u22121 s\u22121 was found.19,63 We observed that as the number of layers increases from the monolayer to the quadlayer, the effective masses and carrier mobilities of the electrons and holes resemble the bulk nature of the CH3NH3PbI3 hybrid perovskites.19,41,51,52,57,62,63Then, we compared the single layer (2D) transport properties3.53NH3PbI3 are shown in \u03b51(\u03c9) and \u03b52(\u03c9). The calculated real \u03b51(\u03c9) static dielectric constant of CH3NH3PbI3 increases from 2.43 to 3.40, monolayer to quadlayer, respectively. In the imaginary part of the dielectric function \u03b52(\u03c9) the wide peak orientation is due to transition of Pb s to I p orbitals. Likewise, the absorption coefficient slightly increases from 14.1 \u00d7 105 cm\u22121 for the monolayer to 16.0 \u00d7 105 cm\u22121 for the quadlayer. Note that the calculated spectra lie in the range of 248 nm to 496 nm for multilayer CH3NH3PbI3. The calculated results are consistent with previously reported experimental results, containing CH3NH3PbI3 thin films with high PbI2 concentration.8,17 As well as the absorption coefficient is significantly more as compared with the bulk CH3NH3PbI3 (2.5 \u00d7 104 cm\u22121 to 8.7 \u00d7 104 cm\u22121).8,52,56,58,59 The high energy peaks at \u223c4 eV occur due to the presence of a large number of interband transitions of the Pb s VBM to I p CBM at the R point.The optical properties of multilayer CH3NH3PbI3 significantly enhance with an increase in photon energy. This reflects that the 1st peak is in the visible region and the 2nd highest peak is in the ultraviolet region of the spectrum. We observed that the maximum light is in the UV region, compared with the visible region. Therefore, the light absorption by the CH3NH3PbI3 surface has been greatly enhanced in the visible (2 eV to 3.26 eV) as well as UV regions.In the optical reflectivity of the multilayer, shown in 43NH3PbI3 hybrid halide perovskite system are investigated comprehensively with varying thickness using the DFT method. The formation energies, band gaps, and effective masses vary with the corresponding mono-, bi-, tri-, and quadlayer. We also observed that from mono- to quadlayer the bandgap decreases because of the transformation from 2D towards 3D. Due to strong antibonding states in the iodine and lead, high carrier mobilities are found with corresponding small effective masses. Besides, the multilayer has good optical properties with an absorption coefficient of about 105 cm\u22121. The calculated absorption spectra lie between 248 nm and 496 nm, which leads to optical activity of the 2D multilayer CH3NH3PbI3 in the visible and ultraviolet regions. We believe that the reasonable absorption in the visible region with high carrier mobilities could make multilayer CH3NH3PbI3 a candidate material for efficient photovoltaic devices.The calculated structural, electronic, and optical properties of the multilayer CHThere are no conflicts to declare.RA-012-D2RA02874C-s001"}
+{"text": "Under density functional theory (DFT) calculation, we applied mechanical strain, i.e., tensile and compressive strain up to 10% in both cases. The mechanical strain engineering technique is useful for a tuned bandgap of 2D MASnBr3 and 2D BAMASn2Br7. The calculated carrier mobility for the electron is 404 cm2 V\u22121 s\u22121 and for the hole is up to 800 cm2 V\u22121 s\u22121 for MASnBr3. For BAMASn2Br7 the highest carrier mobility is up to 557 cm2 V\u22121 s\u22121 for electrons and up to 779 cm2 V\u22121 s\u22121 for the hole, which is 14% and 24% higher than the reported lead-iodide based perovskites, respectively. The calculated solar cell efficiency of 2D MASnBr3 is 23.46%, which is 18% higher than the reported lead-based perovskites. Furthermore, the optical activity of the 2D MASnBr3 and 2D BAMASn2Br7 shows a high static dielectric constant of 2.48 and 2.14, respectively. This is useful to show nanodevice performance. Also, 2D MASNBr3 shows a high absorption coefficient of 15.25 \u00d7 105 cm\u22121 and 2D BAMASn2Br7 shows an absorption coefficient of up to 13.38 \u00d7 105 cm\u22121. Therefore our theoretical results suggest that the systems are under mechanical strain engineering. This is convenient for experimentalists to improve the performance of the 2D perovskites. The study supports these materials as good candidates for photovoltaic and optoelectronic device applications.We have explored the structural, electronic, charge transport, and optical properties of lead-free 2D hybrid halide perovskites, MASnBr 3 (b) BAMASn2Br7 with strain engineering for 0%, 1% , 2%, 4%, 6%, 8% and 10% respectively.The calculated band gap of (a) MASnBr These hybrid halide perovskites being highly reactive with moisture is the main drawback for industrial purposes.9\u201312Nowadays, energy crises and environmental pollution are the main big problems for humans. Many research groups are working on finding alternative solutions for these problems. Globally, educational and industrial purpose scientific communities are now attracted to renewable energy sources, and have developed new technology to overcome such situations and find low-cost solutions. The research community has recently become interested in organic\u2013inorganic hybrid halide perovskites because of their rapid advancement in photovoltaics and optoelectronics over the past ten years.13\u201315 The transformation of 3D to 2D perovskite opens the potential to more clearly analyze electronic, optical, and transport properties.16 These 2D hybrid halide perovskites are a stable candidate for optoelectronic devices. Like light-emitting diodes,17 photocatalysts, field-effect transistors,17\u201319 lasers, etc.20\u201325 Also, these 2D perovskites have flexible structural and compositional properties. Long-term stability characteristics in ambient conditions, more moisture resistance than 3D structure, and tunable photovoltaic properties.26\u201330 2D third-generation hybrid halide perovskites have the structural formula MAZX3 . These 2D perovskites interact well with inorganic layers, same as Ruddlesden\u2013Popper 2D structures,35\u201340 which has structural formula (BA)2MAZ2X7 (BA = CH3(CH2)3NH3, MA = CH3NH3, Z = Pb, Sn, X = Cl, Br, I). Due to the structural flexibility of 2D perovskites, many groups have developed a new technique to find its efficient power conversion efficiency (PCE). 2D material interfacing with 2D perovskites has reached PCE 12.6%.12,15,41 2D lead halide CH3NH3PbI3 and Ruddlesden\u2013Popper (CH3(CH2)3NH3)2CH3NH3Pb2I7 have reached 14.9%, 17%, and 19.7% PCE in theoretical experiments, respectively.42\u201344 Yang et al.'s group reached 20% PCE by interfacing 2D with 3D perovskites.45 Under condition Shockley\u2013Queisser limit, PCE of perovskites was achieved 31% with the theoretical study.46 The 2D lead (Pb) hybrid halide perovskites have been considered good candidates as light-harvesting materials. But has toxicity issues with environmental effects.47 To solve the problem of environmentally and humanly hazardous hybrid halide perovskites based on lead (Pb). Researchers have discovered that substituting alternative divalent elements, such as In, Sn, Sb, Bi, etc., for lead is advantageous for both the environment and human beings. Experimentally, the tin (Sn) based halide perovskites are isostructural compounds of Pb-based halide.31 Sn-based halide perovskites materials contain a narrow optical bandgap high optical absorption coefficient. Also, it contains high charge carrier mobility with low exiting binding energy and good stability.48\u201350 Experimentally, the tin-based halide materials work as a light-harvesting material covering a wide range of visible spectrums. Furthermore, we have found that many groups are working on lead-free tin-based hybrid halide perovskites. The Xu et al. group is tuning the optical band gap of perovskites for light harvester materials.51 For thermoelectric devices, these materials were used.52 Ruddlesden\u2013Popper nonlinear band behaviour due to mismatch s-states of Pb and Sn design a metal alloy.36 As the number of 2D layers increases band, behaviour shows charge carries effect well compared with experimental result.53 Therefore, these studies are useful for developing new building blocks for future photovoltaic and optoelectronic devices.54\u201362Many technologies have developed and extracted the two-dimensional 2D slab by slicing from the 3D framework of hybrid halide perovskites.ZX3 ref.  (MA = CH3NH3SnBr3 as (MASnBr3) (MA = CH3NH3) and Ruddlesden\u2013Popper (CH3(CH2)3NH3)2CH3NH3Sn2Br7 as (BAMASn2Br7) (BA = (CH3(CH2)3NH3)2, MA = CH3NH3) perovskites with their structural, electronic, optical and transport properties. Under mechanical strain conditions, we apply both tensile and compressive strain up to 10%. Mechanical strain condition is a valuable way to tune the electronic and optical properties of 2D halide perovskites because of the flexible structural and composition properties. For the creation of lead-free 2D hybrid halide perovskites for optoelectronic and photovoltaic applications, mechanical strain engineering offers a novel route.In this paper, we give a comparative analysis of two orthorhombic structures of hybrid halide perovskites using density functional theory (DFT) calculations. CH2.ab initio simulation package (VASP) are used for all structural and electrical calculations.63\u201366 The projected augmented wave (PAW) method used the generalized gradient approximation (GGA) exchange\u2013correlation Perdew\u2013Burke\u2013Ernzerhof (PBE) functional.66 The van der Waals (vdW) is included in our calculations. We also include SOC and HSE calculation to find more clear results.67 The plane-wave kinetic energy cut-off is set to 500 eV. The Brillouin zone (BZ) k-point grid (5 \u00d7 5 \u00d7 1) was used for structure optimisation. Also (15 \u00d7 15 \u00d7 1) k-point grid was selected for further electronic properties. All atomic structures of MASnBr3 (MA = CH3NH3) and Ruddlesden\u2013Popper (BA)2MASn2Br7 (BA = CH3(CH2)3NH3, MA = CH3NH3) are converged up to the force 0.0001 eV \u00c5\u22121 and total energy at 10\u22124 eV. To define structural balance, we used the ionic radii based tolerance factor formula t = (RA + RX)/\u221a2(RB + RX) should be in the range between 0.813 and 1.107.5 Optical properties can be estimated through the Kramers\u2013Kronig transformation based on the electronic structure.68\u201371 The optical absorption coefficient can be calculated using the formula:Density functional theory (DFT) and the Vienna 3.3.13 and BAMASn2Br7 monolayer, as shown in 2Br7 monolayers during the literature survey. Here we replaced lead (Pb) with tin (Sn),41,43 which has an ionic radius smaller than Pb and the ionic radius of halogen iodide (I) replaced by bromine (Br). The corresponding lattice parameters are of the 2D MASnBr3, and BAMASn2Br7 monolayer smaller respectively as lead based 2D perovskites.10,41,43Here, we have studied the structural parameter of hybrid halide MASnBrt < 1.0, with consistence formation energy 141 eV for MASnBr3 and 196 eV for BAMASn2Br7.With all these replacements, Sn and Br perovskites structure becomes more stable and does not have much change in the structural phase. Also, for both structures corresponding tolerance factor is in the range of 0.8 < 3 and BAMASn2Br7 in 3, most of the part is contributed with the Br-p states with Sn-s states. While in the conduction band, most of the part is covered by the Sn-p states. Similarly, in the case of the Ruddlesden\u2013Popper BAMASn2Br7 perovskites, most of the part valence band is contributed with the Sn-s and Br-p states. The conduction band is coved with the Sn-p states as shown in 3 and BAMASn2Br7. 3 with PBE (without SOC) and x point 1.14 eV. Which is consistent with previously reported results.52Furthermore, we have calculated the total density of states (TDOS) and projected density of states (PDOS) of MASnBr2Br7 PBE (without SOC) and 2Br7 indirect bandgap is at Z to Y point 2.85 eV. BAMASn2Br7 perovskites have not been much studied, but compared with related Ruddlesden\u2013Popper are like BAMASn2I7 consistent.36,43,58,59 Also, when we replace Pb with Sn and iodine by Br, corresponding lattice parameters decrease with increasing corresponding band gaps.41 In both cases, we have seen that under the SOC study, there is not much effective change in indirect bandgap observed.5,41,43 An orbital contribution point of view in valence band maxima (VBM) is mostly contributed by the Sn-5s and Br-4p orbitals. While conduction band minima, most of the part is contributed with the Sn-5p and 4 s, 4p orbitals of Br atom.In 3.23, 2Br7. Due to flexible structural and compositional properties, mechanical strain is an efficient way to tune electronic properties.5,41 As per literature survey, we found that there are many experimental as well as theoretical techniques have been developed to improve only performance of the system or device. In the literature survey we found that there are no other studies on strain dependent on 2D MASnBr3 and Ruddlesden\u2013Popper BA2MASn2Br7 orthorhombic structures. Lattice strain is promising technique to improve performance of the many 2D materials. This has been useful for both experimental as well as theoretical groups. As per experimental researches studies of hetero epitaxial perovskite thin films shows that lattice strain play an important role to determining the physical properties of the thin films.5,41,60,61 In the case of the MASnBr3 system, we have applied both tensile and compressive strain along the x and y directions from 1% to 10% in the interval of 2%. Under tensile strain, condition bandgap was consistently decreased from 1% to 10% from 1.12 eV to 0.93 eV, as shown in We applied mechanical strain to both systems to moderate electronic properties as shown in 2Br7 perovskites. Here, under conditions of tensile strain, bandgap reduces as strain is increased by 1%, 2%, 4%, 6%, or 8%, going from 2.84 eV to 2.792 eV to 2.70 eV to 2.61 eV to 2.50 eV. Similarly, in compressive strain conditions, band gaps decrease from 1% to 8% to 2.84 eV to 1.96 eV. At 10% both types of strain show metallic behaviour. That means the sustainability of the structure is up to 8%. Further, in Ruddlesden\u2013Popper BAMASn2Br7. Comparison between the unstrained systems with strain systems of nature of the bandgap varies. Due to the presence of antibonding states of Br-p and Sn-s orbitals in valence bands. Non-antibonding states of Br-p and Sn-p orbital's in conduction bands are similar as that of the MASnBr3 perovskites. Overall, the mechanical strain technique is useful for tuning the bandgap and open the path for experimentalists to study thin films' physical and chemical properties.5,6061 In x-axis and y-axis for MASnBr3x-axis and z-axis for BAMASn2Br7. Overall stress was increased from 1% to 10%. Compared with tensile and compressive strain, in both systems, compressive stress is more and tensile stress is less. This change is happened due to contractions and expansion of lattice parameters of the system as shown in 2D barplot in Fig. S1.3 system, the structure's sustainability is up to 10% in both tensile and compressive. While in the case of BAMASn2Br7, the sustainability of the strain is up to 8%. After that, both systems show metallic nature.Using the Hellmann\u2013Feynman theorem3.351 Near the Fermi level, the directional dependent effective mass of both electrons and holes with the slope of x, y, z-axis direction are shown in 3 along x and y directions are 0.36 to 0.057 and for Ruddlesden\u2013Popper BAMASn2Br7 along x and z-direction are 0.33 to 0.146. In both cases, the effective mass of electrons and holes is remarkably more than the previously reported.35,43,62Further, we have a focus on the charge transport properties of both systems. Due to flexible structural and compositional properties, these systems are well sustained in strain conditions, bands experience a high degree of dispersion and have a bandgap near optical band gaps.We also calculate the charge transport carrier mobility and relaxation time using deformation potential theory. Which leads to calculate using formula\u03c4), \u03c4 = (\u03bcm*)/e as shown in e stand for electron charge, h for Planck's constant, m* for effective mass , Ex1 = \u0394V/(\u0394ax/ax) deformation potentials.5 In the case of MASnBr3, the calculated carrier mobility of holes is higher than the electrons. Similar trends were observed in BAMASn2Br7, the hole carrier mobility is higher than the electrons. In compared with lead-iodide-based perovskites, carrier mobility of Sn-bromide-based perovskite is 14% and 24% higher, which are quite larger than the reported experimental and theoretical data.5,35,41,43,62 This anisotropic nature of carrier mobility is due to smaller effective mass and flexible structural properties, affecting the band gaps. This high carrier mobility leads to better device performance. Open a new path for experimentalists to find solar cell efficiency and developed optoelectronic devices.Also carrier mobility dependent relaxation time  as well as ultraviolet region(310\u2013177 nm) of the energy spectrum as shown if 33,35,47 Overall, compared with MASnBr3 and BAMASn2Br7 perovskites with other 2D hybrid halide perovskites. Tunable band gap and optical, charge transport activity useful for photovoltaic and solar cell devices.In both cases, the first peak is at 2.5 eV. After applying both tensile and compressive strain, optical activity orientation is a move towards high energy part 8 eV as shown in 3.53. The Shockley and Queisser (SQ) model depend on the material's bandgap. To calculate the performance of solar cell efficiency, the band gap are required to be in between 1.0 to 1.7 eV. Therefore, the bandgap of 2D MASnBr3 is under SQ limit and band gap of BAMASn2Br7 are higher than the SQ limit. The solar cell parameters like open-circuit voltage (Voc), fill factor (FF), short circuit current density (Jsc), and solar cell efficiency are calculated as per previously reported results.5,41 More information is provided in the ESI\u2020Further, we have investigated the solar cell efficiency performance of 2D MASnBr3 solar cell efficiency is 23.46%. Which is 18% larger than that of the previously reported lead-based 2D MAPbI3 , fill factor (FF), short circuit current density (Jsc), and solar cell efficiency are consistent with the previously reported experimental and theoretical results. Here, tin-based 2D MASnBr3 shows high efficiency up to 23.46%, 18% higher than the lead-based perovskites. In optical activity, a high static dielectric constant shows good device performance. With high absorption coefficient up to 15.25 \u00d7 105 cm\u22121 for MASnBr3 and BAMASn2Br7 absorption coefficient up to 13.38 \u00d7 105 cm\u22121. Therefore overall results of this theoretical study. With mechanical strain engineering may be convenient to the experimentalist support to improve the performance of the 2D perovskites. This study supports these materials as promising candidates for photovoltaic and solar cell device applications.Here, the 2D MASnBrThere are no conflicts to declare.RA-013-D3RA00108C-s001"}
+{"text": "We know that hearing impairment affects a large part of the population. In cases of profound and bilateral hearing loss, children may have problems in speech development, as well as communication and socialization. Cochlear implants have been used as a treatment option in these cases. Today, inner ear MRI is a mandatory test in the preoperative evaluation of these individuals. In our daily routines, we wonder whether MRI can provide not only qualitative, but also quantitative data, with real cochlear linear values built from three dimension images.The aim of the present investigation is to propose a method to obtain MRI cochlear length measures from the temporal bones of cadavers.We assessed three dimensional images from the cochlea of six cadavers. By overlapping digitalized rulers on these images it was possible to measure cochlear length.These measures varied between 17 and 26.5 millimeters.We have concluded that it was possible to measure cochlear length from three dimensional MRI images, by employing the method hereby proposed. Cochlear implants (House 3M\u00ae) were introduced commercially in 1972; these devices stimulate the auditory nerve directly when placed in the cochlea (tympanic ramp).At present, image exams are employed routinely for a diagnostic preoperative assessment of candidates for cochlear implants; the aim is to define the anatomical status of the cochlea. Computed tomography (CT) and magnetic resonance imaging (MRI) are currently used for this evaluation.13Jackler et al.In the mid-80s, MRI was introduced as a new image diagnosis tool. Felix Bloch and Edward M. Purcell - who shared the physics Nobel Prize in 1952 - developed the principles of this technique .Currently, MRI is the image method of choice for assessing spiral canal of cochlea obstructions.15Casselman et al.Silberman et al.Guirado et al.Himi et al.Baumgartner et al.Arnold et al.Gleeson et al.Ketten et al.Using a block inclusion technique for studying a temporal bone of a 76-year old patient for computer analysis, Takagi and Sando17 estimated the cochlear length as being 36.3 mm in a 3D calculation, as opposed to 30.8 mm in 2D graphic reconstruction techniques.New 3D reconstructed MRI techniques have recently been described and compared. Jung et al.Lane et al.We asked whether MRI could provide routine qualitative and quantitative data such as true linear measurements of the cochlea in 3D reconstructed images.The purpose of this study was to propose a technique for verifying cochlear length measurements in magnetic resonance images of cadaver temporal bones.The Research Ethics Committee of the Santa Casa de Miseric\u00f3rdia de S\u00e3o Paulo approved this study on 11 September 2002 (protocol number 191/02).Six adult cadaver temporal bones obtained from the Servi\u00e7o de Verifica\u00e7\u00e3o de \u00d3bitos da Capital de S\u00e3o Paulo from 20 March 2004 to 14 April 2004 were used. These were removed with cutting tools always by the same technician within 12 hours of death and refrigerated at 4\u00b0C in an aqueous 10% formaldehyde solution . The temThe temporal bones were taken to and kept at 4\u00b0C in a 10% formaldehyde solution in the dissection room of the Otorhinolaryngology Department, Santa Casa de S\u00e3o Paulo, before being taken to the Diagnostic Unit of the Santa Casa de Miseric\u00f3rdia de S\u00e3o Paulo for imaging.MRI was done within a week after the temporal bones were received. Fat-suppressed T2-weighted volume sequences (SPIR) were taken , Fig. 2,The 3D reconstructed technique was used to acquire coronal and axial images of the inner ear. The coronal section reconstruction was chosen since it increases visibility of the turns of the cochlea .Fig. 33DMeasurements were taken in six images analyzed using a computer that is part of Philips Medical Systems MRI devices. The length of the cochlea was measured first by using digitized ruler images (in millimeters), which are part of the computer software, projected onto the cochlear images. The length of the spiral canal of cochlea was measured from its closer point to the vestibule to its apex.Given the curvature of the cochlea, it would be impossible to use this method for measuring the spiral canal of cochlea. The solution was to divide the spiral canal into a number of sections and to measure each one; adding up these values yielded the total length of the cochlea.The rulers had predefined 2.0, 1.5 and 1.0 mm markings and were placed over the cochlear images for measurements. Many rulers were required for each image until reaching the apex of the cochlea. The first six rulers (R1 to R6) measured 2 mm; the next rulers had smaller sizes to measure the cochlear contours (R7 to Rx). One of each 3D MRI images for cases 1 to 6 was chosen: that which provided the best view of the turns of the spiral canal of cochlea CT and MRI are the imaging methods used for assessing the spiral canal of cochlea. According to Gleeson et al.14Three-dimensional MRI provided high quality images of the spiral canal of cochlea in our cadaver temporal bone cases.Takagi and Sando (1989)Ketten et al.Our 3D MRI image measurements were not similar to the values measured using CT in the abovementioned studies, which appear to provide measurements that are closer to the true size of the cochlea. In our cases, the size of the cochlea in the images was smaller. Additionally, measurements of cases 2 and 6 were much smaller compared to the remaining cases. This may have been due to an underestimated MRI measurement or true variability in the size of the cochlea in the temporal bones we studied.New 3D reconstructed MRI techniques have been described recently.22A study of a larger series and improved image acquisition techniques may yield more precise cochlear measurements, which may then be used in routinely in otorhinolaryngology. This may provide the means for using MRI to accurately measure the size of the cochlea in implant patients. Procedures may then be tailored individually, varying the size and distribution of electrodes within the cochlea, thus providing improved stimulation across the cochlea.The length of the cochlea in cadaver temporal bones was measured with a technique that uses rulers over 3D reconstructed MRI images of those bones. The results appear to be shorter than those described in the literature."}
+{"text": "We conducted a content analysis of the literature underlying the Centers for Disease Control and Prevention (CDC) Guideline on the Diagnosis and Management of Mild Traumatic Brain Injury Among Children  to determine the extent to which social determinants of health (SDoH) were examined or addressed. The systematic review forming the basis for the Guideline included 37 studies addressing diagnosis, prognosis, and treatment/rehabilitation. We examined those studies to identify SDoH domains derived from the U.S. Department of Health and Human Services' Healthy People 2020 and 2030 websites. No study explicitly mentioned \u201csocial determinants of health,\u201d by name, and few studies addressed SDoH domains as a primary focus (ranging from 0% to 27% of studies across SDoH domains). The most frequently represented SDoH domains, described in an inferential or a descriptive manner, were Education Access and Quality (29.7% of studies), Social and Community Context (27.0% of studies), and Economic Stability (21.6% of studies). Health Care Access (13.5% of studies) was less well represented and no studies (0%) examined Neighborhood and Built Environment. In terms of the CDC clinical questions, SDoH were only examined as predictors of outcome (prognosis) and no studies examined SDoH in relation to diagnosis or treatment/rehabilitation. The Guideline includes some commentary on health literacy and socioeconomic status. Overall, social determinants of health are largely unrepresented as important or meaningful variables influencing the Guideline on the Diagnosis and Management of Mild Traumatic Brain Injury Among Children, or in the studies that informed the Guideline. For example, disparities in pediatric healthcare access and health outcomes have been reported in association with race and ethnicity,8-10 SES,11 English language proficiency,12 and acculturation.14Health equity means that all people should have fair and equal opportunity to enjoy their full health potential15 Moreover, children from low-income households, children from families that receive income assistance, and children born to teenage mothers are at an elevated risk for burn injuries.16There are important associations between SDoH, health inequity, and pediatric injury. For example, social gradients have been found with regard to causes of medically treated adolescent injuries, such that higher income is associated with greater likelihood of sport-related injury whereas poverty is associated with greater likelihood of injuries due to fighting.17\u201323 Approximately 1 in 100 school-aged children in the United States sustain a mTBI each year.24 Emerging research findings have begun to document the clear and expected relevance of SDoH with regard to pediatric mTBI. For example, there is accumulating evidence of racial, ethnic, and socioeconomic disparities in accessing concussion care,25-27 recovery time, and supports received upon returning to school following the injury,29 as well as health literacy .30-32A pediatric injury that is a major source of concern for parents, healthcare providers, school personnel, and legislators is concussion, or mild traumatic brain injury (mTBI).33 The Guideline includes 19 sets of recommendations regarding the diagnosis, prognosis, and management/treatment of pediatric mTBI. The recommendations in the Guideline were developed based on clinical science identified in a major systematic review.34Given the significance of concussion and mTBI as a public health concern, the Centers for Disease Control and Prevention (CDC) undertook a massive and pioneering effort to develop the first clinical management guideline in the United States for pediatric mTBI, which was published as the Centers for Disease Control and Prevention Guideline on the Diagnosis and Management of Mild Traumatic Brain Injury Among Children .33 Authors from multiple disciplines including primary care pediatrics, athletic training, physical therapy, and sports medicine have highlighted the broad applicability and importance of the Guideline.35\u201338The systematic review was organized around six clinical questions related to: 1) diagnosing mTBI; 2) accuracy of routine head imaging; 3) features associated with increased risk of intracranial injury/abnormal head imaging; 4) factors associated with worse outcome in the first year following mTBI; 5) factors associated with worse outcome beyond 1 year following mTBI; and 6) treatments associated with improved mTBI outcome. The systematic review screened 15,150 articles, a total of 2984 full-text articles were reviewed, and 75 articles were ultimately included in the synthesis across the clinical questions . The CDC established a multi-disciplinary workgroup that drafted recommendations relevant to each of the six clinical questions based on the studies identified in the systematic review, along with related evidence, scientific principles, and expert inference.The Guideline does not reference \u201csocial determinants\u201d or \u201chealth equity\u201d by name. However, the Guideline mentions and discusses aspects of SDoH. For instance, the Guideline notes the importance of health literacy and social support as means of promoting better patient outcomes. In addition, family and social stressors (Recommendation 8B), race, ethnicity, and SES (Recommendation 9B) are highlighted as potentially associated with worse outcome following injury and thus healthcare professionals are encouraged to monitor youth with these and other risk factors more closely (Recommendation 11A).34 and relied upon to support the practice recommendations in the Guideline,33 and to extract information pertaining to SDoH and health equity. More specifically, we conducted a content analysis to determine the extent to which the clinical science underlying the Guideline for diagnosing and managing pediatric mTBI has examined or discussed SDoH or health equity. Further, we sought to identify critical knowledge gaps to encourage and suggest future directions to enhance the incorporation of SDoH in pediatric mTBI research and clinical practice.The prevailing Guideline for the diagnosis and management of mTBI in children represents an extraordinary synthesis of a broad and diverse literature leading to specific and practical recommendations for healthcare providers. Leveraging that multi-year effort from multi-disciplinary experts, the purpose of this paper was to carefully review the studies identified in the systematic review33 which were identified through a systematic review.34 We collected the articles identified for four of the six clinical questions from the systematic review . Our focus was on diagnosis and management; thus, we did not examine the studies relevant to the two clinical questions that addressed neuroimaging. A total of 38 studies were identified; one study was excluded because it was a conference presentation, resulting in a final sample of 37 published studies.39\u201375We selected studies underlying the CDC Guideline,5 and Healthy People 20304 websites. Each of these websites lists the same five SDoH domains: Economic Stability, Education Assess and Quality, Health Care Access and Quality, Neighborhood and Built Environment, and Social and Community Context. Healthy People 2020 provided a bulleted list of these five domains, with several key issues and underlying factors within each domain. For example, a key issue listed under Economic Stability is \u201cpoverty.\u201d We included each of the key issues and underlying factors as separate variables to code. We then cross-referenced this list with the information on Healthy People 2030, which provides a separate webpage for each SDoH domain, with an \u201cOverview and Objectives\u201d section that presents brief text descriptions. Three authors  read each text description and extracted any additional key issues or underlying factors that were not included in the Healthy People 2020 bulleted list. For example, in the Health Care Access and Quality domain, health and/or dental insurance was listed in Healthy People 2030, but it was not on the Healthy People 2020 list; therefore, it was added to the coding sheet.We developed a coding sheet to identify SDoH and associated subcategories derived from both the Healthy People 2020The coding sheet also included summary information about the study sample including the sample size, average age, age range, as well as the gender, racial, and ethnic composition of the sample (coders collected the exact sample details or identified that the information was \u201cNot Reported\u201d by the study authors). The coding sheet also included study design, whether studies included exclusionary criteria based on demographic, sociocultural, or health factors, and whether studies mentioned future directions/research needs regarding social determinants or health equity. Lastly, we added summary variables to characterize whether, or the extent to which, each study analyzed or provided information about the following five key health equity variables: race, ethnicity, culture/acculturation, SES, and language. Coders could indicate whether the study: 1) provided no mention of the variable; 2) included the variable as a demographic category only; or 3) examined the variable in depth . The study coding sheet is included in the online supplemental materials.After the coding sheet was developed, four authors  reviewed the 37 articles. Five studies were assigned to all four raters as training articles and the raters met as a group to discuss and calibrate ratings. The remaining 32 articles were assigned to two raters each. Authors performed a content analysis independently by extracting details for each study and completing the coding sheet described above. Discrepancies were resolved by discussion. Lastly, two authors (NEC and AKK) independently determined whether articles addressed SDoH by examining the variables in an inferential or intentional way , or in a descriptive or demographic way . The mean sample age was reported in 14 studies and the average sample age was 12.9 years (standard deviation\u2009=\u20093.2). Samples included individuals ranging in age from 0 to 22 years, although the median age for the lower bound of age ranges was 6 years and the median upper bound was 16 years. Gender was reported in nearly all studies . Racial composition of the samples was only reported in about one-third of studies . Seven of these 11 studies and one additional study that did not report racial composition reported ethnic composition of the samples . Study samples were mostly recruited from hospitals/emergency departments , with very few studies recruiting from high school sports  or specialty concussion clinics . One study (2.7%) reported recruiting broadly from a healthcare system that included the emergency department, hospital admissions, and outpatient clinics. No study explicitly recruited from pediatric primary care. Study designs included prospective cohort , retrospective cohort , retrospective case-control , and randomized controlled trials , with one cross-sectional cohort (2.7%) and one cross-sectional case\u2013control (2.7%).No study explicitly mentioned \u201csocial determinants of health,\u201d by name. The content analysis results related to SDoH domains among the 37 included studies are summarized in No study explicitly mentioned \u201chealth equity\u201d or similar phrasing. The term \u201cdisparities\u201d appeared in three studies, but only one study used the term in relation to health disparities or health equity . The content analysis results related to health equity factors among the 37 included studies are summarized in 75 or if a guardian/caregiver was unable to complete the research consent process in English.71 One study included English speaking children of Spanish speaking parents.51 Many studies excluded children with pre-injury health conditions or disabilities, such as a history of a developmental delay, disability, or disorder,77 including autism spectrum disorder,78 intellectual disability,79 learning disabilities, including attention-deficit/hyperactivity disorder (ADHD),75 enrollment in special education,75 or diagnosed psychiatric or psychological disorder60 including schizophrenia,78 severe psychiatric history including hospitalization,79 or behavioral problems.49 No studies provided rationale or explanation for such exclusionary criteria, nor described such exclusions as a limitation of their study, such that the generalizability of the reported results might be limited. Only one study emphasized the importance of assessing sociocultural factors, such as a youth's pre-injury family background.41Over half of the studies  excluded participants based on demographic, sociocultural, or health factors, namely language proficiency and the presence of pre-injury health conditions/disabilities. Many studies excluded children with limited English proficiency,74 These authors further implore healthcare providers to \u201censure multidimensional support and resources\u201d and attend to such factors as socioeconomic disadvantage when providing clinical care for youth with TBI, specifically referencing those who are at socioeconomic disadvantage, including families with lower parental education, lower SES, and Medicaid insurance.74 A few other studies  mentioned or referred to SDoH or health equity indirectly. For example, one study recommended that future research examine family functioning and parental adjustment in relation to outcome and recovery following pediatric mTBI.73 Another study referred generally to \u201cpreinjury life stressors\u201d and \u201ca history of previous stressful life events\u201d as important to consider in both clinical and research considerations regarding pediatric mTBI.68 One study implied the importance of health literacy by recommending that information and suggested coping strategies be provided to children and their parents following pediatric mTBI.65 Three studies highlighted factors such as disability status 79 and SES57 as important variables to address in terms of sample inclusion and matching criteria.No study explicitly mentioned social determinants of health, by name, as an important framework to guide future mTBI research. One study (2.7%) referenced the need for future research to further explore health disparities in relation to outcome following brain injury, such as disparities in care, adherence to treatment and rehabilitation plans, and limitations in social support among disadvantaged children and families, though the authors specifically mention disparities in outcome following severe TBI, as opposed to mTBI.This content analysis found that SDoH and health equity have been significantly underrepresented in the clinical research literature underlying the CDC Guideline on the Diagnosis and Management of Mild Traumatic Brain Injury Among Children. Studies underlying the Guideline predominantly addressed only a subset of SDoH domains, most commonly Social and Community Context  and Education Access and Quality . The remaining SDoH domains are of importance to pediatric mTBI management but have very little representation in the literature.25-27 Health Care Access and Quality is also an important area for future research given initial evidence of health literacy disparities relating to general concussion knowledge, familiarity with concussion laws, and awareness of concussion symptoms.30-32 Moreover, no studies examined Neighborhood and Built Environment, which is of significant interest when considering the scientific support for exercise-based concussion rehabilitation, which might include, for some youth, the ability to safely access outdoor spaces to be physically active. Regarding the clinical questions that form the basis for the Guideline, no studies addressing diagnosis  and only one study addressing treatment  included SDoH in an inferential or primary fashion. It is important to note that our approach to coding SDoH was liberal and might overrepresent the extent to which social determinants have been examined or woven into this literature.For example, Economic Stability is a major domain of interest given emerging evidence of disparities in accessing concussion care.This content analysis also examined the extent to which health equity and related sociocultural variables were represented or discussed. Health equity was not mentioned explicitly by name, and it was not represented as a primary framework by which researchers designed studies or interpreted results in nearly all of the studies included in the Guideline. Only one study described its findings as revealing disparities in outcomes among disadvantaged youth and encouraged future work to investigate mTBI-related health disparities. Nearly half of the studies did not mention race, ethnicity, language, or culture/acculturation in any context, not even as a demographic characteristic describing the sample composition. Only about one in five studies examined a health equity variable in depth and most frequently this involved SES, such as by including SES as a predictor of outcome. Race and ethnicity were rarely examined in depth (only three studies examined either race and/or ethnicity). No studies underlying the pediatric mTBI Guidelines examined or represented culture/acculturation or language as contributors to health inequity.Just over half the studies underlying the prevailing Guideline utilized exclusionary criteria based on demographic, sociocultural, or health factors. Specifically, studies excluded youth from participating based on English language proficiency and/or the presence of pre-injury health conditions/disabilities. Moreover, no study discussed these exclusionary criteria as a limitation nor provided a rationale or explanation for the exclusion. Language-based exclusionary criteria may reflect resource limitations on researchers' part, such as limited funding to adequately translate informed consent documents in multiple languages or the lack of availability of interpreters to ensure appropriate informed consent procedures. Regardless of potential rationales or practical considerations from a research perspective, these exclusionary criteria represent clear limitations to the generalizability of findings, such that youth from underprivileged backgrounds are under-represented in the literature and the extent to which some of the findings from the literature apply to them is unknown.Lastly, no study explicitly mentioned SDoH, health equity, or similar phrasing as an important framework to guide future mTBI research. Only one study explicitly mentioned the need for future health disparities research regarding clinical care and outcomes, but the authors were referencing severe TBI, not mTBI. A few SDoH and health equity factors were mentioned indirectly, such as authors noting the need for future research to explore family functioning and adjustment following pediatric mTBI, or pre-injury stressful life events in relation to outcome from mTBI.34 It is hoped that more recent literature will increasingly incorporate and examine SDoH and health equity. In turn, the literature base that informs future updated guidelines will more adequately address SDoH and health equity. Moreover, the systematic review that formed the basis for the Guideline did not include a specific question regarding SDoH or health equity, and thus the representation of SDoH and health equity in the larger pediatric mTBI literature may differ, to some degree, from the studies included in this content analysis. Our focus, however, was to review specifically the articles that formed the basis for the development of the current Guideline.It is important to note that the articles included in this content analysis, and that form the basis for the Guideline, were drawn from a systematic review of literature published between 1990 and mid-2015.80-82 However, social considerations such as SES and resource availability are crucial to consider. For many youth, access to an in-home treadmill or exercise bike is not feasible or realistic for economic, space, or other reasons. Additionally, some youth may lack access to fitness facilities with such equipment. Moreover, even suggestions for youth to exercise outdoors may require modification, such as those who live in neighborhoods with limited green space or parks, on or near busy roadways that would not be safe and conducive to jogging, or in neighborhoods where being outside is unsafe in terms of potential for violence.Clinicians treating children with concussion are encouraged to carefully consider how SDoH might be relevant or important during clinical assessment, patient/family education, and during treatment or rehabilitation, and incorporate relevant information into their care, treatment, and rehabilitation plans. For example, for a youth with persistent symptoms following concussion, clinical science strongly supports the use of exercise-based active rehabilitation.The reading level and readability scores of patient education materials is an important consideration. Parental literacy and educational attainment may reduce a family's ability to follow recommendations offered by healthcare providers. It is also important to have access to medical interpreters and to have educational materials and supports available in primary languages for the patients served.83 However, facilitating referrals to specialty clinics is often a complex process and families may be poorly equipped to navigate the complexities of identifying specialty clinics, seeking referrals, and dealing with health insurance policies. Recent research indicates that specialty concussion clinic patients are more likely to: 1) have private insurance and 2) be White.84 Recommendations or referrals for additional forms of treatments to support concussion recovery such as physical therapy or mental health also may present challenges for families with economic constraints or insurance-based limitations. Clinicians are encouraged to proactively support families and facilitate their engagement in care by making direct contact with referral providers to the greatest extent possible.For youth experiencing prolonged recovery following mTBI, referral to specialty care is advised.Youth often require time-limited school accommodations during concussion recovery. However, pursuing and securing accommodations by communicating with school personnel might be more or less challenging for certain caregivers based on factors such as comfort with the school support personnel structure, knowledge of how to contact school nurses or guidance counselors (and comfort in doing so), or trust in the school team that the needs of their children will be prioritized and met.86 Further, research teams are encouraged to partner with school-based athletic trainers, healthcare providers, community-based health clinics, and athletic organizations in underserved communities to focus upon the specific and direct needs of underserved youth.There is a need to emphasize SDoH and health equity in pediatric mTBI research and to refine our understanding of how such factors relate to diagnosing, treating, and managing this injury. From a methodological perspective, concussion researchers should make concerted efforts to use qualitative and community-based participatory research designs and methodologies. These methodologies have proven particularly beneficial in terms of elucidating health disparities as well as conceptualizing and designing ways to intervene upon social determinants to reduce disparities among vulnerable groups.Pediatric mTBI researchers could consider some of the following specific examples for how to address SDoH and health equity in future studies. Focus groups could be conducted to identify potential SDoH needs and barriers to mTBI care. Researchers could directly evaluate the association between SDoH and recovery time from mTBI. Researchers could consider the feasibility of delivering interventions designed to target SDoH. For instance, the readability and reading ease scores of mTBI patient discharge instructions and patient understanding of the instructions might be examined. Studies could examine mTBI treatment and rehabilitation uptake, compliance, and barriers to accessing specialty care. Research designs aimed at informing the nature and scope of interventions designed to improve SDoH, health inequities, and clinical outcomes will be important. Additionally, research focused on uptake and utilization of the Guideline by healthcare providers in regards to SDoH and health equity factors will be important to examine.87Annals of Behavioral Medicine recently published instructions for authors reporting on study sample socioeconomic and sociodemographic characteristics that may serve as a guide for mTBI research.88 Further, researchers are encouraged to measure and describe their samples in terms of relevant SDoH such as health literacy surrounding mTBI, insurance status, access, and barriers to primary care for mTBIs, as well as access and barriers to specialty concussion care.Concussion researchers are encouraged to consistently and routinely measure and report complete demographic information about their samples. It seems particularly important for researchers to characterize gender, racial, and ethnic identities of their participants, as well as measure and report additional sociocultural characteristics such as SES and language proficiency. The website for the EQUATOR  network hosts additional templates for reporting guidelines.89 Encouragingly, there has been increased emphasis and considerable research interest related to screening for SDoH and related risk factors in the context of primary care,90 community health,91 and pediatric emergency medicine92 settings. Still, limitations in the available screening tools and resources have been highlighted along with the need for continued development and refinement of effective and practical tools.93 The American Academy of Pediatrics recommends screening during all patient encounters and provides a website with screening resources.94 It is unknown how frequently, if at all, SDoH are assessed in specialty concussion clinics\u2014and this appears to be an important area of future research. Researchers can examine SDoH and attrition from treatment trials and address disparities accordingly.As of about 2000, SDoH were rarely screened for in pediatric primary care settings.95 Additionally, funders could consider developing targeted mechanisms or opportunities regarding the potential role of and means to address SDoH and health inequities for pediatric mTBI diagnosis and clinical care.Funding agencies and organizations are encouraged to emphasize SDoH in concussion and brain injury research. Funders could consider encouraging researchers to collect and report SDoH in their research studies. For example, the PhenX measures for social determinants of health (SDoH) project, supported by the National Institute on Minority Health and Health Disparities, developed a collection of common data elements to improve the quality and consistency of SDoH data collection.96\u2013101 as well as substantial efforts to develop and validate evidence-supported treatment and rehabilitation approaches for pediatric mTBI, such as exercise-based rehabilitation.102-104 The development and publication of the Guideline was a synthesis of this rapidly evolving clinical and scientific knowledge, with the dissemination and implementation goal of improving the treatment and management of pediatric mTBI. With that said, SDoH and health equity have been significantly underrepresented in the clinical research literature, including the literature underlying the Guideline.Over the past two decades, there has been extraordinary advancement in knowledge about pediatric mTBI. This has included, for example, many studies published examining potential risk or vulnerability factors associated with worse outcome or slower recovery from pediatric mTBI,A major priority for the field is to better understand the role and relevance of SDoH and health inequities for pediatric mTBI diagnosis and clinical management. Moreover, it is a priority to determine the means and methods that can be used to address SDoH and health inequities and to implement those means and methods, with the goal of promoting health equity. Results of this content analysis revealed that SDoH are underrepresented as important, meaningful, or primary variables of interest directly addressed in the studies that formed the basis for the prevailing Guideline for managing pediatric mTBI and the recommendations for diagnosis, prognosis, and treatment contained therein. SDoH have not represented a primary framework by which researchers designed studies or interpreted results. SDoH are relevant and important for the diagnosis and management of pediatric mTBI. We need more work in the area to inform future updated guidelines.34 that was conducted as part of the Centers for Disease Control and Prevention's effort to develop the first clinical management guideline in the United States for pediatric mTBI, which was published as the \u201cCenters for Disease Control and Prevention Guideline on the Diagnosis and Management of Mild Traumatic Brain Injury Among Children.\u201d33 The coding sheet for this content analysis was developed by the authors and is provided in the supplemental materials that accompany this manuscript. Two co-authors independently coded every article and discrepancies were resolved by discussion. The results of this content analysis  are presented in the This review was not pre-registered. This review was designed as a secondary, content analysis of clinical studies that were identified in a large systematic review"}
+{"text": "Transmission of a malignancy from a donor\u2019s organ to the recipient of the graft is a rare event, though it is a severe complication that can result in a poor outcome. Usually, immunosuppressive therapy is discontinued and the allograft is removed. However, treatment of patients with the disseminated cancers implies that after the graft removal and cessation of the immunosuppression, radiotherapy, chemotherapy, or immunotherapy with alpha-interferon (INF-\u03b1) or interleukin-2 (IL-2) are required. The case report presents a clinical case of a transmitted kidney graft with multiple metastases (MTS) in a 31-year-old woman with the spontaneous regression of the metastatic cancer after transplantectomy and cancellation of the immunosuppressive therapy. Obviously, the determining factor is the recognition of the tumor by the effectors of the antitumor immunity due to the human leukocyte antigen (HLA) mismatch between the donor and the recipient. Therefore, cancellation of the immunosuppressive therapy in cases of transferal of a malignancy with a transplanted organ allows the effectors of the immune system to distinguish the tumor as a foreign tissue and effectively eliminate this neoplasm. There were no significant signs of graft rejection in the surrounding renal tissue. Lymphocytes infiltrated only a few tubules. There were no vast vascular changes. The greater omentum was dense, infiltrated with multiple MTS, the adipose tissue was practically not determined on the cut and the cut surface was represented by a grayish-white homogeneous tissue. The MTS found in the omentum were histologically similar to the graft tumor with clear secondary changes, leukocyte infiltrates, and foci of necrosis. Taking into account the uniformity of reactive changes both in the tumor of the transplanted kidney and in the omentum, these changes were regarded as a manifestation of transplant immunity.The serous membrane of the transverse colon was covered with disseminated tumor foci up to 3 mm in diameter; ascites reached 500 mL. In the left half of the abdomen, there was registered a tumor-altered transplanted kidney of 10 cm \u00d7 12 cm, spreading into the pelvic cavity, that was growing up to the anterior abdominal wall, intimately connected with the sigmoid colon, with the left iliac vessels and left appendages of the uterus, growing into the lumbar muscle on the left. Tumor infiltration was observed along the iliac vessels from the aortic bifurcation to the internal opening of the inguinal canal. Histological examination revealed an undifferentiated malignant tumor with significant lymphoid-plasmacytic and leukocyte infiltration.Given the \u201cdonor\u2019s\u201d origin of the tumor, a decision was made not to perform any specific antitumor treatment, and it was expected that the cessation of immunosuppression might be sufficient to cure the patient. One of the arguments in favor of the \u201cdonor\u2019s\u201d genesis of the tumor was the information that the second recipient  of the cadaver kidney from the same donor died 4 months after transplantation from a rapidly progressing \u201cundifferentiated tumor\u201d of the transplanted kidney. Regarding histopathological examination of the graft lesion, no any signs of specific tumor type were revealed, as in the described case .The second patient , who had been transplanted with the second kidney of the same donor, received the immunosuppressive therapy as the standard treatment after allogenic organ transplantation. After transplantation, an advanced tumor was revealed in the patient\u2019s kidney. Histopathological examination of the tumor biopsy showed the undifferentiated neoplasm, and the immunohistochemical staining for cytokeratins 8, 18, 19 did not identify any expression. The resection of the transplanted kidney was not performed. The rapid progression of the malignant disease led to the patient\u2019s death within the two months. Given the results of this case, the other patient  with the transplanted kidney of the same donor was thoroughly examined and the above-described decisions were made due to the detected graft tumor with the same histopathological characteristics .In the postoperative period, febrile fever and signs of intoxication were observed after cancelling immunosuppressive therapy. However, the focus of infection was not detected and high temperature reaction persisted despite massive therapy with a wide range of antibiotics. Regarding this observation, the clinical picture was considered as \u201cresorption-related fever\u201d caused by the destruction of tumor cells affected by the \u201ctransplant immunity\u201d. The patient underwent intermittent hemodialysis within the first four days after the operation. To remove possible tumor decay products, hemodialysis was replaced with hemodiafiltration , which led to an improvement of the patient\u2019s performance status and relief of the symptoms.Fourteen days after the operation, CT examination demonstrated multiple MTS in both lungs ranging in size from 4 mm to 15 mm ; pretracIn the postoperative period, there was noted a spontaneous reduction of the tumor conglomerate and infiltrate in the inguinal area on the left to the size of 1 cm \u00d7 1 cm. Four months later, the CT scan showed an essential positive trend as compared with that of the previous examination . PracticThe subcutaneous conglomerate of nodes in the left iliac region was not detected. In the abdominal cavity against the background of the conglomerate of the small intestine there were noted the nodes of 6.2 cm \u00d7 3.6 cm in diameter at the left pelvic wall. No other changes were found in the abdominal cavity and pelvic cavity. The patient was discharged from the hospital to be supervised by the urologist and oncologist. The patient\u2019s indications included programmed hemodialysis. Further tumor regression was observed in the follow-up examinations, and complete regression of the tumor foci was registered 6 months after the operation .Three years later, another kidney transplantation was performed. Over the next 15 years after the second transplantation with the standard immunosuppressive therapy, no symptoms of any malignant neoplasms were detected.Treatment of patients with advanced cancer that developed during long-term immunosuppressive therapy is a complex and still unresolved problem. The main issue is the use of standard chemo-/radiation therapy or immune-stimulating drugs in these patients after removal of the tumor-affected graft and cessation of immunosuppressive therapy. The regression of tumor foci registered in these cases might be both a consequence of ongoing antitumor therapy and the result of effective immune reactions that could be implemented after discontinued immunosuppression. Previously published data showed that spontaneous regression of the tumor and MTS was observed in approximately half of the initially tumor-affected transplantations after cancellation of the immunosuppressive therapy. The other half of patients received chemo-/radiation therapy since their immune system was considered ineffective in terms of controlling the widespread and extensive MTS , 10.According to some researchers, if spontaneous regression is not observed, the residual neoplasm should be treated with radiation therapy, chemotherapy, or immunotherapy using agents such as INF-\u03b1 or IL-2. After complete tumor regression, the repeated kidney transplantation should be delayed over the following tumor-free period for at least one year \u201313.In the case described, the tumor originated from a kidney allograft. Apparently, the standard immunosuppressive therapy promoted the dormant tumor clones to proliferate in the favorable microenvironment, which ultimately resulted in the development of a primary lesion in the allograft and subsequently, the metastatic spread to the greater omentum and lymph nodes and clear manifestation of the malignant disease.The proof for the assumption that the primary tumor was transmitted by a kidney graft was the fact that another recipient of the same donor\u2019s other kidney developed a tumor in the kidney allograft. In addition, spontaneous regression of tumor MTS after discontinuation of the immunosuppressive therapy and removal of the kidney allograft supported the allogeneic nature of the malignant neoplasm in the above-described patient.The presented clinical case illustrates the tremendous potential of the immune system, which, despite the immunosuppressive factors of the tumor and long-term immunosuppressive therapy, was able to complete a total regression of the MTS with no signs of the disease recurrence for the following 15 years. Obviously, unlike cases with autologous malignant neoplasms, the immunity of this patient had no barriers to recognize an allogeneic tumor and therefore, even the therapeutically suppressed immunity could realize its potential.Obviously, the probability of spontaneous tumor regression in patients after transplant removal and cessation of the immunosuppression is determined by various factors, such as the extent of tumor dissemination, the duration of the immunosuppressive therapy, and the initial status of the immunity. However, we believe that the determining factor is the recognition of the tumor by the effectors of the antitumor immunity due to the human leukocyte antigen (HLA) mismatch between the donor and the recipient. Therefore, cancellation of the immunosuppressive therapy in cases of the transmission of a malignancy with the transplanted organ allows the effectors of the immune system to distinguish the tumor as a foreign tissue and effectively eliminate this neoplasm."}
+{"text": "P < .05). Knowledge levels were satisfactory in this study, and the majority of participants exhibited adequate attitudes and practices related to malaria prevention. However, knowledge differences were observed with regard to the place of residence. Greater emphasis should be directed towards education programs in malaria-endemic areas to ensure complete eradication of malaria.Malaria is a major health problem in Southwestern Saudi Arabia. This study aimed to measure the level of community understanding of malaria transmission, protection, and treatment. A questionnaire-based cross-sectional study enrolled 1070 participants from 2 districts with different malaria prevalence rates in Jazan Province. The response rate was 97.27%. Of the 1070 total; 754 (70.5%) had heard about malaria. Sixty-seven percentage know that fever was the main symptom. Approximately 59.8% did not know that stagnant water is one of the most important locations for mosquito breeding. Nevertheless, we found that 50% of the participants knew that mosquitoes bite at night and 96.9% confirmed that mosquitoes did not bite during the day. The most effective sources of information were distributed leaflets (41.8%) and video awareness (31.9%). The most significant factors affecting participants knowledge were gender, residence, family members, income, and education ( It is a leading cause of mortality and morbidity in many developing countries, where young children and pregnant women are most affected. In 2016, malaria triggered 216 million clinical episodes and 445,000 deaths worldwide. The Global Technical Strategy for Malaria 2016 to 2030 is designed to guide and support all malaria-affected countries. The strategy targeted for the year 2030 an elimination of malaria in at least 35 countries and reduction of malaria case incidence and mortality rate by at least 90%. The revived enthusiasm for scaling up the adoption of validated strategies around the world, however, has rekindled optimism, and even elimination is being pursued where malaria has been reduced to very low levels.,5Despite decades of malaria prevention initiatives, malaria remains a severe public health concern worldwide.,7 Saudi Arabia joined the World Health Organization global malaria eradication effort and the recommendations on malaria prevention and control were updated based on the latest available evidence. Its achievements include biannual meetings and the use of mobile units to assist undocumented refugees arriving from neighboring countries of the Autonomous Country Committee on Malaria Elimination. Saudi Arabia needs to improve vector surveillance along its frontier and expand the number of specialists employed in the entomology and case management of malaria to achieve zero.\u201310The only 2 countries on the Arabian Peninsula that have not yet eradicated malaria are Yemen and Kingdom Saudi Arabia.,11\u201314 It is essential to have excellent surveillance and response systems in place in order to eliminate and keep the malaria problem under control; information systems need to become more \u201cgranular\u201d in order to enable the identification, tracking, classification, and response to each and every case of malaria .\u201316 For a country to be able to eliminate the disease, its healthcare system must have both capable leadership and the ability to penetrate all of the country\u2019s communities . Knowledge should be generated through operational research on tools, methods, and delivery and used to enhance recommendations and future elimination initiatives.\u201314,16,17Malaria elimination is defined as the purposeful interruption of local transmission of a particular malarial parasite species in a defined geographical region as a consequence of deliberate operations. This resulted in a reduction in the incidence of indigenous malaria. It is necessary to maintain preventative measures that have been taken to stop the reestablishment of transmission. For a nation to receive official recognition that the disease has been eradicated, the local transmission of all human malaria parasites must first be stopped. It is necessary to take precautions against the resumption of transmission indefinitely, until the disease has been eradicated.,18,19 Malaria remained at a low level in the Kingdom\u2019s southern regions. These governmental efforts to fight malaria require strong collaboration and pioneering public awareness. Parallel research on knowledge, attitude and practice (KAP) must be conducted to measure public bonding. A previous study of KAP in the Jazan region did not focus on endemic areas and compared them with disease-free areas, as was the case in the present study. In addition, this study includes an objective evaluation of what happened after elimination, and the results will be useful to relevant authorities and decision-makers.Therefore, the current study was designed to measure the level of community understanding of malaria transmission, personal protection, and treatment, to determine the level of KAP in the community for the concepts of control and elimination, and to compare the knowledge of families in 2 regions characterized by different prevalence rates during the period of the National Malaria Elimination Program.Over the last 50 years, great efforts to control malaria in Saudi Arabia have led to a decrease in its prevalence in many parts of the country, mainly by using insecticide spraying, treatment, and personal protection measures. The province of Jazan stretches 300 km along the southern Red Sea coast and 120 km along the border with Yemen. According to the 2010 census, there were 1.37 million inhabitants. A map of Jazan is shown in Figure th village on the list was selected. The same randomization was applied for the selection of households, with 4 hundred households selected in total. We interviewed 2 to 3 people from each household and the total number of people interviewed was 1100.This research was conducted in 2 districts with different malaria prevalence rates in the Jazan Province (Al-Ardah and Samtah Regions).This is a cross-sectional study. Data were collected using a well-structured questionnaire distributed between February and April 2021. A comprehensive workshop on how to use the questionnaire was conducted by the authors. The data collectors were final-year students from the Department of Health Education and Promotion, Faculty of Public Health and Tropical Medicine, Jazan University, Kingdom of Saudi Arabia. For the large population in this study, a total sample of 1100 people was collected assuming differences within \u00b1 1.0 for coefficients and \u00b1 0.02 for Nagelkerke r-squared to derive the statistics that represent the parameters.Based on the sample size guidelines for logistic regression from observational studies with large populations, the minimum sample size to determine the associated factors was 500, assuming differences within \u00b1 0.5 for coefficients and \u00b1 0.02 for Nagelkerke r-squared.,22\u201330 and World Health Organization guidelines.,11,15 The knowledge questions were about the causes of malaria, the primary vector, symptoms, preventive measures, personal means for prevention, and knowledge about mosquito behavior. The main question for measuring knowledge of malaria consisted of 4 options: no information, little information, medium information, and good information. This assumption was based on assessments of the data collectors. Heard about malaria and know about its transmission is the minimum requirement to categorize respondents into little information. If the respondent knew about the transmission and symptoms, they were placed in the moderate information category. If the respondent\u2019s knowledge extended to the treatment of malaria, it was placed in the good information category. Based on these choices, the knowledge prevalence rate was calculated, and this variable was used as the dependent variable in multivariate logistic regression analysis. Data on mosquito breeding areas were also collected. The questionnaire also had a section that asked about people\u2019s habits, such as where they slept, how they cleaned their water containers, covered their tanks, used insecticides, and shut their windows.The structured questionnaire included demographic characteristics in the first part . The second part contained questions about knowledge, attitudes, and practices, which were designed based on previous studiesThe questionnaire was translated into Arabic and pretested in a pilot study (n = 30) before the final analysis. The respondents for the pilot study were selected from 2 villages (1 village from each region), and 15 respondents were chosen from 15 households in each village. Trained interviewers conducted interviews with responsible adults in their respective families. Only 1 individual per household was interviewed in the pilot study. Verbal permission was granted to all participants. For quality management, questionnaire administration and data collection were tracked regularly. Data obtained from the pilot study were not included in the final analysis.Data were entered into an Excel file and transferred to the SPSS statistical software version 20.0 , where all descriptive and inferential statistics were obtained. The reliability of the questionnaire was tested using a Cronbach alpha of 0.713. Binary logistic regression modeling was performed with knowledge as the dependent variable  and demographic factors as independent variables. As few respondents were in the moderate and good categories for knowledge, we placed them with the little category in 1 group and compared them with those with no information category in the binary logistic regression modeling. Dummy variables and reference groups for each independent variable were generated using SPSS software. Model fitting was evaluated using the-2 log-likelihood, Cox & Snell R-Square, Nagelkerke R-Square, and Hosmer and Lemeshow tests.\u201cThe study was conducted in accordance with the Declaration of Helsinki, and approved by the Institutional Review Board (or Ethics Committee) of the Department of Health Education and Promotion, Faculty of Public Health and Tropical Medicine, Jazan University, Saudi Arabia. (Protocol code FSP-2/194 on 10/12/2020)\u201d.The data collection process had a 97.27% response rate. Table Of the 1070 household members surveyed, 754 (70.5%) had heard of malaria Fig. . Figure Table As observed, 59.8% (n = 640) of them did not know that stagnant water was one of the most critical places for mosquito breeding. Nevertheless, 50% knew that mosquitoes bite at night, and 96.9% (n = 1037) confirmed that mosquitoes do not bite during the day, as shown in Table Statistics regarding preferred educational resources and the most effective campaigns are listed in Table Table Knowledge of the fight against malaria campaigns was reported by 52.5% (n = 562) of respondents, as shown in Table Further analysis using binary logistic regression with recategorized knowledge was used as the dependent variable. Respondents with no information n = 316) were categorized and coded as 0. While 754 (70.5%) respondents who had heard about malaria were classified and coded as 1, the independent variables included in the logistic regression model were gender, age, residence, family members, region, marital status, income, type of home, and education. Beta coefficients, p-values, adjusted, and crude odds ratios (OR) are reported in Table  were catThe goal of the current study was to compare the knowledge of households in 2 regions with different malaria prevalence rates to assess how well the community understood malaria transmission, personal protection, and treatment. It also assessed the extent to which the community understood the concepts of control and elimination. This is the first study in Saudi Arabia to include baseline evidence and an evaluation of knowledge, attitudes, and practices during the malaria elimination phase in Jazan, rural Saudi Arabia.,16 Understanding community perspectives and practices is an essential component of a successful malaria control program. In most affected nations, the possible contribution of KAP studies to malaria research and control has received little attention. The findings of this study are directly reflected in public awareness. Of the 1070 household members surveyed, 754 (70.5%) had heard of malaria. These results are in agreement with those of previous studies conducted in Tanzania, western Sierra Leone, and Saudi Arabia. This high level of knowledge about malaria may be due to the work that the Saudi authorities in charge of public health have done on a national level. Learning about malaria is insufficient; rather, it should be viewed as a foundation for understanding a wide variety of malaria-related topics such as transmission, symptoms, prevention, and therapy. More than 2 to 3rd of the respondents in this study were correctly answered the questions related to the malaria symptoms and the method of transmission. However, there was misconception with regard to the vector that transmitted malaria as only 1 to 3rd of the respondents selected mosquitoes as a pathogen vector for malaria. The findings of this study lend credence to those found in earlier research carried out in Nigeria,,34,35 Guinea, Tanzania, Ethiopia, and India.Despite the existence of effective preventive strategies, the burden of malaria is increasing in many countries.,10 It should be noted that there was confusion between malaria symptoms and dengue fever, which is also prevalent in these regions, among many participants in the Al-Ardah region. However, mosquito prevention does not affect disease control. However, this can lead to severe problems in patients who assume that dengue fever may be treated with antipyretics and drinking water.,27This study revealed that most people in both regions had sufficient knowledge of the malaria symptoms. The most frequent symptom, fever, was recognized by more than 2 to 3rd of participants (67%). This finding is consistent with previous research in endemic countries with tropical and subtropical climates. The present study showed variable findings on these measures, such as sleeping behavior, water container cleaning, opening water tanks, use of pesticides, closing windows, rainwater monitoring, cultivation of small shrubs, and regular checking for malaria. This may be because most people rely on malaria prevention programs to defend themselves from malaria infections. These inconsistent statistics should be considered by health authorities, and further actions should be taken based on the deficits depicted in these measures. In the current study, a significant proportion of participants incorrectly identified the primary control measures. Moreover, 72.7% and 63.2% of the participants did not recognize bed nets and insecticides as essential measures for malaria prevention and control, respectively. These are considered to be high figures. Only 20.7% of Americans currently use personal malaria-protection measures. This might be explained by the outstanding efforts of the Ministry of Health, which reduced malaria to minimum levels in previous years. Responses regarding knowledge of malaria symptoms and signs showed that only 8% had good knowledge, and 47% needed more information about the disease. A 2005 study found that participants in India had a moderate-to-high degree of knowledge and successfully implemented preventative actions. Only 4% of participants in a cross-sectional study of 1330 households in rural Nepal conducted in 2004 to 2005 demonstrated any knowledge of the significance of using insecticide-impregnated mosquito nets, and only 23% of these people actually used nets. This Nepali study revealed that participants had a minimal understanding of preventive measures.Public health is critical for the elimination of malaria.,38Our results are encouraging for malaria treatment. Eighty-two point eight Of the respondents, 82.8% preferred to do what doctors had instructed them correctly. Only 3.6% of participants sought treatment from traditional healers. Previous surveys in Southeast Asian rural communities have shown that over half of the residents prefer to manage their health without attending health centers. The improved conduct documented in this study may be attributed to the provision of health facilities to all Saudi citizens and access to their services. This behavior helps eliminate malaria, as mosquitoes cannot transfer infected blood to healthy people and break the disease transmission cycle.,39 that used descriptive patterns only for malaria KAP.Exciting results were obtained from logistic regression modeling of knowledge levels across both regions. Sex, age, home, family members, region, income, and education were independent variables used in the logistic regression model. All of these variables had an important impact on malaria awareness, except for age and marital status. The inclusion of all the variables in the model substantially changed the crude OR. This modeling analysis provided an additional advantage over previous studiesA significant difference in the knowledge level of malaria was observed between the villagers from Al-Ardah and Samtah. This could be explained by the differences in climate between the 2 regions. The weather in the Al-Ardah region is tropical and the rainy season is summer. Al-Ardah rains significantly during autumn. This climatic difference between the 2 regions leads to differences in mosquito breeding rates. On the other hand, Al-Ardah is a valley and mountainous area, whereas Samtah is a plain area. Therefore, the environment in the Al-Ardah region is considered suitable for mosquito breeding, as swamps, rain, and water pools are the most important factors that help the spread of mosquitoes. The higher number of health facilities recorded in Samtah may explain this finding.This study presents the first evidence of KAP regarding malaria, and it was conducted in large rural populations that were randomly chosen from well-populated villages in the southwestern region of Saudi Arabia. This region is known to have a comparatively high risk of exposure to imported malaria. However, possible limitations of the research include the use of a biased tool to assess the KAP of participants, as well as the cross-sectional design of the study, which makes it difficult to establish any causative relationship. Both aspects of the study could be considered drawbacks. Despite this, it was a strategic strategy to evaluate the baseline information on KAP in relation to the treatment and prevention of malaria.The vast majority of respondents in this study heard about malaria and they know much about its symptoms and mode of transmission. However, there was misconception with regard to the causative agent and there were variations related to the knowledge between the different regions. As a result, we suggest that a collection of local strategies should be developed to encourage the implementation of preventative and control measures that aim to minimize the exposure to, occurrence of, and spread of malaria as well as to encourage positive attitudes and the most effective preventative practices that are currently available. Additional research is necessary to validate and broaden the scope of the findings of this study.The authors are grateful to the participants of this study. The authors also extend their appreciation to the Deanship of Scientific Research, Jazan University, for supporting this research through the Research Unit Support Program (Support Number: ISP22-7).Conceptualization: Ibrahim Siddig Abdelwahab, Ibrahim Elhassan, Osama Albasheer.Data curation: Ibrahim Siddig Abdelwahab, Ibrahim Elhassan, Osama Albasheer, Nasir Ali.Formal analysis: Ibrahim Siddig Abdelwahab.Investigation: Waleed Madkhali.Methodology: Ibrahim Siddig Abdelwahab, Ibrahim Elhassan, Mohammed Alshehri.Project administration: Ibrahim Siddig Abdelwahab, Osama Albasheer.Resources: Nasir Ali, Yahya Al-Jabiri, Bassem Oraibi, Ahmed Altraif, Nasser Hakami, Mohammed Alshehri, Mohammad Abu Shaphe, Rashid Beg, Meshal Alshamrani.Software: Ibrahim Siddig Abdelwahab, Ibrahim Elhassan, Yahya Al-Jabiri, Ahmad Sahly, Rashid Beg, Meshal Alshamrani.Supervision: Ibrahim Siddig Abdelwahab, Osama Albasheer, Manal Taha, Nasir Ali, Yahya Al-Jabiri, Waleed Madkhali, Ahmad Sahly, Bassem Oraibi, Ahmed Altraif, Nasser Hakami, Mohammed Alshehri, Mohammad Abu Shaphe, Rashid Beg, Meshal Alshamrani.Validation: Manal Taha, Nasir Ali, Yahya Al-Jabiri, Waleed Madkhali, Ahmad Sahly, Bassem Oraibi, Ahmed Altraif, Nasser Hakami, Mohammed Alshehri, Mohammad Abu Shaphe, Rashid Beg, Meshal Alshamrani.Visualization: Manal Taha, Nasir Ali, Yahya Al-Jabiri, Waleed Madkhali, Ahmad Sahly, Bassem Oraibi, Ahmed Altraif, Nasser Hakami, Mohammed Alshehri, Mohammad Abu Shaphe, Rashid Beg, Meshal Alshamrani.Writing \u2013 original draft: Ibrahim Siddig Abdelwahab, Ibrahim Elhassan, Osama Albasheer, Manal Taha, Nasir Ali, Yahya Al-Jabiri, Waleed Madkhali, Ahmad Sahly, Bassem Oraibi, Ahmed Altraif, Nasser Hakami, Mohammed Alshehri, Mohammad Abu Shaphe, Rashid Beg, Meshal Alshamrani.Writing \u2013 review & editing: Ibrahim Siddig Abdelwahab, Ibrahim Elhassan, Osama Albasheer, Manal Taha, Nasir Ali, Yahya Al-Jabiri, Waleed Madkhali, Ahmad Sahly, Bassem Oraibi, Ahmed Altraif, Nasser Hakami, Mohammed Alshehri, Mohammad Abu Shaphe, Rashid Beg, Meshal Alshamrani."}
+{"text": "Linear IgA bullous dermatosis (LABD) is characterized by linear deposition of IgA at the dermoepidermal junction. It is associated with medications, malignancies, and vaccinations.A 48-year-old man with type 2 diabetes mellitus, nonalcoholic fatty liver disease, and stage IV melanoma  presented to our Dermatology clinic with 2\u00a0days of a pruritic, blistering rash.He was diagnosed with stage IIIC acral lentiginous melanoma in 2018. He underwent a partial amputation of the involved digit and adjuvant nivolumab therapy for 1\u00a0year. In 2021, he developed lung metastases. He was treated with nivolumab and ipilimumab, followed by nivolumab monotherapy. Repeat imaging showed worsening lung disease. Given his progression despite anti\u2013programmed cell death protein 1 immunotherapy, he was enrolled in a trial in June 2022 of weekly subcutaneous nemvaleukin alfa monotherapy (NCT04830124).A). Direct immunofluorescence test revealed linear deposition of IgA along the basement membrane, confirming a diagnosis of LABD (B). Nemvaleukin alfa was used.He initially tolerated nemvaleukin well, with grade I injection site pain, fatigue, and myalgias resolving within 3\u00a0days of each cycle. After the seventh cycle, he developed a rash, keratoconjunctivitis, and dyspnea on exertion. Examination revealed edematous, crusted erosions and scattered papules and pustules on the flanks and buttocks, as well as ill-defined annular plaques with vesicles on the upper extremity .\u00a0He deni of LABD , B. NemvOver the next 2\u00a0weeks, the rash spread to the chest and groin, then improved spontaneously. Nemvaleukin alfa was reinitiated. Two days later, the rash recurred, presenting with erythematous annular plaques on the left upper extremity. He was prescribed clobetasol ointment 0.05% twice daily and dapsone 50\u00a0mg/d for 10\u00a0days. Nemvaleukin alfa was continued, and the rash resolved over the next 2\u00a0weeks. Ultimately, he developed brain metastases, resulting in discontinuation of nemvaleukin.Drug-induced LABD can have cutaneous presentations ranging from plaques to bullae with positive Nikolsky\u2019s sign, making the diagnosis challenging. IL\u20132-associated LABD has only been reported once in the English literature in a 1996 case of a patient with metastatic renal cell carcinoma who developed LABD after the second cycle of aldesleukin, a recombinant IL-2 therapy.,+ T cells without binding regulatory T cells. To date, the most common side effects of nemvaleukin alfa have been fevers and neutropenia.Although recombinant IL-2  has been US Food and Drug Administration-approved for metastatic melanoma since 1998, its use declined after the introduction of programmed cell death protein 1, V-Raf murine sarcoma viral oncogene homolog B, cytotoxic T lymphocyte associated protein 4, and mitogen-activated protein kinase inhibitors, which revealed superior survival rates and more favorable toxicity profiles.Nemvaleukin alfa is being studied in patients with other solid organ tumors (NCT05092360 and NCT02799095). With wider applications of modified IL-2 molecules, it is important for clinicians to be aware of its potential skin toxicities to allow for successful diagnosis and management of these complex and high-need patients.None disclosed."}
+{"text": "Lead (Pb) and furan are toxic agents, and persistent exposure may impair human and animal reproductive function. We therefore explored the effects of Pb and furan on male rat hypothalamic-pituitary-gonadal reproductive status, oxidative stress, inflammation, and genomic integrity. We found that co-exposure to Pb and furan reduced the activities of testicular function enzymes, endogenous antioxidant levels, total sulfhydryl group, and glutathione. Sperm abnormality, biomarkers of oxidative stress, inflammation, and p53 expression were increased in a dose-dependent manner by treatment with furan and Pb. Typical rat gonad histoarchitecture features were also damaged. Conclusively, co-exposure to Pb and furan induced male reproductive function derangement by decreasing the antioxidant defences in rats, increasing abnormalities in spermatozoa morphology, and reducing reproductive hormone in circulation. These pathophysiological alterations, if persistent, might provide a permissive environment for potentiating reproductive dysfunction and infertility. Recent findings revealed that exposure to environmental factors such as heavy metals\u2014lead (Pb), cadmium, chromium, manganese, mercury, and nickel; and furan, a by-product of food processing involving heat treatment, can predispose men to infertility.The factors that promote infertility include socioeconomic and behavioural characteristics, tobacco smoking, insulin resistance, sleep apnoea, reduced level of gonadotropins and testosterone, and underlying medical conditions, and can cause reproductive dysfunction. Environmental pollution from Pb can occur from mining activities, Pb-based gasoline, pipe, ammunition, vehicle exhaust, electronic wastes, erosion of natural wastes, re-processing Pb-battery, natural fires, and sea salt spray. When Pb enters the body, either through ingestion of contaminated feed or water, it is distributed to two chief compartments depending on whether the exposure is acute or chronic. Acute Pb exposure is distributed to readily exchangeable pools including the blood, liver, kidney, and brain, while chronic exposure will accumulate Pb in deep tissue reservoirs, including the bones. The World Health Organization (WHO) and the United States Centre for Disease Control and Prevention (CDC) set the permissible blood level of Pb (<10 \u03bcg/dL in adults and 5 \u03bcg/dL in children). Despite these reference values, Pb toxicities have roused concerns globally. The toxicological impacts of Pb exposure have been investigated at low and high concentrations. Some studies report that the blood concentration of Pb in adults (>100 \u03bcg/dL) and children (approximately 80\u2013100 \u03bcg/dL) can cause cognitive impairment. A minimal concentration below 3 \u03bcg/dL of Pb, caused cognitive deterioration and maladaptive behaviour in humans and animals, implying that there is no acceptable baseline for Pb exposure. Also, exposure to Pb has been attributed to reproductive health debility. A rise in seminal plasma or blood Pb levels  negatively correlate with reductions in sperm count and density, decreased ejaculate volume, immature and abnormal spermatozoa, and drop in total sperm count of exposed individuals, respectively.Pb is a heavy metal known to impact spermatogenesis. In a 2011 report, the European Food Safety Authority (EFSA) detected a high level of furan in coffee brands, including roasted bean coffee (3660 ng/g), unspecified coffee (2016 ng/g), roasted ground coffee (1936 ng/g), and instant coffee (394 ng/g). Also, low levels of furan were detected in food products such as soft drinks (0.8\u20131.2 ng/g), tea (1\u20131.7 ng/g), wine and liquors (1.3 ng/g), vegetable fats (1.5\u20131.7 ng/g), and fruit juice (2.2\u20134.6 ng/g). Furan toxicity is mediated by cytochrome P450 2E1 involved in furan metabolism, to the reactive intermediate, cis-but-ene-1,4-dialdehyde (BDA). BDA interacts with amino acids, proteins, and DNA to induce toxicities in significant organs of the body, thus making furan a global public health issue. The foregoing necessitated the European Commission to ask the EFSA Panel on Contaminants in the Food Chain to conduct a scientific evaluation on the risk to humans of furan in foods. The calculated margin of exposure obtained by the EFSA indicated a health concern. More worrisome is the documentaries from the US Food and Drug Administration (US FDA), EFSA, National Toxicology Program (NTP), and International Agency for Research on Cancer (IARC) that furan is carcinogenic to humans.Furan is a by-product of food processing and preservation technique involving heat treatment including cooking, roasting, baking, pasteurisation, and sterilisation. Formation of furan occurs by Maillard reaction; a non-enzymatic response of amino acids, peptides, and proteins with reducing sugars and vitamin C. Hence, there is a need to monitor the degree of toxicities following combined exposure to Pb and furan since humans and animals quickly ingest them through their diets and drinking water. A feasible means of investigating the harmful effects of environmental toxicants in experimental models is to monitor the process of oxidative stress-mediated by increased production of reactive oxygen species (ROS) with a concurrent decline in endogenous antioxidants, signalling in favour of pro-inflammation mediators and loss of genomic integrity. We hypothesised against this background that accidental exposure to Pb and furan may significantly impair male rats' reproductive health. Therefore, the present study was aimed at investigating the combined effects of Pb and furan on the integrity and functionality of the hypothalamic-pituitary-testicular axis in rats by examining sperm functionality, testicular function biomarkers, reproductive hormones, genomic stability, and oxido-inflammatory biomarker in rats. We observed that co-exposure to Pb and furan altered hypothalamic-pituitary-testicular system function by primarily exacerbating oxidative-inflammatory stressors, and genomic instability.In developing countries, exposure of humans and experimental animals to Pb and furan may emanate from antiquated facilities for transporting municipal water from pump stations to residential homes. Increases in anthropogenic activities including Pb-ore processing, Pb-battery recycling, ammunition, vehicle exhaust, e-wastes and bush burning, and poor-quality control practices on the preservation of food products also contribute to these exposure routes2O2), 5,5-dithio-bis-2-nitrobenzoic acid, Griess reagent, 1-chloro-2,4-dinitrobenzene (CDNB), trichloroacetic acid, and bovine serum albumin were purchased from Sigma (USA). Disodium hydrogen phosphate, sodium carbonate, sodium hydroxide, sodium-potassium tartrate, and sodium chloride were obtained from BDH Ltd. (UK) and William Hopkins Ltd. (UK). Acid phosphatase, alkaline phosphatase (ALP), lactate dehydrogenase (LDH) kits were obtained from Randox Laboratories Ltd. (UK). Enzyme-linked immunosorbent assay (ELISA) kits for the assessment of p53 and tumour necrosis factor-alpha (TNF-\u03b1) were obtained from Elabscience Biotechnology (China), and luteinizing hormone, follicle-stimulating hormone, prolactin, and testosterone kits from Fortress Diagnostics (UK). All other chemicals used for these experiments are of analytical grade.Lead acetate, furan, epinephrine, glutathione (GSH), thiobarbituric acid, hydrogen peroxide (H. The sample size was estimated with the G* Power software (v3.1.9.4) . To avoid type \u2160 and \u2161 errors and an effect size of 0.40 (larger effect) at 0.05 alpha error of probability for one-way analysis of variance (ANOVA) was employed, giving a total sample size of 125 at 95% power. Out of 125 estimated experimental animals, 30  male Albino Wistar rats (approximately 177 g body weight [BW]) were purchased and housed in the Department of Biochemistry University of Ibadan. The rats were acclimatised for seven days under natural daily photoperiod; fed and provided clean water ad libitum.The 3Rs  guidelines for care and use of experimental animals were adopted in this study as previously described was prepared by dissolving 213 \u03bcL furan in corn oil and administered per os (p.o.) according to the BW of rats. PbAc stock solution (0.1 mg/mL) was prepared with 50 mg PbAc in 50 mL of distilled water. Subsequently, 1, 10, and 100 \u03bcg/L were prepared by adding 0.02, 0.2, and 2 mL of PbAc stock and reconstituted to 2 L with distilled water. Experimental rats water intake was estimated daily, and replenished to maintain a daily supply of 300 mL. Rats in the control group received corn oil, the furan group received furan (8 mg/kg BW) p.o., the PbAc group received PbAc (100 \u03bcg/kg BW) in drinking water p.o, the furan+PbAc1 group received furan (8 mg/kg BW) and PbAc (1 \u03bcg/kg BW), the furan+PbAc2 group received furan (8 mg/kg BW) and PbAc (10 \u03bcg/kg BW), and the furan+PbAc3 group received furan (8 mg/kg) and PbAc (100 \u03bcg/kg BW) .Treatment of the rats for 28 successive days was initiated following the study's approval by the University of Ibadan Animal Care and Use Research Ethics Committee . The furan stock solution (8 mg/kg)via retro-orbital venous plexus, and subsequently sacrificed by cervical displacement. The blood was left to coagulate and then centrifuged  to obtain the serum for sex hormones estimation. The testis, epididymis, and hypothalamus were immediately excised, weighed and processed for biochemical analysis and histopathology. The relative weight (%) of the organ  were calculated accordingly: (weight of the organ)/(weight of the body)\u00d7100.After 28-days of treatment, the rats were fasted (24 h), whole blood collected into non-heparinised tubes g) to obtain a clear supernatant (mitochondria fraction) collected and frozen in aliquots pending biochemical analysis.Using a Teflon homogeniser, the organ samples used for biochemical assays were prepared by homogenising in 0.1 mol/L of phosphate buffer at pH 7.4. The homogenates were cold centrifuged , follicle-stimulating hormone (FSH), prolactin, and testosterone level using ELISA kits according to the protocols of the manufacturers. The data were acquired with a Microplate Reader Spectra Max 340 .. Activities of testicular function biomarkers\u2014acid and alkaline phosphatases (ACP and ALP)\u2014were assessed by the method as reported\u201325. Glucose-6-phosphate dehydrogenase (G6PD) was determined by Wolf et al method. Also, lactate dehydrogenase-X was assessed by Vassault method. Biomarkers of oxidative stress including the activity of superoxide dismutase (SOD) was evaluated by the method of Misra and Fridovich; total sulfhydryl (TSH) concentration was evaluated by Ellman process; catalase (CAT) activity was measured according to the protocols of Clairborne using H2O2 as a substrate as previously reported; glutathione-S-transferase (GST) and glutathione peroxidase (GPx) were estimated by the methods of Habig and Rotruck et al, respectively. Reduced GSH was determined following Jollow et al method, while xanthine oxidase (XO) was quantified by Bergmeyer et al method. Also, malondialdehyde (MDA), a biomarker of lipid peroxidation, was quantified by the method of Okhawa and Yagi, and reactive oxygen and nitrogen species (RONS) concentration was measured by the method of Owumi and Dim. Furthermore, biomarkers of inflammation, including nitric oxide (NO) and myeloperoxidase (MPO), were estimated by the protocols of Green et al and Granell et al, respectively. TNF-\u03b1 and a biomarker of genomic integrity (p53) were estimated by ELISA kits according to the manufacturer's protocol.The tissue homogenate of the rats after treatment was subjected to biochemical analysis, and total protein concentrations were determined by Bradford's method, and haematoxylin and eosin stained tissue slides prepared. The prepared slides were coded and examined under a Carl Zeiss microscope (Germany). On inspection, images were taken using a Zeiss Axiocam 512 camera by a pathologist unaware of the various treatment groups from which the slides were prepared.Microscopic assessment of the testes was performed following standard dehydration and paraffin embedding methodt-tests were used to analyse the data generated to determine the significance level of the mean body weight of rats before and after treatment. One-way analysis of variance (ANOVA) followed by Tukey's post hoc test was used to determine the significant difference across the different experimental cohorts . The results are expressed as means\u00b1SD of replicates, and statistically significant differences were set at a value of P<0.05.Paired Student's P>0.05) the mean percentage body weight change of rats . In addition, PbAc and furan treatments slightly decreased (P<0.05) the mean body weight of the animals compared to the control in the order: furan+PbAc3 < furan+PbAc2 < furan < furan+PbAc1 < PbAc < control, although this trend was not statistically significant within the exposure time of 28 days. Similarly, PbAc and furan treatment slightly altered the mean organ weight and relative organ weight of the hypothalamus, testis, and epididymis relative to the control (Table 1). The result further revealed that the water intake in the control group was not affected but was altered in groups treated with furan and PbAc . The results indicated that PbAc and furan slightly altered the mean body weight and organosomatic indices of the experimental rats post 28 days exposure.The effects of Pb and furan on the hypothalamic-pituitary-gonadal axis were investigated using male Wistar Albino rats. PbAc and furan were administered to rats for 28 consecutive days and the changes in mean body weight, organ weight, and water intake were examined. It was revealed that treatments with PbAc and furan slightly waned (P<0.05) percentages of sperm motility and viability, as well as sperm count compared to the control group. Conversely, sperm abnormality indices, including abnormality of the head (%), irregularity of the mid-piece (%), abnormality of the tail (%), and total abnormality (%) were significantly increased (P<0.05) in groups treated with furan and/or PbAc compared to the control (Table 2). In addition, the activities of ALP, LDH, and G6PD in cohorts of rats treated with furan and PbAc at 10 and 100 \u03bcg/kg BW were significantly waned (P<0.05) compared to furan alone group, while the activity of ACP in cohorts of rats treated with furan and PbAc at 1, 10 and 100 \u03bcg/kg BW were significantly reduced (P<0.05) compared to furan alone group .The effects of Pb and furan on reproductive function and integrity of rats were examined in this study. Both individual exposure to furan or PbAc and combined exposure to furan and PbAc led to significantly decreased (P<0.05) the levels of LH, FSH, and testosterone while significantly increasing (P<0.05) the level of prolactin (an indication of reproductive toxicity). Compared to furan alone group, the level of LH was significantly (P<0.05) lowered in group treated with furan and PbAc at 100 \u03bcg/kg BW; the level of FSH was significantly (P<0.05) reduced in groups treated with furan and PbAc at 1, 10, and 100 \u03bcg/kg BW, while the levels of testosterone and prolactin were significantly (P<0.05) altered in groups treated with furan and PbAc at 10 and 100 \u03bcg/kg BW . Taken together, the above results indicated that the administration of PbAc and furan synergistically altered the integrity and functionality of the reproductive system of male rats.Furthermore, alterations in the basal level of gonadotropin-releasing hormones such as LH and FSH, as well as other reproductive hormones such as testosterone and prolactin, were observed in this study. The results revealed that treatments with PbAc and furan, either individually or in combinations, significantly decreased (P<0.05) the levels of SOD, CAT, GPx, GST, GSH, and TSH in the hypothalamus, testis, and epididymis of male rats compared to the control group rats. This effect was further potentiated (P<0.05) in cohorts of rats co-treated with furan and Pb at multiple concentrations . Compared to furan alone group, the activities of SOD were significantly (P<0.05) decreased in groups treated with furan and PbAc at 10 \u03bcg/kg BW in the testis; furan and PbAc at 10 and 100 \u03bcg/kg BW in the epididymis, and furan and PbAc at 100 \u03bcg/kg BW in the hypothalamus. In addition, the activities of CAT were significantly (P<0.05) waned in groups treated with furan and PbAc at 1, 10, and 100 \u03bcg/kg BW in the testis; furan and PbAc at 10 and 100 \u03bcg/kg BW in the epididymis and hypothalamus compared to furan alone group . The activities of GPx in cohort of rats were significantly (P<0.05) lowered in groups treated with furan and PbAc at 10 and 100 \u03bcg/kg BW in the testis, epididymis and hypothalamus compared to group treated only with furan. The results further divulged that the activities of GST were also significantly (P<0.05) diminished in groups treated with furan and PbAc at 10, and 100 \u03bcg/kg BW in the testis, epididymis and hypothalamus compared to furan alone group . The levels of reduced-GSH were significantly (P<0.05) decreased in groups treated with furan and PbAc at 10 and 100 \u03bcg/kg BW in the testis and epididymis and PbAc at 1, 10, and 100 \u03bcg/kg BW in the hypothalamus compared to group treated only with furan. In addition, the results revealed that the activities of TSH were significantly (P<0.05) reduced in groups treated with furan and PbAc at 10, and 100 \u03bcg/kg BW in the testis and hypothalamus, and PbAc at 1, 10, and 100 \u03bcg/kg BW in the epididymis compared to furan alone group . In comparison to the control, we observed that treatment with Pb and furan significantly increased the tissue levels of XO, MPO, LPO, and RONS (P<0.05). The activities and levels of these oxidative mediators were further increased as the concentration of Pb was raised  . The results implied that PbAc and furan treatments impaired the redox system along the hypothalamic-pituitary-gonadal axis in male rats.The impacts of PbAc and furan treatment on the redox balance of the hypothalamic-pituitary-gonadal axis of male rats were evaluated. Exposure of Pb and/or furan significantly decreased (P<0.05) the levels of NO and TNF-\u03b1 in the hypothalamus, testis, and epididymis of rats compared to the control group . In addition, PbAc and furan markedly raised the tissue levels of p53 in the testis, epididymis, and hypothalamus, indicating the potential of cell cycle arrest, DNA repair and/or apoptosis . This indicates that treatments with PbAc and furan orchestrated inflammation and genomic instability in the hypothalamus, testis, and epididymis of rats.Excessive oxidative stress could induce inflammation and genomic instability if not minimized. In this study, we examined the effects of PbAc and furan on pro-inflammatory mediators and p53 levels in rats' hypothalamus, testis, and epididymis. The results showed that PbAc and furan mitigated tissue homeostasis by significantly increasing  are shown in . Specifically, the infiltration of cytokines in tissues may result in a cytokine storm, which in turn stimulates the hypothalamic-pituitary-adrenal axis to release adrenocorticotropic hormones (ACTH) and subsequently glucocorticoids. Glucocorticoids promote gluconeogenesis, amino acid mobilisation, and fat breakdown. These macromolecules are needed to maintain the size and weight of the examined organs, and their utilisation to abate Pb and furan stress may lead to tissue atrophy observed in our and others' studies .We investigated whether combined exposure to Pb and furan synergistically exacerbate oxidative stress, inflammation, and genomic stress in rat hypothalamic-pituitary-testicular system. The alteration in mean body weight and organ to body weight ratio indicates the onset of tissue dysfunction emanating from Pb and furan exposure may be associated with ROS and RNS accumulation. Increased RONS can trigger cell death if left unchecked. Co-exposure to Pb and furan suppressed LH, FSH, and testosterone production, while prolactin level increased. This inverse correlation may have been brought about by the action of ROS and pro-inflammatory mediators on the hypothalamic-pituitary-testicular system. Accumulation of ROS and pro-inflammatory cytokines induces ACTH expression that drives glucocorticoid production. Glucocorticoid negatively influences GnRHs and dysregulates the biochemical and physiological processes that drive spermatogenesis and steroidogenesis. The aftermaths of such negative regulation include increased sperm abnormality observed in this study corroborating previous report. A limitation of this study is a lack of data on GnRH and its antagonist gonadotropin-inhibitory hormone (GnIH), which plays an active role in reproductive function. GnRH regulates FSH and LH release from the anterior pituitary enhancing reproductive effectiveness. These essential sex regulatory/inhibitory hormones from the gonads are regulated via the hypothalamus-pituitary-gonadal (HPG) axis and experimental data can be insightful to reproductive function.The hypothalamic-pituitary-testicular system is tightly regulated, and the functions of the male reproductive system depend on the maintenance of the level of gonadotropin-releasing hormones (GnRH) including LH and FSH. LH is a glycoprotein that stimulates testicular Leydig cells to synthesise testosterone and control spermatogenesis. In addition, FSH rouses sperm production in the Sertoli cells. Prolactin is known to exert a short loop of negative feedback on LH and FSH in oxidative and inflammatory stress. Reduction in LH and FSH levels adversely reduces spermatogenesis and the activities of testicular enzymes, and the observed reduction in these enzyme activities indicate a defect in rat reproductive status. Also, LDH is widely expressed in the testis, and drives several processes necessary for male fertility and sperm function. For instance, LDH activity furnishes the Embden Meyerhof-Parnas pathway with NAD+ needed to sustain the processes of glycolysis and ATP production in the flagellum. In addition, G6PD in the hexose monophosphate shunt generates NADPH -a reducing equivalent- and the sugar moiety (ribose 5-phosphate) to maintain spermatogenesis and steroidogenesis. While ALP expressed in the Leydig cells and SSCs of the testis regulate several metabolic activities during spermatogenesis and testicular steroidogenesis. Decreases in these enzyme activities indicate testicular atrophy, degeneration of the seminiferous epithelium, and dysfunction in the hypothalamic-pituitary-testicular system. Therefore, we validate the hypothesis that reduced reproductive health of male rats is associated with the suppression of ACP, ALP, G6PD, and LDH activities.Furthermore, the integrity of these cells to drive spermatogenesis depends testicular ACP, ALP, G6PD and LDH activities. ACP provides phosphate during the development, and maturation of testicular Sertoli and Leydig cells.We observed that animals co-exposed to Pb and furan exhibited a significant reduction in the GSH and TSH levels and the activities of antioxidant enzymes, such as CAT, SOD, GST, and GPx. This observation implies inadequate antioxidants necessary to scavenge harmful free radicals and prevent the induction of oxidative stress in the examined tissues. The increased levels of LPO and RONS and the activities of XO and MPO in the examined tissues of rats co-exposed to Pb and furan occasioned RONS production that may overwhelm the antioxidant capability. Consequently, the exposure led to oxidative damage in the testes, epididymis, and hypothalamus. ROS mediates toxicity by inducing lipid peroxidation, formation of protein crosslinks, and oxidative DNA damage. These metabolic processes invariably inhibit essential processes involved in spermatogenesis and steroidogenesis, reducing sperm quality and male fertility. Also, increased TNF-\u03b1 levels in the hypothalamus, testis and epididymis of rats are indicative of tissue injury. TNF-\u03b1 in the company of other pro-inflammatory cytokines such as IL-1\u03b2, IL-6, and IL-8 stimulate hypertrophy of the Sertoli and Leydig cells as well as the infiltration of pro-inflammatory cells into the spermatocytes and spermatids. In addition, the accumulation of ROS and pro-inflammatory mediators may trigger loss of genomic integrity by oxidative DNA damage. Excessive inflammation and oxidative stress are associated with activating p53 to regulate important cellular and molecular decisions, including cell cycle arrest, DNA repair, and apoptosis. We observed that Pb and furan treatment triggered the activation of rat p53 in the hypothalamus, testis, and epididymis that may have occurred to avert defective DNA replication into the spermatogonial nuclei during spermatogenesis. Histopathology findings support the altered morphology, tissue histoarchitectural defect and functionality caused by Pb and furan treatment. Treatment with Pb and furan increasingly exacerbated tissue damage in the co-treated groups stemming from increased Pb concentration in the drinking water. In addition, Pb exposure is well established to be toxic via several mechanisms on reproductive dysfunction.Overproduction of NO induces nitrosative stress, which reportedly modifies cellular proteins, lipids, and nucleic acids after exhausting antioxidant defence systemsvia the induction of oxidative stress, inflammation, and loss of genomic stability. The probable mechanisms of toxicity of Pb and furan include altering signalling networks including extracellular signal-regulated kinase, nuclear factor kappa B and the repression of signalling networks including nuclear factor erythroid 2-related factor-2 as depicted in our proposed toxicity mechanism . More stringent measures should be taken to regulate indiscriminate anthropogenic activities and monitor food processing methods that are known to exacerbate Pb and furan toxicity. Moreover, such inadvertent exposure also have damaging effect on neurobehavioral-coordination as outlined in our latest findings.Taken together, our study shows that co-exposure to Pb and furan altered the integrity of the hypothalamic-pituitary-testicular system"}