Non-scarring hair loss is a common problem that affects both male and female patients. Since any disturbances in the hair follicle cycle may lead to hair shedding, or alopecia, it is not surprising that the possible role of vitamin D in alopecia was investigated in many studies. Vitamin D has been shown to have many important functions. A growing body of evidence shows that vitamin D and its receptor are responsible for maintaining not only calcium homeostasis but also skin homeostasis. Moreover, vitamin D could also regulate cutaneous innate and adaptive immunity. This paper presents a review of current literature considering the role of vitamin D in alopecia areata, telogen effluvium, and female pattern hair loss. The majority of studies revealed decreased serum 25-hydroxyvitamin D levels in patients with different types of non-scarring alopecia, which could suggest its potential role in the pathogenesis of hair loss. According to the authors, vitamin D supplementation could be a therapeutic option for patients with alopecia areata, female pattern hair loss, or telogen effluvium. However, further studies on a larger group of patients are required. Moreover, they observed that 1,25(OH)2D alone or in conjunction with lipopolysaccharide (LPS) induced cathelicidin antimicrobial peptide expression and release of antimicrobial activity in neutrophils. It has been proposed that induction of antimicrobial peptide expression by 1,25(OH)2D could be involved in the suppressive effects of ultraviolet B (UVB) radiation on innate immunity and suggested the potential of its analogues in treatment of opportunistic infections [2D has been found to suppress antigen presentation by dendritic cells, [2D has also been shown to suppress the T helper type 17 (Th17) related cytokine expression [2D also induces apoptosis in activated B cells, suppresses B-cell proliferation, and decreases plasma cell and class-switched memory B-cell generation [Vitamin D plays an important role in human health. Its main source is photosynthesis in the skin, whereas lower amounts are derived from nutrition and diet supplements . The rolfections . Furtherc cells,  and to mc cells, . 1,25 is formed in the kidneys [Skin is a target organ for vitamin D not only because this compound is mostly synthesized in the epidermal keratinocytes and dermal fibroblasts from 7-dehydrocholesterol upon exposure to UVB radiation, but also because skin is able to respond to the active metabolites of vitamin D . As ment kidneys .It was demonstrated that keratinocytes are capable of metabolizing vitamin D to the active form because they possess the activity of 1-α-hydroxylase and 25-hydroxylase . Other cVDR gene and Hairless gene (Hr) may be involved in the same genetic pathway that regulates postnatal hair cycling [Hr expression [Vitamin D exerts its action through the vitamin D receptor (VDR) ,19,20,21 cycling ,25,27. Mpression .2D in the regulation of postnatal hair follicle cycling [9k knockout pups born of calbidin-D9k knockout females fed with a low-calcium and vitamin D-deficient diet developed transient alopecia during the first postnatal catagen. However, alopecia was not present when these pups were suckled by mothers fed with a diet containing calcium and vitamin D [Since the role of VDR in the hair follicle cycle was confirmed, the potential role of vitamin D was the focus of many studies. It has been demonstrated that VDR knockout mice have increased levels of the active form of vitamin D. However, the possible toxic effect of 1,25-dihydroxyvitamin D on hair follicles was not proven . Recentl cycling . They foitamin D .Considering that any disturbances in the hair follicle cycle may lead to hair loss, it is not surprising that the possible role of vitamin D in alopecia was investigated in many studies. The aim of this paper is to review the current literature considering the role of vitamin D in non-scarring alopecia.Alopecia areata (AA) is a common form of hair loss, characterized by sharply demarcated, round to oval, skin-coloured patches of non-scarring alopecia ,31. StudLiterature data suggest that vitamin D, due to its immunomodulatory effect, may be involved in the pathogenesis of AA ,16,33.Decreased serum 25(OH)D levels in AA in comparison to healthy subjects were reported in many studies ,35,36,37Mahamid et al. suggested that serum 25(OH)D levels < 30 ng/mL were associated with AA occurrence . D’Ovidi2D or 25(OH)D concentrations in 42 patients with AA and the extent of hair loss, number of patches, disease duration, and nail involvement [Interestingly, data concerning the correlation between serum 25(OH)D levels and clinical disease parameters are inconsistent. Yilmaz et al. did not find any correlation between 1,25(OH)olvement . This waolvement . In contolvement . This coolvement ,35,37. Iolvement . Howeverolvement ,35,36,37olvement ,33. In aolvement ,36, studolvement ,37 and solvement . This caolvement ,33,34,36Since the majority of studies were conducted on adult patients, the study by Unal et al., is of special interest. The authors demonstrated vitamin D deficiency in paediatric patients with AA and in the control group. However, they reported significant inverse correlation between serum 25(OH)D levels and disease severity, duration, as well as the number of bald patches . The autThe potential role of VDR in the pathogenesis of AA was also assessed. Fawzi et al. revealed significantly lower levels of tissue and serum VDR in AA than in a control group. An important negative correlation was observed between the extent of AA and tissue VDR . The resDespite the fact that many studies reported deficiency or insufficiency of vitamin D in patients with AA, few reports evaluate the topical application of vitamin D analogues as a therapeutic option. Therefore, data reported by Kim et al., appear interesting . The autLater, the effectiveness of topical application of 0.005% calcipotriol was evaluated in 22 adult patients with AA and scalp involvement lower than 40%. After three months of therapy, 59.1% of patients had hair regrowth; no response was observed in 36.4% of patients and worsening in 0.04%. A better clinical effect was observed in patients with a single bald patch. Among all patients, 91% had vitamin D deficiency. Interestingly, faster hair regrowth was observed in patients with lower serum 25(OH)D levels . SimilarTo sum up, the observed decreased serum 25(OH)D levels in AA patients suggest its role in the disease pathogenesis. It was demonstrated that vitamin D shifts the Th1 immune response toward Th2. Since AA is mainly Th1-mediated, the vitamin D deficiency may be involved in the development of hair loss ,35. The Female pattern hair loss (FPHL) is one of the most common types of alopecia in women. Clinically, it is characterized by diffuse hair shedding with maintained frontal hairline . Recent Moneib et al. reported a significantly lower serum 25(OH)D levels in patients with FPHL than in controls . The majIt has also been pointed out that women with positive family history of FPHL and vitamin D deficiency or insufficiency are more prone to develop FPHL in comparison with women with sufficient serum 25(OH)D levels . Contrar2D, which results in enhancement of VDR gene expression [Although a decreased serum 25(OH)D concentration was demonstrated in FPHL, the role of VDR in FPHL was assessed in a single study. Fawzi et al.  reportedpression .At present, there are no studies evaluating the oral supplementation of vitamin D or its topical application in patients with FPHL.The results described above suggest a possible involvement of vitamin D in pathogenesis of FPHL. However, the studies were limited by the small number of enrolled patients and different inclusion criteria. To elucidate the role of vitamin D in FPHL, further large-scale studies are required .Telogen effluvium (TE) is a common clinical problem defined as a non-scarring, diffuse hair loss that usually occurs 3 months after exposure to a triggering factor and is usually self-limiting. Causative factors considered in the pathogenesis include: stress, febrile states, drugs, endocrine abnormalities, and nutritional disturbances .In a study conducted by Rasheed et al., the authors compared serum 25(OH)D levels in female patients with chronic TE, FPHL, and healthy controls. They revealed significantly lower serum 25(OH)D levels in comparison to the control group. The lowest level was observed in patients with the most severe hair loss. According to the authors, the results may indicate that vitamin D is involved in TE . SimilarDue to the limited number of studies and different methodologies it is difficult to compare the results. However, since nutritional disturbances are one of the triggering factors causing hair loss, the possible role of vitamin D or its deficiency should be studied further.The studies revealed important alterations in serum 25(OH)D levels in patients with different types of non-scarring alopecia. In the majority of reports, the mean serum levels of 25(OH)D were decreased when compared to the healthy control, which might suggest a causal role of this decrease in the pathogenesis of hair loss. However it cannot be excluded that observed low serum 25(OH)D levels are secondary to the disease. Emotional stress related to severe hair loss might discourage patients form going outdoors as much as those without this condition. Therefore their potential for vitamin D synthesis might be reduced. Despite different mechanisms of AA, FPHL, and TE, it seems a reasonable therapeutic strategy to monitor serum 25(OH)D levels and introduce vitamin D supplementation in case of its deficiency or insufficiency in patients with AA, TE, and FPHL. It is worth noting that due to different methods used to evaluate the serum concentration of 25(OH)D, caution should be warranted when comparing the results. Other limitations of the studies include unequal study groups, different inclusion criteria, and the possible influence of seasonal variability of serum 25(OH)D concentration, since the samples were collected during different seasons. Therefore, there is an urgent need to standardize the laboratory measurement of vitamin D status that would be accurate, comparable over time, localization, or laboratory ,57. Furt


The number of people with type 2 diabetes mellitus (T2DM) has been increasing worldwide due to aging, urbanization, dietary, and lifestyle changes. It was estimated that there were 415 million people with diabetes aged 20–79 years in 2015, and the number was predicted to rise to 642 million by 2040 [β, RAGE, and Nox1 could attenuate the development of atherosclerosis in diabetic mice [In recent years, there are several advances in this field. Diabetes patients usually have hyperglycemia, hyperlipidemia, and insulin resistance; all of them are risk factors for macrovascular diseases. PKCs, RAGE, and ROS may mediate the effects of hyperglycemia and hyperlipidemia on cardiovascular systems. Knockout of PKCtic mice , 3. To mtic mice , and CVD2S) contributed to high glucose- (HG-) induced sFlt-1 production via activating ADAM17 in adipocytes. Exogenous H2S donor NaHS has a potential therapeutic value for HG-induced sFlt-1 production. S. Ye et al. found that the traditional Chinese medicine Didang Decoction intervention could upregulate the expression of cAMP-1 and PKA and downregulate the expression level of MLCK and PKC, thus ameliorating vascular endothelium injury in high-fat diet followed by the small dose of STZ-induced diabetic animal model.In this issue, T. Hu et al. found that decreased expression of CSE and 3-MST and the subsequently decreased production of hydrogen sulfide  and 6.9  in those with lower extremity PAD, respectively.Lower extremity peripheral arterial disease (PAD) is a common type of PAD in patients with T2DM, which can be noninvasively and objectively diagnosed by using the ankle-brachial index (ABI), and is also a marker of arterial atherosclerosis at other sites . J. Ma eCoronary revascularization is a safe and effective therapy for patients with single and multivessel coronary artery disease. Patients with diabetes mellitus contribute to about 25% of patients undergoing coronary revascularization . CoronarIn conclusion, there is ongoing progress in the pathophysiology, glycemia control, and risk factors of diabetic macrovascular complications shown in this special issue. Several other fields are also showing progress, such as weight loss surgery, but these are not discussed in the present issue. We believe that the present issue could bring some latest progress in this field and will be of interest to the readers.


Several studies have found that, despite a decrease in the overall amplitude of the late positive potential (LPP) with repeated presentation of the same picture, emotional stimuli continue to elicit a larger LPP than neutral ones. These findings seem to support the hypothesis that the affective modulation of the LPP reflects a mandatory process and does not rely on stimulus novelty. However, in these studies participants were asked to merely look at the pictures, without carrying out any additional task (free-viewing), making picture emotionality the most salient aspect of the stimulus, despite its repetition. The current study aimed to examine the impact of an explicit categorization task on the emotional processing of repeated pictures. To this purpose, ERPs to novel and repeated pictures were measured during free-viewing as well as during an explicit categorization task, where the emotional content of the pictures was task-irrelevant. The within-subject comparison between the free-viewing and task context revealed that the overall LPP habituated more rapidly in the free-viewing condition, but, more importantly, the LPP affective modulation was unaffected by task requirements during both novel and repeated presentations. These results suggest that the affective modulation of the LPP reflects an automatic engagement of cortico-limbic motivational systems, which continues to take place regardless of stimulus novelty and task context. Such stimuli include emotional stimuli  and novel stimuli.Our perceptual system serves the adaptive function of identifying features in the sensory environment that might be of potential relevance to behavior. While most scenes, people, or objects encountered everyday can be ignored, some of them elicit patterns of brain responses because of their intrinsic Emotional processing has been widely investigated by presenting affective pictures that are effective cues in evoking a broad range of emotional reactions, varying in intensity, and involving both pleasant and unpleasant affect . One of Previous studies found that during explicit categorization tasks , in whicSimilarly, when attention was manipulated by stimulus repetition, both pleasant and unpleasant pictures continued to elicit larger LPPs than neutral pictures even after massive repetition of the same stimulus exemplars , 2016. OIn the present study, we aimed to examine the impact of an explicit categorization task on the emotional processing of repeated pictures. To this purpose, picture repetitions  occurred either in a free-viewing context or during a categorization task (within-subject design), with pictures depicting any means of transportation serving as targets, and pictures depicting contents other than means of transportation (including pleasant and unpleasant cues) serving as non-target stimuli.When repetition occurred in a free-viewing context, emotional cues were the most salient aspect of the stimulus to process. Thus, in the present study we may expect a more evident impact of repetition on the affective modulation of ERPs when participants were engaged in a categorization task in which attention was not specifically directed toward affective information, compared to when repetitions occurred in a free-viewing condition. On the other hand, an opposite scenario could be predicted based on early oddball studies , which sParticipants were 33 students  from the University of Bologna who volunteered to take part in the study. All participants had normal or corrected to normal vision, and none of them reported current or past neurological or psychopathological problems. They had no previous experience with the materials used in this experiment. The experimental protocol conformed to the Declaration of Helsinki and was approved by the Bioethics committee of the University of Bologna. Due to technical problems, one female participant was excluded from behavioral analyses, and three participants  were excluded from the EEG analysis.1 were, respectively (numbers in parentheses are standard deviations), 6.7 (0.49) and 6.0 (0.71) for pleasant pictures, 5.2 (0.63) and 3.5 (0.41) for neutral pictures, and 2.2 (0.77) and 6.1 (0.80) for unpleasant pictures  = 742.1, ps < 0.0001, ps < 0.0001). A second ANOVA on the arousal scores yielded a significant main effect of arousal, F = 169.2, ps < 0.0001, ps < 0.0001), with no difference between pleasant and unpleasant pictures (p = 0.21). In addition, 280 pictures depicting a variety of means of transportation  were selected from public-domain images available on the Internet. All pictures were equated in brightness and contrast , using the gamma parameter of the Weibull fit  was identical between the two sessions. In each session, pictures were arranged in seven blocks of 80 trials each. In each block, 20 pictures belonged to the category of means of transportation, whereas the remaining 60 trials were equally representative of pictures depicting people in pleasant, neutral, and unpleasant contexts. A series of five blocks were presented that constituted the habituation phase (B1 to B5), during which 2 pleasant, 2 unpleasant and 2 neutral pictures were repeated 10 times in each block . Two blocks of only novel pictures were presented , respectively, before and after the habituation phase. Pictures of means of transportation were always novel pictures throughout the study, as the same exemplar was never repeated.For each participant, two different sets of pictures were selected to be presented in two different experimental sessions, that took place one after the other, within the same day. The experimental structure  or not. In this session , pictures of people served as non-target stimuli. In the other session, participants were only required to simply look at each picture without an explicit response task , such that there was not an instructed discrimination among picture categories.The specific set of pictures serving as novel or repeated varied across participants, such that, across subjects, all pictures were used in all conditions. The order of picture presentation was pseudo-randomized with the restriction that no more than two times consecutively the same picture or a picture of the same valence occurred.After completing the informed consent form, participants were seated in a chair in a sound-attenuated, dimly lit room, and the EEG sensor net was attached. In the categorization task, participants were instructed to respond whether the presented image contained the target category (any means of transportation) or not, by pressing one of two buttons on the keyboard (“z” or “m”) as fast and accurately as possible. For half of the participants, the letter-button mapping was reversed. In the free-viewing context, participants were instructed that a series of pictures would be presented and that each picture should be viewed the entire time it was on the screen. Half of the participants performed the categorization task at first followed by the free-viewing condition; the order was reversed for the other half. Between the two sessions there was a 15 min-break. Participants sat in front of a computer monitor with their head supported by a chinrest. The distance between the eyes and the monitor was 100 cm for all subjects. Stimuli were presented on a 21″ CRT monitor at 800 × 600 pixel-resolution and 85 Hz refresh rate, subtending 22.6° horizontal × 17.1° vertical degrees of visual angle. E-Prime software  synchronEEG was recorded at a sampling rate of 512 Hz from 256 active sites using an ActiveTwo Biosemi system. An additional sensor was placed below participant’s left eye, to allow for detection of blinks and eye movements. The EEG was referenced to an additional active electrode located near Cz during recording. Off-line analysis was performed using Emegs . EEG dat2 p), indicating the proportion between the variance explained by one experimental factor and the total variance, has been calculated and is reported. During the categorization task, analysis of reaction times (RTs) was performed only on trials associated with correct responses. Responses were scored as correct if the appropriate response latencies were not shorter or longer than two standard deviations from the individual mean. Mean RTs and Accuracy scores (%) were analyzed in a two-way ANOVA with Block and Picture Content as within-subject factors.The LPP amplitude during picture viewing was submitted to a repeated-measures analysis of variance (ANOVA) with Picture Content , Block , Task , as within-subject factors. Greenhouse-Geisser corrections were applied where relevant. The partial eta squared statistic  elicited when viewing novel and repeated (average of 50 repetitions of the same picture exemplar) pictures in free-viewing (upper panel) and during a categorization task (lower panel). A striking difference between the free-viewing and the task condition was in terms of the overall ERP amplitude  which was much more emphasized during the task context, perhaps reflecting the recruitment of different brain regions, or an amplitude change in a subset of those generators between the two experimental conditions  = 66, p < 0.0001, F = 10, p < 0.0001, F = 40, p < 0.0001, F = 13.6, F = 5, p < 0.0001, F = 8.6, p < 0.0001, Main effects of Content, F = 48, p < 0.0001, Fs > 17, ps < 0.0001, F > 63, ps < 0.0001, F < 1). When instead means of transportation served as targets, the LPP amplitude was significantly larger compared to non-target neutral people, F = 29, p < 0.0001, p > 0.05). The affective modulation of the LPP, with larger positive amplitude during viewing of both pleasant and unpleasant, compared to neutral, pictures, was not affected by the viewing context  > 155, p < 0.0001, F = 77, p < 0.0001, Fs = 135, p < 0.0001, Fs = 69, p < 0.0001, F = 9, p < 0.01, The interaction Content × Task was mainly due to the effect of task relevance on the LPP prompted by pictures of means of transportation. Compared to the free-viewing condition, the task prompted a centro-parietal positivity enhancement, which was larger for pictures depicting means of transportation, Figure 3). The difference in the LPP amplitude between emotional and neutral pictures significantly decreased with picture repetition , but remained stable during the viewing of neutral pictures. In the novel phase that followed blocks of repeated pictures, the LPP was equivalent in magnitude to that elicited in the first novel phase, with emotional pictures again prompting a larger LPP than neutral pictures, quadratic trend F = 74, p < 0.0001, The Content × Block interaction showed the habituation of the LPP affective modulation over blocks of repeated pictures , as the linear decrease in the LPP amplitude across blocks of repeated pictures (B1 to B5) was less pronounced when participants were engaged in the categorization task, compared to the free-viewing condition . The emotional content of the pictures was effective in modulating RTs only during the viewing of novel pictures (both N1 and N2), with slower RTs for unpleasant, compared to neutral and pleasant pictures, Fs > 27, p < 0.0001, F < 1). The overall accuracy during the categorization task was high (average 98%), and no main effect or interaction was significant.The free-viewing of emotional pictures is typically associated with a larger positive potential (LPP) measured over centro-parietal sites, compared to when pictures depict neutral contents, and this cortical modulatory pattern is only moderately affected by stimulus repetition, even with a high number of repetitions of the same stimulus exemplars . In the A within-subject comparison between free-viewing and categorization task conditions revealed stimulus-specific modulatory effects as a function of task relevance, that were particularly evident in the LPP component over the centro-parietal scalp region. Pictures depicting means of transportation elicited larger LPPs when they were target stimuli, compared to when they were simple neutral stimuli in the free-viewing condition, where they prompted similar LPP amplitudes to those measured for other neutral contents . Moreover, consistent with previous studies , the LPPThe impact of the task was also evident on the rate of habituation of the overall centro-parietal positivity, which was somewhat milder compared to that of the free-viewing condition. This overall pattern of results is consistent with several oddball studies that investigated the P3 component during active and passive (or ignore) conditions . AccordiOn the other hand, while the task affected the overall centro-parietal LPP and its reduction across repetitions, the within-subject comparison between the free-viewing and task context revealed that the LPP affective modulation, and its slight habituation, were similar across the two conditions. The novel phases of the present study, in which only new pictures were presented, also allowed us to evaluate whether a free-viewing context weakened the impact of emotional pictures, as measured by ERPs, compared to when participants directed attention to picture content in order to perform a decision task. Some previous studies have compared the impact of different types of task contexts on the LPP affective modulation , mostly Previous studies on stimulus repetition found that while participants were asked to simply look at the pictures (free-viewing context) the affective modulation of the LPP was only slightly affected by picture repetition, and persisted even after massive repetition , 2017b. Behavioral data also provided the opportunity to investigate repetition effects on behavioral interference triggered by emotional stimuli. As expected, when pictures were novel, RTs were slower during the viewing of unpleasant, compared to neutral, pictures, showing an attentional capture effect . More reIn the present study, pleasant and unpleasant pictures elicited a larger LPP compared to neutral stimuli consistent with several previous studies . In addiIn summary, these findings indicate that evaluative processes strongly engage the motivational systems during novelty processing, as reflected by the affective modulation of the LPP, and that this motivational engagement is only slightly affected by picture repetition. Critically, repetition effects on the LPP affective modulation did not vary as a function of the task, indicating that the evaluative process, and therefore, the engagement of motivational systems, is mandatory. On the other hand, the overall LPP amplitude seems to reflect attention allocation, as it is not only enhanced for novel and target stimuli, but also shows a slower habituation pattern during the categorization task compared to the free-viewing condition, similar to the P3 during active and passive oddball paradigms. The dissociation between the overall LPP amplitude and its modulation as a function of stimulus emotion was also evident in other previous studies. For example, directing attention toward a specific picture category, and thus making it a target, increased the overall amplitude of the LPP, leaving its affective modulation nearly unaffected by target status . SimilarTaken together, these findings are consistent in showing that the enhanced LPP for emotional compared to neutral pictures reflects automatic engagement of motivational systems, which occurs regardless of stimulus novelty and task context . On the MC and VF designed the research; VF and SM performed the research; VF, SM, and MC analyzed the data; VF, SM, and MC wrote the paper.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.


Age-related macular degeneration (AMD) is referred to as the leading cause of irreversible visual loss in developed countries, with a profound effect on the quality of life. The neovascular form of AMD is characterized by the formation of subretinal choroidal neovascularization, leading to sudden and severe visual loss. Research has identified the vascular endothelial growth factor (VEGF) as an important pathophysiological component in neovascular AMD and its intraocular inhibition as one of the most efficient therapies in medicine. The introduction of anti-VEGF as a standard treatment in wet AMD has led to a great improvement in the prognosis of patients, allowing recovery and maintenance of visual function in the vast majority of cases. However, the therapeutic benefit is accompanied by a difficulty in maintaining the treatment schedule due to the increase in the amount of patients, stress of monthly assessments, as well as the associated economic burden. Therefore, treatment strategies have evolved from fixed monthly dosing, to individualized regimens, aiming for comparable results, with fewer injections. One such protocol is called “pro re nata”, or “treat and observe”. Patients are given a loading dose of 3 monthly injections, followed by an as-needed decision to treat, based on the worsening of visual acuity, clinical evidence of the disease activity on fundoscopy, or OCT evidence of retinal thickening in the presence of intra or subretinal fluid. A different regimen is called “treat and extend”, in which the interval between injections is gradually increased, once the disease stabilization is achieved. This paper aims to review the currently available anti- VEGF agents – bevacizumab, ranibizumab, aflibercept, and the aforementioned treatment strategies. Although 80 per cent of the patients have non-neovascular, or atrophic AMD, the neovascular form of the disease is responsible for nearly 90 per cent of the severe, central visual acuity loss associated with AMD [1]. The advances in the medical research have identified the Vascular Endothelial Growth Factor (VEGF) as a key pathophysiological factor in the development of neovascular AMD, with an essential role in angiogenesis, vascular permeability, and inflammatory response [4]. Furthermore, the innovations in the diagnostic techniques, such as Spectral Domain Optical Coherence Tomography (SD-OCT) allow high quality visualization of disease morphology, correct diagnosis, and efficient follow-up [11]. The introduction of anti VEGF intravitreal injections has opened a new therapeutic window in the management of wet AMD, thus efficiently blocking the pathophysiological process of AMD, with a restoration of retinal morphology and the maintenance of its function. Injections are considered safe, well tolerated, with few adverse reactions [1]. In the past years, anti VEGF injections have become the standard treatment for wet AMD, accounting for better results than the previous choices, such as photodynamic therapy (PDT) and laser photocoagulation. Currently, three drugs – bevacizumab, ranibizumab, and aflibercept work well, so as to achieve a rapid resolution of exudative signs in most patients [10].Age related macular degeneration (AMD) is referred to as the leading cause of severe, irreversible blindness in developed countries worldwide, with a profound effect on the quality of life of affected individuals, as well as on the health care systems, due to the increase of life expectancy, number of reported cases and expensive treatments [4].However, the first option was pegaptanib sodium, a selective VEGF isoform 165 inhibitor, approved by the FDA in 2004 for the treatment of neovascular AMD. Although the VISION study proved its therapeutic benefit, which was better than PDT, visual acuity remained low and it was soon exceeded by the next anti VEGF, ranibizumab. Therefore, pegaptanib is no longer recommended in the treatment of wet AMD [6].Bevacizumab is a full-length recombinant monoclonal antibody, which binds all isoforms of VEGF, and was approved by the FDA in 2004 for the intravenous treatment of metastatic colorectal cancer. The SANA study showed promising results after two or three bevacizumab intravenous doses, with a mean gain of 14 ETDRS letters at 24 weeks. The first case of intravitreal bevacizumab was reported one year later, with good results after just one month and no adverse effects. It soon became widely used in the treatment of wet AMD, due to its good results, safe profile and reduced cost, but in an OFF LABEL manner [11].Ranibizumab is a monoclonal antibody fragment, with a hundred times higher affinity than bevacizumab, for all VEGF isoforms, approved by the FDA in 2006, for the monthly intravitreal treatment of wet AMD. The MARINA study compared it to sham injections, with positive results: patients gained a mean 6.6 ETDRS letters after 2 years, compared to a mean loss of 14.9 ETDRS letters in the sham group. The ANCHOR study compared intravitreal ranibizumab to PDT. At one year, the mean gain in the first group was 11.3 ETDRS letters, while the PDT group lost a mean 9.5 letters. Given the similarities between bevacizumab and ranibizumab, in the matter of structure, results and side effects, and the significant price difference (50 $ vs. 2000 $ per injection), the CATT study was initiated, aiming to compare the two substances. There was no significant difference both at one and two years, between monthly bevacizumab and ranibizumab  or as needed – which will be discussed later – bevacizumab and ranibizumab. Also, the GEFAL and IVAN studies found similar results at one and two years respectively. The CATT and IVAN studies showed no statistical significant differences in the local side effects, such as endophthalmitis, uveitis, retinal detachment, vitreous hemorrhage, traumatic cataract, and systemic side effects, such as atherothrombotic events between the two drugs. However, there have been more frequent gastrointestinal hemorrhages and infections among patients treated with bevacizumab [11].Aflibercept, the newest treatment option, is a fusion protein, with a high affinity for VEGF-A, VEGF-B and PlGF , approved by the FDA in 2012, for the treatment of wet AMD, in a bimonthly regimen, after a loading phase of three monthly doses. The VIEW 1 and 2 studies proved that bimonthly 2 mg aflibercept is non-inferior to monthly 0.5 mg ranibizumab, with similar ocular and systemic adverse events, and a slightly lower cost. Aflibercept has also shown positive results in non-responders, previously treated with bevacizumab or ranibizumab [5].Taking into consideration that ranibizumab is approved and recommended for monthly intravitreal injections for long periods of time, its price tag is high, and the number of patients is constantly increasing, the burden on both the health care systems and the patients is extremely high. Therefore, we began asking ourselves whether similar results could be obtained, with fewer injections needed for each patient. Thus, we could lower the costs, the risks, the natural evolution towards geographical atrophy, and avoid over-treating patients [8].The first option that was taken into account was the bimonthly treatment. However, the PIER and EXCITE studies provided modest results. In the PIER study, patients underwent a loading phase of 3 monthly ranibizumab injections, followed by a quarterly retreatment, compared to sham injections. At 24 months, patients experienced a mean loss of 2.2 ETDRS letters from baseline and a significant decrease in the best-corrected visual acuity (BCVA) achieved during the loading phase. The EXCITE study compared monthly to quarterly ranibizumab, after a loading phase. The difference between the two groups at month 12 was 4.5 ETDRS letters, with morphological differences noticeable on OCT. Therefore, the quarterly treatment was abandoned due to its poor results, both anatomic and functional [7]. The PRN regimen was first investigated in the small PrONTO study. During the two years of follow-up, the 37 patients were assessed on a monthly basis, and received intravitreal 0.5 mg ranibizumab injections whenever a mean visual acuity loss of 5 ETDRS letters or a 100-micron increase in the central retinal thickness were noted. The visual results were very good, comparable with those of the pivotal studies, ANCHOR and MARINA: the mean gain was + 11 ETDRS letters, compared to baseline, achieved with far fewer injections: 9.9 over two years [10]. The SUSTAIN study examined the efficacy and safety of ranibizumab administered to 513 patients on a PRN basis, following three monthly loading doses. Retreatment criteria were the same as in the PrONTO study; BCVA increased during the 12 months of follow-up, with a mean gain of 3.6 letters from baseline, and a mean 5.6 injections administered. However, most of the visual increase was achieved during the loading phase, followed by a slight decrease afterwards [8]. The results of the larger SAILOR study (n = 2378) were not as favorable, with a slight decline in the visual acuity over time, due to the quarterly visit protocol. Therefore, patients were probably undertreated [10]. HORIZON is a two-year extension study, following patients who had completed the ANCHOR, MARINA, or FOCUS trials, in order to evaluate the long term safety and efficacy of intravitreal 0.5 mg ranibizumab as needed. The results showed a decline in BCVA gains achieved with a previous monthly treatment, thus highlighting the strict need for a continuous follow-up and rigorous retreatment criteria. The best results achieved on a PRN regimen were obtained in the HARBOR study, which is the only one to include the SD- OCT monitoring. Participants (n = 1098) received 0.5 or 2 mg ranibizumab, monthly vs. as needed, after three monthly loading doses. At 12 months, results showed no significant difference between the 0.5 and 2 mg groups, while at 24 months, the mean change in BCVA from baseline was + 9.1 letters in the monthly 0.5 mg injection group, compared to + 7.9 letters in the 0.5 mg PRN group, which received a mean 13.3 treatments during the two years period. Therefore, this study confirmed that the monthly 0.5 mg ranibizumab provided optimum results, while there was no significant disadvantage in using a PRN protocol, provided that patients were strictly monitored, using SD-OCT technology [11]. Back to the CATT study, data specific for year two showed a mean difference between the monthly and PRN ranibizumab groups of only 2.4 letters and 29 microns in the central retinal thickness, with a mean 22-23 monthly injections, compared to a significantly lower 12-14 in the PRN group. Also, more geographic atrophy cases were reported in the monthly treatment group [8].Another less frequent treatment regimen is the PRO RE NATA (PRN), or Treat and Observe. It consists of a three monthly injection loading phase, followed by a monthly follow-up and retreatment as needed. The retreatment criteria include visual acuity loss without other reasons, hemorrhage, or edema upon fundus examination, leakage on fluorescein angiography, or increased central retinal thickness, due to intra or subretinal fluid, on OCT examination [9]. The advantages of this regimen are decreased costs, fewer visits and therefore reduced burdens for both patients and physicians. Also, given the fact that therapeutic VEGF suppression varies among patients, between 26 and 69 days, with a maximum at 36–38 days, the TAE protocol manages to blend with each patient’s response pattern. The TAE protocol also seeks to reduce macular hemorrhages, which are sometimes reported in PRN treated patients, sometimes untreated for longer periods of time [8]. Spaide and Freund were the first to describe a TAE regimen with good results. A few patients were followed for three years, averaging 7 ranibizumab treatments yearly and a mean visual improvement of 4 lines. Oubraham et al. reported better results with the TAE regimen, compared to PRN: +10.8 vs. +2.3 ETDRS letters at 12 months, achieved with 7.8 and 5.2 injections, respectively. Gupta et al. compared bevacizumab to ranibizumab on a TAE regimen, with similar results at 18 months. The LUCAS study also compared bevacizumab to ranibizumab on a TAE basis, with no loading phase. The results at one year were found to be equivalent, with a mean gain of 8.0 and 8.2 letters respectively, and a similar number of visits. Freund et al. found that the mean interval between retreatments settled at around 10 weeks [6]. The largest recent study documented was the 3 year treatment outcomes for 196 patients with neovascular AMD, treated with bevacizumab or ranibizumab on a TAE regimen. On average, the patients received 17 injections over the 3-year period; participants gained a mean 13.6 ETDRS letters, the central retinal thickness decreased by about 75 microns, irrespective of the drug used, as measured on SD-OCT, and the mean interval between visits was 13 weeks. Also, the visits were 50% less than in PRN studies [9]. Another recent study compared 0.5 mg ranibizumab administered to 60 patients in a TAE vs. TAO regimen, during a three year period. There were, however, no significant differences among the visual activity outcomes, central retinal thickness (as measured by SD-OCT) and the number of injections between the two treatment strategies [2].Finally, the Treat and Extend (TAE) protocol is the most recent less frequent regimen, gaining more and more popularity; about 78% of the American retina specialists reportedly used this approach in 2013. Patients are treated with monthly injections, until no signs of choroidal neovascularization (CNV) activity are observed on the slit lamp examination and OCT. Signs of disease activity include intra, subretinal fluid or hemorrhages in the macula. Follow-ups are then extended by two-week intervals as long as no CNV activity is detected. However, if exudation is present, treatment intervals are shortened by 2 weeks. Thus, the anti VEGF treatment is administered at each visit, regardless of the disease activity, but the increasing intervals between visits allow fewer injections [3].While debates continue over which treatment protocol provides the best results, new substances are being developed, which will probably change the way these patients are managed. Fovista®, a Platelet Derived Growth Factor inhibitor, binds pericytes on new vessels, thus inhibiting their growth; conbercept promises similar results to aflibercept’s, but at a lower price; squalamine and pazopanib eye drops are currently in phase II trials [To sum up, anti VEGF therapy has removed wet AMD from the list of incurable diseases and the optimum treatment choice should provide a reasonable balance between cost and benefit. Although treat and extend seems to provide the most effective results, there is still insufficient evidence to determine the best treatment option and more studies are needed to compare protocols. However, there are still unanswered questions, such as the following: do we need an individualized treatment protocol for aflibercept? And what is to be done with non responders? Regardless, the best results are only achieved with early, correct diagnosis and treatment initiation, followed by strict follow-up.


BMPR2, ACVRL1, CAV1, ENG, and SMAD9.Pulmonary arterial hypertension (PAH) is a group of vascular diseases that produce right ventricular dysfunction, heart failure syndrome, and death. Although the majority of patients appear idiopathic, accumulated research work combined with current sequencing technology show that many gene variants could be an important component of the disease. However, current guidelines, clinical practices, and available gene panels focus the diagnosis of PAH on a relatively low number of genes and variants associated with the bone morphogenic proteins and transforming Growth Factor-β pathways, such as the To provide an expanded view of the genes and variants associated with PAH, we performed a systematic literature review. Facilitated by a web tool, we classified, curated, and annotated most of the genes and PubMed abstracts related to PAH, in which many of the mutations and variants were not annotated in public databases such as ClinVar from NCBI. The gene list generated was compared with other available tests.Our results reveal that there is genetic evidence for at least 30 genes, of which 21 genes shown specific mutations. Most of the genes are not covered by current available genetic panels. Many of these variants were not annotated in the ClinVar database and a mapping of these mutations suggest that next generation sequencing is needed to cover all mutations found in PAH or related diseases. A pathway analysis of these genes indicated that, in addition to the BMP and TGFβ pathways, there was connections with the nitric oxide, prostaglandin, and calcium homeostasis signalling, which may be important components in PAH.Our systematic review proposes an expanded gene panel for more accurate characterization of the genetic incidence and risk in PAH. Their usage would increase the knowledge of PAH in terms of genetic counseling, early diagnosis, and potential prognosis of the disease.The online version of this article (doi:10.1186/s12881-017-0440-5) contains supplementary material, which is available to authorized users. Pulmonary arterial hypertension (PAH) is a group of vascular diseases that are characterized by a progressive increase in pulmonary vascular resistance and pulmonary arterial pressure with secondary vascular and right ventricular remodeling. PAH leads to right ventricular dysfunction, heart failure syndrome, and finally, premature death . PAH is BMPR2)  OR mutation [TIAB] OR polymorphisms [TIAB] OR polymorphism [TIAB]).” The analysis was restricted to abstracts published in English. We used a web tool developed in our group called Pub-Term (http://www.ncbi.nlm.nih.gov/home/develop/api.shtml) to acquire abstracts for a given query. PubTator [We used the PubTerm tool to revise, annotate, and classify genes according to the evidence of mutations in PAH. The PubTerm tool developed in our research group used the PubMed programmatic interface (PubTator  was used(i) annotation error, (ii) unrelated, (iii) negative evidence, (iv) related but not mutated, (v) mutation evidence in PAH, (vi) other genetic alterations in PAH, and (vii) genetic evidence in PAH-related disease. The details of these criteria are shown in Table We used the option “Term View” within PubTerm to revise the abstracts per gene. The abstracts per gene were manually reviewed and the corresponding gene was categorized by level of importance into: Homo sapiens within the term view in PubTerm. The definitive sentences in the abstract were marked and included in the notes per gene. The annotations could be revised and retrieved in the PubTerm tool .For genes that occurred in the abstracts more than 10 times, we revised the abstracts in descending order by date until a definitive relation with PAH was found  or a clear annotation error was determined. For genes that occurred in less than 10 abstracts, in which their possible relationship with PAH was not clear, a manual review of all related abstracts was performed to identify a potential relationship. Only human genes were reviewed, which were filtered by using “Hs.” for All investigators designed the search. One investigator acquired the data (DR) in PubTerm and the initial annotations were done by two investigators (DR and JGP). The annotations of the revised genes were further reviewed by two more investigators (VT and GGR). Any disagreements were resolved by discussion . No attempts were made to contact the authors of the original articles for information. Investigators were not blinded to the journal, author, or institutions.We used the NCBI recommended reference mRNA sequence annotation and the isoform 1 to obtain exon counts and positions. For well-known PAH genes, we used ClinVar annotations  to mark experimental evidence of mutations or other evidence of genetic alterations . DAVID/EnrichR performs an over-representation test using databases of biological terms listing the terms that significantly overlap with the list of genes. From the generated lists of biological terms, we grouped terms based on the similarity of gene content using hierarchical clustering where the resulted clusters were clearly associated with a consensus topic. This analysis was performed separately for terms related to Gene Ontology (GO) and non-GO terms commonly related to pathways.We used DAVID  and EnriBMPR2, ACVRL1 , ENG (endoglin), and those of the SMAD family. We did not find mutations in SMAD1, BMP4, BMP2, ID1, or SMAD5, which had 19, 16, 15, 11, and 11 associated abstracts, respectively. In addition, the phenylalanine hydroxylase gene, whose official gene symbol is PAH, was annotated in all abstracts as a gene and as a disease, it was therefore classified as an annotation error. After the revision, from the 15 genes that were found in 10 or more abstracts, five of these genes  were found to have some evidence of mutations in PAH, one gene (NOS3) was found to be mutated in a related disease, and one gene, serotonin transporter (SLC6A4), was classified as negative evidence.From 2004 to 2015, our electronic search identified 405 abstracts involving 389 different genes mentioned in the query for PAH mutations. Figure For the 222 genes that appeared in fewer than 9 abstracts, a full abstract revision was performed and a full-text read was done if the abstract was unclear. From these 222 genes, 16 genes were found to have evidence of mutations. Thus, a total of 21 genes were found to have evidence of mutations. Table EDNRA, NOS2, SERPINE1, SIRT3, TRPC6, TBX4, THBS1, and TOPBP1 were not included in any of the reviewed genetic panels . For example, angiotensin II receptor type 1 (AGTR1), a potent vasopressor hormone and an important regulator of cardiovascular hemodynamics , appeareCBLN2, CYP1B1, GNB3, HLA-DQB1, HLA-DRB1, HTR2B, PTGIS, SOD2, and TGFB1). Two of the genes were related to the major histocompatibility complex [We also found nine genes that had other genetic evidence in PAH  co-occurring with PAH terms or names that were confused with the acronyms of diseases, methods, or chemicals.Thirteen genes found in people with PAH had no significant polymorphism or associated mutations. Another 45 genes were wrongly annotated; these genes had “The classification of all genes can be found as Additional file To provide an overview of the location of the mutated regions for planning genetic testing strategies, we collected all the mutations as reported in ClinVar that were related to single nucleotide variants, small deletions, and insertions for the 21 genes identified with specific mutations. For genes with mutations that were not registered in ClinVar, we obtained the mutations from original publications: 64 additional mutations in 19 genes. The complete list of mutations is provided in Additional file BMPR2 gene and along the TGFβ signaling pathway, which include SMAD4, SMAD9, CAV1, and ENG. This was confirmed in our analysis. However, we also found possible connections with the prostaglandin signaling, nitric oxide, and calcium homeostasis, which are discussed below.We performed a composed functional analysis of genes with experimental evidence of mutations and other evidence of mutations for PAH to appreciate the different correlated genes and the altered signaling pathways that occurred from these mutations; the consequences of the mutations were considered at a phenotypical level Fig. . For thiBMPR2, BMPR1B, EIF2AK4, CAV1, KCNK3, and ENG where BMPR2 has a key role in the etiological factor of the disease. Currently, genetic testing is a suggestion, but not a recommendation because of the level of evidence that supports the use of genetic testing in the diagnostic approach. In addition, genetic counseling is an issue that must be analyzed from an ethical perspective to allow the patient to make appropriate decisions.Heterozygous BMPR2 mutations account for approximately 75% of hereditable PAH and up to 25% of presumably sporadic PAH cases , while tBMPR2, ACVRL1, ENG, CAV1, and SMAD4). None of the kits included genes that have been associated with PAH, such as AGTR1, TBX4, EDN1, EDNRA, NOS2, SERPINE, SIRT3, THBS1, TOPBP1, or TRCP6. For example, AGTR1 has been related to later age PAH at diagnosis [TBX4 has been related to childhood-onset of PAH [EDN1, EDNRA, and NOS2 have been related to susceptibility to develop the disease and its clinical course as well as susceptibility to systemic sclerosis-related PAH [TOPBP1 and TRCP6 have been linked to cellular protection and proliferation, respectively [SERPINE1 has been linked with a risk of venous thromboembolism [SIRT3 was related to mitochondrial dysfunction [THBS1 has been identified [Through PubMed and Internet searches, we found 12 available kits to identify genetic alterations related to PAH. Some of these kits are also applied to diverse cardiovascular disorders, such as dilated cardiomyopathy, arteriovenous malformations, and congenital defects. On average, these kits considered around eight genes, which were compared with the 21 genes identified , ALK1 (ACVRL1), and CAV1, are also very important. ALK1 and ALK2 function to activate other proteins and genes in the TGFβ and BMPR2 pathways [CAV1 has been extensively reported with many confirmed mutations specific for PAH [CAV1 could be related to BMPR2 mutations in PAH [Mutations associated with the genes in the TGFβ signaling pathway, such as pathways . CAV1 ha for PAH , 44. This in PAH , 45. Durs in PAH , 47.CAV1, AGTR1, EDN1, and KCNA5, were related to the regulation of homeostasis and ion controls. As a consequence, one of the pathways shown in the analysis was for the regulation of cytosolic calcium; many of the altered genes in PAH code for the transmembrane proteins that are responsible for calcium transport. As shown in Fig. EDN1, EDNRA, and SMAD9, are also associated with the nitric oxide biosynthesis process. For example, EDN1 and EDNRA are that are common in homeostasis control in the cell or in nitric oxide biosynthesis processes. Besides, these genes are associated with oxidative processes; for example, EDNRA, EDN1, and CAV1 are related to the synthesis of different oxidoreductases that control oxidative stress within cells. One of the most novel genes associated with oxidative stress is SIRTUIN3, which protein is involved in the post-translational modification of several proteins complexes involved in the antioxidant cellular and mitochondrial system [Finally, genes related to redox homeostasis and calcium handling were also classified. In the analysis, genes with specific mutations for PAH, such as l system . Surprisl system .BMPR2, ACVRL1, ENG, EDN1, SMAD9; then, if no mutations are found, SMAD4, KCNA5, CAV1, BMPR1B, EIF2AK4, and KCNK3 may follow; and so on with the remaining genes.The results of our systematic review propose that other genes whose prevalence or incidence have not widely studied may be important in the pathogenesis of PAH. In particular, the pathway analysis and the biological function of genes support our view. For research, it may be sensible to assess all genes. For clinical purposes, or if cost is an issue, a series of panels may be used; the first including well-known and more likely mutated genes like The limitations of this study relate to the PAH classification, which has recently been modified; several reports could be potentially included in our analysis that used previous classifications of PAH. Based on the results of our systematic approach, we explored the relative risk associated with mutations and the occurrence of PAH. However, because of the heterogeneity of reports, populations, and some case reports, the associations were sometimes difficult and confusing to determine. Few studies reported associations between genetic alterations and the manifestation of PAH. We were also limited by the abstracts annotations provided by third party tools like PubTator  where, oIn summary, the gene list we provided, in combination with recent sequencing methods and larger studies, may contribute to accurately estimate the relative genetic incidence and risk of PAH. This would expand the knowledge of PAH in terms of genetic counseling, early diagnosis, incidence of mutations, clinical assessment, therapeutic approach, and prognosis of the disease. A future study could validate this gene-targeted panel test in PAH patients.


The purpose of this review is to document the various types of astrocytoma that occur in the fetus and neonate, their locations, initial findings, pathology, and outcome. Data are presented that show which patients are likely to survive or benefit from treatment compared with those who are unlikely to respond.One hundred one fetal and neonatal tumors were collected from the literature for study.Macrocephaly and an intracranial mass were the most common initial findings. Overall, hydrocephalus and intracranial hemorrhage were next. Glioblastoma (GBM) was the most common neoplasm followed in order by subependymal giant cell astrocytoma (SEGA), low-grade astrocytoma, anaplastic astrocytoma, and desmoplastic infantile astrocytoma (DIA). Tumors were detected most often toward the end of the third trimester of pregnancy. A number of patients were considered inoperable since their tumor occupied much of the intracranial cavity involving large areas of the brain. High-grade astrocytomas were more common than low-grade ones in this review. Fetuses and neonates with astrocytoma have a mixed prognosis ranging from as low as 20 % (GBM) to a high of 90 %. The overall survival was 47/101 or 46 %. Glial cells are the supportive elements of the central nervous system (CNS) . They inGlioma (astrocytoma) is the leading CNS tumor in children. Low-grade gliomas are the leading pediatric CNS tumors overall and are responsible for approximately one third of cases , 63. HigLow- and high-grade astrocytoma differs significantly in terms of their histological grading, site of origin, treatment, and prognosis. The various types of tumors within the group of low-grade astrocytoma are classified as WHO grade I and grade II , 39. TheFetal and neonatal brain tumors are rare comprising only 0.5–1.5 % of all childhood CNS tumors . AstrocyRetrospective review of 101 cases including 57 fetuses and 44 neonates with astrocytoma was performed. They were collected from the literature, mostly from the archives of the National Library of Medicine (PubMed MEDLINE) and from our institution. The study was confined to fetuses with astrocytoma that were detected prenatally by imaging studies or discovered at birth and neonates with tumors occurring during the first 2 months of life. Only patients with adequate clinical and pathological data and where outcomes of pregnancy and survival were given were accepted for review. The following data were collected and tabulated: maternal age, gravida, initial presentation, and obstetric management, and the fetal data—sex of the fetus, weeks of gestation, birth weight, tumor location and dimensions, histology and grade, treatment, and outcome. Diagnosis of the various tumors was established in patients included in the study by histological confirmation of biopsy or surgical and/or postmortem specimens. Time period of patient accrual was from 1970 to 2015. Length of follow-up varied from 1 week to 1 year.One hundred one fetuses and neonates, presented with astrocytoma Tables , and 9. Overall, the main presenting findings were macrocephaly and an intracranial mass followed by hydrocephalus and intracranial hemorrhage Tables , and 9. Arises from the cerebral hemispheres and basal nuclei of fetuses, stillbirths, and infants , 68, 72.The cerebral hemisphere was the most common site of origin, namely, 34/45 or 76 % Table . On grosFetal and neonatal GBMs are genetically different from their adult counterparts and show a low frequency of known genetic defects .Fetuses with GBM may be born moribund or die soon after birth , 30, 49.Subependymalgiant cell astrocytoma (SEGA) is one of the unique forms of astrocytoma. It occurs in brains of patients with tuberous sclerosis (TSC) and in those without the disease. Most are found during the first and second decades of life and only occasionally in the perinatal period [l period –60, 63 , rather than manifestations of their CNS pathology , 33, 89.Table SEGAs occurred as outgrowths from within or beneath the walls of lateral ventricles. They obstructed the foramen of Monro or blocked the third ventricle producing hydrocephalus , 56, 58.Twelve of 18 SEGA patients were treated; 5 were not. One nontreated neonate was lost to follow-up. Nine of 11 infants treated with either surgical resection alone or surgery plus evorolimus chemotherapy survived , 60, 64.Low-grade astrocytoma—pilocytic astrocytoma, grade I, arises throughout the neural axis. The spinal cord, cerebral hemisphere, and optic nerve were the tumor locations in this review. Half occurred in the spinal cord [nal cord , 53, 77,nal cord , 83, 90,nal cord   surgical resection as the only form of therapy , 69, (2)Desmoplasticcerebral astrocytoma/desmoplastic cerebral ganglioglioma of infancy (DIAGA) occurs most often during the first year of life than at any other time [her time , 83, 86.her time , 75, 86.An ongoing discussion in the literature exists regarding nosology and grading of this group of tumors , 39. TheIn addition, there is some disagreement about the WHO grading classification , 39. TheCharacteristic histologic findings are a cellular, spindle cell tumor with a prominent collagenous  stroma composed of bipolar and spindle-shaped cells having an eosinophilic cytoplasm and cells with long processes , 83, 86 Eight of ten patients who were treated by surgery alone were cured , 81, 88.DIAGA is one of the astrocytoma groups usually associated with a relatively good prognosis overall provided that a complete resection was done; there was an 80 % survival for this group of tumors [f tumors , 45, 86 Astroblastoma is a rare, controversial, supratentorial, tumor first described by Bailey and Bucy in 1930 [ in 1930 . Current in 1930 . It is e in 1930 , 66, 87 The dominant histologic feature is the characteristic perivascular pseudorosette , 66. TwoThe main entity in the differential diagnosis of astroblastoma is the well-differentiated ependymoma , 61, 66.Treatment of astroblastoma is unsettled, largely because the rarity of the tumor precludes definitive therapeutic studies. Anaplastic histology is an important prognostic factor. Surgical resection is regarded as the preferred treatment modality; it plays a major role in managing the well-differentiated astroblastoma . The preGemistocyticastrocytoma (GA) is a member of the diffuse astrocytoma group [ma group , 41, 54.ma group , 48, 54 Certain histologic criteria are required to make the diagnosis of GA. The main one is that a significant number of neoplastic gemistocytic astrocytes must be present in the tumor, at least 20 %, since collections of well-differentiated GA cells can mimic reactive astrocytes . AlthougAll three GA patients in this review had large hemispheric tumors detected by prenatal ultrasound or on the day of birth. The first patient in Table The only survivor, patient 2 (tumor WHO grade 2), was a full-term male with a right occipital lobe primary  Table 88. MicrosGA shows a tendency to behave aggressively more than other grade I and II astrocytomas. Some may progress to anaplastic astrocytoma and eventually GBM .).Astrocytoma is the foremost neuroglial tumor occurring throughout infancy and childhood and is derived from and composed of astrocytes showing varying degrees of differentiation. As a group, astrocytomas differ in their gross and histological features as well as in their site of origin and clinical manifestations , 68, 93 Most perinatal astrocytomas occur outside the posterior cranial fossa and above the tentorium Tables . When itAstrocytoma typically presents in the fetus and neonate as an intracranial mass, which is the initial observation on prenatal or postnatal sonograms; hydrocephalus macrocephaly and intracranial hemorrhage, in that order, are additional presenting findings Tables , and 8.Tumors vary considerably in their microscopic appearance. Histologically, they range from benign (low-grade) to malignant (high-grade) tumors Table . Here, tTreatment is primarily surgical, like other brain tumors in this age group . Sometim


Dictyostelium to macrophages. Nramp1 regulates iron efflux from the phagosomes, thus starving pathogenic bacteria for iron. Similar studies for zinc or copper are scant, due to the large number of copper and zinc transporters. In Dictyostelium, zinc and copper transporters include 11 and 6 members, respectively. To assess the role of zinc or copper in Dictyostelium, cells were grown under conditions of metal depletion or excess and tested for resistance to Legionella pneumophila infection. Iron shortage or overload inhibited Dictyostelium cell growth within few generations. Surprisingly, zinc or copper depletion failed to affect growth. Zinc or copper overloading inhibited cell growth at, respectively, 50- or 500-fold the physiological concentration, suggesting very efficient control of their homeostasis, as confirmed by Inductively Coupled Plasma Mass Spectrometry quantification of cellular metals. Legionella infection was inhibited or enhanced in cells grown under iron shortage or overload, respectively, confirming a major role for iron in controlling resistance to pathogens. In contrast, zinc and copper depletion or excess during growth did not affect Legionella infection. Using Zinpyr-1 as fluorescent sensor, we show that zinc accumulates in endo-lysosomal vesicles, including phagosomes, and the contractile vacuole. Furthermore, we provide evidence for permeabilization of the Legionella-containing vacuole during bacterial proliferation.Iron, zinc, and copper play fundamental roles in eucaryotes and procaryotes, and their bioavailability regulates host-pathogen interactions. For intracellular pathogens, the source of metals is the cytoplasm of the host, which in turn manipulates intracellular metal traffic following pathogen recognition. It is established that iron is withheld from the pathogen-containing vacuole, whereas for copper and zinc the evidence is unclear. Most infection studies in mammals have concentrated on effects of metal deficiency/overloading at organismal level. Thus, zinc deficiency or supplementation correlate with high risk of respiratory tract infection or recovery from severe infection, respectively. Iron, zinc, and copper deficiency or overload affects lymphocyte proliferation/maturation, and thus the adaptive immune response. Whether they regulate innate immunity at macrophage level is open, except for iron. The early identification in a mouse mutant susceptible to mycobacterial infection of the iron transporter Nramp1 allowed dissecting Nramp1 role in phagocytes, from the social amoeba  Both ATP7A and the CTR protein p80 are localized in the plasma membrane and transitorily in phagosomes , but no data are available on their localization in phagosome and their potential involvement in host-pathogen interactions.The Dictyostelium phagocytosis and defense mechanisms against bacterial pathogens, and given the large number of transporters for these metals, we have followed in this paper a holistic approach, based on cultivation of wild type cells or Nramp1 knockout mutant in a minimal medium depleted of, or overloaded with either zinc, copper, or iron. The rationale is that extensive growth in media deprived of or with high content of a given metal, should results in either metal deficiency or overload in cells, thus potentially altering their resistance to pathogens. We show here that iron, but not copper or zinc deficiency affects cell growth. Metal excess results in inhibition of cell growth, with the cells being much more sensitive to iron than to copper or zinc overloading. Cells grown under iron depleting or overloading conditions, show, respectively, resistance to or exacerbation of L. pneumophila infection, whereas Legionella intracellular growth is unaltered by depletion or overloading with copper or zinc. Thus, it appears that zinc and copper, in contrast to iron, play a minor role in Dictyostelium resistance against Legionella. We provide also evidence for very tight regulation of copper and zinc homeostasis in Dictyostelium, which can in part explain these results, and for accumulation of free zinc ions in vesicles of the endo-lysosomal pathway and in the contractile vacuole.To assess a role for zinc or copper in Dictyostelium discoideum parental strain AX2 and the Nramp1-KO mutant were used. The Nramp1-KO mutant or cells producing Nramp1-RFP and NrampB-RFP were generated previously in the lab . AX2 cells producing CshA-RFP or RFP alone were generated by electroporation  were used. Pre-cultures of bacteria were maintained in LB agar.Legionella pneumophila Corby producing GFP or Ds-red Express were provided by Michael Steinert and grown on buffered charcoal yeast extract agar 3  and resuspended at a final concentration of 5 × 107 cells/ml. Aliquots of 50 μl were plated on agar plates buffered with Soerensen phosphate buffer at a density of about 3 × 105 cells per cm2, and incubated at 23°C , 2.5 g of KH2PO4, 1 g of Na2HPO4 2(H2O), 18 g of agar in 1 l of water) for K. aerogenes, and further grown overnight at 22°C. AX2 cells were grown as previously described in the minimal media listed in Table A single colony of bacteria was picked from plate and bacteria were grown in LB medium overnight with shaking. The next day, 50 μl of the bacterial culture were spread in each well of a 24-well N-agar  for 5 cells were plated in 96-well tissue culture plates. For every condition, cells were tested in triplicates for a series of time points ranging from 0 to 96 hpi. As control, AX2 cells grown in axenic medium and resuspended in low fluorescence medium  containing 5 μg/ml chloramphenicol were used. Freshly collected L. pneumophila Corby producing GFP were added to Dictyostelium cells at a MOI (Multiplicity Of Infection) of 1:1 and immediately centrifuged at 600 g for 10 min at room temperature to synchronize infection. Plates were incubated at 25°C and samples were collected every 24 h .L. pneumophila producing GFP or Ds-red Express were plated in black 96-well tissue culture μ-plates  and incubated at 25°C. Confocal series images were taken daily on an inverted Zeiss LSM800 with AiryScan  equipped with a Plan-Apochromat 63x/1.40 DIC Oil- immersion objective. For excitation, a 488 nm Diode laser was used and its emission collected with a 495–519 nm filter for GFP and a 592–614 nm filter for RFP. Phase contrast was recorded simultaneously.Cells infected with E. coli (MOI 100:1) were used. Cells were washed and resuspended in Soerensen phosphate buffer and incubated for 30 min with 5 μM Zinpyr-1 fluorescent probe . Non-incorporated probe was removed by washing with Soerensen buffer and the cells were plated on glass coverslips. Images were acquired as previously described.For monitoring zinc intracellular localization, cells producing Nramp1 or NrampB-RFP or AX2 cells mixed with TRITC-labeled 8 cells were washed five times in Soerensen phosphate buffer and pelleted in Eppendorf tubes. Digestion was performed by adding 1 ml of concentrated HNO3 (70%) to each sample. After complete dissolution, samples were further digested by applying microwave heating . After digestion, the volume of each sample was brought to 3 ml with ultrapure water, filtered with 0.45 mm filter and analyzed by ICP-MS, using a Thermo Scientific ELEMENT 2 ICP-MS  . Addition to M2 or M3 medium of extracellular zinc chelator ZX1 or copper chelator trientine hydrochloride at concentrations varying between 50 and 500 μM also failed to affect cell growth.To assess whether cell growth would be affected by prolonged incubation in medium without added zinc or copper or with high, but non-toxic, concentrations of zinc or copper, namely 20- or 200-fold excess, respectively, cells were re-diluted in the same medium, shortly before reaching the stationary phase, and cell growth measured over 480 h. As shown in Figure Presuming there are no other sources of these metals in the medium, at any cell doubling the intracellular amount of iron, zinc, or copper should halve. Initially, cells contain the metals engulfed with the axenic medium and might undergo some duplication rounds, as observed for iron Figure , but thi7 cells still contained about 84 or 3.1 ng of zinc or copper, respectively, i.e., about 87 and 51% of the amount present in cells growing in standard minimal medium or AX2 medium. In cells incubated with a 20 or 200-fold excess of zinc or copper, respectively, the cellular concentration increased of only 2.7- and 2.4-fold, respectively, compared to FM or AX2 medium . In the nominally metal-free M2 and M3 medium, 0.58 and 0.028 μM zinc or copper, respectively, were detected, amounting to 7.26 and 4.67% the concentration of zinc or copper, respectively, present in the standard FM medium , and decreasing thereafter   and incubated for 40 min at 25°C. Uptake was measured as increase of the percentage of fluorescent  cells. When cells grown in M1 were assayed, only about 23% of them were fluorescent, rising to 38% 40 min post-infection  made of amino acids, glucose, a few selected vitamins and trace elements  membrane even up to later stages of infection. Association with the ER has been already described at early stages of infection, presumably induced by Legionella for acquiring new membrane for the LCV  and later of higher molecular mass (about 76 kDa), but the bacteria remain packed together and do not egress into the cytosol, suggesting that, though permeable to proteins, the LCV membrane does not allow exit of the bacteria before cell lysis. Vacuole disruption shortly before cell lysis has been reported (Creasey and Isberg, Legionella intracellular growth. For both, the infection rate was slower, though statistically not significant or borderline, in cells grown in nominally zinc or copper-free medium compared to cells with 5-fold more intracellular copper. If any, this would suggest that a higher intracellular zinc or copper concentration is not toxic for Legionella. In the case of zinc, Dictyostelium cells with the lowest zinc concentration were blocked at mound stage, suggesting that a suboptimal zinc concentration is deleterious for development. Zinc ions have been shown to enhance pre-stalk to stalk differentiation (Kubohara and Okamoto, In sharp contrast to iron, variation in cellular zinc or copper content over a range of, respectively, 3- to 5-fold did not significantly affect timing or extent of Dictyostelium phagosomes upon bacterial phagocytosis. Thus, both zinc and copper are found in Dictyostelium phagosomes, consistent with a potential intoxicating role against pathogens, as suggested for macrophages (Babu and Failla, By using the iron fluorescent reporter calcein, we showed previously that iron is rapidly depleted from endo-lysosomal vesicles via Nramp1 (Buracco et al., E. coli, which are not alive, whereas Botella and coworkers used living bacteria. If this would be the reason, then it would not be phagocytosis per se that triggers zinc accumulation, but a cellular response to the ingested living bacteria. Alternatively, the different results can be explained with the different zinc reporters used, Zinpyr-1 in this paper and FluoZin-3 in the Botella et al. (We have no evidence for phagocytosis inducing a burst of zinc in endocytic vesicles, as shown in macrophages with non-pathogenic bacteria (Botella et al., a et al.  paper. Ia et al. . FurtherDictyostelium resistance to pathogens, at least with L. pneumophila. Whether this holds true for other pathogens, such as mycobacteria, is under investigation.Our data suggest that, in contrast to iron, changes in cellular zinc or copper play a minor role in Dictyostelium cells to cope with conditions of zinc or copper starvation or overloading. To which extent this is typical of Dictyostelium cells or is a common feature of other cells, including macrophages, is unknown. The experimental approach described here offers now the opportunity to identify, in a transcriptomic study, the genes responsible for this high homeostatic adaptation to zinc and copper deprivation or intoxication. From the site of the pathogen, the same approach can be used to identify potential mutants in metal dependent virulence genes.A shortcoming of our experimental approach is the apparently very high efficiency of Dictyostelium support this caution, offering a mechanistic explanation for the iron effects on infection that holds true also for macrophages. Concerning copper, but in particular zinc, further studies are required with macrophages isolated from animals exposed to metal deficiency or overloading. Identifying the genes responsible in Dictyostelium for the tight regulation of zinc and copper homeostasis and for their transport in phagosomes can shed some light on cellular zinc and copper store and mobilization, and the genetic basis of antimicrobial metal transport, also in macrophages.There is an analogy between the conditions used in this paper and those encountered with humans affected by transition metal deficiency or overloading, due to nutritional or environmental problems. In particular, iron or zinc deficiency is a global nutritional problem, with negative health consequences, including anemia, impaired immune function and neurological diseases. Dietary iron supplementation to prevent micronutrient deficiency in areas with a high burden of infectious diseases is a double–edged weapon, as it can exacerbate morbidity and mortality from infectious diseases (Oppenheimer, Dictyostelium KO mutants and the plasmids for RFP-fused proteins and helped with some experiments; CA was responsible for the bioinformatic analysis of the metalloproteome. EB cloned the cshA gene, generated, and characterized cells expressing CshA-RFP. SBu and SBo wrote the manuscript; all approved the paper.SBu and SBo designed the experiments; SBu conducted the experiments, acquired, and analyzed the data; BP generated the 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.


Herd 1 had persistently high urinary ZEN and STC concentrations due to the presence of contaminated rice straw. Herd 2, the second female JB fattening herd , received the same dietary feed as Herd 1, with non-contaminated rice straw. Urine samples were collected from Herd 1, two weeks after the contaminated rice straw was replaced with uncontaminated rice straw (Herd 1N). Identified metabolites were subjected to principal component analysis (PCA) and ANOVA. The PCA revealed that the effects on cattle metabolites depended on ZEN and STC concentrations. The contamination of cattle feed with multiple mycotoxins may alter systemic metabolic processes, including metabolites associated with ATP generation, amino acids, glycine-conjugates, organic acids, and purine bases. The results obtained from Herd 1N indicate that a two-week remedy period was not sufficient to improve the levels of urinary metabolites, suggesting that chronic contamination with mycotoxins may have long-term harmful effects on the systemic metabolism of cattle.The objective of this study was to evaluate the metabolic profile of cattle fed with or without zearalenone (ZEN) and sterigmatocystin (STC)-contaminated diets using a gas chromatography-mass spectrometry metabolomics approach. Urinary samples were collected from individual animals ( Contamination of agricultural commodities with mycotoxins, which are secondary metabolites of fungi, is a major problem in agriculture and livestock production worldwide . The conFusarium spp. and Aspergillus spp., and their secondary metabolites in urine using our established liquid chromatography-tandem mass spectrometry (LC-MS/MS) monitoring technique [Recently, we reported the presence of mycotoxin contamination in the dietary feed (rice straw) of a cattle herd by demonstrating the presence of zearalenone (ZEN) and sterigmatocystin (STC), which are produced by two groups of fungi, echnique ,5. Moreoechnique ,4,6.Because organisms must maintain homeostasis, metabolic profiles vary continuously with changes in their physical condition. Metabolomics can provide a quantitative description of endogenous metabolites of low molecular mass present in biological samples such as urine, plasma, tissue, and organs, or in the whole body of an organism ,8. MetabConversely, techniques such as mass spectrometry combined with gas chromatography (GC/MS) ,18, liquIn the present study, we used GC/MS to identify 55 non-targeted endogenous metabolites related to the metabolism of ATP generation, amino acids, thyroid hormones, neurotransmitters, glycine-conjugation, organic acids, and purine bases, as well as dietary plant components. All identified data was analyzed by ANOVA and/or principal component analysis (PCA) to determine the effects of dietary mycotoxin contamination.Unfortunately, metabolic data from one sample of Herd 2 were not obtained because the derivatization step failed; therefore, data for this sample were not included in the PCA analysis. In the PCA analysis shown in Data for both Herd 1 and Herd 1N clustered on the upper left-to-right of the plot, and those for Herd 2 fell in the lower left of the plot (PC1 accounted for 36.3% of the variance and PC2 accounted for 14.4%). These results indicated that the metabolite profiles of Herd 1, 1N, and 2 clearly differed. Therefore, based on these results, clustering along PC1 represented the effects of mycotoxin (ZEN and STC) contamination intensity within the dietary feeds, and PC2 represents the effects common to the mycotoxin contamination group. The component loadings that significantly contributed to the clustering of each group along PC1 and/or PC2 are listed in Metabolites associated with ATP generation metabolism , organic acid , amino acids , glycine-conjugates , purine bases , and dietary plant-derived metabolites  significantly contributed to different clusters for the three cattle herds on PCA . We usedMetabolomics can be efficiently utilized to clarify several simultaneous effects in a single examination, and can identify temporal variation in the metabolic responses of living organisms to physiological and pathological stress conditions ,25,26. APrevious studies investigating the effects of ZEN have involved measuring and comparing levels of a single or several biochemical markers. Seeling et al.  reportedPreviously, ZEN exposure was shown to induce oxidative stress, and some common changes in systemic metabolism, including effects on cell membrane metabolism, protein biosynthesis, glycolysis, and gut microbiota metabolism in rodents . AdditioIn the present study, seven detected metabolites were associated with gluconeogenesis, and significantly/strongly contributed to the separation along PC1 (positively), and four detected metabolites were along PC2 (negatively) in the PCA. Additionally, significant differences were observed for both urinary lactate and lactose among the three groups by ANOVA analysis. Our results indicate that mycotoxin exposure may affect gluconeogenesis/energy metabolism in cattle, as reported in experimental animals . CarbohyBased on the results of the PCA, urinary pantothenic acid, myo-inositol, aconitic acid, and isocitric acid were found to differ significantly between contaminated and control herds. Animals require pantothenic acid to synthesize coenzyme-A (CoA) . CoA is In the present study, a significant difference in methyl succinic acid, hydroxyisovaleric acid (HIVA), and uric acid was observed between the contaminated and control groups. The formation of 3-HIVA in mammals has been demonstrated in association with leucine degradation, and is normally present in small quantities in urine . In a prIn the present study, significant differences in the essential amino acids, threonine, and lysine, and taurine were observed, resulting in the separation of contaminated and control groups along with PC1 in PCA. These results suggest that mycotoxin exposure alters amino acid metabolism in cattle. Threonine is converted to pyruvate via threonine dehydrogenase, and an intermediate in this pathway can undergo thiolysis with CoA to produce acetyl-CoA and glycine. Lysine is metabolized in mammals to produce acetyl-CoA, via initial transamination with α-ketoglutarate. Because both threonine and lysine are essential amino acids, they must be obtained from the diet, or are biosynthesized within the rumen in cattle. As the diet composition of the individual herds was almost the same, except for the contaminated rice straw, variation can be attributed to the different levels of mycotoxin exposure. Previous studies have shown that ZEN can inhibit protein synthesis in experimental animals ,36, whicIn the present study, levels of tyrosine and oxoproline were significantly different between contaminated and control herds. Notably, tyrosine is an important amino acid in many proteins, peptides, and even enkephalins, and is the precursor of thyroid hormones . OxoprolIn the present study, levels of tryptophan, phosphocolamine, and serine significantly differed between the contaminated and control herds along PC1 of the PCA. Additionally, a significant difference was observed in phosphocolamine among the three herds. Tryptophan is a precursor of the neurotransmitters serotonin and melatonin. Phosphocolamine is an ethanolamine derivative that is used to construct two different types of phospholipids; glycerophospholipid and sphingomyelin. Serine, a non-essential amino acid, is important in metabolism, and participates in the biosynthesis of purines and pyrimidines. In addition, it is the precursor of several amino acids, including glycine, cysteine, and tryptophan in bacteria, and plays an important role in the catalytic function of many enzymes. 5-hydroxytryptamine is a messenger generated from tryptophan, which can produce pleasurable emotions and affects nearly all types of brain activity . PreviouIn the present study, three metabolites associated with glycine conjugates, hippurate, butyrylglycine, and methylbutyrylglycine, significantly contributed to the separation of contaminated and control herds along with PC1. The results of a previous study suggested that both hippurate and glycine conjugates are normally present in the urine of healthy cattle, and that the increased urinary excretion of glycine conjugates is related to detoxification . HippuraUrine metabolites, such as purine derivatives, have been used to estimate levels of microbial protein synthesis in the rumen . In the In the present study, the levels of nine identified metabolites associated with feed materials differed significantly between contaminated and control herds, including adonitol, threitol, cinnamate, hydroxyphenyllactate, glycolic acid, indol-3-acetic acid, deoxytetronic acid, threonic acid, and gluconic acid. Additionally, significant differences were observed in the levels of galacturonic acid, hippuric acid, and glycolic acid among the three herds. Adonitol is a crystalline pentose alcohol formed via the reduction of ribose, which occurs in the cell walls of Gram-positive bacteria. Cinnamate is found naturally in a variety of plants. It is thought that hydroxyphenyllactate is derived from roughage. Glycolic acid is found in some sugar-crops. Indol-3-acetic acid is the most common naturally occurring plant hormone of the auxin class. Therefore, the above results may reflect the difference in dry matter content in dietary feed between the contaminated and control herds, and the subsequent nutrient utilization by the animals. Conversely, galacturonic acid is a sugar acid- oxidized form of galactose, and is the main component of pectin. Pectin consists of a complex set of polysaccharides, which are present in most primary cell walls of plants. As noted, because urinary galactose excretion contributed to the separation of contaminated and control herds, this may affect/reflect the significant differences in urinary galacturonic acid excretion observed in the present study. Hippuric acid is a carboxylic acid found in the urine of horses and other herbivores. Although rumen microorganisms may synthesize benzoic acid from a variety of sources, benzoic acid is not normally synthesized by animals, and ingested benzoic acid is excreted as hippuric acid in the urine of herbivorous animals following conjugation with glycine . HippuriIn conclusion, the results of the present study indicate that the contamination of cattle feed with multiple mycotoxins, as confirmed by the measurement of ZEN and STC, may result in altered metabolic processes, including ATP generation, amino acid, and purine base metabolism. The results obtained from Herd 1N indicate that the two-week remedy period for contaminated feed was not sufficient to modify most of the urinary metabolites, suggesting that chronic contamination of mycotoxins may have long-term adverse effects on systemic metabolism in cattle. This suggests that monitoring endogenous metabolites in urine samples (metabolomics) is useful for evaluating the effects of cattle exposure to multiple mycotoxins. Further field studies are required to develop a basic tool to evaluate the impact of chronic exposure to low-dose mycotoxins on cattle health and productivity. Additionally, because of the limitations of using GC/MS as the analytical tool for metabolomics analysis, extensive sample treatment and analytical validation must be performed.Animals were cared for according to the Guide for the Care and Use of Laboratory Animals .N-methyl-N-(trimethylsilyl)-trifluoroacetamide with 1% trimethylchlorosilane (MSTFA + 1% TMCS) was purchased from Thermo Scientific . O-methylhydroxylammonium chloride (methoxylamine hydrochloride), pyridine, 2,2-dimethyl succinic acid, myristic acid d27, pesticide-analytical-grade chloroform and hexane, and high-performance liquid chromatography (HPLC)-analytical-grade methanol were purchased from Wako Pure Chemical . Urease was purchased from Sigma-Aldrich .n = 6 per herd) 2 h after the morning feed by massaging the perineum. Additional urine samples were collected from Herd 1, 2 weeks after replacing their rations with newly un-contaminated rice straw (Herd 1N). ZEN and STC concentrations in the dietary straw, ZEN, and the concentrations of its metabolites; α-Zearalenol (α-ZOL), and β-Zearalenol (β-ZOL), in urine samples of cattle derived from each experimental group collected on the same day, including the results of the dietary straw and urine samples collected at 42 or 34 days before , have been partially reported previously , and are shown in g for 10 min at room temperature to remove debris and frozen at −30 °C until GC/MS analysis.Two herds of female Japanese Black cattle  maintained for fattening for meat in Kagoshima Prefecture, Japan, were used in this study. Herd 1 is known to have persistently high urinary ZEN and STC concentrations due to the contamination of rice straw. Herd 2 had the same dietary feed as Herd 1, but were fed with rice straw not contaminated with ZEA and STC. The content of the feed given to each herd is described in After thawing at room temperature, the urine samples were centrifuged  at 10,000 rpm for 10 min at 4 °C. Aliquots of urine samples (100 μL) were transferred into 2-mL polypropylene (PP) microtubes, and 100 μL urease (2 mg/mL) was added. Samples were incubated for 30 min at 37 °C with shaking. One-milliliter of methanol cooled with ice was added to the urine samples, which were then shaken on a vortex mixer, and centrifuged at 10,000 rpm for 10 min at 4 °C. Next, 900 μL of the upper layer was placed in another 2-mL PP microtube, spiked with 2,2-dimethyl succinic acid and myristic acid d27 for use as the internal standards, and then evaporated to dryness under a gentle stream of nitrogen at room temperature. Derivatization was performed in two steps: oximation and silylation. To this end, the dried residue was dissolved in 10 μL of a pyridine solution of methoxylamine hydrochloride (40 mg/mL) by means of continuous shaking at 30 °C for 90 min, then silylation was carried out with 90 μL of MSTFA + 1% TMCS at 37 °C for 30 min. After derivatization, 100 μL of hexane was added to each sample, which were additionally diluted 10 times with hexane, and rapidly injected into the GC/MS.Metabolites were analyzed on an Agilent Technologies 6890 Series gas chromatograph equipped with a 5973 MSD mass selective detector and a DB 5-ms capillary column . The injected sample volume was 1 μL. The injector and detector temperatures were 250 °C and 290 °C, respectively; the oven temperature program was as follows: 60 °C for 1 min, increased to 325 °C at 10 °C/min, and then held at 325 °C for 12 min. In the present study, we replicated the GC/MS analysis with the same urine to avoid experimental fluctuations and misinterpretations.Creatinine concentrations in the urine were determined using a commercial kit  according to the manufacturer’s instructions and measured using a clinical autoanalyzer .http://www.r-project.org/). p < 0.05 was considered significant.Metabolite peaks on the GC/MS chromatogram were drawn from the baseline to obtain the peak area, deconvoluted, and aligned using MetAlignTM . Data obtained from peak areas for individual target metabolites and normalized by the peak area of 2,2-dimethyl succinic acid as an internal standard were additionally normalized by the creatinine levels measured in individual cattle. The normalized values were analyzed by one-way analysis of variance (ANOVA). Then PCAs were performed for the metabolites that differed significantly by ANOVA among all groups, in order to evaluate differences in toxic effects among the groups. PCA score plots were used with component loading to evaluate how specific metabolites were affected by exposure to mycotoxins. Statistical analyses were performed with the R programming language (


This study investigates the effect of fat diet on gene expression profile in rat liver via protein-protein interaction mapping analysis.Nonalcoholic fatty liver disease (NAFLD) is a prevalent condition of liver in the world. This progressive metabolic disease is representative with fat accumulation in the patients’ liver that can led to advance stages, namely, cirrhosis and eventually cancer. Differentially expressed genes of NAFLD rat liver after 2, 4 and 6 weeks fat diet feeding were analyzed via GEO2R and protein-protein interaction network by Cytoscape v3.6.0. and the related plug-ins. The important genes were assigned based on degree and betweenness centrality analysis and enriched using ClueGO+CluePedia Plug-in.  GAPDH, PRDM10, TP53, AKT1, INS, ALB, SRC, MAPK1, ACLY, ACACA, DECR1, ACACB, MBOAT4, TNF, EHHADH and JUN genes were introduced as key genes related to the fat diet fed NAFLD rats. Fatty acid biosynthesis and four other terms were introduced as the main related ones to the essential genes. The introduced critical genes and the related terms may describe NAFLD molecular condition and its progression to the other severe metabolic diseases. Moreover, these potential biomarkers may be monitored for diagnosis and treatment approaches after validation investigations.  About one-third of the world population are affected by Nonalcoholic fatty liver disease (NAFLD) . The disProtein-protein interaction network analysis of significantly changed genes in expression is conducted in this study. The data is from the Gene Expression Omnibus (GEO) in whichp≤ 0.05) along with the log transformation of fold change as well as considering the correction test of Benjamini & Hochberg . Thousands of genes with other relevant information are detectable via GEO2R, however, here only the genes among the top 250 ranked significant ones (p≤ 0.05) with the fold change cutoff of ≥ 2 were chosen for further analysis. Accordingly, the R script provided by GEO2R was applied in the R Studio Software environment for conducting this evaluation as well, using GEO query and limma R packages from the Bioconductor project  were used as suitable tools for gene screening. The key genes based on degree values and amounts of betweenness centralities determined for each networks and results are tabulated in the The statistical analysis for gene ontology are as follow: Kappa statistic is used for analysis how much the biological terms are grouped together as clusters. Kappa Score: 0.4, Min level of ontology: 3 Max level of ontology: 8, Number of min gene per term: 3 and 3, Percentage of min gene per term: 5, the correction method: Bonferroni step down, Enrichment/depletion test for the terms: 2-sided enrichment/depletion based on hypergeometric method.Fatty liver disease is the most prevalent type of liver disorder worldly that can lead to other kinds of serious disorders such as cirrhosis, diabetes, heart attack and stroke . MoleculO-acyltransferase 4 (MBOAT4) is a hydrophobic enzyme that esterifies long-chain fatty acids to the target proteins (In the first step, combination of all of the key genes can be introduced as a panel including GAPDH, PRDM10, TP53, AKT1, INS, ALB, SRC, MAPK1, ACLY, ACACA, DECR1, ACACB, MBOAT4, TNF, EHHADH and JUN genes. This panel independent of feeding time is corresponded to the effect of fat diet on the NAFLD gene expression profile especially the key genes. Since there are five common genes for each group, it can be concluded that about 50-60% of the introduced genes for individual groups are common genes. In the second glance, it is clear that AKT1, SRC and MAPK1 are the specific crucial genes that refer to the early effect of fat feeding. Finally, MBOAT4, TNF and JUN are appeared in the last time course feeding. Moreover, the roles of the 16 key genes in NAFLD rats after feeding fat diet were investigated via gene ontology analysis. For achieving important pathways, the restricted investigation was applied to GO enrichment. The findings are shown in the proteins . This enproteins . It is rproteins . Investiproteins . Based oproteins , 2. Thisproteins . Since fproteins . Therefoproteins . The thiproteins . Negativproteins . MAPK1 aproteins , 41. Rolproteins , 43. Aceproteins . Albuminproteins . This brproteins . Relatioproteins . Since iproteins , The finAmong query genes and their related ones, 16 genes were introduced as key elements in relationship with five essential pathways which are involved in the NAFLD rat in fat diet feeding course. Most of the enriched genes refer to four weeks fat diet feeding group which represent time course of feeding. It seems that the introduced genes and pathways play significant roles in pathology of NAFLD and its progress to the other diseases in particular cancer.