Purpose. Use of microperimetry (Mp-1), correlating with Humphrey perimetry (30-2 program), in patients affected by primary open-angle glaucoma (POAG) with perimetric defects, in order to obtain an evaluation of the accuracy of the results obtained by Mp-1. Materials and Methods. In this study 40 eyes of 25 patients affected by POAG with perimetric defects were included. All patients underwent microperimetry test by Nidek Mp-1 . Mean sensitivity values expressed in decibel (dB) of all tested dots and mean values for each quadrant obtained by microperimetric test were correlated with corresponding quadrants obtained by static perimetry analysis. Data were analyzed by Pearson's correlation and Bland-Altman analysis. Results. Interpolated data showed that mean sensitivity values in all spots tested by Mp-1  may be significantly correlated with mean total values obtained by Humphrey 30-2 perimetry , with correlation coefficient of 0.556. Conclusions. Topographic visualization of the perimetric alteration by microperimetry allows retesting areas with reduced sensitivity which are topographically visualized and displayable on the ocular fundus examination, avoiding worsening of the functional defect by better modulation of the antiglaucoma therapy and therefore it allows better monitoring of the pathologic functional damage. Glaucoma is classically recognized as the main ocular condition causing irreversible blindness occurring in 70 million subjects worldwide. It consists in an ocular neuropathy progressing into an over loss of retinal ganglion cells (RGC) and their axons which determines the crucial organic-functional damage .For the diagnosis of glaucoma, the detection of alteration occurring at the optic nerve head level, abnormalities of the retinal nerve fiber layer (RNFL), visual field loss, and the presence of elevated intraocular pressure (IOP) is fundamental.Currently, various assessment and functional tests are used for the diagnosis of the glaucoma . Among functional tests, the automated (computerized) perimetry is considered the gold standard for both diagnosis and follow-up because of its elevated sensitivity to detect perimetric abnormalities using full threshold test or SITA-Standard with Humphrey perimeter evaluating a sufficient dot density, distributed within “the 30° area.”Nevertheless, the computerized automated perimetry is not able to identify the area and quantify the stability of the fixation, particularly in partially sighted subjects. The fundamental issue is the impossibility of controlling ocular movements and displaying ocular fundus in real time during the test.Conversely, microperimetry (MP) is a new-generation morphofunctional technique that does not show this limit, allowing performing an electronic view field contemporarily to the display of stimulated dots within patient's ocular fundus.The sensitivity map is generated by observing in a continuative way the ocular fundus and projecting light stimuli in retinal areas selected by the operator. In this way, it becomes possible to correlate specific dots within the ocular fundi with the eventual alterations of retinal sensitivity and thus to associate objectively the morphologic retinal changes ophthalmoscopically observed and the subsequent functional alterations .Currently, the use of microperimetry to study glaucoma progression consists in the analysis of peripapillary retinal sensitivity through the use of light stimuli projected radially to the optic disc (12 meridians) .In this way, microperimetry could be able to detect and monitor the damage of RNFL in subjects affected by glaucoma.The possibility of evaluating contemporarily both retinal sensitivity and the direct observation of ocular fundus allows limiting microperimetric analysis to selected areas that would show defects localized within the retinal nerve fiber layer (RNFL), leading to the identification of functionally abnormal areas and allowing an eventual follow-up evaluation. In addition, microperimetry is able to detect and monitor precocious and specific RNFL impairment and, when it is conspicuous, could lead to a progressive enlargement of the physiological optic papilla excavation and visual field impairment. Therefore, the early evaluation of morphofunctional alterations at this level is crucial in the diagnosis and prevention of the glaucomatous disease.The aim of the study was the application of microperimetry, correlating it with Humphrey perimetry 30-2, in patients with primary open-angle glaucoma with perimetric defects “localized” at the papillary level in order to get an assessment of the accuracy of the results obtained by Mp, which could then be used routinely for early diagnosis of the disease and close follow-up in the prevention of damage caused by glaucoma Figures .In this study, 40 eyes from 25 patients  affected by chronic open-angle glaucoma or focal papillary perimetric abnormalities, comparing them with Humphrey 30-2 perimetry threshold test, have been included. The exclusion criteria were represented by (i) the opacity of dioptric lens, (ii) previous ocular surgery , and the presence of type II diabetes, blood hypertension, and other systemic disorders.Each patient underwent an ophthalmologic visit including Best Corrected Visual Acuity (BCVA), anterior segment examination, fundus examinations , and gonioscopy to classify anterior chamber angles according to Shaffer system. Furthermore, the same operator performed a tonometric curve during the same day to each patient.Each patient was tested with microperimetry Nidek Mp-1  applied to conventional static perimetric analysis.The hardware is composed of an IR fundus camera  allowing both a dynamic visualization and a real-time retinal examination for a 45° exam angle.The fundus camera is equipped with a lens system for correcting patient's refractive defect in a range of −12.5/+16 diopters; because of this system the invasiveness and patient's discomfort are reduced during the exam, and the accuracy functional evaluation and focus of the retinal image are improved. fundus camera  allowing the acquisition of high-quality digital retinoic picture where it is possible to select retinal spots  and a system of eye-tracking which is able to compensate instinctive ocular movements occurring during test performance allowing testing the fixation throughout the functional test.In addition to that, this tool is equipped with a colorAll the tests were performed in the same room with low luminance, by the same skilled operator, minimizing potential biases.2-white background, magnitude of the stimulus Goldmann III white-100 ms, initial attenuation of 20 dB, threshold strategy (4-2-1), 77 tested dots, and single cross fixation sight 1° . All patients showed BCVA values >8/10 (Snellen optotype); slit-lamp exam of anterior chamber was normal and cup/disc ratio was <0.3 tested by direct ophthalmoscopy.Gonioscopy performed on all subjects detected a 4° angle according to Shaffer.IOP after tonometric curves resulted in no change during combined therapy with more than one antiglaucoma drug, with mean value of 14 mmHg ± 3 mmHg.Interpolated results showed mean value of sensitivity for each tested dot with Mp-1 of 11.98 dB ± SD 4.31. They may be correlated with mean total values or with mean values of the corresponding quadrant tested by Humphrey 30-2 perimetry (17.95 dB with SD 4.32), with correlation coefficient of 0.556 .The comparison for corresponding quadrants with regard to the defects observed resulted to be statistically significant for parapapillary sectors, with values of H3-M3  and H4-M4 , with 0.762 and 0.456 , respectData were obtained by Bland-Altman analysis. As shown in Tables  Humphrey 30-2 perimetry is considered the gold standard for the diagnosis and follow-up of glaucomatous diseases. The analyses of our outcomes led to considering that conventional perimetry using Mp-1 microperimeter in patients affected by chronic glaucoma is accurate because of the statistically significant correlation with Humphrey 30-2 perimetry.In the last years, microperimetry has been widely used in case of macular disorders, evaluating retinal sensitivity of central area , 3. Its This correlation is even more marked for the campimetric inferonasal quadrant  and superonasal quadrant , well known to be primarily affected by loss of ganglion cells.The relevance of obtained results is related to the accuracy of this new analysis in evaluating functional glaucoma damage. Furthermore, it is a morphofunctional procedure that is able to evaluate the sensitivity threshold with topographic accuracy, as it is possible to establish with accuracy the retinic dots that are stimulated by a system of direct retinal visualization.This aspect is relevant because Mp-1 would result as a follow-up test, highly useful to monitor glaucoma. The traditional electronic Humphrey 30-2 perimetry consists in identifying stimulated retinic areas in their geometric position compared to the patient's fixation point. This type of test does not provide any correlation between ocular fundus test and the retinal sensitivity in a specific point, as it is not possible to have a direct observation of ocular fundus and to identify where the light stimuli will be projected.Topographic visualization of the perimetric defect detected by microperimetry would allow retesting zones with reduced sensitivity  which are topographically visualized and displayable on the ocular fundi, avoiding worsening of the functional defect by a better modulation of the antiglaucoma therapy and allowing better monitoring of the pathologic functional damage.Therefore, we may conclude that Mp-1 microperimetry is a sensitive method that provides detailed topographic maps easy to read and could be used for glaucomatous patient's follow-up correlated with methods that are widely standardized in the glaucoma semeiotic.


CCL11 is probably responsible for the association. The association of a protective haplotype with a 10-year delay in the onset of Alzheimer’s disease and the identification of a CCL11 variant with possible functional roles in this association might allow the future development of immunomodulators with the potential to halve disease incidence.Alzheimer’s disease is the most common type of dementia, and it is characterized by a decline in memory or other thinking skills. The greatest risk factor for Alzheimer’s disease is advanced age. A recent genome-wide study identified a locus on chromosome 17 associated with the age at onset, and a specific variant in  It is one of the leading causes of death around the world and is associated with substantial direct and indirect costs for families and societies. The vast majority of AD cases have a late onset ; the disease is rare among younger individuals. Incidence of AD is known to increase with age, and age is the most important risk factor for the development of AD. The ability to delay the age at onset (AAO) of this disease through preventive or therapeutic approaches would have significant benefits, but no therapies have been successful in achieving this important goal , 2. A re.  used genAPP), Presenilin-1 (PSEN1) and Presenilin-2 (PSEN2). However, the genes involved in AAO variance mostly remain to be identified; linkage and candidate gene studies have established several possible associations with different loci, but the only consistently replicated modulator of AD AAO, in both familial and sporadic cases, is also the strongest genetic risk locus for the development of the disease (APOE) [. [APOE Ɛ4 allele with earlier onset and identified associations with CR1, BIN1 and PICALM. Burden analyses showed that APOE contributed 3.7 % of AAO variation; the other nine loci studied contributed 2.2 % when considered together [AAO is highly heritable in AD families. In early-onset cases, mutations in three genes are known to account for around half of familial cases : Amyloide (APOE) . More reAPOE) [.  performetogether .. [PSEN1 mutation (p.E280A) who are part of an extended family and who have been subjected to careful prospective phenotyping. By analyzing the genome sequence of individuals with different AAOs, the authors [CCL11 (which encodes the chemokine eotaxin-1) associated with a 10-year delay in AAO of AD [Many of these studies suffered from confounders, such as genetic and phenotypic heterogeneity, population stratification or inaccurate phenotyping. In fact, AAO of a disease is often a very difficult parameter to record with precision when assessed retrospectively. Lalli et al.  reduced  authors  identifiAO of AD .ABCA7, CLU, CR1, MS4A4E/MS4A6A, CD33, EPHA1, HLA-DRB5, HLA-DRB1, INPP5D, MEF2C and TREM2). Pathway analyses of GWAS data have identified the immune response as important in AD, and an integrated network analysis of genome and transcriptome data identified the immune and microglia module as significant for AD and TYROBP as the driver gene for this module [What seemed like a farfetched idea a few years ago is now a well established fact in AD: inflammatory and immune responses have a significant role in its development and progression. Several of the genetic loci associated with AD risk contain genes with known roles in inflammation, the complement system and the immune response in general , the CD33 risk allele influences triggering receptor expressed on myeloid cells 2 (TREM2) and the TREM1 risk allele is associated with a decreased TREM1/TREM2 ratio. Interestingly, the authors [TREM locus. TREM1 expression was found to increase with advancing age in younger but not in older individuals, and TREM1 variants were found to affect TREM2 expression in younger but not older people [Microglial activation and monocyte/macrophage-mediated inflammatory responses are currently particularly interesting areas of research on AD. To evaluate the relationship between known AD risk loci, Chan et al.  recently authors  also uncr people .APP, which suggests that manipulating the amyloid pathway could be a successful approach to reducing AD [The ability to delay the AAO of AD through preventive or therapeutic approaches would have significant benefits. A 2012 study found a protective variant in ucing AD . One canCCL11 in the most recent study by Lalli et al. [. [Similarly, the variant found in i et al.  also hast al. [.  p.A23T)TCCL11 inAPOE Ɛ4 allele not only modulates the AAO of AD in a sporadic setting, but also in carriers of presenilin mutations, and second, the identification of the protective effect of APP p.A673T in the Icelandic population showed that amyloid β has crucial roles in both early- and late-onset AD [It is important to understand how the recent findings from isolated and endogamous cohorts will translate to other populations, and what the implications are for the understanding of late-onset AD. Although single rare variants will not have high population-attributable risks, they will be extremely important for individual risk prediction and for the understanding of biology and pathobiology. Likewise it will be important to understand how findings made in early-onset familial cases will translate to the most common and sporadic late-onset AD. Two observations support significant commonalities between these two forms of disease, suggesting that factors important for early-onset and familial cases will also be important for late-onset and sporadic AD: first, the onset AD .CCL11 and AAO in the studied Colombian kindred [The mechanisms through which eotaxin and other peripheral immune molecules contribute to the disease process are still to be determined, but the identification of an association between  kindred , if vali


AbstractJasusedwardsii, is a temperate species of spiny lobster with established well managed fisheries in Australia and New Zealand. It has also been under consideration as a species with aquaculture potential. Agonistic behaviour has important consequences under aquaculture conditions that encompass direct effects, such as damage or death of protagonists, and indirect effects on growth that relate to resource access, principally food and refuge. This study aimed to identify and characterize behaviours and to make a preliminary investigation of their occurrence under tank culture. Juvenile Jasusedwardsii were examined in a flow-through seawater system using a remote video camera system. Twenty-nine behaviours were divided into three sub-groups: aggressive (11), avoidance (6) and others (12). Aggressive behaviours included attacks, pushing, lifting, clasping and carrying an opponent. Avoidance behaviours included moving away in a backwards-, forwards- or side-stepping motion as well as with more vigorous tail flips. These behaviours were components of twelve behavioural groups that described contact, attack and displacement between individuals. Activity was crepuscular with two clear peaks, one in the morning and the other in the evening. The occurrence of behavioural groups was not different between the morning and evening. The frequency of aggressive behaviours was not affected by changes made to stocking density or access to food. The implications of agonistic behaviours are discussed further in relation to developing aquaculture.The Southern rock lobster,  Jasusedwardsii, is a temperate species with established and well managed fisheries in Australia and New Zealand . The carapace was marked to identify each individual. The floor of each tank was marked with a 100 × 100 mm grid so distance and direction of movements could be assessed more easily.Behaviours were examined in two rectangular 25L (width 300 × length 500 × depth 250 mm) tanks that were part of a flow-through seawater system. The flow rate was 7.4 L minAn initial assessment of activity was made on two groups of five animals that were monitored continuously for seven days. Focal sampling  was usedPageBreakStocking density compared standard with a higher density: density was doubled from the standard density of 33.5 to 67.0 ind. m-2 by dividing the tanks in half . The effect of feed availability was investigated by comparing one with four feeding stations: the standard single feeding station in the centre of the tank or using four feeding stations simultaneously, one in each corner of the tank .Four new groups of five animals were used to investigate the occurrence of the behavioural groups. Occurrence was the number of times a behavioural group was observed as the first behaviour exhibited during each period of observation: each occurrence of behavioural unit was given a score of 1, observations were made over five days and data adjusted to one hour of observation . The eff-1). Mean group occurrence was calculated for each behavioural group as total occurrence divided by the number of individuals (five) and standardised to an hour of observation, it was expressed as occurrence per individual per hour (occ. Ind-1 h-1). Non-parametric statistical analysis using the Kruskal-Wallis test followed by multiple comparison using all pairwise comparisons was conducted due to preliminary nature of the data and the lack of homogeneous variance according to Levene’s test. Statistical analysis was conducted using SPSS Statistics version 22.For each experiment the same sampling regime was used: eight 15 min time blocks, four from the morning (05:00–06:45) and four from the evening (18:00–19:45) periods of peak activity, were analysed and behavioural group recorded against individuals . In contrast to Homarusamericanus, Jasusedwardsii exhibited several forms of contact, termed touch, between individuals that were part of low and medium intensity contact behavioural groups and that were not obviously agonistic. When alone Panuliruscygnus would sometimes straighten and hold their legs horizontally and suddenly settle in a behaviour termed splay , this meant that at the higher density the number of animals that did not exhibit any behaviour doubled. Although this possible density-effect requires further investigation, Jasusedwardsii may have behavioural mechanisms that will allow them to be held communally at high densities. Excellent survival (>90%) was achieved at up to 200 ind. m2 in small Jasusedwardsii juveniles (2 g), although weight gain over 118 days increased by as much as 80% when stocking density was decreased from 200 to 50 individuals per m2  and the absence of contact or attack behavioural groups helps explain why the long-term holding experiments were relatively successful. The main issue is clearly related to feed formulation and nutrition and not communal holding of rock lobsters . Further research into characterising the agonistic behaviours of various spiny lobster species with potential for commercial aquaculture is warranted. Better characterisation of agonistic behaviours in culture will assist in testing and improving husbandry management strategies which may include the use of chemical cues, better feed management and shelter designs.Twenty-nine behaviours were detected and characterised into three sub-groups in this study: aggressive (11), avoidance (6) and others (12). Among the behaviours recorded, the occurrence of medium and high intensity attacks and displacement events were minimal. This suggested


It is estimated there are 2.9 million diabetic patients in the United Kingdom, and around 5%-7% of patients have diabetic ulcers. This number will continue to increase globally. Diabetic ulcers are a major economic burden on the healthcare system. More than £650 million is spent on foot ulcers or amputations each year, and up to 100 people a week have a limb amputated due to diabetes. In T1DM, the level of IL-2 is reduced, and hence, wound healing is in a prolonged inflammatory phase. It is not known if IL-2 topical cream can shorten the healing process in T1DM patients.The objective of this study is to understand the pathophysiology in type 1 diabetes (T1DM) and investigate possible future treatment based on its clinical features. The hypothesis is that IL-2 cream can speed up wound healing in NOD mice and that this can be demonstrated in a ten-week study. An experiment protocol is designed in a mouse model for others to conduct the experiment. The discussion is purely based on diabetic conditions; lifestyle influences like smoking and drinking are not considered.Skin incisions will be created on 20 nonobese diabetic (NOD) mice, and IL-2 topical cream will be applied in a 10-week study to prove the hypothesis. Mice will be randomly and equally divide into two groups with one being the control group.T1DM patients have a decreased number of T regulatory (Treg) cells and interleukin 2 (IL-2). These are the keys to the disease progression and delay in wound healing. Diabetic ulcer is a chronic wound and characterized by a prolonged inflammatory phase.If the experiment is successful, T1DM patients will have an alternative, noninvasive treatment of foot ulcers. In theory, patients with other autoimmune diseases could also use IL-2 topical cream for treatment. The incidence of one major cause of chronic wounds, the diabetic ulcer, is increasing globally. There are an estimated 2.9 million diabetic patients in the United Kingdom; around 5%-7% suffer from diabetic ulcers, and 67% have one or more risk factors. These numbers are expected to increase. Up to 85% of amputations are preceded by foot ulcers, and a recent World Health Organization report pointed out that a reduction in amputations could be achieved through success in reducing ulcer incidence . AccordiType 1 diabetes (T1DM) is an autoimmune disease the cause of which remains unknown. In T1DM patients, β-cells in the pancreas are destroyed by autoimmune responses, resulting in a lack of insulin production. Scientists generally believe that it arises either by genetic or environmental factors, and those factors contribute three criteria for developing T1DM. First, β-cell-reactive T cells are being activated, and hence, a proinflammatory response is induced. Last and most importantly, the immune regulation of autoreactive responses fails .+ T cell and Fas on the β-cell, and eventually the β-cell antigen (MHC II molecule) will be released.The enterovirus is believed to be the most common viral infection causing T1DM. Other viruses like rotavirus, mumps, cytomegalovirus, and Epstein-Barr virus might be able to trigger autoimmune responses or associate with T1DM, as they have the ability to infect or lyse β-cells directly . During The reason why the infection is chronic is that specific antibodies from persistent infections can target Coxsackievirus (one of the RNA enteroviruses) and adenovirus receptors and FcγRII and III to enhance the infection of monocytes and macrophages. Antibody-dependent enhancement of infected monocytes and macrophages could increase the spread of the virus to β-cells and continuously stimulate autoimmunity.reg) cells are reduced due to an ineffective regulatory system. Hence, viruses keep replicating and persistently result in high concentrations.Initial viral infection in children is thought to be beneficial, as their bodies develop adaptive immunity. However, in T1DM patients, T regulatory  during antigen binding. This could increase the binding of IL-2, which is vital in T-cell proliferation. The IL2α chain CD25) is part of the IL-2 receptor and expresses during the predevelopment stage of the T and B lymphocytes. Chromosome 10 contains more than one susceptibility locus; they are called IDDM10. One of the regions is 10p15-p14, where the IL2α chain (CD25) is encoded. Mutation in this region would possibly lead to an IL-2 receptor α deficiency and hence affect the FOXP3 protein. Tag single-nucleotide polymorphisms were analysed, and it was discovered that the deficiency is likely due to linkage disequilibrium  is part .+ T cells express IL-22, while islet cells express the IL-22 receptor to activate the signal transducer and activator of transcription (STAT) 3 [Various T cells are activated when MHC I and II are expressed by a virus or β-cell. At the same time, exposure of proinflammatory cytokines on a β-cell drives the β-cell to upregulate IL-8 and chemokine (c-c motif) ligand 5 (CCL5), which draw target cells to migrate to the infection site by chemotaxis. In normal practice, CD4reg cells are the critical immune cells in T1DM immune modulation. These cells are important, as they monitor and kill autoreactive T cells to prevent pathological self-reactivity. In patients with T1DM, Treg cells no longer effectively control the islet autoreactive T cells and consequently, the body loses immune tolerance and continuously activates B cells and effector T cells. However, the nature of the dysfunction remains unclear [T unclear .reg cells. Its specific contribution in the differentiation and function of Treg cells remains uncertain. Several experiments have been done in animals and humans to test the effects of a defective FOXP3 or a deficiency in FOXP3. Evidence shows that FOXP3 is critical in maintaining self-tolerance by suppressing self-reactive T cells. One may expect that the faulty FOXP3 gene is crucial in contributing to T1DM. It is true that patients with FOXP3 deficiency will develop immunodysregulation polyendocrinopathy enteropathy X-linked syndrome (IPEX). More than 80% of IPEX patients acquire T1DM at very early onset [IDDM10, which encodes the IL2α chain (CD25), is the causative reason. CD25 is so important because it is the key cytokine for the fitness and function of FOXP3+ Treg cells. In other words, CD25 maintains the stability of and upregulates FOXP3. It is believed that it involves a direct pathway linking IL-2 signalling to the expression of the FOXP3 gene through STAT proteins [reg cell survival. In T1DM patients levels of IL-2 are reduced, and one can deduce that this will affect FOXP3 effects on Treg cells. It is believed that the loss of expression of FOXP3 not only results in a loss of regulatory function but could also be associated with the conversion of Treg cells into potential effector T cells and with the secretion of IFN-γ and IL-17. These cytokines may further reduce the secretion of IL-2 and form a vicious circle. Another suggestion is that the effector could become resistant to suppression [H1 and TH17 is the evidence of failure of immune suppression. The numbers increase throughout T1DM’s development.FOXP3 is a transcriptional factor which acts as lineage specification factor of Tproteins . Studiesreg cell function but is perhaps not involved in the expression of FOXP3 [reg cells appear unusual. Investigation shows that performance of suppression by Treg cells is weaker in the higher ratio of effector T cells [reg cell functions are impaired in patients with T1DM. Perhaps this affects the wound-healing processes.Cytotoxic T-lymphocyte–associated protein 4 (CTLA-4) is a protein receptor on the T-cell surface that downregulates possibly by recruiting phosphatase to the TCR. There is controversy about the main functions of CTLA-4; it could be involved in another essential part of the Tof FOXP3 . Serum lof FOXP3 . The rea T cells . DiffereAlthough the T1DM pathophysiology is different from T2DM, both types of diabetes share many clinical features . The mechanism is not fully discovered yet; a paper suggested insulin therapy might be a primary inducer. In a cultured hepatocytes model, significant insulin resistance was developed under prolonged exposure to insulin, and this is similar to the setting of insulin therapy . Apart fIt is believed that there are inflammatory events around adipose tissue and immune responses to elevated blood glucose. Those events can be observed in all tissues that process energy homeostasis  . Eventuareg cells and CD4+ T cells dramatically decreases with increasing levels of obesity [reg cells are not just involved in immune response; they could also increase the circulation of blood glucose.Adipose tissues per se can induce immune responses. Although the mechanism that initiates the inflammation is unknown, inflammation is associated with an accumulation of immune cells in obese patients. It is deduced that hypertrophy is responsible, as cellular contents might leak into cellular spaces and promote an inflammatory response or induce adipocyte death . Earlier obesity . The impIncreased tumor necrosis factor α (TNF-α) has been detected in the adipose tissue of obese mice and is recognized as one of the immune characteristics . This haMeanwhile, hyperglycemia provokes a similar immune response and activates different cytokines, transcriptional factors, and ROS in the islets. Glucose can stimulate nonenzymatic glycation and generate advanced glycation end products (AGEs), one of the factors in the stimulation of the pattern recognition receptor, RAGE. Once RAGE is activated, the production of nuclear factor-κB (NF-κB), ERK1, ERK2 (MAPK) and ROS will be induced . All theThus far, few activation mechanisms of IL-1β have been mentioned; these are induction via TLR, IAPP, and ROS. In addition to induction, IL-1β could be upregulated under high glucose concentration, as more Fas is likely to be expressed on β-cells, and Fas could induce such secretion.IL-1 acts as a sensor of metabolic stress and activates immune response in β-cells, insulin-sensitive tissue, and blood vessels . In low Diabetic nephropathy is a typical complication in both types of diabetes. It is a progressive and chronic kidney disease (CKD) in the glomerulus and is characterized by hyperfiltration . MicroalAn experimental wound model has been applied in mice and studied with different analyses. It included study of the kidney glomerular tuft area, histology and immunofluorescence, tissue gnostics quantification, and polymerase chain reaction . AlthougStaphylococcus aureus; it is believed that the infection is responsible for increased morbidity and mortality [The physical conditions of wounds have also been reviewed. Dryness, rashes, microangiopathy, and even calciphylaxis were considered to correlate with the severity and duration of the CKD state . Drynessortality . InfectiAtherogenesis is considered a threat to healing by contributing prolonged inflammation and inadequate blood and oxygen supply to tissue. Although it alone does not cause ulceration, it can be serious enough to cause amputation of affected limbs . Its chaNitric oxide (NO) has an imperative role in protecting blood vessels by regulating vasodilation, as well as mediating molecular signals and thus preventing leukocyte and platelet interaction. However, in diabetic patients, hyperglycemia, FFA, and insulin resistance are believed to be the major cause of reduced level of NO. Hyperglycemia and FFA can increase the generation of ROS and hence ROS inactivates NO by disturbing the phosphatidylinositol 3 kinase pathway via the activation of protein kinase C. Meanwhile, FFA could further weaken endothelial function by reducing endothelium-dependent vasodilation. When the liver deals with excess FFA, it enriches production of very-low-density lipoprotein. Plaque is easily formed in diabetic patients because plasma coagulation factors and lesion-based coagulants are elevated, whereas thrombomodulin and protein C decline. A reduced level of NO and endothelial dysfunction favor the migration of vascular smooth muscle cells into nascent atherosclerotic lesions.Although the individual role of C-reactive protein (CRP) in atherogenesis is still under investigation, it could contribute to atherogenesis together with LDL. It is reported that the CRP level is elevated during inflammation and under hyperlipidemic conditions, and it presents throughout all stages and colocalizes in activated complements in humans . CRP is Diabetic neuropathy is the most common diabetic complication in both T1DM and T2DM, with 45%-60% of ulcerations believed to be purely neuropathic , capillaDamaged nerves to intrinsic foot muscles lead to an inequity between flexion and extension of the affected foot. Skin breakdown and ulceration are progressively shown, since foot deformities cause abnormal bony prominences and build pressure points. In addition, neuropathy reduces the functionality of sweat and oil glands. As a result, the foot loses the natural ability to moisturize the skin and hence becomes dry. Often, dry skin increases susceptibility to microorganisms and subsequently to the developing of infection. Patients with severe peripheral neuropathy may also suffer from neuropathic edema in the lower legs. This is possibly related to vasomotor changes and arteriovenous shunting . It is gDiabetic neuropathic ulcers are most likely to be seen on a patient’s palm and the soles of the feet. In general, a diabetic foot ulcer is more predominant. Ulcers do not happen spontaneously; they can begin with minor skin infections or cuts. Wound healing is a complex process involving blood clotting, inflammation, proliferation, tissue remodelling, and eventually wound closure; the phases overlap each other. Diabetic wounds are chronic wounds in which the wounds are trapped in the inflammation phase and do not heal within three months .Wound healing is a complicated process and involves the nervous and immune systems. The first stage, the inflammatory phase, begins with a passive leakage of circulating leukocytes from damaged blood vessels towards the wound site. T cells and Langerhans cells secrete cytokines and chemokines in response to the injury . VariousLike other chronic wounds, a diabetic ulcer is characterized by the imbalance of proinflammatory and anti-inflammatory factors. This feature possibly arises due to macrophage dysfunction. In diabetic mice, macrophages isolated from a wound showed momentous impairment in efferocytosis; in other words, cell debris was not cleared up effectively, resulting in an accumulation of apoptotic cells. Fas ligands drove the apoptotic cell burden to enhance proinflammatory but weaken anti-inflammatory cytokine response . AlthougThere are three important events in the proliferative phase: angiogenesis, formation of granulation tissue and the extracellular matrix (ECM), and reepithelialization. Angiogenesis is the most critical of these events and is the key process of successful wound healing. New blood vessels are essential to deliver nutrients and oxygen to support the growth of cells and tissue and the formation of a wound matrix .Cellular hypoxia, a major issue in chronic wounds, is one of the consequences when angiogenesis is blocked or retarded. It is thought that angiogenesis could be affected by diabetic neuropathy to some degree. Various cells need oxygen to survive and function. For example, fibroblasts and epithelial cells require oxygen to migrate, and collagen fibril crosslinking requires oxygen to carry out hydroxylation. Infection is likely to occur with insufficient oxygen, as the bactericidal potency of leukocyte oxidative phosphorylation is changed.Formation of new granulation tissue and reepithelialization are the mechanisms to restore the function of the skin and bring about permanent closure of the wound gap. Granulation tissue is formed in the dermis, performed by fibroblasts, whereas reepithelialization is carried out in the epidermis by keratinocytes. Both mechanisms correlate with each other closely to bring about the outcome.Reepithelialization begins with the migration of keratinocytes. Upon arrival at the injured site, keratinocytes modify or dissolve cell-cell and cell-matrix adhesions by releasing collagenases (MMP1) and different types of MMPs. These changes facilitate the formation of a laminin V and collagen IV-rich basement membrane through provisional matrix substrates. Debris and blood clots are also dissolved during this progression to benefit the migration of other cells. Keratinocytes themselves produce growth factors and basement membrane proteins to accelerate reepithelialization . ECM is Normal fibroblasts come from different parts of the body and migrate to the wound site. The primary source is the surrounding healthy dermis, while bone marrow progenitor cells and circulated fibrocytes are alternative sources. These cells migrate and join together in response to stress fibers and various growth factors promoting the migration. Fibroblasts use the fibrin crosslinking fibers, formed in the later period of the inflammatory phase, to migrate across the wound, and eventually they adhere to fibronectin. Fibroblasts synthesize, bind, and align collagen fiber into the wound bed in the contraction step . ContracThere are a few difficulties to consider in the proliferative phase. First, MMPs are excessively synthesized in diabetic patients; the high level of activity could cause unnecessary collagen loss. A high level of MMP1 is vital in wound healing; however, too much MMP8 and 9 could be dangerous. A longitudinal study was conducted on T2DM patients with foot ulcers investigating the impact of MMP levels on recovery. The study recorded levels of MMPs and tissue inhibitors of metalloproteinases around the wound edge and concluded that reduced MMPs do not contribute to good healing . The secreg cells have been discussed in detail above. In T1DM, the level of IL-2 is reduced, and hence, wound healing is in a prolonged inflammatory phase. If optimum IL-2 dosage is given to patients, perhaps it can increase the Treg cell survival rate and response; this likely can be achieved through immunotherapy. Immunotherapy is often used on cancer patients, and research on treating patients with T1DM is ongoing. In 2013, experimental results showed that a low IL-2 dose could increase the number of Treg cells; however, a high dosage might induce β-cell impairment in humans. IL-2 was given by injection in the experiment, and some T1DM patients had serious injection site reactions [reg cells in inflamed nonlymphoid tissue. Although the effect was more remarkable in tissue with ongoing autoimmunity such as the pancreas, results also showed that IL-2 can upregulate CD25 expression and suppress IFN-γ in skin. Moreover, no harmful effects were detected in other cells, which means that the risk of generalized immune suppression is low. These two experiments demonstrated that IL-2 could be a safe therapeutic choice for treating inflammation. In terms of the IL-2 effects on wounds, experiments have been done on Adriamycin-treated [Impaired function and a decrease in numbers of Teactions . Similar-treated  and Lewi-treated  mice andEscherichia coli for synthesis. The first aim is to study the effect of IL-2 cream on the wound-healing process to prove the hypothesis. The second aim is to test the cream’s pharmacokinetics and determine the effective dosage (with no serious side effects).The hypothesis is that IL-2 cream can speed up wound healing in NOD mice and that this can be demonstrated in a ten-week study. Inspired by the success of insulin cream, it is decided that IL-2 will be given in cream form at a low dosage. A human IL-2 gene could be modified by genetic technology and will subsequently be inserted into A ten-week-old male NOD mouse’s hair will be removed before the surgery. During the surgery, the mouse will be anesthetized by open-mask method and circular dorsal skin incisions at least 1 cm in diameter will be created on the left- and right-hand sides in parallel see . The lefAn aseptic technique will be used to prevent bacterial infection. After surgery, a thin protective dressing will immediately be placed on the wound site. Throughout the experiment, all mice will be fed the same standard mouse diet and water and will live in a pathogen-free and temperature-controlled animal facility with a 12-hour light and dark cycle until sacrificed. Mice will be euthanized 24 hours after the last treatment.Twenty male mice will be randomly and equally divided into two groups. In one group, a thin layer (0.1 g) of IL-2 cream will be applied on each wound every 24 hours; the other group will be the control group and will receive typical wound care. The cream will first be applied 24 hours after the surgery. Both groups will have typical wound care, which includes changing dressings and cleaning the wounds daily with saline water.As in a previous study, recombinant human IL-2 will be given in a 25,000 IU dosage per day  for the reg cells will be isolated by magnetic cell separation and analysed by fluorescence-activated cell sorting, chosen for studying the surface expression of CD4+ and CD25+ of the Treg cells [2 after the mice are sacrificed [Both groups will have the following measurements unless otherwise specified. Two blood samples, a total of 0.3 mL, will be taken from the tail vein before surgery and every 24 hours after daily care. One of the samples will be used to check the level of insulin (pmol/L) in the serum and the white blood cells (K/uL) and the differential of white blood cells, based on 100 cells counted in plasma. Cytokines in serum will specifically be detected by cytokine array for quantitative measurement and will be observed for changes in the amount. Teg cells . A woundeg cells . Maximalcrificed .Body weight will be measured daily in grams by an electronic balance. A pharmacokinetic test will be conducted by high-pressure liquid chromatography to measure the concentration of IL-2 in plasma in IU/mL .t test with a 95% confidence interval will be calculated to demonstrate the effect of IL-2 cream in both shallow and deep wounds as well as the difference between the two groups.A 2-tailed unpaired Student The number of immune cells and cytokine trends is expected to be similar to that in normal wound-healing progress. The IL-2 level should reach its peak after 24 hours of administration, while the pharmacokinetics are unknown and should be investigated. It is hoped that the insulin level will increase as well. H&E staining should show a shorter epithelial gap and granulation tissue, and the total epithelial closure of the wound should be bigger in contrast to the control group.To limit the suffering from the incisions as much as possible, the mice will be given anesthesia before surgery, and the surgeries will be completed within 15 minutes. A mortality risk exists, as this is an invasive operation, but the risk should be low because the surgery does not involve injury to vital organs. Moreover, proper wound care will be given to minimize wound deterioration and infection. All mice in this experiment will receive humane care throughout the study and euthanasia at the end of the study.IL-2 has been proven effective in cancer patients and has become one of the current treatments. One can see that there is a therapeutic value in conducting a similar experiment. The experiment is unlikely to be replaced by computer models or conducted on humans at this stage, so mouse models are chosen. Dosages will be given in a safety range based on similar experiments and may be adjusted after weekly evaluation of health and progress.As this is a pilot experiment and no previous data exists, only small numbers of mice are used. The sample size should be big enough to show the differences and prove the hypothesis. Male mice are studied to prevent unnoticed pregnancy in female mice.This project is currently on hold as funding has not been secured yet.Lifestyle changes or surgical intervention could significantly improve ulcers in T2DM patients. However, clinical procedures might not be as effective on T1DM patients. Considering the success of imiquimod cream for treating melanoma and the latest results of IL-2 therapy in diabetic patients, it is realistic to develop topical immunotherapy. So far, no related experiment has been done on animal models or humans. If this experiment is successful, T1DM patients will have an alternative, noninvasive treatment. Since the use of topical cream doesn’t require much education, it will be suitable for both young and elderly patients and will enhance compliance. In addition, the risk of infection and serious skin reaction could be reduced in comparison to intravenous treatment. It is assumed that this treatment can be safely used without contraindications with oral medicine. Last, it can protect skin from dryness as other creams do and can maintain a moist environment for the wound. In theory, patients with other autoimmune diseases could also use IL-2 topical cream for treatment.


Consistent with this hypothesis, we show that SBF can regulate promoters with E2F binding sites in budding yeast.Although cell cycle control is an ancient, conserved, and essential process, some core animal and fungal cell cycle regulators share no more sequence identity than non-homologous proteins. Here, we show that evolution along the fungal lineage was punctuated by the early acquisition and entrainment of the SBF transcription factor through horizontal gene transfer. Cell cycle evolution in the fungal ancestor then proceeded through a hybrid network containing both SBF and its ancestral animal counterpart E2F, which is still maintained in many basal fungi. We hypothesize that a virally-derived SBF may have initially hijacked cell cycle control by activating transcription via the DOI:http://dx.doi.org/10.7554/eLife.09492.001 Living cells grow and divide with remarkable precision to ensure that their genetic material is faithfully duplicated and distributed equally to the newly formed daughter cells. This precision is achieved through a series of steps known as the cell cycle. The cell cycle is ancient and conserved across all Eukaryotes, including plants, animals and fungi. However, some of the core proteins present in animals and fungi are unrelated. This raises the question as to how a drastic change could have occurred and been tolerated over evolution.In animals and plants, a protein called E2F controls the expression of genes that are needed to begin the cell cycle. In most fungi, an equivalent protein called SBF performs the same role as E2F, but the two proteins are very different and do not appear to share a common ancestor. This is unexpected given that fungi and animals are more closely related to one another than either is to plants.Medina et al. searched the genomes of many animals, fungi, plants, algae, and their closest relatives for genes that encoded proteins like E2F and SBF. SBF-like proteins were only found in fungi, yet some fungal groups had cell cycle regulators like those found in animals. Zoosporic fungi, which diverged early from the fungal ancestor, had both SBF- and E2F-like proteins, while many fungi later lost E2F during evolution.So how did fungi acquire SBF? Medina et al. observed that part of the SBF protein is similar to proteins found in many viruses. The broad distribution of these viral SBF-like proteins suggests that they arose first in viruses, and a fungal ancestor acquired one such protein during a viral infection. As SBF and E2F bind similar DNA sequences, Medina et al. hypothesized that this viral SBF hijacked control of the cell cycle in the fungal ancestor by controlling expression of genes that were originally controlled only by E2F. In support of this idea, experiments showed that many E2F binding sites in modern genes are also SBF binding sites, and that E2F sites can substitute for SBF sites in SBF-controlled genes. Future experiments in zoosporic fungi, which have animal-like and fungal-like features, would provide a glimpse of how a fungal ancestor may have used both SBF and E2F. These experiments may also reveal why most fungi have retained the newer SBF but lost the ancestral and widely conserved E2F protein.DOI:http://dx.doi.org/10.7554/eLife.09492.002 The networks regulating cell division in yeasts and animals are highly similar in both physiological function and network structure  Cross e. For exaCLN1 and CLN2, which also inactivate Whi5 to complete a positive feedback loop  during cell cycle entry  and animal (Metazoa) lineages would suggest that this regulatory complex, rather than the fungal SBF-Whi5 complex, was present in the last eukaryotic common ancestor . The seqTo address this question, we examine conserved and divergent features of eukaryotic cell cycle regulation. In contrast to previous work that considered a protein family of cell cycle regulators in isolation , we studOur results indicate that LECA likely had complex cell cycle regulation involving at least one Cdk, multiple cyclin families, activating and inhibitory E2F transcription factors, and pRb-family pocket proteins. Identifying the LECA repertoire helps establish that the emergence of SBF-Whi5 is abrupt and distinguishes fungi from all other eukaryotes. We show that basal fungi can have both ancestral E2F-pRb and fungal SBF-Whi5 components. Thus, fungal evolution appears to have proceeded through a hybrid network before abruptly losing the ancestral components in the lineage leading to Dikarya. This supports the hypothesis that network structure, rather than the individual components, has been conserved through the transition to fungi and argues against the case of convergent evolution.cis-regulatory elements targeted by the ancestral cell cycle regulator E2F, much like extant viral oncogenes.Our data confirm that SBF shows homology to KilA-N, a poorly characterized domain present in prokaryotic and eukaryotic DNA viruses. Thus, SBF is not derived from E2F  and likely emerged through horizontal gene transfer in the fungal ancestor. We show that SBF can regulate promoters with E2F binding sites in budding yeast. We then use high-throughput in vitro binding assay data to elucidate the shared nucleotide preferences of E2F and SBF for DNA binding. These data suggest that a viral SBF may have initially hijacked cell cycle control, activating transcription via the Recent work shows that the last eukaryotic common ancestor (LECA) already had a complex repertoire of protein families . Indeed,Arabidopsis thaliana, Homo sapiens, Schizosaccharomyces pombe, and Saccharomyces cerevisiae. These HMMs were then used to query the sequenced eukaryotic genomes for homologs of both fungal and animal cell cycle regulators  outside of Fungi, we did find animal-like cell cycle regulators in Archaeplastida, Amoebozoa, SAR, Haptophyta, Cryptophyta, Excavata and Metazoa . For exae.g., Cdk7-9, which regulate transcription and RNA processing, and Cdk5 (yeast Pho85), which regulate cell polarity, nutrient regulation, and contribute to cell cycle regulation in yeast (Members of the Cdk1-3 family (i.e. CdkA in plants) are also broadly conserved across eukaryotes, suggesting they were the primary LECA cell cycle Cdks ; see Materials and methods and Fonticula alba) and because Microsporidia are fast-evolving fungal parasites with reduced genomes  can have both fungal and animal cell cycle regulators, which likely represents the ancestral fungal hybrid network. SBF-Whi5 in budding yeast plays a similar role to E2F-pRb in animals, which suggests that these pathways were functionally redundant in an ancestral hybrid network. This redundancy would lead to the evolutionary instability of the hybrid network and could explain why different constellations of components are present in the extant zygomycetes and basal fungi , which is homologous to the KilA-N domain . Our rathomologs . We willTo further test the possibility that SBF was a gene duplication of E2F/DP and evolution was so rapid that sequence identity was lost, but structural and functional homology to E2F/DP was maintained, we looked for possible evidence of structural homology. The DNA-binding domains of SBF/MBF  and E2F/S. cerevisiae in the absence of DNA, originally suggested that Mbp1 recognizes its MCB  binding site via a recognition helix  are found in prokaryotes (85%) with a smaller fraction (10%) found in eukaryotes and a smaller fraction found in DNA viruses (5%). The KilA-N domains in prokaryotes appear to be either integrated by or derived from prokaryotic DNA viruses (i.e. bacteriophage), and thus, we will treat them as such. Within the eukaryotes, all known KilA-N domains are found in fungal genomes with three notable exceptions.Trichomonas vaginalis, a parasitic excavate with 1000+ KilA-N domains  and Rhodius prolixus ('triatomid bug'). The one KilA-N domain in A. pisum genome has a top blast hit to eukaryotic DNA viruses (e.g. Invertebrate Iridescent Virus 6). The three KilA-N domains in R. prolixus have top blast hits to prokaryotic DNA viruses (e.g. Enterobacteria phage P1). The diverse and sparse distribution of KilA-N domains throughout the eukaryotic genomes is consistent with their horizontal gene transfer into hosts from eukaryotic DNA viruses and/or via engulfed bacteria that were infected with prokaryotic DNA viruses. In fact, the horizontal transfer of genes between Mimivirus and their eukaryotic host, or the prokaryotic parasites within the host, has been shown to be a more frequent event that previously thought  and 'Zygomycetes') and built a phylogenetic tree . There aOf all the members of the SBF+APSES family, the most likely candidate to be a 'founding' TF is SBF, as it is the only member present in all fungi . In buddCLN2 promoter, which has three binding sites for SBF  in a nucleosome-depleted region  to hijack cell cycle control in the fungal ancestor, it must have been able to both bind E2F regulatory regions and then activate the expression of genes under E2F in a cell cycle-regulated fashion. The overlap between the conserved E2F and SBF consensus sites suggests that ancestral SBF could bind E2F regulatory regions . Howeverd region . Removald region . We replon  assays  of humanSGCGSN) and SBF-only (NNCRCGNN) motifs indicate that differential specificity between E2F1 and SBF is mediated by the nucleotides in the 3rd and 4th positions (underlined) before the invariant CG at the 5th and 6th positions. E2F has a strict preference for G in the 4th position, where as SBF has a strict preference for C in the 3rd position  with our empirically-defined DNA binding sites from PBM assays   . In the id state .Clearly, something similar happened during cell cycle evolution. It appears that the replacement of the E2F-pRb transcription regulatory complex with the SBF-Whi5 complex proceeded via a hybrid intermediate that preserved its function. In the hybrid intermediate, E2F-Rb and SBF-Whi5 may have evolved to be parallel pathways whose functions overlapped to such an extent that the previously essential E2F-Rb pathway could be lost in the transition to Dikarya. Interestingly, many basal fungi (e.g. Chytrids) have preserved rather than lost this hybrid intermediate, which suggests that each pathway may have specialized functions. Chytrids exhibit both animal  and fungal features  whose synthesis needs to be coordinated with cell division. The preservation of the hybrid network in chytrids could then be explained if animal-like and fungal features are regulated by the E2F-Rb and SBF-Whi5 pathways respectively . Once FuThe origin of the hybrid network at the base of Fungi is abrupt and may have been initiated by the arrival of SBF via virus. Many tumor viruses activate cell proliferation. For example, the DNA tumor viruses Adenovirus and SV40 highjack cell proliferation in part by activating the expression of E2F-dependent genes by binding pRb to disrupt inhibition of E2F . While tArabidopsis thaliana, Homo sapiens, Schizosaccharomyces pombe, and Saccharomyces cerevisiae, to build the profile-HMMs used to detect homologs. Our profile-HMM search used a stringent E-value threshold of 1E-10 to detect putative homologs in the 'best' filtered protein sets (where available) of our 100+ eukaryotic genomes (see We used Profile-Hidden Markov Models (profile-HMMs) to detect homologs for each of the families studied, using the HMMER 3 package . Profileomes see .Our profile-HMM for E2F/DP family only detects E2F or DP, where as our profile-HMM for SBF/MBF family only detects SBF/MBF (or APSES). The same protein was never identified by both profile-HMMs because the sequence profiles and the structure are non-homologous. In the case of basal fungi, which have both E2F/DP and SBF/MBF, all proteins classified as an E2F/DP had clear homology to E2F or DP  to align the sequences of eukaryotic protein family members . We thenà la Bayes aLRTs (aBayes) were calculated to determine branch support from two independent PhyML 3.0 runs. For Bayesian inference we used PhyloBayes (rather than the more frequently used MrBayes) because it allows for site-specific amino-acid substitution frequencies, which better models the level of heterogeneity seen in real protein data (https://github.com/danlwarren/RWTY)  with 100 pseudo-replicates. PhyML 3.0 phylogenetic trees were obtained from five independent randomized starting neighbor-joining trees (RAND) using the best topology from both NNI and SPR moves. Non-parametric Shimodaira-Hasegawa-like approximate likelihood ratio tests (SH-aLRTs) and parametric ein data . We perfen/RWTY) . The besà la Bayes aLRTs (aBayes) for PhyML. These different support metrics complement each other in their advantages and drawbacks. SH-aLRT is conservative enough to avoid high false positive rates but performs better compared to bootstrapping  posterior probability for the Bayesian inference, (2) rapid bootstrap support  for RAxMtrapping . aBayes trapping .CLN2pr-GFP-CLN2PEST constructs were all derived from pLB02-0mer  (BSgenome.Hsapiens.UCSC.hg38) and the annotation package org.Hs.eg.db from Bioconductor (v3.2)  . Only rer (v3.2) . In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your submission has been evaluated by Diethard Tautz (Senior editor), and three reviewers, one of whom is a member of our Board of Reviewing Editors.Thank you for submitting your work entitled "Punctuated evolution and transitional hybrid network in an ancestral cell cycle of fungi" for peer review at The reviewers have discussed the reviews with one another and the Reviewing editor has drafted this decision to help you prepare a revised submission.Summary:This paper addresses the evolution of cell cycle specific transcription factors and cyclins in fungi. All of us feel that the cyclin part was less interesting, more speculative, and poorly motivated. Your story about evolution of SBF-like transcription factors is the more interesting aspect. Unfortunately, the key discovery discussed in the paper, namely the origin of SBF from DNA viruses was reported long ago by Koonin and this aspect of the paper is therefore not entirely novel. More interesting is the finding that basal fungi appear to have both SBF and E2F/Rb like factors, confirming that E2F/Rb must have been replaced by SBF early on in the fungal lineage. In a way, one already knew that this must be case since the E2F system is present in plants as well as animals, implying that it must have been present in the common ancestor of fungi and animals, which are closer to each other than plants. We already knew therefore that E2F must therefore have been lost early in the fungal lineage. However, placing when this took place is certainly interesting and an important advance in our understanding.All of us were unimpressed by the experiment that E2F site from a histone promoter in mammals can replace the SBF sites in the CLN2 promoter. Results are also consistent with the obvious alternative hypothesis that SBF and E2Fs are distant homologues and the binding specificity has simply been conserved during evolution. We don't see how any experiment along these lines could be said to support the hijacking hypothesis (to the exclusion of the obvious competing hypothesis). Furthermore, even if SBF and E2F are non-homologous, they have overlapping binding specificity. Since sequence specific transcription factors allow degeneracy (or wobble positions) and bind to short DNA sequence families (motifs), these experiments show only that the E2F sites that were chosen probably overlap the specificity of SBF enough that they can be regulated. Therefore, these experiments really add nothing to the hijacking model, except to confirm the observation that the two transcription factors have overlapping specificity.Overall, the paper has one really key novel observation, namely the detection of the E2F/Rb system in basal fungi and this is certainly worth publishing. However, you need to show that there really are both classes of proteins in the basal fungi. You need to consider and rule out alternative explanations. For example, are you simply assigning some of the homologues in the basal fungi to be E2Fs and some to be SBF/MBFs because they have reached the limit of homology detections?The problem is that this key finding has been wrapped up in a paper that merely repeats Koonin's findings and addresses the evolution of fungal cyclins in a manner that is not particularly revealing. We would recommend a far shorter paper focusing on the one key finding.Other Major Comments:Note some of these points may not be relevant when you shorten this article. Nonetheless, these comments may help if you decide to wrap up the remaining content for another journal:Reviewer 1:With regard to your models in Reviewer 3:1) For example, they need to show convincing evidence that the diversity of the SBF/MBFs falls outside of the diversity of the E2Fs, and that the viral proteins fall within the SBFs and outside of the E2Fs. Figure 6a shows a phylogeny of SBFs, but it doesn't include the E2Fs.Even more important, they need to show that there really are both classes of proteins in the basal fungi. They need to consider and rule out alternative explanations. For example, are they simply assigning some of the homologues in the basal fungi to be E2Fs and some to be SBF/MBFs because they have reached the limit of homology detections?2) The structural evidence could represent key supporting data to show that E2Fs and SBFs really are two different families. However, is not presented in a very convincing way."it is possible that fungal SBF is a duplicated and hyper-evolved E2F. However, the number and topology of secondary elements  that stabilize and pack around the core helix-turn-helix are different."Does the number and topology of secondary structural elements always stay the same in homologues? If not, how does this support the hypothesis that they are non-homologous?"Crystal structures of the DNA binding domains of E2F and SBF show that they are not structural homologues  see ."Here the authors just state that E2F and SBF are not structural homologues. What is it about the structures in both proteins in the basal fungi. The authors should show and explain the evidence that the putative homologues in the basal fungi are likely to adopt one structure and not the other. The alignment in 3) Even if the structural evidence is sound, and the proteins are non-homologous, the authors specific argument rests on the identification of I think the authors need to consider their evolutionary model much more carefully and design experiments that actually test it.eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Diethard Tautz as the Senior Editor.Thank you for submitting your work entitled "Punctuated evolution and transitional hybrid network in an ancestral cell cycle of fungi" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.The reviewers see improvements in your revision but are still far from being satisfied. Ordinarily, this level of criticism on a revised submission would be considered unacceptable, however, we have decided to provide you with an opportunity to substantially revise the manuscript one last time. If you have questions about our instructions, please write and ask for clarification. Of course, given the concerns we have expressed we would understand if you choose to submit to a different journal. Please advise as to your course of action as soon as possible.I encourage you to shorten this further (as per multiple reviewers' request).Reviewer #1:The authors did a good job of clarifying the structural data and present a much more convincing case that the two proteins are non-homologous.However, they did very little to address my other concerns.Rather than doing a detailed, focused analysis to convince the reader of their hypothesis that the basal fungi contain both transcription factors, and that the SBFs are inherited from a single horizontal transfer event, they simply bolded the basal fungi species names in an alignment. They did not show any phylogenetic analysis that would rule out competing evolutionary scenarios  or technical issues (such as homology detection problems). In response to my comments about the latter, they simply stated that their HMMs don't make any classification errors:"Our profile-HMM for E2F/DP family only detects E2F or DP, where as our profile-HMM for SBF/MBF family only detects SBF/MBF (or APSES)."In fact, I agree with the authors that the HMMs are probably very accurate, but why not show data to support this claim? not present) is a statistical process and always has an element of uncertainty.  Since the detection of these homologs in the basal fungi and no SBFs in other eukaryotes is the key result of this paper, why not make a convincing case to support it (instead of the unnecessary The authors don't seem to appreciate (or have chosen to ignore) the fact that remote homology detection  which of them SBF is likely to regulate. The observation that SBF can bind the histone binding site (on its own) really adds nothing.I think the most important thing for the authors to add is a phylogenetic analysis showing that the data support the hypothesis of a single transfer event in the ancestral fungi.As a secondary point, I guess if they really want to include the histone binding site experiment, they should include it in the context of a survey of other known E2F and SBF binding sites, and consider to what extent SBF could have taken over the sites. Reviewer #2:A primary concern of the previous round of review was the distinction between E2F and SBF/MBF homologs in basal fungi. The new figure showing the alignment with strong conservation within each family is convincing to me that the distinction drawn is not caused by a limit in homology detection. Adding E2F family proteins to Overall, this work extends the earlier hypothesis that fungal SBF was transferred from viruses, by narrowing down the phylogenetic context of that transfer and identifying extant species with both SBF and E2F homologs. Much work must be done to characterize the degree to which those species truly have a "hybrid" network, but the present work lays an intriguing foundation. To me, the alternative hypothesis of rapid divergence between the SBF and E2F families is less parsimonious than horizontal transfer of SBF, although it is possible.eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Diethard Tautz as the Senior Editor.Thank you for submitting your article "Punctuated evolution and transitional hybrid network in an ancestral cell cycle of fungi" for consideration by The manuscript needs minor revisions.Essential revisions:Reduce the number of figures and make them less busy and higher quality (visible). Is each figure really essential? Can it be treated as a supplement to the primary figure (see how this is done for other papers we publish)? People are much less likely to read a paper with many busy figures.Reviewer #1:The authors have greatly improved the paper, and have addressed my concerns about technical issues related to the homology detection, as well as included phylogenetic analysis that shows the fungal proteins to be monophyletic, consistent with their model of a single transfer event. They have also added a reasonable bioinformatics analysis that shows the overlap in specificity between SBF and E2F.Reviewer #2:Overall, this work extends the existing viral-origin hypothesis for SBF, primarily by identifying species carrying both SBF and E2F. The additional analyses in this round of review further support this hypothesis. The authors more detailed analysis of HMM performance  gives moAt this point, I have no substantive concerns. We thank you for the opportunity to revise and resubmit our manuscript.We have modified the main text to address reviewer comments and we expanded details of data analysis in Methods. We also modified the Figures:1) We reorganized the names and eukaryotic species tree in 2) We split old 3) We split old 4) We improved and updated new 5) We deleted old Summary:This paper addresses the evolution of cell cycle specific transcription factors and cyclins in fungi. All of us feel that the cyclin part was less interesting, more speculative, and poorly motivated. Your story about evolution of SBF-like transcription factors is the more interesting aspect. Unfortunately, the key discovery discussed in the paper, namely the origin of SBF from DNA viruses was reported long ago by Koonin and this aspect of the paper is therefore not entirely novel. More interesting is the finding that basal fungi appear to have both SBF and E2F/Rb like factors, confirming that E2F/Rb must have been replaced by SBF early on in the fungal lineage. In a way, one already knew that this must be case since the E2F system is present in plants as well as animals, implying that it must have been present in the common ancestor of fungi and animals, which are closer to each other than plants. We already knew therefore that E2F must therefore have been lost early in the fungal lineage. However, placing when this took place is certainly interesting and an important advance in our understanding.We thank the reviewers for recognizing the significance of our manuscript .The Whi5-SBF pathway in yeast was only recently established as functionally analogous to the Rb-E2F pathway in mammals . These authors were careful to classify Whi5-SBF as "functional analogues" rather than homologs because Whi5-SBF lacked sequence identity to Rb-E2F. However, neither these authors nor the cell cycle community made the connection between viral KilA-N domains, SBF, hybrid networks, and early fungal evolution -- despite the fact that Koonin and colleagues had already published on the viral origins of APSES .eLife .Our manuscript is respectful of the existing literature. We cite multiple papers that already noted that Rb-E2F is likely ancestral because it is found in both plants and animals . However, none of these papers addressed the evolution of SBF versus E2F in the fungal lineage or established the example of cell cycle rewiring via a hybrid network. To this end, we think that our manuscript will be of great interest to the broader community that reads All of us were unimpressed by the experiment that E2F site from a histone promoter in mammals can replace the SBF sites in the CLN2 promoter. Results are also consistent with the obvious alternative hypothesis that SBF and E2Fs are distant homologues and the binding specificity has simply been conserved during evolution. We don't see how any experiment along these lines could be said to support the hijacking hypothesis (to the exclusion of the obvious competing hypothesis).We have rewritten our experimental section to better clarify the purpose of our experiments in budding yeast. By the time we motivate our experiments in subsection “SBF ancestor could regulate E2F-target genes”, we already established that SBF is not a distant homolog of E2F new  and thatFurthermore, even if SBF and E2F are non-homologous, they have overlapping binding specificity. Since sequence specific transcription factors allow degeneracy (or wobble positions) and bind to short DNA sequence families (motifs), these experiments show only that the E2F sites that were chosen probably overlap the specificity of SBF enough that they can be regulated. Therefore, these experiments really add nothing to the hijacking model, except to confirm the observation that the two transcription factors have overlapping specificity.Briefly, the point of these experiments was to determine whether budding yeast SBF  is able to recognize and bind a canonical E2F site. We clearly state that the known consensus motifs of SBF from budding yeast and E2F from animals have overlapping binding specificity, which is the observation that originally prompted our hijacking hypothesis. However, given that a single base pair difference can abrogate DNA binding, it was not obvious to us a priori whether yeast SBF would be able to bind a canonical E2F site. To our knowledge, no one has measured SBF binding to an E2F site. Hence, we felt obligated to test SBF binding to an E2F consensus site in vivo.Overall, the paper has one really key novel observation, namely the detection of the E2F/Rb system in basal fungi and this is certainly worth publishing. However, you need to show that there really are both classes of proteins in the basal fungi. You need to consider and rule out alternative explanations. For example, are you simply assigning some of the homologues in the basal fungi to be E2Fs and some to be SBF/MBFs because they have reached the limit of homology detections?Arabidopsis thaliana, Homo sapiens, Schizosaccharomyces pombeS. pombe, and Saccharomyces cerevisiae, to build the profile-HMMs used to detect E2F/DP and SBF/MBF homologs. All homologs recovered through profile-HMM search were further validated (or rejected) using an iterative search algorithm (Jackhmmer) against the annotated SwissProt database using the HMMER web server .We used Profile-Hidden Markov Models (profile-HMMs) to detect homologs for each of the families studied, using the HMMER 3 package . Profile-HMMs are some of the most accurate and sensitive methods for detection of remote homologs . Starting with a set of diverse yet reliable protein homologs is fundamental for detecting remote protein homology and avoiding “model poisoning” . To this end, we used reliable training-set homologs from the cell cycle model organisms Our profile-HMM for E2F/DP family only detects E2F or DP, where as our profile-HMM for SBF/MBF family only detects SBF/MBF (or APSES). The same protein was never identified by both profile-HMMs because the sequence profiles and the structure are different; see discussion of E2F and SBF sequence-structure below. In the case of basal fungi, which have both E2F/DP and SBF/MBF, all proteins classified as an E2F/DP are clear homologs of E2F or DP  As described above in our response to the summary critique, we are confident that we do not have errors in detection of homology in basal fungi.2) The structural evidence could represent key supporting data to show that E2Fs and SBFs really are two different families. However, is not presented in a very convincing way. "it is possible that fungal SBF is a duplicated and hyper-evolved E2F. However, the number and topology of secondary elements  that stabilize and pack around the core helix-turn-helix are different." Does the number and topology of secondary structural elements always stay the same in homologues? If not, how does this support the hypothesis that they are non-homologous? "Crystal structures of the DNA binding domains of E2F and SBF show that they are not structural homologues  see ." Here tIn response to the reviewer's questions, we have extended and clarified how the structural evidence supports the hypothesis that E2F/DP and SBF/MBF are two different families. We now write:"The DNA-binding domains of SBF/MBF  and E2F/DP  are structurally classified as members of the winged-helix-turn-helix (wHTH) family, which is found in both prokaryotes and eukaryotes . Although the DNA-binding domains of E2F/DP and SBF/MBF are both classified as wHTH proteins, they show important differences in sequence, overall structure, and mode of protein-DNA complex formation that lead us to conclude that it is highly unlikely that they are orthologs.S. cerevisiae in the absence of DNA, suggested Mbp1 recognizes its MCB  binding site via a recognition helix . A recent structure of PCG2, an SBF/MBF homolog in the rice blast fungus Magnaporthe oryzae, in complex with its MCB binding site does not support this proposed mode of DNA binding . In striking contrast to many wHTH structures, in which the recognition helix is the mediator of DNA binding specificity, the wing of PCG2 binds to the minor groove to recognize the MCB binding site. The two glutamines in the wing  are the key elements that recognize the core MCB binding motif CGCG  see , left. Itif CGCG , right. 3) Even if the structural evidence is sound, and the proteins are non-homologous, the authors specific argument rests on the identification of both proteins in the basal fungi. The authors should show and explain the evidence that the putative homologues in the basal fungi are likely to adopt one structure and not the other. The alignment in We now highlight the E2F/DP proteins from basal fungi (bold font) in the sequence alignment of new The reviewers see improvements in your revision but are still far from being satisfied. Ordinarily, this level of criticism on a revised submission would be considered unacceptable, however, we have decided to provide you with an opportunity to substantially revise the manuscript one last time. If you have questions about our instructions, please write and ask for clarification. Of course, given the concerns we have expressed we would understand if you choose to submit to a different journal. Please advise as to your course of action as soon as possible.We thank you for giving us the opportunity to revise our manuscript. We include new data and analysis to address the Reviewer comments. Our new co-author, Dr. Raluca Gordân (Duke), has extensive experience generating and analyzing high-throughput in vitro DNA binding data of eukaryotic transcription factors, e.g. E2F . We modified the Figures as follows:1) We now show the E-values of E2F (E2F_TDP.hmm) and SBF (KilA-N.hmm) HMM models against all predicted ORFS in our surveyed genomes new .2) Old 3) Old 4) Old 5) New Figure 10 analyzes the DNA binding specificity of human E2F1 and budding yeast SBF generated via high-throughput in vitro binding assays. We show there are E2F-only motifs, SBF-only motifs, and "common" motifs that can be bound by both E2F and SBF. We then scan human promoters (known to be regulated by E2F) with these in vitro E2F and SBF binding specificities to show that many human E2F binding sites could be bound by SBF.6) Old I encourage you to shorten this further (as per multiple reviewers' request).To address our Reviewer comments, the manuscript now includes new text, data, and figures. We moved towards more data and analysis rather than less. Given the level of skepticism from one of our Reviewers, we think it is important to show all the data and analysis to future readers. We note that our main text has 4950 words, which is below the suggested maximum of 5000.Reviewer #1:The authors did a good job of clarifying the structural data and present a much more convincing case that the two proteins are non-homologous. However, they did very little to address my other concerns. Rather than doing a detailed, focused analysis to convince the reader of their hypothesis that the basal fungi contain both transcription factors, and that the SBFs are inherited from a single horizontal transfer event, they simply bolded the basal fungi species names in an alignment. They did not show any phylogenetic analysis that would rule out competing evolutionary scenarios  or technical issues (such as homology detection problems). In response to my comments about the latter, they simply stated that their HMMs don't make any classification errors: "Our profile-HMM for E2F/DP family only detects E2F or DP, where as our profile-HMM for SBF/MBF family only detects SBF/MBF (or APSES)." In fact, I agree with the authors that the HMMs are probably very accurate, but why not show data to support this claim?  The authors don't seem to appreciate (or have chosen to ignore) the fact that remote homology detection  is a statistical process and always has an element of uncertainty.  Since the detection of these homologs in the basal fungi and no SBFs in other eukaryotes is the key result of this paper, why not make a convincing case to support it , and convince readers that there have been no errors of remote homology detection or misclassification by HMM models.To address our Reviewer's concern with homology detection, we plot the Pfam HMM E-values of E2F_TDP.hmm and KilA-N.hmm (the SBF DNA binding domain from Pfam) of all predicted proteins for any given genome in our new S. cerevisiae genome, Our controls  are found in prokaryotes (85%) with a smaller fraction (10%) found in eukaryotes and a smaller fraction found in DNA viruses (5%). The KilA-N domains in prokaryotes appear to be either integrated by or derived from prokaryotic DNA viruses (i.e. bacteriophage), and thus, we will treat them as such. Within the eukaryotes, all known KilA-N domains are found in fungal genomes with three notable exceptions.Trichomonas vaginalis, a parasitic excavate with 1000+ KilA-N domains . Mimiviruses are giant viruses known to infect simple eukaryotic hosts, such as Acanthamoeba and possibly other eukaryotes . The second and third exceptions are found in two insects, Acyrthosiphon pisum ('pea aphid') and Rhodius prolixus ('triatomid bug'). The one KilA-N domain in A. pisum genome has a top blast hit to eukaryotic DNA viruses (e.g. Invertebrate Iridescent Virus 6). The three KilA-N domains in R. prolixus have top blast hits to prokaryotic DNA viruses (e.g. Enterobacteria phage P1). The diverse and sparse distribution of KilA-N domains throughout the eukaryotic genomes is consistent with their horizontal gene transfer into hosts from eukaryotic DNA viruses and/or via engulfed bacteria that were infected with prokaryotic DNA viruses. In fact, the horizontal transfer of genes between Mimivirus and their eukaryotic host, or the prokaryotic parasites within the host, has been shown to be a more frequent event that previously thought .The first exception is  domains . The T. To gain further insight into the possible evolutionary origins of the SBF subfamily via horizontal gene transfer, we aligned diverse KilA-N sequences from the Uniprot and PFAM database to the KilA-N domain of our most basal fungal SBF+APSES sequences (Zoosporic fungi (“chytrids”) and “Zygomycetes”) and built a phylogenetic tree new . There aCritique #3: Convince the reader that E2Fs were not horizontally transferred into basal fungi.We now include new Similarly, they have not changed the presentation of the experiment demonstrating that the SBF can bind to an E2F binding site. In their response, they claim that this experiment is important because "a single base pair substitution in the SBF motif can reduce gene expression". However, they have still not showed which substitution that was. Therefore the argument is misleading: all substitutions in transcription factor binding sites are not equivalent, and this experiment would only be interesting if the substitution between the histone binding sites and the SBF consensus was one of those that was likely to reduce expression. If it is, why not show this and explain it clearly?Our statement that “a single base pair substitution in the SBF motif can reduce gene expression” was meant as a general statement. For many transcription factor binding sites, even changing a single base pair may result in decrease binding and subsequently decreased gene expression. In our experiment, the change in the SBF site was much more substantial than a single base pair mutation: we replaced the SBF sites (TCACGAAA) of CLN2  with a known E2F binding site (GCGCGAAA) from the promoters of the histone cluster genes . There are, of course, other possible E2F DNA binding sites that we could have used in our experiment; we picked this one because it is a well-characterized E2F binding site.To circumvent this sampling bias, we now analyze data from high-throughput protein-binding microarray (PBM) assays  of human E2F (E2F1) and budding yeast SBF (Swi4). PBM assays measure, in a single experiment, the binding of recombinant proteins to tens of thousands of synthetic DNA sequences, guaranteed to cover all possible 10-bp DNA sequences in a maximally compact representation (each 10-mer occurs once and only once). We used these PBM data to generate DNA motifs for E2F and SBF , and to compute, for each possible 8-bp DNA sequence, an enrichment score (or E-score) that reflects the specificity of the protein for that 8-mer. E-scores vary between -0.5 and +0.5, with larger values corresponding to higher affinity binding sites . As shown in new Figure 10A, E2F1 and Swi4 can bind a set of common motifs. For example, the E2F binding site variant that we tested in budding yeast , is one of the sites commonly bound in vitro by E2F and SBF.Most notably, the in vitro PBM data show that there are specific motifs that can be bound only by E2F or only by SBF. To identify the key nucleotide differences between E2F-only and SBF-only binding, we created motifs of E2F-only and SBF-only sites. The consensus E2F-only (NNSGCGSN) and SBF-only (NNCRCGNN) motifs indicate that differential specificity between E2F1 and SBF is mediated by the nucleotides in the 3rd and 4th positions (underlined). Thus, to address our Reviewer's comment, E2F has a strict preference for G in the 4th position, whereas SBF has a strict preference for C in the 3rd position .We now include new Figure 10 and this discussion in the main text.Furthermore, since E2Fs regulate many cell cycle target genes, it would be much more convincing if the authors simply surveyed the known E2F binding sites and estimated  which of them SBF is likely to regulate. The observation that SBF can bind the histone binding site (on its own) really adds nothing.We scanned the promoters of known E2F target genes from the human genome  with our empirically-defined DNA binding sites from PBM assays  to predict putative E2F-only, SBF-only, and common sites . As expected, there are many predicted E2F-only and common (E2F & SBF) sites that can be bound by E2F in these known target genes. However, we can also find many potential SBF-only binding sites in these promoters. We extended our analysis to 290 known E2F target genes in the human genome to test the generality of SBF cross-binding to E2F sites . Most E2F target promoters could be bound by SBF, which supports the hijacking hypothesis where an ancestral SBF might have taken control of several E2F-regulated genes.We now include new Figure 10 and this discussion in the main text.We note that our observation that SBF can bind an E2F binding site in vivo, leading to cell cycle regulated gene expression, is critical for showing that our results based on high-throughput in vitro data are also true in a cellular (in vivo) environment.I think the most important thing for the authors to add is a phylogenetic analysis showing that the data support the hypothesis of a single transfer event in the ancestral fungi.Please see new text and As a secondary point, I guess if they really want to include the histone binding site experiment, they should include it in the context of a survey of other known E2F and SBF binding sites, and consider to what extent SBF could have taken over the sites. Please see new text and Figure 10.Essential revisions:Reduce the number of figures and make them less busy and higher quality (visible). Is each figure really essential? Can it be treated as a supplement to the primary figure (see how this is done for other papers we publish)? People are much less likely to read a paper with many busy figures.We removed 2 figures  and decreased the level of detail from one figure. When necessary, we uploaded figures of higher quality (500 dpi), such that all species/proteins in large phylogenies (e.g. Cyclins) can be viewed using magnify/zoom function.Here’s a list of the major changes:1) We changed old 2) We changed old 3) We moved panels (B) and (C) from old Figure 10 into