Studies have reported inconsistent results concerning the existence of associations of folate intake and serum folate levels with prostate cancer risk. This study sought to summarise the evidence regarding these relationships using a dose–response meta-analysis approach.In January 2014, we performed electronic searches of PubMed, Embase, and the Cochrane Library to identify studies examining the effect of folate on the incidence of prostate cancer. Only prospective studies that reported effect estimates with 95% confidence intervals (CIs) of the incidence of prostate cancer for more than 2 categories of folate were included.Overall, we included 10 prospective studies reporting data on 202,517 individuals. High dietary folate intake had little or no effect on prostate cancer risk . The dose–response meta-analysis suggested that a 100 μg per day increase in dietary folate intake has no significant effect on the risk of prostate cancer . However, high serum folate levels were associated with an increased risk of prostate cancer . The dose–response meta-analysis indicated that a 5 nmol/L increment of serum folate levels was also associated with an increased risk of prostate cancer .Our study indicated that dietary folate intake had little or no effect on prostate cancer risk. However, increased serum folate levels have potentially harmful effects on the risk of prostate cancer.The online version of this article (doi:10.1186/1471-2458-14-1326) contains supplementary material, which is available to authorized users. Prostate cancer is the second most common non-skin cancer among men globally, and with markedly higher incidence rates in developed countries, 2. OverThe results of a previous prospective study indicateIn 2012, Wien used a sIn this study, we attempted a large-scale examination of the available prospective studies to determine the association between dietary folate intake or serum folate levels and prostate morbidity. We also performed a dose–response meta-analysis to quantify the risk of prostate cancer associated with incremental increases in dietary folate intake and serum folate levels for the general population.This review was conducted and reported according to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis Statement issued in 2009 , with no language restrictions for studies in humans. We included all studies investigating an association between dietary folate intake or serum folate levels and prostate cancer incidence. Our core search included the following terms: (“folate” OR “folic acid”) AND (“prostate cancer” OR “prostate neoplasm” OR “prostate carcinoma”) AND (“cohort” OR “cohort studies” OR “nest case–control studies”). If a site-specific dataset had been published more than once, we used the most recent publication. We reviewed the reference lists of the identified reports, reviews, meta-analyses, and other relevant publications to find additional pertinent studies. The medical subject heading, methods, population, study design, exposure, and outcome variables of these articles were used to identify relevant studies.A study was eligible for inclusion if the following criteria were met: (1) the study had a prospective observational design (prospective cohort or prospective nested case–control study); (2) the study investigated the association between dietary folate intake or serum folate levels and the risk of prostate cancer; and (3) the authors reported effect estimates  and 95% confidence intervals (CIs) for comparisons between high and low dietary folate intake or serum folate levels (with more than 2 categories). We excluded all retrospective case–control studies because various confounding factors could bias the results.The literature search was independently undertaken by 2 authors (RW and YZ) using a standardised approach. Any inconsistencies were resolved by discussion with the primary author (YHZ) and a consensus was reached.The following data elements were collected: name of the first author or study group, publication year, country, study design, assessment of folate levels, sample size, age at baseline, effect estimate, comparison categories, follow-up duration, and covariates in the fully adjusted model. We also extracted the numbers of cases per person or per person-year, effect of the different exposed categories, and 95% CIs. For studies that reported several multivariable adjusted RRs, we selected the effect estimate that was maximally adjusted for potential confounders.The Newcastle-Ottawa Scale (NOS) was used to evaluate methodological quality, 20. The2 statistic to measure the proportion of total estimate variation that was attributable to study heterogeneity; I2 values of 25%, 50%, and 75% were used as cut-off points for low, moderate, and high degrees of heterogeneity, respectively[We examined the relationship between dietary folate intake or serum folate levels and the risk of prostate cancer based on the effect estimate  and its 95% CI published in each study. We first used the random-effects model to calculate summary RRs and 95% CIs for the highest folate levels compared to the lowest folate levels, 22. We ectively. Subgrouectively. Severalectively and Beggectively were alsThe results of the study selection process are shown in Figure Five prospective cohort studies–12 invol2 = 0.0%; P = 0.959). The findings of the dose–response meta-analysis also suggested no association between the risk of prostate cancer and a 100 μg/day increment of dietary folate intake  different cooking methods may moderate the effect of dietary folate intake and (2) different types of prostate cancer might provide a biased view of the study question. Previous studiesSubgroup analyses indicated that the harmful effect of increased serum folate levels was more evident in studies with follow-up periods of less than 15 years than in those with longer follow-up periods. The reason for this difference could be that studies with longer follow-up periods (greater than 15 years) did not reach statistical significance owing to the low incidence of prostate cancer. This conclusion may be unreliable because smaller cohorts were included in each subset. Similar explanations may apply to subgroup analyses based on other potential biases factors.Two strengths of our study compared with previous meta-analyses should be highlighted. First, only prospective studies were included, which should eliminate selection and recall bias. Second, the dose–response analysis included a wide range of dietary folate intake levels and statuses, which permitted an accurate assessment of the dose relationship between dietary folate intake or status and prostate cancer risk.The limitations of our study are as follows: (1) publication bias is possible in meta-analyses of published studies; (2) data on prostate cancer type were not available, and thus, we could not differentiate the effects of dietary folate intake or status by prostate cancer type; and (3) the analysis used pooled data , which restricted us from performing a more detailed relevant analysis and obtaining more comprehensive results.In conclusion, the findings of this study suggest that increased dietary folate intake was not associated with prostate cancer risk. However, increased serum folate levels were associated with an increased risk of prostate cancer. These findings need to be confirmed in future studies. Future studies should (1) ascertain the specific type of prostate cancer and analyse effects by type, (2) consider the effects of the cooking method when assessing dietary folate intake and its effects on clinical outcomes, and (3) consider the effects of alcohol consumption and smoking status.Checklist S1. PRISMA Checklist. (DOC 70 KB)Additional file 1:Additional file 2: Table S1: Quality scores of prospective cohort studies using Newcastle-Ottawa Scale. (DOC 44 KB)


Wegener's granulomatosis or granulomatosis with polyangiitis is a necrotizing vasculitis affecting both arterioles and venules. The disease is characterized by the classical triad involving acute inflammation of the upper and lower respiratory tracts with renal involvement. However, the disease pathology can affect any organ system. This case presents Wegener's granulomatosis presenting with facial nerve palsy as the first manifestation of the disease, which is rarely reported in medical literature.  Wegener's granulomatosis (WG) is an idiopathic systemic form of vasculitis characterized by the presence of necrotizing granulomas and vasculitis in the upper airways, lower airways, and kidneys. Being a systemic disease, it may additionally involve any organ system. Etiology of this disease is proposed to be autoimmune , 2. InciA 50-year-old nondiabetic, nonhypertensive male patient was referred from a primary health centre to our institute with a provisional diagnosis of bell's palsy of left side. He had a history of acute onset deviation of angle of mouth to the right and difficulty in closure of left eye 2 days back. There was no history of any preceding fever, local trauma, ear related problem, cough, hemoptysis, chest pain, skin rash, or any hypoesthetic patches over the body. Patient's past history was not significant except for 2-3 episodes of upper respiratory tract infection with persistent rhinorrhea for 2 months in the last one year. General physical examination was notable for bilateral pitting pedal edema. Neurological examination revealed left sided lower motor neuron type of 7th nerve palsy (House-Brackmann grade 5) . The resLaboratory investigations showed mildly elevated erythrocyte sedimentation rate (ESR) (52 mm in 1st hour) and deranged renal function tests . The rest of the routine hematological and biochemical investigations including blood sugar and thyroid profile were normal. Blink reflex study was performed which showed absent ipsilateral R1 and R2 responses on left side stimulation while normally presenting contralateral R2 response. On right side stimulation, ipsilateral R1 and R2 responses were normal while contralateral R2 response was absent. These findings were corroborative of left sided facial nerve palsy. MRI brain did not reveal any abnormality, thus ruling out any structural cause of facial nerve palsy. Viral markers for HIV, Hepatitis B, and Hepatitis C were nonreactive. Chest X-ray was normal. Routine microscopy of urine revealed 3+ proteinuria and red blood cells (RBCs) casts. Accordingly 24-hour urinary protein was sent which came out to be 2.2 g/day. Based on these investigations, patient's history was retrospectively explored. Patient further reported the backdrop of intermittent swelling of feet with mild arthralgia involving both knee and small joints of hands in previous 6 months. On further testing, rheumatoid factor (RF) and antinuclear factor (ANF) were found to be negative. However, c-ANCA was detected positive against proteinase-3 (titre 1 : 640). Kidney biopsy was done  according to EUVAS (European Vasculitis Study Group) protocol for the management of primary small and medium vessel vasculitis. Initially he received 3 pulses of daily intravenous methyl-prednisolone (1 gm) followed by intravenous cyclophosphamide at a dose of 15 mg/kg every 2 weeks for the first 3 pulses, followed by infusions every 3 weeks for the next 3 pulses. We also put him on a remission-maintenance therapy with a combination of low-dose glucocorticoid (0.25 mg/kg) and azathioprine (2 mg/kg).Patient's clinical features were closely followed and a urine analysis performed 15 weeks after starting immunosuppressive therapy revealed only 1+ proteinuria (260 mg/24 hours) without any RBCs. By this time patient improved significantly from his systemic involvement with normalization of renal function parameters along with complete recovery from facial nerve paralysis.Wegener's granulomatosis or granulomatosis with polyangiitis is an uncommon, systemic disease that mainly affects the head and neck region, the lungs, and the kidneys. Besides, a significant proportion of patients present with peripheral nervous system (PNS) involvement in the form of mononeuritis multiplex due to vasculitis or with central nervous system (CNS) involvement due to infiltrating granulomatous lesions 10–45%) . The com5% . The Unilateral facial nerve palsy in our case was a probable manifestation of vasculitis leading to ischemic cranial neuropathy. Alternatively, the cranial neuritis could be a result of immune-mediated inflammatory process. Cadoni et al.  substantIsolated facial cranial neuritis as the first presentation of the disease is rarely reported. Although, there have been a few reports of bilateral facial nerve palsy. Nikolaou et al. reported a case of a young female who presented with bilateral serous otitis media followed by bilateral sensorineural hearing loss and bilateral facial nerve palsy . WegenerPrognosis of neuropathies associated with Wegener's granulomatosis depends mainly on early diagnosis and prompt initiation of treatment –10.Wegener's granulomatosis is an autoimmune disease and can be lethal if left untreated. Long term remission can be achieved in up to 90% of the cases with the help of therapeutic agents like steroids and other immunosuppressant drugs such as cyclophosphamide, azathioprine, and methotrexate. The presentation of Wegener's granulomatosis can be varied and the patients may initially present as an isolated cranial neuritis. Physicians should be aware of the atypical and varied presentations of this disorder, which may thus help in early diagnosis and initiation of treatment. Lower motor neuron type of facial paralysis is not always synonymous with bell's palsy and secondary causes should always be ruled out by a thorough clinical examination and a comprehensive investigation panel.


The cerebral haemodynamic status of idiopathic generalized epilepsy (IGE) is a very complicated process. Little attention has been paid to cerebral blood flow (CBF) alterations in IGE detected by arterial spin labelling (ASL) perfusion magnetic resonance imaging (MRI). However, the selection of an optimal delay time is difficult for single-delay ASL. Multi-delay multi-parametric ASL perfusion MRI overcomes the limitations of single-delay ASL. We applied multi-delay multi-parametric ASL perfusion MRI to investigate the patterns of postictal cerebral perfusion in IGE patients with absence seizures. A total of 21 IGE patients with absence seizures and 24 healthy control subjects were enrolled. IGE patients exhibited prolonged arterial transit time (ATT) in the left superior temporal gyrus. The mean CBF of IGE patients was significantly increased in the left middle temporal gyrus, left parahippocampal gyrus and left fusiform gyrus. Prolonged ATT in the left superior temporal gyrus was negatively correlated with the age at onset in IGE patients. This study demonstrated that cortical dysfunction in the temporal lobe and fusiform gyrus may be related to epileptic activity in IGE patients with absence seizures. This information can play an important role in elucidating the pathophysiological mechanism of IGE from a cerebral haemodynamic perspective. Idiopathic generalized epilepsy (IGE) is characterized by typical absence seizures, generalized tonic-clonic seizures, juvenile myoclonic epilepsy and paroxysmal generalized spike and wave (GSW) discharges on electroencephalography (EEG)Arterial spin labelling (ASL) is a non-invasive magnetic resonance perfusion approach to the measurement of cerebral blood alterations by magnetically labelling the inflowing water proton spins in the arterial blood proximal to the tissue of interest as a freely diffusible tracer457891078128However, most ASL techniques in previous studies have acquired images at a single-delay time point. The main limitation of single-delay ASL is that the delay time between labelling in the feeding arteries and arrival of labelled blood in tissue  can have a large effect on the perfusion signals16The recently developed method of multi-delay multi-parametric ASL perfusion MRI allows the limitations of single-delay time point studies to be overcome. It has several potential advantages over existing single-delay ASL perfusion MRI, including improved accuracy of CBF quantification and imaging of multiple haemodynamic parameters 15215O PET study revealed a mean global increase of 14.9% in CBF in association with typical absences. In addition to the global increase, a focal increase in thalamic blood flow of 3.9 to 7.8% following hyperventilation-induced typical absences with GSW discharges was noted23The cerebral haemodynamic status is a very complicated process before, during, and after the epilepsy status of IGE. Therefore, characterization of the cerebral perfusion patterns in patients with IGE at different periods of epilepsy is necessary to better understand the fundamental underlying mechanisms of IGE. To date, the cerebral perfusion patterns of IGE patients have largely been derived from these studies using PET222324The above studies indicate complex changes of CBF during different phases of IGE, and the perfusion pattern of CBF does not always involve an ictal increase and interictal decrease. Therefore, the purpose of the present study was to conduct an exploratory study for characterizing the patterns of postictal cerebral perfusion in IGE patients with absence seizures using multi-delay multi-parametric ASL perfusion MRI to: (1) investigate whether there are significant differences in the parameters of ATT and CBF using voxel-based whole brain analysis between these patients and healthy control subjects, and (2) evaluate the relationships between these identified neuroimaging findings and illness duration, as well as the age at onset.All 21 IGE patients with absence seizures and 24 healthy control subjects underwent ASL perfusion MRI at 0.5 to 30 days (mean ± standard deviation (SD): 7.6 ± 5.9 days) since the last epilepsy episode had occurred. The characteristics of the participants are presented in Patients with IGE showed prolonged ATT in the left superior temporal gyrus versus healthy control subjects , Fig. 1.r = −0.504, p = 0.020)  , Fig. 2.= 0.020) .In the present study, we identified postictal cerebral perfusion alterations in IGE patients with absence seizures relative to healthy control subjects using multi-delay multi-parametric ASL perfusion MRI. The results indicate that the CBF at 1,400 ms among the 4 PLD times was the most sensitive for the detection of CBF changes in IGE patients with absence seizures, with a significantly increased CBF in the left parahippocampal gyrus, bilateral fusiform gyri and left middle temporal gyrus. IGE patients exhibited prolonged ATT in the left superior temporal gyrus. Furthermore, the prolonged ATT in the left superior temporal gyrus was negatively correlated with the age at onset in IGE patients. These findings suggest that IGE with absence seizures is likely associated with cortical haemodynamic abnormality, providing insight into the pathophysiological mechanism of the complex disease.Increased CBF was previously reported during the postictal phase of typical absence seizures29303133Whether GSW discharges in patients with IGE originates in the cortex, subcortex  or simultaneously in a cortical-subcortical network remains a matter of debate273941In addition, the present work used ATT to quantify the variability of cerebral perfusion patterns in IGE patients with absence seizures. ATT is another haemodynamic parameter measured by quantitative ASL with multiple-delay time sampling and represents the duration of the labelled blood flowing from the labelling region to the vascular compartment of the imaging sections161743et al. believed that not all GSW discharges are generated by the same mechanisms and that some brain regions become more active whereas others become less active2930313248Our results revealed that IGE patients with absence seizures showed prolonged ATT in the left superior temporal gyrus. This finding is consistent with a recently published study that found that patients with late-onset epilepsy have significantly prolonged ATT in widespread brain regions, but the greatest prolongation is predominantly distributed in the temporal and frontal lobesThere are several limitations to our study that should be acknowledged. First, owing to the small sample size, our results must be confirmed by future studies with a larger sample size. The current study demonstrates the feasibility of using multi-delay multi-parametric ASL perfusion MRI to characterize cerebral perfusion patterns in IGE patients. Second, in our cohort of patients, the different seizure subtypes of IGE are mixed together, including typical absence seizures and absence seizures accompanied by other seizure subtypes of IGE. Thus, these findings should be interpreted with caution because the different seizure subtypes of IGE could lead to inconsistent results. Third, ASL perfusion MRI was performed using a wide time window ranging from 0.5 to 30 days with a mean time of 7.6 days since the last epilepsy episode occurred. Given the complexity of cerebral haemodynamic changes, the cerebral perfusion patterns may vary with different time points after seizures, particularly in the postictal phase. In addition, the through-plane blurring in the single-shot acquisition can affect the 3D-GRASE data and the subsequent perfusion estimates. Finally, simultaneous EEG was not combined with ASL perfusion MRI to analyse the relationship between cerebral haemodynamic changes and GSW discharges in patients with IGE.Taken together, this study revealed the postictal alterations of regional cerebral perfusion in the brains of IGE patients with absence seizures using multi-delay multi-parametric ASL perfusion MRI with the parameters of ATT and CBF at approximately one week since the last epilepsy episode occurred. Our results demonstrated abnormal perfusion patterns during the postictal phase and provided further evidence that cortical dysfunction in the temporal lobe and fusiform gyrus may be related to epileptic activity in IGE patients with absence seizures. This information can play an important role in elucidating the pathophysiological mechanism of IGE from a cerebral haemodynamic perspective.Twenty-one consecutive IGE patients with absence seizures  and 24 age and sex-matched healthy control subjects  were recruited to undergo MRI using a protocol specifically designed for the present study. IGE patients were diagnosed and classified based on clinical examination and EEG according to the International League Against Epilepsy (ILAE) classificationThe inclusion criteria for the patients were as follows: (1) clinically diagnosed absence seizures with or without generalized tonic-clonic seizures and/or juvenile myoclonic epilepsy; (2) experienced more than two spontaneous epileptic seizures; (3) no evidence of secondary generalized seizures, such as tumours, encephalitis, and vascular malformation; (4) no structural or signal abnormalities on conventional brain MRI; (5) normal neurological examination, except for GSW mixtures or generalized spikes with normal alpha rhythms on EEG; (6) patients who had prior antiepileptic treatment received no medication for at least 48 h before the MRI; and (7) right-handedness. The exclusion criteria were as follows: (1) a history of head trauma, psychiatric or other neurological disorders; (2) a history of addictions; (3) a history of partial seizures; (4) self-reported falling asleep during ASL perfusion MRI scanning; (5) excessive head motion exceeding 1.5-mm translation and 1.5° rotation; and (6) any contraindications to MRI scanning. In our cohort, eighteen patients were taking antiepileptic drugs, including valproate, levetiracetam, lamotrigine, oxcarbazepine or some combination. In addition, 3 patients had never taken any medications.All healthy control subjects were right-handed. No familial or personal histories of neurological or psychiatric diseases were noted. Head trauma or lesions on brain MRI were not noted in healthy control subjects. This study was approved by the West China Hospital Clinical Trials and Biomedical Ethics Committee of Sichuan University, and written informed consent was obtained from all of the participants. The study protocol was performed in accordance with the approved guidelines.3; 26 slices covering the whole brain; 12 pairs of tag/control images were acquired for each delay with a total scan time of 5.9 min. An M0 image was acquired using TR = 5,000 ms and PLD = 4,000 ms (scan time = 15 s). ASL perfusion images were acquired with background suppressed, but M0 image was acquired without background suppression.The patients and healthy control subjects were imaged using a 3-T Siemens Trio Tim system with a 12-channel phased-array head coil. Multi-delay multi-parametric ASL perfusion MRI was performed using a pCASL pulse sequence with background-suppressed single-shot 3D GRASE readouts. The imaging parameters were as follows: 4 PLD times, 1,400/1,800/2,200/2,600 ms; labelling pulse duration, 1,500 ms; TR, 3,500/3,500/3,500/3,500 ms; TE, 22 ms; voxel size, 3.44 × 3.44 × 5 mmet al.et al.18SPM8  and a self-complied Matlab program based on SPM8  were used for image processing. Motion correction was performed first for all control and label images of the same series using the M0 image as a reference. After motion correction, the mean perfusion difference, ΔM (i), in the images was generated for each PLD. The weighted delay (WD) was calculated using the images of ΔM (i) by The CBF at each delay was calculated using the following equation:−1) is the longitudinal relaxation rate of blood at 3T, α ( = 0.8) is the tagging efficiency, τ  is the duration of the labelling pulse, and w(i) is the PLD . The final CBF was the mean of the estimated CBF at each PLD. All of the ASL images of the IGE patients and healthy control subjects were spatially normalized to the Montreal Neurological Institute (MNI) template space with 79 × 95 × 68 dimension and were smoothed using a 6-mm full width at half maximum Gaussian kernel using SPM8. A representative example of the ATT and mean CBF map of an IGE patient and healthy control subject is shown in where λ ( = 0.9 g/ml) is the blood/tissue water partition coefficient, R . To investigate the relationship between the abnormal cerebral perfusion patterns  and clinical characteristics, the regional values of ATT and CBF in the identified brain areas were extracted using an automated tool . Pearson’s correlation was employed to evaluate the relationships between these neuroimaging findings and illness duration, as well as the age at onset (p < 0.05). The statistical analyses were performed using SPSS 20.0 software .The two-sample t-test was used for the group comparisons of the ATT and CBF differences in the whole brain between the IGE patients and healthy control subjects. The height threshold for the SPM analysis was set to the false discovery rate (FDR) corrected How to cite this article: Chen, G. et al. Patterns of postictal cerebral perfusion in idiopathic generalized epilepsy: a multi-delay multi-parametric arterial spin labelling perfusion MRI study. Sci. Rep.6, 28867; doi: 10.1038/srep28867 (2016).


COLQ was inherited IBD. COLQ encodes the collagenous tail of acetylcholinesterase, the enzyme responsible for termination of signal transduction in the NMJ. Sequences from COLQ revealed a variant in exon 14 (c.1010T>C) that results in the substitution of a conserved amino acid (I337T) within the C-terminal domain. Both affected puppies were homozygous for this variant, and 16 relatives were heterozygous, while 288 unrelated Labrador Retrievers and 112 dogs of other breeds were wild-type. A recent study in which 2 human CMS patients were found to be homozygous for an identical COLQ mutation  provides further evidence that this mutation is pathogenic. This report describes the first COLQ mutation in canine CMS and demonstrates the utility of SNP profiles from nuclear family members for the identification of private mutations.Congenital myasthenic syndromes (CMSs) are heterogeneous neuromuscular disorders characterized by skeletal muscle weakness caused by disruption of signal transmission across the neuromuscular junction (NMJ). CMSs are rarely encountered in veterinary medicine, and causative mutations have only been identified in Old Danish Pointing Dogs and Brahman cattle to date. Herein, we characterize a novel CMS in 2 Labrador Retriever littermates with an early onset of marked generalized muscle weakness. Because the sire and dam share 2 recent common ancestors, CMS is likely the result of recessive alleles inherited identical by descent (IBD). Genome-wide SNP profiles generated from the Illumina HD array for 9 nuclear family members were used to determine genomic inheritance patterns in chromosomal regions encompassing 18 functional candidate genes. SNP haplotypes spanning 3 genes were consistent with autosomal recessive transmission, and microsatellite data showed that only the segment encompassing  CHRNE, COLQ, RAPSN, and DOK7Skeletal muscle contraction is stimulated by the emission of the neurotransmitter acetylcholine (ACh) by the motor neuron, and terminated by acetylcholinesterase (AChE) in the neuromuscular junction (NMJ). Disruption of signal transmission within the NMJ resulting from presynaptic, synaptic, or post-synaptic defects causes congenital myasthenic syndromes (CMSs), heterogeneous neuromuscular disorders characterized by skeletal muscle weakness and fatigue. Mutations causing CMSs in humans have been identified in 18 genes to date, with a majority of cases attributed to CHAT)CHRNE)Naturally-occurring CMSs are rarely described in veterinary medicine; when they do occur, they are usually in animals between 6 to 12 weeks of age, appear to be familial, and are characterized by severe generalized skeletal muscle weakness. The first report of canine CMS was in the Jack Russell Terrier in 1974 Diagnosis of CMS is challenging and relies on clinical evaluation, morphological studies of muscle and peripheral nerves, electrodiagnostic studies, the absence of serum antibodies against muscle AChRs, demonstration of AChR deficiency, and most recently, molecular genetic studies. We have identified a novel canine CMS in a family of Labrador Retrievers. Affected littermates exhibited signs clinically distinct from neuromuscular disorders previously characterized in the breed: exercise-induced collapse (EIC) COLQ.While genome-wide association studies (GWAS) are an efficient approach for the identification of recessive alleles, they require several unrelated affected individuals The dogs evaluated in this study were members of a Labrador Retriever family. The dam and sire were clinically normal and both tested clear for the EIC Whole blood was drawn from family members by their primary care veterinarians. Buccal swabs were collected from additional Labrador Retrievers, both related and unrelated to the affected dogs. DNAs from Labrador Retrievers and other breeds previously collected for unrelated studies were also available. Informed owner consent and all samples were obtained in accordance with protocols approved by the Clemson University Institutional Review Board (IBC2008-17). Owner consent was obtained for post-mortem tissue collection from both affected puppies at TAMU. Genomic DNA was isolated using Gentra Puregene Blood Kit (Qiagen).Electrodiagnostics on the affected second puppy, including electromyography (EMG), measurement of motor nerve conduction velocity (MNCV), and measurement of the decrement in the compound muscle action potential (CMAP) following repetitive nerve stimulation, were performed on the left side under general inhalational anesthesia using a Nicolet Viking Select EMG/evoked potential system . Insulated stainless steel needle electrodes were used for both nerve stimulation and recording from muscle, while a platinum subdermal electrode (Grass-Telefactor) was employed as a ground. MNCV of the peroneal and ulnar nerves was determined by dividing the distance between proximal and distal stimulation sites by the difference in latency of the corresponding CMAP recorded from the extensor digitorum brevis muscle and palmar interosseous muscles, respectively, after supramaximal stimulation . Amplitude (peak to peak) was measured from CMAPs derived from stimulation at the proximal and distal stimulation sites. Repetitive nerve stimulation parameters included stimulation frequencies of 1, 2, 3, 5, 10, 20, or 50 Hz.Specimens collected post-mortem from the second puppy included the infraspinatus, extensor carpi radialis, triceps brachii, biceps femoris, quadriceps, and cranial tibial muscles on the right side. The muscles were frozen in isopentane pre-cooled in liquid nitrogen and stored at –80°C until further processing. Light microscopic evaluation of histological and histochemical stains and reactions was performed according to standard protocols Specimens from the radial and peroneal nerves were immersion fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer before shipment to the laboratory. Upon receipt, nerves were post-fixed in 1% aqueous osmium tetroxide for 3 h to 4 h and then dehydrated in a graded alcohol series and propylene oxide. After infiltration with a 1:1 mixture of propylene oxide and araldite resin for 4 h, nerves were placed in 100% araldite resin overnight and then embedded in fresh araldite resin. Thick sections (1 µm) were cut and stained with paraphenylediamine prior to light microscopic evaluation.For immunohistochemical localization of motor end-plates, serial cryosections (8 µm) were obtained from the external intercostal muscle of 1 affected Labrador Retriever, archived frozen muscle of a previously diagnosed Jack Russell Terrier with CMS due to AChR deficiency (neuromuscular disease control), and a normal dog (wild-type control). Sections from each dog were incubated with the esterase reaction for identification of presumptive motor end-plates or Alexa Fluor 594 α-bungarotoxin  for localization of AChRs at the motor end-plate. Serial sections were evaluated with light microscopy (esterase reaction) and fluorescent microscopy (red fluorescence) and localization of stainings compared.125I-α-bungarotoxin (125I-αbgt) followed by sequential addition of high titer rat-anti-AChR antibody and precipitation with goat anti-rat IgG. The precipitate was pelleted, washed, and quantitated in a gamma counter. The amount of AChR complexed with 125I-αbgt was quantified and expressed in terms of moles of 125I-αbgt precipitated per gram of tissue. The concentration of in-situ antibody-AChR complexes was determined by precipitation with goat anti-rat serum in the presence of normal rat serum. Quantitative serum AChR antibody concentrations were determined as previously described using an immunoprecipitation radioimmunoassay procedure AChR was extracted from external intercostal muscle specimens of both affected puppies by a procedure modified from that of Lindstrom and Lambert DOK7), 5 , 6 (ALG14), 9 (SCN4A), 10 (GFPT1), 11 , 13 (PLEC1), 18 (RAPSN), 20 (LAMB2), 23 (COLQ), 25 , 28 (CHAT), and 36 (CHRNA1) Eighteen candidate genes were selected based on their involvement in human CMSs. Candidate genes were distributed across 13 canine chromosomes: 3  with 0.4 µM of each primer and 50 ng of DNA. Products were purified using the Qiax II Gel Extraction Kit (Qiagen) or an ExoSAP master mix consisting of 0.5 units of Exonuclease I (New England BioLabs) and 0.25 units of SAP (Promega). Primer sequences and purification methods for each amplicon are given in Primers were designed to amplify all coding regions and splice sites of COLQ variant was accomplished using the endonuclease BtsI, which recognizes the following sequence and cutsite (∧): 5′ …NN∧CACTGC…3′. The digestion was performed using BtsI CutSmart (New England BioLabs) with 2.5 µL 10X buffer, 0.40 µL BtsI, 1 µg DNA, and water adjusted accordingly for a total reaction volume of 25 µL. Products were resolved on a 2% agarose gel.Detection of the Neurological examination was consistent with a generalized neuromuscular disease with marked short-strided tetraparesis that worsened with exercise. Postural reactions were preserved with the exception of hopping which was diminished in all limbs when the puppies were made to bear full weight. Spinal reflexes including the patellar, cranial tibial, and flexor withdrawals were reduced in all limbs. A pyridostigmine bromide challenge resulted in worsening of muscle weakness.EMG was performed on the left side of the body and included the supraspinatus, infraspinatus, triceps, biceps, extensor carpi radialis, superficial and deep digital flexor, interosseous, biceps femoris, rectus femoris, cranial tibial, and gastrocnemius muscles. Abnormal EMG activity was not identified in any muscle group. Peroneal and ulnar MNCV was likely age appropriate  Myofiber size was appropriate in all muscles evaluated and no specific abnormalities were identified, making a congenital myopathy unlikely. Multifocal areas of esterase reactivity were identified, but it could not be determined from this reaction if staining correlated with localization of motor end-plates. In the Labrador Retriever with CMS, esterase staining was evident in multiple locations but did not fully correlate with AChR labeling . There wThe concentration of AChR was determined from external intercostal muscle samples collected origin to insertion following euthanasia of both affected Labrador Retriever puppies. The AChR concentration was decreased in both puppies . AChR antibodies were not detected bound to muscle AChRs or in the serum.COLQ, while the region on chromosome 25 includes both CHRNG and CHRND.Over 172,000 SNPs across 40 canine chromosomes were genotyped for each of 9 nuclear family members. Allele frequencies in regions on the 13 chromosomes harboring candidate genes were evaluated for a pattern consistent with a recessive trait . SNP hapCOLQ, providing evidence that the segment on chromosome 23 is IBD . To determine whether the aforementioned segments of chromosomes 23 and 25 were inherited IBD, we genotyped polymorphic microsatellite markers in each region. Genotypes revealed homozygosity in the affected dogs for markers flanking 3 is IBD . AffecteCHRNG. A single nonsynonymous variant in exon 4 of CHRND did not segregate with a recessive phenotype. Sequence data for COLQ revealed 3 nonsynonymous variants, only 1 of which segregated with a recessive trait. The exon 14 variant, c.1010T>C, predicts the substitution of isoleucine with threonine at residue 337  COLQ are IBD, whereas those flanking the other 2 identified candidate genes are not.Through the examination of SNP allele frequencies in the Labrador Retriever family, we identified 2 chromosomes harboring CMS candidate genes that showed an inheritance pattern consistent with autosomal recessive transmission. Whereas human forms of CMS are often caused by compound heterozygosity, low levels of genetic diversity within purebred dog populations make simple recessive alleles more common. Linebreeding in this Labrador Retriever family makes it likely that the sire and dam inherited the mutation from a common ancestor and that the affected puppies are homozygous for a chromosome segment transmitted IBD. Analysis of polymorphic microsatellites showed that the regions flanking COLQ in the Labrador Retrievers revealed a missense mutation that predicts the replacement of a conserved hydrophobic isoleucine with a hydrophilic threonine in the C-terminal domain. ColQ has 3 domains: an N-terminal proline-rich attachment domain (PRAD), a collagenic central domain, and a C-terminal domain. The PRAD serves to attach the ColQ strand to an AChE tetramer. The collagen domain assembles the triple helix, while the C-terminal domain is involved in both the formation of the triple helix Sequencing of COLQ and have different functional consequences depending on their location In humans, mutations responsible for EAD have been identified in each domain of COLQ c.1010T>C mutation; however, our approach does not exclude the possibility that another mutation exists in a novel CMS gene.Linebreeding practices expedite the appearance of recessive diseases in purebred dog populations. The availability of genetic tests for the detection of carrier dogs allows for selective breeding to prevent widespread dissemination of the deleterious allele to the breed while maintaining genetic diversity. Because only 2 affected littermates were available for study herein, GWAS techniques could not be applied. The analysis of chromosomal inheritance patterns indicated a single functional and positional candidate gene and led to the discovery of the COLQ and was determined to be pathogenic through a prediction program While this manuscript was in revision, Matlik et al. reported that an identical mutation  was homozygous in 2 human CMS patients with EAD Table S1CHRNG, CHRND, and COLQ. Primers were designed to amplify exons and splice sites. ExoSAP indicates the use of the ExoSAP protocol and Gel X indicates the use of the gel extraction protocol for post-PCR clean-up.Primers (5′-3′) for amplification of (PDF)Click here for additional data file.Table S2Candidate regions based on allele frequencies. Genomic regions known to harbor CMS candidate genes were screened for case allele frequencies of 1.0 and control allele frequencies of between 0.14 and 0.50. CHR = chromosome number; SNP = SNP name; BP = chromosome position; A1 =  allele 1; F_A = frequency of allele 1 in affected dogs (cases); F_U = frequency of allele 1 in unaffected dogs (controls); A2 =  allele 2.(PDF)Click here for additional data file.Table S3COLQ 14 variant. Digestion with Bts1 was used to genotype the 2 affected dogs, 56 other members of the Labrador Retriever pedigree, 288 unrelated Labrador Retrievers, and 112 dogs representing 65 other breeds.Dogs screened for the (PDF)Click here for additional data file.


We have previously shown that liposomes composed of egg-derived phosphatidylglycerol (PG), with a mixed fatty acid composition , inhibit the proliferation and promote the differentiation of rapidly dividing keratinocytes, and stimulate the growth of slowly proliferating epidermal cells. To determine the species of PG most effective at modulating keratinocyte proliferation, primary mouse keratinocytes were treated with different PG species, and proliferation was measured. PG species containing polyunsaturated fatty acids were effective at inhibiting rapidly proliferating keratinocytes, whereas PG species with monounsaturated fatty acids were effective at promoting proliferation in slowly dividing cells. Thus, palmitoyl-arachidonyl-PG (16∶0/20∶4), palmitoyl-linoleoyl-PG (16∶0/18∶2), dilinoleoyl-PG (18∶2/18∶2) and soy PG (a PG mixture with a large percentage of polyunsaturated fatty acids) were particularly effective at inhibiting proliferation in rapidly dividing keratinocytes. Conversely, palmitoyl-oleoyl-PG (16∶0/18∶1) and dioleoyl-PG (18∶1/18∶1) were especially effective proproliferative PG species. This result represents the first demonstration of opposite effects of different species of a single class of phospholipid and suggests that these different PG species may signal to diverse effector enzymes to differentially affect keratinocyte proliferation and normalize keratinocyte proliferation. Thus, different PG species may be useful for treating skin diseases characterized by excessive or insufficient proliferation. Keratinocyte proliferation and differentiation are precisely regulated processes which are essential for proper formation and function of the epidermis of the skin to serve as a physical and water-permeability barrier Previously, our laboratory has shown the existence of a novel cell signaling module composed of the glycerol transporter, aquaporin-3 (AQP3) and phospholipase D2 (PLD2). Phospholipase D (PLD) is a lipid-metabolizing enzyme that can catalyze both phospholipid hydrolysis to produce phosphatidate and a transphosphatidylation reaction using primary alcohols, such as glycerol, to generate phosphatidylalcohols Egg PG is comprised of multiple PG species, with different acyl groups identifying the different PG species. Thus, egg PG exhibits the following fatty acid composition : 16∶0 (34%) 16∶1 (2%), 18∶0 (11%), 18∶1 (32%), 18∶2 (18%) and 20∶4 (3%) (Avanti Polar Lipids website). As a first step to define the mechanism underlying the normalization effect of egg PG, we sought to identify the PG species most effective at altering keratinocyte proliferation, with the assumption that the same species of PG would exert both effects on proliferation (inhibition of rapidly proliferating keratinocytes and enhancement of slowly growing cells). Cell proliferation was examined in order to screen a large number of PG species, although as the initial step in differentiation, growth arrest  often reflects effects on other differentiation processes, such as involucrin levels as we have shown previously Dihexanoylphosphatidylglycerol (DHPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylglycerol (DSPG), palmitoyl-oleoylphosphatidylglycerol (POPG), dioleoylphosphatidylglycerol (DOPG), palmitoyl-arachidonoylphosphatidylglycerol (PAPG), palmitoyl-linoleoylphosphatidylglycerol (PLPG), dilinoleoylphosphatidylglycerol (DLPG), soy-derived PG (soy PG), egg-derived PG, and dilinoleoylphosphatidylpropanol (DLPP) were all obtained from Avanti Polar Lipids, Inc. . The composition of egg PG is provided in the Introduction. Soy PG is composed of 16∶0 (17%), 18∶0 (6%), 18∶1 (13%), 18∶2 (59%), and 18∶3 (5%) (Avanti Polar Lipids website). Calcium-free minimal essential medium and antibiotics were obtained from Biologos, Inc. . Bovine pituitary extract and epidermal growth factor were purchased from Life Technologies, Inc. . ITS+ was supplied by Collaborative Biomedical Products .2 at 37°C in plating medium All animal studies were performed under a protocol approved by the Georgia Regents Institutional Animal Care and Use Committee and adhere to the standards described in the Guide for the Care and Use of Laboratory Animals. Mouse epidermal keratinocyte cell cultures were prepared from ICR strain CD-1 outbred neonatal mice 1–3 days of age as described in detail in 3H]Thymidine incorporation into DNA was assayed as a measure of keratinocyte proliferation as described in 3H]Thymidine at a final concentration of 1 µCi/mL, was added to the cells for an additional 1-hour incubation. Reactions were terminated using ice-cold 5% trichloroacetic acid and unincorporated radiolabel removed by washing. Cells were solubilized in 0.3 M NaOH, and the radioactivity incorporated into DNA quantified by liquid scintillation spectroscopy., which results in even spacing of the transformed concentrations. Cell proliferation rate  was transformed using ln(CPM). Because in some experiments cells were quickly proliferating, while in other experiments the cells were not, we grouped experiments that had different responses to egg PG using finite mixture regression models and the flexmix package detailed in 2 effect, which reflects the PG species change in the quadratic regression coefficient. The first effect relates to the overall difference in means between the PG species, although this difference may be dose dependent as reflected by the last two effects. Together, these three effects reveal how PG species change the overall relationship between cell proliferation [ln(CPM)] and PG concentration. The mixed model analysis was conducted using the Proc Mixed procedure in SAS 9.13. P≤0.05 was deemed as significant.We used a mixed model analysis of variance separately for each of the four grouped clusters identified above to determine how the different PG species affected the relationship between cell proliferation and PG concentration. The model used for this analysis of variance included PG species as a fixed effect that potentially interacted with ln(concentration), and random coefficients for each culture/experiment. This analysis accounts for the different responses for each individual culture/experiment. We focused on three effects from this model: (1) the PG species effect, which reflects the PG species change in intercept; (2) the PG species by ln(concentration) effect, which reflects the PG species change in the linear slope; and (3) the PG species by ln(concentration)3H]thymidine incorporation by 36%, suggesting a likely pro-stimulatory effect of saturated fatty acids as well (Since previous data indicated an ability of egg PG to inhibit the proliferation of rapidly dividing and stimulate the growth of slowly proliferating keratinocytes  as well . Since nPLPG (16∶0/18∶2), DLPG (18∶2/18∶2), and PAPG (16∶0/20∶4), each possessing at least one polyunsaturated fatty acid, were selected as PG species containing fatty acids comprising a minor fraction of those in egg PG. These PG species, prepared as liposomes, were directly provided to the primary mouse keratinocytes at doses as described above. Synthetic PLPG, DLPG, and PAPG were particularly effective at inhibiting keratinocyte proliferation in a dose-dependent manner . The iniWe also tested a synthetic PG with short acyl chains, which should be more soluble in aqueous solution and thus more easily applied, and determined that there was no significant effect of DHPG (6∶0/6∶0) on keratinocyte proliferation . This reOur results with PLPG, DLPG, and PAPG suggested an important role of polyunsaturated fatty acids in PG’s antiproliferative effects. However, these synthetic PGs are expensive to produce. Considering the potential medical application of this phospholipid to hyperproliferative skin disorders, we sought to determine the effects on keratinocyte proliferation of soy PG, which is a less expensive PG mixture containing a large percentage of polyunsaturated fatty acids, including linoleic (18∶2) and linolenic (18∶3) acids . Consistent with the effects of synthetic PG species containing polyunsaturated fatty acids, soy PG also significantly inhibited DNA synthesis in a dose-dependent manner . We nextIt was not technically feasible to conduct all of the tests of PG species reported above in one single set of experiments. Likewise, it was impossible to treat all keratinocytes at exactly the same degree of confluence across the multiple experiments required, as the primary cultures were prepared from neonatal mice of slightly different ages resulting in somewhat different plating efficiencies and thus confluence at treatment. Because epidermal keratinocytes are contact inhibited, these differences in confluence likely resulted in the observed disparate proliferation under control conditions. Consequently, to allow comparison among all the experiments, egg PG was tested as a comparator in each experiment. Note that a total of 38 separate experiments from 38 different keratinocyte preparations/primary cultures were performed, each with its own egg PG control to allow comparison among experiments with different degrees of confluence. Based on the response to egg PG, all of the results could be grouped into four clusters as shown in In a previous study, we used liposomes composed of dioleoyl- or dipalmitoyl-phosphatidylpropanol (DOPP or DPPP) as a phospholipid control for PG As shown in PG is garnering attention as a physiologically active phospholipid with potential involvement in cell signaling. Older studies from the Elias laboratory suggested the existence of a PG-activated protein kinase (PK-P) in human leukemia cells and human spleen Our previous study provided evidence for the existence in primary mouse keratinocytes of a novel lipid signaling pathway, for which PG is a key effector in regulating keratinocyte proliferation and differentiation  and DOPG (18∶1/18∶1), stimulated mouse keratinocyte proliferation, while PG species containing polyunsaturated fatty acids, such as the arachidonic and linoleic acids in PAPG (16∶0/20∶4), PLPG (16∶0/18∶2) and DLPG (18∶2/18∶2), inhibited mouse keratinocyte proliferation. It is perhaps not surprising that PG species with different acyl groups have different signaling functions since in the lung saturated PG cannot block the anti-inflammatory effects of surfactant protein A on lipopolysaccharide (LPS)-treated macrophages while unsaturated PG can The efficacy of linoleic acid-containing PGs is intriguing considering the fact that this fatty acid is the predominant species in the epidermis. Thus, Marcello and colleagues found that linoleic acid represents over 20% of the fatty acid species in the epidermis For each PG species we tested, we also tested at the same time within the same cell preparation the effects of egg PG as a comparator. By performing an egg PG dose response in all experiments, a later assessment among experiments could be accomplished. As demonstrated previously in vitro, the expense of these PGs could potentally preclude their use as a treatment for psoriasis. Therefore, we also investigated the ability of soy PG, a mixture of PG species containing a high proportion of polyunsaturated fatty acids, to inhibit keratinocyte proliferation. This lipid also has the advantage of being a natural product, and our results in vitro indicated its efficacy . Pattern recognition receptors can be activated not only in reponse to pathogen-associated molecular patterns but also damage-associated molecular patterns arising from cell injury  different fatty acids had disparate actions and (2) phospholipids similar to PG but lacking the glycerol headgroup (phosphatidypropanol) were less efficacious than the corresponding PG in promoting proliferation or differentiation. To our knowledge these findings also represent the first (and so far only) demonstration of the ability of different species within a given phospholipid class to induce opposite effects in intact cells. Future investigation is ongoing to determine the downstream targets of the AQP3-PLD2-PG signaling pathway and the mechanism by which the different PGs exert these disparate effects. Since abnormal keratinocyte proliferation and differentiation characterize a wide range of skin diseases, the findings of this study may open new avenues for treatment of these diseases. Furthermore, our results suggest that different PG species may be useful for treating various skin diseases characterized by excessive or insufficient proliferation.


CCL28-induced growth modulation was used as marker of chemokine responsiveness, and 38 patients were then classified as chemokine-responsive. The effects of exogenous CCL28 (growth inhibition/enhancement/no effect) thus differed among patients and was also dependent on the presence of exogenous hematopoietic growth factors as well as constitutive AML cell cytokine release. The effect of CCR1 inhibition in the presence of chemokine-secreting mesenchymal stem cells also differed among patients. Chemokine-responsive AML cells showed altered expression of genes important for (i) epigenetic transcriptional regulation, particularly lysine acetylation; (ii) helicase activity, especially DExD/H RNA helicases; and (iii) angioregulatory proteins important for integrin binding. Thus, chemokine responsiveness is part of a complex AML cell phenotype with regard to extracellular communication and transcriptional regulation. Chemokine targeting in chemokine-responsive patients may thereby alter AML cell trafficking and increase their susceptibility toward antileukemic treatment, e.g., conventional chemotherapy or targeting of other phenotypic characteristics of the chemokine-responsive cells.Acute myeloid leukemia (AML) is an aggressive and heterogeneous bone marrow malignancy, the only curative treatment being intensive chemotherapy eventually in combination with allogeneic stem cell transplantation. Both the AML and their neighboring stromal cells show constitutive chemokine release, but chemokines seem to function as regulators of AML cell proliferation only for a subset of patients. Chemokine targeting is therefore considered not only for immunosuppression in allotransplanted patients but also as a possible antileukemic strategy in combination with intensive chemotherapy or as part of disease-stabilizing treatment at least for the subset of patients with chemokine-responsive AML cells. In this study, we characterized more in detail the leukemia cell phenotype of the chemokine-responsive patients. We investigated primary AML cells derived from 79 unselected patients. Standardized  Acute myeloid leukemia (AML) is an aggressive disease characterized by infiltration of malignant myeloblasts in the bone marrow; the only curative treatment is intensive chemotherapy potentially in combination with allogeneic stem cell transplantation . Howeverin vitro studies suggest that chemokines function as growth regulators in leukemic hematopoiesis only for a subset of AML patients, and a wide range of both CCL and CXCL chemokines can then modulate leukemia cell proliferation . Samples were collected after written informed consent. AML blasts were derived from 79 consecutive patients . Six patients had AML relapse Table .Prior to the study, we decided to include 80 unselected patients ; based on a previous study , we woul9/L) of leukemia cells among the peripheral blood leukocytes (The study included only patients with a high percentage (>80%) and a high absolute number (>15 × 10ukocytes . A cell ukocytes . Cells wukocytes .3H-thymidine incorporation (3H-thymidine incorporation) in transwell cocultures with bone marrow mesenchymal stromal cells  , and all recombinant cytokines were supplied by PeproTech . All exogenous cytokines were added at 20 ng/mL, i.e., corresponding to an excess of the added cytokine. Our methods for flow-cytometric characterization of AML cell viability . Amount and quality of the biotin-labeled cRNA were controlled both by NanoDrop spectrophotometer and Agilent 2100 Bioanalyzer, before 750 ng of biotin-labeled cRNA was hybridized to the HumanHT-12 V4 Expression BeadChip according to the manufacturer’s instructions. The chip targets 47,231 probes derived primarily from genes in the NCBI RefSeq database (Release 38).U-test was used to compare value distributions among different patient groups, and χ2 tests (Pearson’s χ2 test and the likelihood ratio) were used to determine correlations between different categories. Additionally, Kendall’s tau-b correlation tests were performed to determine the agreement between cytokine expression levels in cell supernatants and the mRNA levels of these cytokines. P-values <0.05 were regarded as statistically significant.Bioinformatical analyses were performed using the J-Express 2012 software . For hierarchical clustering of the cytokine secretion, all values were median-normalized and log(10)-transformed. Complete linkage and Pearson correlation were used as linkage method and distance measurement, respectively. The statistical analyses were performed with the IBM Statistical Package for the Social Sciences (SPSS) version 23 . The Mann–Whitney in vitro culture in medium alone . The presence of 20 ng/mL exogenous CCL28 during culture did not significantly alter AML cell viability  when comparing the overall results (data not shown). Thus, exogenous CCL28 does not have any major effect on the regulation of spontaneous in vitro apoptosis for primary human AML cells.There was a wide variation among the 79 AML patients with regard to the viability of the leukemic cells after 40 h of 3H-thymidine incorporation assay, where the nuclide was added after 6 days and nuclear activity assayed 24 h later. A median value of at least 1,000 counts per minute (cpm) in triplicate cultures was defined as detectable proliferation, whereas a significant alteration of 3H-thymidine incorporation was defined as (i) an increase/decrease corresponding to at least 20% of the control culture, and the absolute value of this change being at least 2,000 cpm for patients with detectable proliferation or alternatively, (ii) a change from detectable to undetectable proliferation or vice versa , stem cell factor (SCF), or FMS-like tyrosine kinase 3 ligand (Flt3L). A detectable proliferative response for at least one of these three growth factors was detected for 42 of the 56 patients, and the results for these patients are summarized in the hierarchical cluster analysis presented in Figure CCL28 can bind to the CCR3 and CCR10 receptors –15. We u6 cells/mL, 48 h of in vitro culture). Detectable CCL28 release was observed for only 18 patients , whereas the other chemokines were released for most patients (Table in vitro apoptosis), differentiation , karyotype, and Flt3 or NPM1 mutations (data not shown). Finally, there was no significant association between constitutive CCL28 release and spontaneous/autocrine in vitro AML cell proliferation, and constitutive CCL28 release was seen both for patients with and without CCL28-associated growth modulation.The constitutive release of 11 chemokines was investigated for all 79 patients . The chemokine levels for each individual patient was median-normalized and log(10)-transformed prior to Pearson clustering in J-Express. The analysis is presented in Figure It can be seen from Figure Bone marrow failure with pancytopenia in peripheral blood is common at the time of diagnosis for AML patients . Furtherin vitro model, where primary human AML cells and normal bone marrow MSCs derived from a healthy donor were separated by a semipermeable membrane during coculture  of <0.01. The identified genes  as an immunosuppressive treatment. GVHD has antileukemic effects, and immunosuppression due to severe GVHD will reduce this effect. However, at least for patients with chemokine-responsive AML cells, the use of chemokine targeting may represent a therapeutic alternative that reduces the GVHD-associated antileukemic reactivity but at the same time has an additional antileukemic effect by itself against residual AML.Chemokine receptor blockers can be used as an immunosuppressive strategy, e.g., as GVHD therapy in AML patients receiving allogeneic stem cell transplantation . ChemokiAcute myeloid leukemia is a heterogeneous and aggressive disease, and in a previous study of another smaller patient population, we observed that exogenous CCL28 could modulate cytokine-dependent AML cell proliferation for a minority of patients. For this patient subset, CCL28 was one out of several chemokines with growth-modulating effect . SeveralCCL28 is a chemokine involved in normal hematopoiesis . We now We included 79 patients in the present study, and they represent a consecutive and thereby unselected group, except that all of them showed relatively high levels of circulating AML cells. This strategy was used to allow a standardized preparation of highly enriched AML cells by gradient separation alone, thereby avoiding the risk of inducing functional alterations in the cells by more extensive separation procedures . The usein vitro culture; (ii) an intermediate subset of clonogenic progenitors (often <1%); and (iii) a small minority of AML stem cells  a majority of more mature cells undergoing stress-induced apoptosis during em cells , 26. Stus often <%; and  in a more physiological model of AML/MSC cocultures; this model was used to study chemokine effects in a local cytokine network with a major influence of bone marrow stromal elements. Our results show that even in a cytokine environment formed by the constitutive cytokine release and the cytokine-mediated cross talk between primary AML and bone marrow MSCs, the CCR1/CCR3-mediated effects on AML cell proliferation are divergent, and the different effects of CCR1/CCR3 inhibition on AML cell viability and proliferation can be explained by the hierarchical organization of the AML cell population . The viain vitro, studies in chronic lymphocytic leukemia (CLL) have shown that altered regulation of in vivo cell trafficking can contribute significantly to the antileukemic effects of certain drugs  showed an altered expression of genes included in the GO-terms lysine acetylation/integrins/helicases. As discussed above, there is a functional interaction between CCL28 and integrins, but there is also a biological interaction between the integrin system and lysine acetylation. First, histone acetylation is important for regulation of gene expression , includiHelicases are also important for transcriptional and translational regulation and thereby seem to represent a functional overlap with lysine acetylation. First, a major part of the helicases with altered expression are members of the DEAD box family of RNA helicases, they belong to the helicase superfamily 2 together with the DEAH helicases and are often referred to as DExD/H box RNA helicases , 53. Thein vivo models  to see whether chemokine targeting alters AML cell trafficking/homing and thereby increases their chemosensitivity.Chemokines have growth-modulating effects on the AML cells only for a minority of patients; and these patients are then responsive to several chemokines. However, this chemokine responsiveness is only a part of a more extensive leukemia cell phenotype that includes several partly interacting cellular functions, i.e., altered extracellular communication (chemokines/integrins/acetylation) and transcriptional regulation  compared with the other non-responsive AML cells. Furthermore, the direct effects of chemokine targeting on primary human AML cells are difficult to predict because they depend both on differences among patients and on the overall local cytokine network; this is probably true both if chemokine targeting is tried as an antileukemic strategy or as an approach to treat immune-mediated complications in allotransplanted AML. A careful evaluation of the AML cells will possibly be important to avoid the risk of leukemia enhancement and thereby increased relapse risk; this would probably be important if the treatment is tried both as an antileukemic and an immunosuppressive strategy. A possible approach to avoid this risk may be combined targeting of other aspects of this complex phenotype. However, based on the lessons from recent studies in CLL where chemokines are important for the supportive function of neighboring stromal cells by keeping the CLL cells within their permissive microenvironment, one would emphasize that AML cells from chemokine-responsive patients should be further tested in relevant ØB conceived and designed the experiments; AB performed the experiments; AB and HR analyzed the data; and ØB and AB 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.


Drosophila have focused on opportunistic human pathogens. Here, we performed a screen of 2,613 mutant Drosophila lines to identify host genes affecting susceptibility to the natural insect pathogen Metarhizium anisopliae (Ma549). Overall, 241 (9.22%) mutant lines had altered resistance to Ma549. Life spans ranged from 3.0 to 6.2 days, with females being more susceptible than males in all lines. Speed of kill correlated with within-host growth and onset of sporulation, but total spore production is decoupled from host genotypes. Results showed that mutations affected the ability of Drosophila to restrain rather than tolerate infections and suggested trade-offs between antifungal and antibacterial genes affecting cuticle and gut structural barriers. Approximately, 13% of mutations where in genes previously associated with host pathogen interactions. These encoded fast-acting immune responses including coagulation, phagocytosis, encapsulation and melanization but not the slow-response induction of anti-fungal peptides. The non-immune genes impact a wide variety of biological functions, including behavioral traits. Many have human orthologs already implicated in human disorders; while others were mutations in protein and non-protein coding genes for which disease resistance was the first biological annotation.Fungi cause the majority of insect disease. However, to date attempts to model host–fungal interactions with  Drosophila melanogaster has been the model of choice to develop ideas about innate immunity and host–pathogen interactions1Drosophila activates a wide range of inducible reactions when microbes enter the hemocoelToll pathwayThe fruit fly Metarhizium anisopliae that are naturally pathogenic to Drosophila. Aside from playing a crucial role in natural ecosystems, Metarhizium spp. are being developed as alternatives to chemical insecticides, and as a model for understanding how fungi infect insectsDrosophila model system to investigate the interplay between host components and fungal strategies to circumvent these components. However, other than the well-characterized activation of the Toll pathwayDrosophila’s interactions with naturally occurring fungal pathogens.Fungi cause the majority of insect diseaseDrosophila’s ability to resist infection by M. anisopliae ARSEF549 (Ma549), utilizing Minos-element insertion lines that were constructed in isogenic backgrounds. Unlike viruses and bacteria that normally infect through the oral route, M. anisopliae breaches the cuticle reaching directly into the hemocoel using a combination of mechanical pressure and an array of cuticle-degrading enzymesM. anisopliae much more amenable to screening than opportunistic human fungal pathogens that have to be injected into the hemocoel to cause infection, and allowed us to study the whole suite of host defenses that the fly is able to mount. By focusing on pathogen life history traits and fly survival rather than just monitoring antimicrobial peptide (AMP) transcription, as many studies do8Drosophila’s resistance to a natural fungal pathogen, and will allow the identification of gene networks that may be specific to M. anisopliae infection, to insect pathogenic fungi in general, or to a variety of opportunistic mammalian pathogens.Here we report the results of a screen for mutations affecting Drosophila Minos insertion mutant collection  had altered resistance to Ma549. This indicates a large mutational target for disease resistance, consistent with pleiotropy and many biological processes affecting this traitAge-matched flies from each mutant line were infected topically with spores of Ma549, and survival was monitored. A total of 91 mutant lines demonstrated enhanced resistance, while 150 demonstrated lowered resistance compared to wild-type flies, and these were defined as being “resistant” or “susceptible”, respectively . The aveConnectin gene), and seven were in long non-protein coding genes, 22.4% of which, including six of the non-protein coding genes, had unknown functions. The Drosophila genome has a few hundred non-coding RNAs but they are poorly characterizedhsr-omega, has functions affecting protein synthesis and oogenesis. A total of 17 inserts were in regions with no annotated genes within 5 kb of the Minos insertion site and either have long-range effects on the neighboring gene(s) or affect an un-annotated gene in the more immediate vicinity. Thirteen lines have inserts located in overlapping genes and four additional lines had inserts in an intergenic region <2kb from two flanking genes, and could affect either or both genes.These 241 lines included 180 with insertions in the coding regions of genes and 19 lines with insertions in upstream promoter regions. Three lines have insertions 2-5kb upstream of a gene and five lines have insertions within 2 kb downstream of the 3’UTR of a gene. These genes are listed in Minos inserts did not decrease general robustness of susceptible flies or increase robustness of resistant flies. Climbing ability is another physiological index commonly applied to flies, and we assessed the correlation between time of death and climbing ability for a subset of 54 randomly selected mutant lines , latent period (the lag time between inoculation and sporulation), and sporulation capacity . Spore production is a measure of pathogen transmission potential and therefore pathogen fitness2) of the variation in life span is explained by variation in fungal load at this time point. CFU’s per insect ranged from <5 in the most resistant flies to over 8,000 in susceptible flies, showing that susceptible host genotypes were much less able to restrain fungal growth. A time course of CFU counts confirmed that wild type (LT50 4.3 days) and resistant flies delayed fungal growth compared to susceptible flies , host life span .Host genotype impacted the onset of Ma549 sporulation which strongly correlated with life span ; correla50 was 1.08 days less than males). Females disrupted in CG3940 died faster than males but the difference fell short of significance (p = 0.06046). We found no significant differences between virgin and mated wild type females and between virgin and mated wild type males in susceptibility to Ma549.To identify sexual dimorphism, we measured disease resistance separately for males and females in the 54 randomly selected lines . We obseDrosophila genes that affect susceptibility to Metarhizium have human counterparts. Overall, 54 lines (22.4%) with altered susceptibility have inserts in genes with no known function in Drosophila. Nine of these genes are evolutionary conserved and have human counterparts with no phenotype found, i.e., altered susceptibility to Metarhizium is the first phenotype described for them.Gene ontology (GO) analysis revealed diverse categories of genes that confer increased susceptibility or resistance to Ma549 . Most  expression is up-regulated by Metarhizium-infected fliespipe (stock numbers 24732 and 29054) and spheroide (29227), involved in activating the Toll pathway, and core Toll signaling pathway components Pellino (26071), Gprk2 (26097) and ush (23467), did not affect susceptibility to Ma549 although they are known to be involved in production of DrosomycinDrs-GFP, a classical read-out of activation of the Toll pathwayDrs-GFP flies disrupted in the critical Toll pathway gene dif show no GFP fluorescence in response to Ma549, but nor were they more susceptible to Ma549  of our 241 candidate genes , overlapped with candidate innate immune response genes identified in previous screens using viruses81415161718 and ush 467, did to Ma549 . Real tiMetarhizium when functioning normally  is necessary for encapsulationRhoGEF3 increased susceptibility of Drosophila to Ma549, suggesting commonalities in defense against fungi and larger parasitoids. During encapsulation, plasmatocytes deposit a protein layer onto the parasite derived from their extracellular matrix proteins (ECM)peroxidasin, that functions in both phagocytosis and encapsulationcaupolican and a clathrin-binding protein (likeAP180) that regulate phagocytosis against C. albicansCryptococcus neoformansAlthough the Toll pathway is ineffective against Ma549, there was substantial evidence that other components of defense provide resistance. A group of susceptible lines were mutated in cuticle biosynthesis genes, and presumably limit cuticle penetration by normally . An inseheartless), an endopeptidase (CG11843), an ATPase involved in cell motility (CG14838), a peroxidase (CG5873) and two genes with unknown functions (CG14331 and CG32198) increased susceptibility to Serratia marcescensCpr97Eb and an unknown gene CG31323 increases resistance to S. marcescenssrpk79D increases resistance to Listeria monocytogenesDisrupting an FGF receptor , early development, muscle and nervous system development and function, chemosensation and vision, and metabolism; 25.2% of the mutations represent diverse pathways affecting morphogenesis and neurogenesis. Thirteen genes from susceptible lines (and eight genes from resistant lines), have human orthologs with describers that include an aspect of temperament , or cognitive development including learning.Drosophila survival to bacteria28CkIIalpha, associated with circadian rhythms in a previous studyOctbeta2R, Oamb) and dopamine (Dop1R1) receptors, which work together in Drosophila learning, and form memories for sugar and other foodsdpr4) or near (dpr15) members of the Ig family that regulate taste perception and proboscis extensionAstA-R1, AstA-R2 and AstC-R2), were mutated in the allatostatin signaling pathway that directly regulates food intake by adult DrosophilaOctbeta2R, Oamb and Dop1R1 mutated lines were not significantly altered in starvation tolerance; dpr4 , dpr15  and AstA-R2  lines were significantly more resistant and AstA-R1 was significantly less  resistant to starvation.Nutritional factors and circadian rhythms are known to affect Drosophila and mammalsDrosophila have human counterparts associated with diseases, with the obesity category being one of those prominently associated with susceptibility , sensory inputs, and immunity . Starvation tolerance may depend on metabolic state, but Ma549 resistance in lines affected in basic metabolic processes also did not correlate with starvation resistance  . AnalyzeSpn, Arrest and Px previously implicated in reduced aggressive behavior in fliesDrosophila’s Synaptotagamins , increased resistance. Synaptotagamins are responsible for multiple types of Ca2+-induced exocytosis, but their exact biological roles in mammals and Drosophila are controversial36CG31760 and the glutamate Ca2+ ion channel CG11155 also increased resistance consistent with Ca2+ having an unknown function inhibiting Drosophila’s defense against Ma549. Two other genes (CG13793 and Neurexin IV), associated with exocytosis and neurotransmitter transport were disrupted in resistant lines as was Still life, a Rho GTPase regulator of the neural cytoskeleton.Six behavioral genes were found in Ma549-resistant mutant flies. Of particular note, mutations in Drosophila lines, resistant lines were disrupted in genes with pleiotropic effects on other complex traits such as morphogenesis and response to stimuli. Not surprisingly, resistant lines were disrupted in fewer (three) positive regulators of processes than susceptible lines (eleven). Three resistant lines were disrupted in negative regulators of multicellular growth (Ac76E), transcription (pgc) and translation (Arrest). As previously mentioned Arrest is also implicated in moderating aggression illustrating the pleiotropic nature of many genes implicated in disease resistance.As with susceptible Drosophila lines and their susceptibility to Ma549 does not affect total spore production on individual flies because Ma549 sporulates only after host death. Instead, trade-offs effect latent periods which are longer on resistant hosts, so a Metarhizium strain that evolves to kill these as quickly as susceptible lines should have higher lifetime fitness. However, transgenic Metarhizium strains that killed very quickly came at a cost of decreased spore production39Ma549 life history stages show apparent density-dependent effects; greater growth within the host was correlated with shorter life spans and earlier onset of reproduction. The most commonly accepted model assumes that within-host growth is a critical parameter in the evolution of virulence, but that high virulence is traded-off against increased host mortality cutting short the infectious period for transmission between hosts7Metarhizium strains, Ma549 does not use toxins and kills principally by invasive growth and depleting the host of nutrientsDrosophila. Fly lines that succumbed quickly to Ma549 all had higher fungal loads than wild type flies 3.5 days post-infection, suggesting that the principal defect in these flies is that they are less able to restrain Metarhizium growth. In contrast, many Drosophila mutants succumb to bacterial infections because of defects in tolerance rather than resistance8pvf1, involved in wound repairdystrophin increase susceptibility because these genes are normally modulators of both tolerance and resistance. This could occur if reduced damage repair in the vicinity of invasive hyphae facilitates rapid fungal growth. However, none of the resistant lines appeared to tolerate Ma549 better than the wild type because their delayed mortality coincided with delayed rapid proliferation of Ma549.Unlike some other Drosophila lines. However, the concept of trade-offs is central to many evolutionary hypotheses for limited lifespan and optimal allocation of resourcesDrosophila and in particular determine how nutrients and energy are distributed among organs during different phases of bacterial and fungal infectionsThe first line of defense that prevents microbial invasion into the hemocoel is structural, and comprises the external cuticle (for fungi), the gut peritrophic matrix (for bacteria), and the tracheal lining. Mutations in some genes that affect the gut matrix caused Ma549 infected flies to live longer implying trade-offs between resistance to fungal diseases and increased susceptibility to gut pathogenic bacteria. Possibly, knocking out antibacterial defenses in the gut allows resources to be diverted to anti-fungal defenses in the cuticle and hemolymph. There are alternative possibilities such as antagonism between Ma549 and commensal bacteria if the latter flourish more in some Drosophila typically involves injecting the pathogen of interest into female flies because of their larger size and relative resistance to injection injury when compared with male fliesDrosophila to defend against bacteriaModeling fungal or bacterial infections in Drosophila are resistant to most pathogenic fungi and bacteria1Metarhizium spp. to function as professional entomopathogens. Tolerance to products of the Toll pathway, e.g., drosomycin, will prevent clearance of Ma549 from the hemocoel, which likely dooms the fly. Nevertheless, the degree of functioning of the cellular immune response against Ma549 was somewhat surprising. Metarhizium blastospores can evade hemocytes by producing a hydrophilic collagen (Mcl1) coat51Metarhizium often produces a melanization reaction in the cuticle at the site of entryMetarhizium spp., mainly due to toxic effects from L-DOPA oxidation products53Even when fungal pathogens are experimentally introduced directly into the fly hemolymph, wild-type flies are still capable of eliminating infection because they activate a wide range of immune mechanisms1Drosophila mutants in the obesity category included orthologs to human genes impacting sensory inputs and immunity, and nine have human orthologs that also influence temperament   at 24 ± 3 °C. The single insertion site in each line has been mapped and verified by resequencing of flanking DNAw1118stock number 5905; y1w67c23 stock number 6599) were employed as controls.The Drs-GFP, M. anisopliae (ARSEF 549) was obtained from the USDA Entomopathogenic Fungus Collection . Fungal cultures were moved from −80 degree stock tubes 10 days before each bioassay and grown on potato dextrose agar at 27 °C. Plasmid construction and transformation for GFP fluorescent Ma549 strains was described previously50 values from individual lines were compared with the mean LT50 of the experimental cohort treated the same day. Each cohort ranged from 100 to 200 lines. A fly line with an LT50 value ±5% different than the average LT50 was retested with a new cohort. Out of 2,613 lines, 866 were identified as putative candidates. In the secondary screen, putative candidate lines where compared with their control isogenic background line , and the LT50 values were analyzed using Welch’s T-test. The survival data from candidates were also compared to controls each day post infection using a Log-rank (Mantel-Cox) test (SPSS). Fly lines with significantly lower survival than the wild type (p ≤ 0.05) were identified as susceptible lines and lines with significantly longer survival (p ≤ 0.05) than wild type were identified as resistant lines.During bioassay, flies were maintained at 27 °C, ~85% humidity, on food made without Tegosept and propionic acid. We bioassayed ~30 flies (2–4 days old) per mutant line with conidia from 10 day old Ma549 plates see . For theMinos element insertion locations, genes affected by insertions, gene ontology, and gene expression levels were obtained from Flybase (http://flybase.org), Blast2Go Tool (https://blast2go.com), and FlyAtlas (http://flyatlas.org). In addition, we manually looked for genes that may be affected by the Minos element if the insertion site is within 2 kb from a gene. Gene ontology enrichment analysis was conducted using the DAVID programDetailed information on http://www.flyrnai.org) to identify human orthologs and disease traits, and analyze alignments and scores. The DIOPT-DIST search tool in DRSC includes information from the NCBI OMIM database and genome-wide association studies. A low score (≤2) was excluded unless it was the only match score.We used the DRSC platform  per mutant line were sub-divided into five fly vials each containing 5 ml of 1% agar and maintained at 27 °C, ~85% humidity. Dead flies were recorded every 8 hours for 72 hours. LTy1w67c23 background and so the y1w67c23 line was used as a control. To measure sexual dimorphism, ~25 females or males per line were tested per tube and the infection methods were as described in the fungal infection assay. Each experiment was replicated three times and LT50’s were calculated using SPSS 22.0.We randomly selected 29 susceptible and 25 resistant strains , to testTo measure fungal growth in the haemolymph, 3.5-days post inoculation, 5 flies per sex were individually homogenized with 45 μl of 0.1% Tween 80. As most females from susceptible lines were dead by 3.5 days, we only analyzed data collected from the males of these lines. For resistant lines, the entire 45 μl homogenate was spread onto selective medium plates. For susceptible lines, 5 μl of a 10-fold dilution of the homogenate was spread onto plates. Colony forming units (CFUs) were counted after 7 days. For latent period and sporulation capacity, ten flies per sex harvested within 12 hours of death, were individually transferred into tubes containing a damp cotton ball and time of appearance of spores (latent period) was recorded. After 20 days, 500 μl of 0.1% Tween 80 was added to each tube, the tubes were vortexed (1 minute), and spore counts per individual fly were made using a hemocytometer. Results are the average of 10 flies per line.To assess climbing ability, a group of ~25 flies were transferred without anesthesia to a 50-mL conical tube. The flies were tapped to the bottom of the tube as a timer was simultaneously started. Images were taken at three, six and nine seconds and subsequently processed to determine the height of individual flies at each time point. Height and time values were then used to generate the slope of the line. Three replicates per line were performed and compared with the wild type control using Student’s t-test.Correlations between LT50 survival values and starvation, CFU counts, latent period, sporulation capacity, and climbing ability, were analyzed using GraphPad Prism 6 .How to cite this article: Lu, H.-L. et al. Identification of Drosophila Mutants Affecting Defense to an Entomopathogenic Fungus. Sci. Rep.5, 12350; doi: 10.1038/srep12350 (2015).