Altered functional neuroanatomy of high-order cognitive processing has been described in very preterm individuals  compared to controls in childhood and adolescence. However, VPT birth may be accompanied by different types of adverse neonatal events and associated brain injury, the severity of which may have differential effects on brain development and subsequent neurodevelopmental outcome. We conducted a functional magnetic resonance imaging (fMRI) study to investigate how differing degrees of neonatal brain injury, detected by neonatal ultrasounds, affect the functional neuroanatomy of memory processing in VPT young adults. We used a verbal paired associates learning task, consisting of four encoding, four cued-recall and four baseline condition blocks. To further investigate whether differences in neural activation between the groups were modulated by structural brain changes, structural MRI data were also collected. We studied 12 VPT young adults with a history of periventricular haemorrhage with associated ventricular dilatation, 17 VPT individuals with a history of uncomplicated periventricular haemorrhage, 12 individuals with normal ultrasonographic findings, and 17 controls. Results of a linear trend analysis demonstrated that during completion of the paired associates learning task right frontal and right parietal brain activation decreased as the severity of neonatal brain injury increased. There were no statistically significant between-group differences in on-line task performance and participants' intelligence quotient (IQ) at assessment. This pattern of differential activation across the groups was observed particularly in the right middle frontal gyrus during encoding and in the right posterior cingulate gyrus during recall. Structural MRI data analysis revealed that grey matter volume in the right superior temporal gyrus, right cerebellum, left middle temporal gyrus, right globus pallidus and right medial frontal gyrus decreased with increasing severity of neonatal brain injury. However, the significant between-group functional neuroanatomical differences were not directly attributable to the detected structural regional differences. Children and adolescents who were born before 33 weeks' gestation  show poorer performance compared to controls on a variety of memory tasks, including working, spatial and episodic memory in childhood and adolescence Damage to the hippocampus, possibly due to hypoxia-ischemia, has been postulated to underlie memory deficits in VPT populations The most common form of brain injury following VPT birth is periventricular haemorrhage (PVH), which is well-recognized on neonatal cranial ultrasounds The greatest degree of neonatal insult, i.e. PVH+VD, is likely to cause the greatest disruptions in brain growth in VPT individuals In addition, individuals who experienced PVH+VD are the most vulnerable to developmental compromise To our knowledge, no study to date has investigated whether the functional neuroanatomy of memory processing in VPT individuals varies according to their degree of neonatal brain injury. Verbal paired associates learning tasks are used to assess the episodic memory processes We used fMRI with a verbal paired associates learning task we previously used Ethical approval for the study was granted by King's College Hospital Research Ethics Committee. All participants gave their written informed consent to undergo assessments.Between 1979 and 1984, 368 infants born very preterm  were admitted to the neonatal unit at the University College London Hospital (UCHL), and survived to be discharged. All VPT individuals received neonatal ultrasound scans daily for the first 4 days, at 1 week, and weekly until discharge from the hospital At 14–15 years, 269 individuals of the original cohort agreed to be assessed. Results of the adolescent assessment have been previously published The current study included 19 of those VPT individuals who had previously participated in fMRI studies and 22 newly–recruited VPT individuals. VPT study participants were chosen on the basis of their neonatal ultrasonographic findings. These were classified according to previously specified criteria All VPT participants were dextral, as assessed by clinical neurological examination at 14–15 years of age. Exclusion criteria were: severe head injury, stroke, epilepsy and multiple sclerosis, severe eyesight impairment, hearing and/or motor impairment, metal implants or a fitted pacemaker, operations to the head or the spine, claustrophobia, and pregnancy for female participants.Term-born control data were drawn from healthy individuals previously studied and were selected according to age, handedness and gender in order to be comparable to the VPT participants All participants were English native speakers.2(3) = 0.45, p>0.05], SES  and educational level , but there were significant between-group differences in age at assessment . Post-hoc comparisons showed that the PVH+VD group was significantly older than the normal VPT  and the control groups .One VPT participant with a history of psychiatric illness was excluded from the analyses. Twelve VPT young adults with a history of PVH+VD (newly recruited), 17 with a history of UPVH (9 previously studied and 8 newly recruited), 12 VPT young adults with normal ultrasonographic findings (10 previously studied and 2 newly recruited), and 17 term-born controls were studied . Information about sex, age at assessment, educational level and socio-economic status (SES), categorized according to a standard occupational classification  = 1.42, p>0.05] and gestation at birth  between the three VPT groups.Neonatal data i.e. birth-weight (grams) and gestation at birth (weeks) were previously collected for VPT study participants only. There were no significant differences in birth-weight , verbal  and performance  IQ between the PVH+VD, UPVH and normal VPT groups and controls.ANCOVA, controlling for age at assessment, revealed no statistically significant differences in full scale . A one-way repeated-measures ANOVA showed a significant effect of learning, measured in terms of number of correctly recalled words in the cued-recall phase of the task across the four blocks, for all study participants .ANOVA revealed that there were no significant between-group differences in the mean number of correct responses given during the recall condition of the task , and right posterior cingulate gyrus .To explore the relationship between birth-weight and SSQ values in brain regions where functional between-group differences were observed, multiple linear regression analysis was performed. Results suggested that birth-weight did not have a significant unique contribution to the prediction of the SSQ values in the right middle frontal gyrus  and in right posterior cingulate gyrus (BA 30) , where PVH+VD < UPVH < normal VPT < controls during encoding and recall, respectively.Multiple linear regression results indicated that structural between-group differences i.e. structural grey matter volume in the right superior temporal gyrus (BA 22), right cerebellum, left middle temporal gyrus (BA 21), right globus pallidus and right medial frontal gyrus  were not significantly associated with functional differences in right middle frontal gyrus (BA 46) [F This study demonstrates that the adult neuroanatomy of mnemonic processing is modulated by the severity of neonatal brain injury. This occurs in a linear fashion, with those individuals with a history of periventricular haemorhage with associated ventricular dilatation (PVH+VD) displaying the greatest extent of right hypofrontality and decreased right parietal activation during performance of a verbal paired associates learning task compared to the other three groups. In addition, VPT individuals with a history of uncomplicated periventricular haemorrhage (UPVH) show decreased activation compared to VPT individuals with normal ultrasound findings, who, in turn, show reduced activation compared to controls.The study groups  displayed a comparable on-line performance and achieved comparable scores on measures of IQ and they did not signicantly differ in terms of educational level at assessment. These data are in line with previous fMRI studies describing altered brain activation in VPT young adults in the absence of significant between-group differences in on-line task performance or measures of IQ at assessment During encoding, our results identified altered regional brain activation in VPT young adults with differing degrees of neonatal brain injury, where PVH+VD < UPVH < normal VPT < controls, in the right dorsolateral prefrontal cortex (DLPFC), in a cluster with local maxima in right middle frontal gyrus (BA 46), extending ipsilaterally to the middle frontal gyrus (BA 10), and to the inferior frontal gyrus (BA 46). Studies investigating the neuroanatomy underlying the encoding of paired-associates which are subsequently remembered have suggested the involvement of the right DLPFC, possibly due to its role in organizing information in working memory, thereby strengthening associations among items in episodic memory During recall, the same pattern of differential activation across the study groups i.e. PVH+VD < UPVH < normal VPT < controls was observed in a cluster with local maxima in the right posterior cingulate gyrus (BA 30), extending bilaterally to posterior cingulate (BA 30 and 31). Lesion studies suggest that damage to the posterior cingulate gyrus may result in loss of verbal episodic memory Taken as a whole, the results of the current study show that regional brain activation decreases with increasing severity of neonatal brain injury in regions mediating verbal paired-associate learning (task-specific regions: posterior cingulate gyrus) and more extensively, in regions subserving working memory, an executive component of the verbal paired associates learning task (DLPFC). Hypoactivity in the DLPFC has been described in developmentally delayed populations, such as individuals with attention deficit hyperactivity disorder (ADHD) during executive-type tasks Nevertheless, in spite of attenuated activation in these regions, we observed similar levels of on-line task performance in VPT young adults with differing degrees of neonatal brain injury and controls. This may be because the studied task was relatively easy to perform, as we wished to ensure all participants would be able to complete it during the scanning session. Behavioural differences may become apparent with increasing cognitive load, when VPT individuals with differing degrees of neonatal brain injury may fail to optimally engage task specific areas and more general ‘executive’ brain areas, which could result in impaired task performance When investigating the relationship between participants' structural volume in the regions listed above where significant between-group differences were observed and functional MRI results, we did not detect a statistically significant association. This is in contrast to previous studies in similar samples, where we observed that structural differences between the groups accounted for a small percentage of the variance of fMRI results However, we cannot rule out that differences in neuronal activation between the groups may be associated with cytoarchitectonic changes in the brain regions where structural between group differences were observed  A limitation of this study is that the fMRI data reported here may not be generalizable to VPT populations – for example VPT young adults who have compromised cognitive function were not studied. Additionally, we have not collected any data on other factors that may have contributed to fMRI differences among the study groups, such as family history of psychiatric disorders and maltreatment during development. Other limitations include the age difference between VPT participants with a history of neonatal brain injury  and VPT participants with normal ultrasonographic findings and controls. The majority of the participants belonging to these two latter groups (see Methods sections) were scanned at an earlier time point i.e. approximately 5 years before the PVH+VD and UPVH groups, due to logistic reasons. We, however, controlled for age at assessment in the fMRI data analyses. Further limitations arise from the use of a previously scanned normal VPT and control group and relate to possible implications of scanner variation over time. Nevertheless, fMRI experiments measure changes in BOLD signal, which is less affected by hardware changes as it relies on an innate contrast mechanism between experimental conditions and a baseline. Quality control of the fMRI data acquired throughout the time of the two studies was also carried out to identify variations of BOLD signal intensity using an automated data processing scheme and Shewhart charting To summarise, the results of this study suggest that increased severity of early brain injury is associated with decreased neural recruitment in regions mediating verbal paired-associate learning (i.e. posterior cingulate gyrus) and in regions postulated to be involved in more general ‘executive-type’ processing (i.e. DLPFC). Although the current study did not detect significant differences in on-line task performance between the groups, the sub-optimal neural engagement of task-specific and of ‘executive’ brain regions in VPT individuals, and especially in those with severe early injury, poses the question whether behavioural differences may become apparent with increasing cognitive load, which will be the focus of future studies.


The normal startle response is a form of physiological myoclonus. Its anatomic origin is probably the brain stem. Pathologic startles are defined as reproducible exaggerated startle responses to trivial and not surprising stimuli. Symptomatic forms of an exaggerated startle response can be due to a variety of brain stem disorders. We have, however, found scant data about an exaggerated startle reflex induced by Lyme neuroborreliosis. We therefore report the case of a patient with this unusual presentation.A 69-year old Caucasian man presented with a two-week history of a pronounced startle myoclonus, as well as a four-week history of double vision, gait disturbance and severe lancinating pain in his upper thoracic region. Neurological examination showed an excessive startle reaction of his upper trunk evoked by visual and tactile stimulation, a positive sign of Lhermitte, mild right-sided palsy of his sixth and seventh cranial nerve, moderate dysarthria, very brisk deep tendon reflexes, pallhypesthesia of his legs, and an atactic gait disturbance. A diagnosis of a Lyme neuroborreliosis was confirmed by cerebrospinal fluid examination. Under intravenous treatment with ceftriaxone, our patient improved considerably with complete remission in a follow-up at two months.This case illustrates the chameleon role that neuroborreliosis likes to play: although the wide spectrum of different symptoms that neuroborreliosis can present with has been described, to the best of our knowledge this is the first case report about a symptomatic form of a pathologic startle response as the predominating sign of Lyme neuroborreliosis. The normal startle response is a form of physiological myoclonus. Its clinical presentation usually consists of an involuntary jerky movement  evoked by a sudden and unexpected acoustic stimulus. Its anatomic origin is probably the brain stem. PatholoA 69-year old Caucasian man presented with a two-week history of pronounced startle myoclonus elicited by unexpected visual or tactile stimuli to his upper body, which had led to a fall shortly before admission. In addition, he had a four-week history of double vision, gait disturbance and severe lancinating pain in his upper thoracic region. His medical history included arterial hypertension, a mild left-sided stroke due to a lacunar infarction of the basal ganglia six years ago and severe bilateral hypacusis requiring hearing aids. On admission, a neurological examination showed an excessive startle reaction of his upper trunk evoked by visual and tactile stimulation of the mantle region, positive sign of Lhermitte, mild right-sided palsy of his sixth and seventh cranial nerves, moderate dysarthria, pre-existing latent hemiparesis of his left side, irregular moderate postural tremor of both hands, mild dysmetria of both legs, very brisk deep tendon reflexes, pallhypesthesia of his legs, and an atactic gait disturbance.Borrelia burgdorferi with a specific antibody index (CSF/serum) of 8.2 for immunoglobulin G and 4.0 for immunoglobulin M [A magnetic resonance imaging (MRI) scan of his head showed diffuse unspecific signal alterations compatible with pre-existing vascular lesions. An MRI scan of his cervical spine demonstrated no pathology except for degenerative alterations of his cervical column. Results of an electroencephalogram were normal. His cerebrospinal fluid (CSF) showed a predominantly lymphocytic pleocytosis (500 cells/μL), a raised protein level (2370mg/L) and a positive immunoglobulin titer for  <0.300). All theOur patient improved considerably after a few days of an intravenous treatment with ceftriaxone of 2g per day, which was continued for two weeks. His lancinating pain syndrome, cranial nerve palsies and atactic gait disappeared. His exaggerated startle reaction to visual and tactile stimuli rapidly diminished. On discharge, the only remaining symptom was a clinically irrelevant myoclonus of his right sternocleidomastoid muscle after a tactile stimulus of the vertex. Two months later, our patient had completely recovered. The lymphocytic pleocytosis of his CSF had dropped to 11 cells/μL  elicits a startle response that mainly consists of bilateral flexion of his arms. His legs are not involved. In the subsequent diagnostic tests, there is no reaction after a visual stimulus but a non-habituating startle reaction after tactile stimulation on his left arm. Segment 2. Seven days after antibiotic treatment, there is no startle response after tactile stimulation on his chest or arms. However, a myoclonic response of the right sternocleidoid muscle after a gentle tap on the vertex can still be observed, which was no longer seen in the follow-up at two months.Click here for file


Root-end preparation and restoration with an endodontic material are required when nonsurgical endodontic retreatment has failed or is impossible. The present clinical study reports the treatment outcomes of periradicular surgery using calcium-enriched mixture (CEM) cement.A prospective outcome study of periradicular surgery using CEM was conducted on 14 permanent teeth with persistent apical periodontitis. Using a standardized surgical protocol, 2-3 mm of the root apex was resected; approximately 3 mm deep root-end cavities were ultrasonically prepared and filled with CEM cement. All patients were available for recall.Clinical and radiographic examination revealed complete healing of periradicular lesions, i.e. regeneration of periodontal ligament and lamina dura in 13 teeth (93% success) during a mean time of 18 months; moreover, the teeth were functional and asymptomatic.Favorable treatment outcomes in this prospective clinical study suggested that CEM cement may be a suitable root-end filling biomaterial. When conventional root canal treatment (RCT) has failed, non-surgical retreatment is the preferred option in the mainstream of cases. In a number of cases, several factors such as a complex root canal system or previous procedural accidents may impede the success of nonsurgical retreatment. In such cases, periradicular surgery would be the treatment of choice in order to save the tooth .After root-end resection and preparation, the root canal filling material is placed within the created cavity to close the path of communication between infected root canal system and periradicular tissues. Using a root canal filling material with ideal properties will have an immense effect on the treatment outcomes of the surgery. An ideal root canal filling material should be non-absorbable, non-corrosive, non-cytotoxic, not affected by moisture, dimensionally stable, biocompatible, antibacterial, radiopaque, cost-effective, easily manipulated, adhesive to the dentinal walls, and able to create a tight seal as well as to induce cementogenesis -4. NumerMineral trioxide aggregate (MTA) was introduced to create an effective seal between the root canal system and the periradicular tissues , 7. As aCalcium-enriched mixture (CEM) cement was also introduced as root-end filling biomaterial. It proved to have coronal as well as retro-sealing ability equal to MTA , 15, 16 The purpose of this prospective clinical study is to describe the clinical and radiographic outcomes of periradicular surgery in human permanent teeth using CEM cement as a root-end filling biomaterial.Patients from both genders were required to have i) a symptomatic permanent tooth; ii) an acceptable root canal filling; and iii) a periradicular lesion of endodontic origin. Patients with moderate or severe marginal periodontitis, active systemic disease, physical or mental disability, or those who were pregnant or nursing were excluded. In cases with inadequate coronal restoration, a new coronal restoration was provided before surgical treatment. All recruited subjects were informed of the possible complications. Written informed consent was signed by all patients. Twelve patients  were treated in an endodontic clinic . MedicalIn the operative session, a 400 mg tablet of Ibuprofen was prescribed to prevent post-treatment pain and discomfort . An antiA sulcular incision followed by a full thickness mucoperiosteal buccal flap provided adequate access to the region; a Luebkhe-Ochsenbein flap design was preferred for incisors for aesthetic reasons . Buccal All radiographs were taken with Suni Ray digital charge-coupled device (CCD) intraoral sensor  with bisecting method and evaluated in a room with a dimmed light; after radiographic confirmation of proper placement of the biomaterial, the reflected tissues were repositioned, sutured with polyvinylidene fluoride   and compClinical and radiographic criteria according to the “Quality Guidelines” of European Society of Endodontology (ESE) for favorable outcomes were as follows: absence of pain, swelling and other symptoms, satisfactory healing of soft tissue, no sinus tract, no loss of function and radiological evidence of repair of apical periodontitis including reformation of the periodontal ligament space . ClinicaA total of 14 mandibular or maxillary teeth in twelve patients were treated . The aveThe mean time for recall was 18 months (12-26 months). There was no clinical sign of inflammation and/or infection except in one case . Cases 4 and 14 were maxillary left second and third molar, whose surgical approach was difficult; treatment outcomes were, however, satisfactory and the lesions successfully healed. The case illustrated in Although non-surgical retreatment is successful in most case of endodontic failure, there are cases in which periradicular surgery is necessary to save the tooth. The success of periradicular surgery is, in part, dependent on the selected root canal filling material. In a recent systematic review, investigators found a success rate of 77.8% for periradicular surgery at 2-4 years and a rate of 71.8% at 4-6 years . AnotherThe high clinical success rate may be the result of a number of physical and biochemical characteristics. A crucial characteristic for an ideal root canal filling material is the ability to stimulate periradicular tissue regeneration, in particular, cementogenesis over the material , 9. PrevA randomized controlled animal study demonstrated that both CEM cement and MTA induced periradicular tissue healing regeneration including the production of cementum and new bone, when used as root-end filling biomaterials . In an iThe egress of bacteria and/or their byproducts into the periradicular tissues results in tissue inflammation. Root canal filling materials are meant to eradicate any remnant bacteria and to seal communications between the root canal and tooth and its external environment . ConsequThe considerable success rate of this clinical study indicates that CEM cement may be considered an acceptable endodontic biomaterial for periradicular surgeries. Further randomized clinical trials with long-term follow-up and larger sample size are recommended.


To the Editor: Amebic keratitis is an aggressive infection usually associated with soft contact lenses, and its poor outcome can lead to a corneal graft , bacteria (16S rDNA), and herpesvirus (DNA polymerase), were negative. However, culture of contact lens storage case liquid in Page-modified Neff ameba saline enriched with heat-killed Candidatus Babela massiliensis . The second organism was a new, unnamed, gram-negative, ciprofloxacin-susceptible (20 μg/mL) Alphaproteobacterium bacillus with 99% 16S rDNA sequence similarity (GenBank accession no. HM138368) with Candidatus Odyssella sp. and Acanthamoeba endosymbiont KA/E9 (A. polyphaga mimivirus (Culturing this ameba in sterile peptone–yeast extract–glucose broth showed that it hosted 4 organisms. The first organism was a new intracellular Deltaproteobacterium provisionally named Acanthamoeba spp. are ubiquitous in tap water (,,Acanthamoeba spp. and M. chelonae are well-described agents of keratitis, other ameba-resistant organisms have not been associated with keratitis. However, 1 of the 2 new ameba-resistant bacteria isolated from the contact lens storage case liquid had a 16S rDNA sequence similar to that of the endosymbiont of an Acanthamoeba species isolated from the corneal sampling of a patient with keratitis in South Korea (Although These data indicate the ubiquity of these emerging organisms and raise questions about roles of the ameba host and the symbiont as causative agents of keratitis. We therefore estimate that any of the organisms reported or any combination of these agents could have been involved in the keratitis that developed in the patient. Because the minute quantity of corneal scraping material prevented molecular detection, the relative contribution of each organism could not be specified.This case illustrates that culturing ameba from ocular specimens and contact lens storage case liquid is mandatory for determining the diversity of pathogens potentially responsible for ameba-associated infections, such as keratitis, in patients who wear contact lenses. Ameba-resistant organisms have complex reciprocal interactions with the host, reminiscent of the mafia behavior.>70% alcohol for hand disinfection before contact lens manipulation.The patient’s practice of not drying her hands after cleansing them with tap water and diluting the contact lens cleansing liquid with tap water may have provided a route for contamination of the contact lens fluid with ameba-resistant, tap water–borne organisms. Patients should be informed of the risk for keratitis caused by water-borne, amebal-resistant pathogens. They should also be educated to avoid contact with tap water when manipulating contact lenses, to dry hands after washing them with water and soap, or to use antiseptics containing


Emerging evidence suggests that cancer stem cells account for the initiation and progression of cancer. While many types of cancer stem cells with specific markers have been isolated and identified, a variety of differences among them began to be appreciated. Cancer stem cells are hierarchical populations that consist of precancerous stem cells, primary cancer stem cells, migrating cancer stem cells and chemoradioresistant cancer stem cells, playing different roles in cancer initiation and progression. Here we propose a new concept "horizontal hierarchy of cancer stem cells" to distinguish them from vertical hierarchy cancer stem cells, cancer transient-amplifying cells and cancer differentiated cells, and summarize our current understanding of these subsets of cancer stem cells with the aim to open up novel therapeutic strategies for cancer based on this understanding. Cancer is a kind of abnormal tissue that develops the ability of unlimited growth and the resistance to various survival stresses. Recently, accumulating experimental evidence supports that cancer stem cells account for the initiation and progression of cancer, which challenges the classical stochastic model of cancer development. The canThe essential features of cancer stem cells are self-renewal, multi-differentiation and tumorigenic capacity. Cancer Cancer cells with features of stem cells were discovered by Rudolf Virchow in the mid-19th century, who found that some cancer cells had the histological characteristics, proliferation and differentiation capacity similar to embryonic cells . In 1937+CD38- leukemia cells had the capacity of self-renewal and multi-differentiation similar to hematopoietic stem cells, and developed tumor more quickly than CD34-CD38+ leukemia cells. Thus they concluded that CD34+CD38- subpopulations were the initiating cells of leukemia. This was the first experimental evidence of cancer stem cells. Later, Al-Hajj et al. [+CD24- breast cancer stem cells from breast cancer patients in 2003, thus providing the first experimental evidence of solid tumor stem cells. After that, more types of solid tumor stem cells were isolated with specific surface markers  is generally considered a type of precancerous lesion of breast invasive ductal carcinoma (IDC). The precancerous stem cells in DCIS stage are confined within the duct, but develop invasive capacity upon hypoxia or other stimuli, contributing to the progression of DCIS to IDC. Therefore, precancerous stem cells develop into primary cancer stem cells, and neoplastic ductal is not precanerous lesion but cancer foci . Second The next question is the origin of precancerous stem cells. Several studies suggested that cancer initiating cells may be responsible for the development of precancerous stem cells. Wang et al  reportedAdditionally, Barker et al  and Zhu The malignant transformation of normal stem cells was also discovered in mesenchymal stem cells. Røsland et al  showed tMoreover, human embryonic stem (hES) cells can transform into abnormal stem cells. Werbowetski-Ogilvie et al  identifi+, c-kit+, FcγRIII/II+, Gr1int) demonstrated enhanced self-renewal capacity through the down-regulation of the transcription factor CCAAT/enhancer binding protein-α(C/EBP-α) and were capable of efficiently generating leukemia in recipient mice. Krivtsov et al [Progenitor cells may be another origin of cancer initiating cells. Jamieson et al.  reportedov et al  reportedSelf-renewal is the most essential feature of normal stem cells and cancer stem cells. NotablyTaken together, adult stem cells, embryonic stem cells, progenitors with unlimited self-renewal capacity, and induced pluripotent stem cells are the potential origins of cancer initiating cells.Metastasis is a very important feature of malignant tumors, accounting for 90% death of tumor patients. Given iAccording to cancer stem cell hypotheses, cancer stem cells are ideal seeds of metastasis. Stem cells are indeed ideal carrier of gene mutations and their accumulation. First, the initiating cell must be a cell with extensive divisions and the mutations will be not lost after several divisions. Second, the initiating cell must have long life with strong resistance to different external stress. In contrast, a mature differentiated cell is subject to senescence and death and can not be the initiator of cancer.+CXCR4+ subsets determined the migrating phenotype of pancreatic cancer, although both CD133+CXCR4+ and CD133+CXCR4- pancreatic cancer stem cells could form pancreatic cancer when transplanted into athymic mice. An inhibitor of CXCR4 could significantly reduce the metastasis in group CD133+CXCR4+ mice. Furthermore, removal of CD133+CXCR4+ subset from CD133+ cancer stem cells could disrupt the metastasis of pancreatic cancer, but did not affect tumorigenesis in primary organ. Collectively, these data suggest that CD133+CXCR4+ cancer stem cells determine the metastasis and represent the migrating cancer stem cells of pancreatic cancer.But not all cancer stem cells have the characteristic of migration. Hermann et al  reported+ but not CD90- liver cancer cells were able to form tumor. Notably, CD90+CD44+ subpopulations had stronger capacity of tumorigenesis and metastasis than CD90+CD44- subpopulations, and the proportion of CD90+CD44+ subpopulations in metastasis increased compared to primary cancer. Therefore, CD90+CD44+ subpopulations might be the migrating cancer stem cells of liver cancer.Furthermore, Yang et al  reportedlowCD24high and CD44highCD24low subpopulations from five breast cancer tissues and applied serial analysis of gene expression to reveal that CD44highCD24low subpopulations expressed high level of mesenchymal markers N-cadherin, Vimentin, Fibronectin, Zeb2, Foxc2, Snail, Slug, Twist1 and Twist2, and low level of E-cadherin. They further transplanted human mammary epithelial cells constitutively expressing either Snail or Twist into immune-deficient mice and found that both of them had more efficiency of tumorigenesis, and the number of CD44highCD24low subpopulations is elevated. Therefore, they concluded that EMT might be responsible for the generation of migrating cancer stem cells.However, current studies on migrating cancer stem cells are very limited, mainly due to the lack of specific migrating markers to isolate migrating cancer stem cells from primary cancer stem cells. It has been established that epithelial to mesenchymal transition (EMT) is involved in migration and metastasis, thus providing some clues on how to isolate migrating subpopulations from primary cancer stem cells. Mani et al  isolatedZhang et al.  discoverBased on these studies it is a potential approach to utilize mesenchymal markers to isolate migrating cancer stem cells from primary cancer stem cells.One significant feature of cancer is its relapse after chemotherapy and radiation. This is because a few of cancer cells evolve with the capacity of resistance to chemotherapy and radiation. Whether primary cancer stem cells can evolve into chemoradioresistant cancer stem cells is not well known but recent studies provided indirect evidence for the existence of chemoradioresistant cancer stem cells.Todaro et al -100 repoATP-binding cassette superfamily is one type of multi drug resistant proteins, which can pump chemotherapy drugs out of the cell and lead to chemoresistance,102. ABCDespite these reports demonstrating the relationship between cancer stem cells and chemoresistance, further studies are crucial to provide direct evidence supporting the existence of chemoresistant cancer stem cells, which may help develop alternative strategy for chemotherapy and targeted therapy.Diehn et al  reportedUp to now, precancerous stem cells, primary cancer stem cells and migrating cancer stem cells have been proven to exist in the progression of cancer ,52,61,62Primary cancer stem cells may play the most important role in the progression of cancer and recurrence. Thus the transformation from precancerous stem cells to primary cancer stem cells is a crucial step in tumorigenesis. When primary cancer stem cells acquire migrating capacity through different mechanisms such as EMT, they metastasize to distant organs and cause metastatic cancer. Therefore, migrating cancer stem cells may be originated from primary cancer stem cells and this transition may be a key step of metastasis. In order to escape from chemoradiotherapy, primary cancer stem cells may develop into chemoradioresistant subsets, which is an important reason of chemoradioresistance and cancer recurrence after traditional chemotherapy and radiation therapy. Whether chemoradioresistant cancer stem cells can transform into migrating cancer stem cells is still not known.In summary, based on the above discussion we propose the model shown in Figure The authors declare that they have no competing interests.LHG, CC, YH, PYF, ZXH all contributed to the development of the concept, literature review, discussions, and writing of the manuscript. All authors have read the manuscript and agree to its submission.


Roles of myeloid cells in critical tumourigenesis steps such as vascularization and invasion were revealed by genetic and pharmaceutical approaches. We discovered that the physiological migration of neutrophils controlled tumour invasion by conditioning the collagen matrix and forming the metastatic niche, as detected by two-photon confocal microscopy and second harmonic generation. Administration of VEGFR inhibitors blocked tumour vascularization and a localized tumour growth but enhanced migration of neutrophils, which in turn promoted tumour invasion and formation of micrometastasis. This demonstrates the in vivo cooperation between VEGF signalling and myeloid cells in metastasis and provides a new mechanism underlying the recent findings that VEGFR targeting can promote tumour invasiveness. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.Inhibition of VEGF signalling effectively suppresses localized tumour growth but accelerates tumour invasiveness and micrometastasis by unknown mechanisms. To study the dynamic and reciprocal interactions between tumour cells and their microenvironment during these processes, we established a xenograft model by injecting tumour cells into the blood circulation of transparent zebrafish embryos. This reproducibly results in rapid simultaneous formation of a localized tumour and experimental micrometastasis, allowing time-resolved imaging of both processes at single-cell resolution within 1 week. The tumour vasculature was initiated  Vascular endothelial growth factor (VEGF) is an essential mediator of vascularization under both physiological and tumour conditions. Due to the control function of tumour vascularization in development of both primary and secondary tumours + Gr1+ myeloid cells, which include neutrophils, macrophages and myeloid-derived suppressor cells, promote tumour progression independent of the adaptive immune system, and that these cells render tumours refractory to angiogenic blockade by VEGF antibodies in vivo kinetic studies of their actual roles in tumour progression remain challenging. Therefore, non-invasive visualization of the kinetic interaction between tumour cells and their microenvironment at high resolution will largely improve our understanding of basic cancer biology and will help to design new therapeutic strategies.Tumour progression is controlled by dynamic and reciprocal interactions between malignant tumour cells and non-malignant cells in their microenvironment Danio rerio, has been an important animal model for cancer, immune and stem cell research over the last decade in vivo analysis of tumour progression and the interactions between tumour cells and the host microenvironment The zebrafish, We have established a rapid, reproducible zebrafish embryonic xenograft model for simultaneous formation of a localized tumour and experimental micrometastasis by intravascular injection of tumour cells into the blood circulation of zebrafish embryos. With non-invasive high-resolution imaging we characterized the critical steps of tumour progression, including tumour vascularization and tissue invasion. Using this model, we found that myeloid cells are involved in these tumour processes, and especially that neutrophils condition the collagen matrix to facilitate metastatic niche formation and tumour invasion. Importantly, we show that VEGFR inhibitors suppress localized tumour growth but, in contrast, promote tumour invasion and micrometastasis formation by enhancing neutrophil migration.mN-phenylthiourea  was applied to prevent pigment formation from 1 day post-fertilization (dpf). For Pu.1 knockdown as previously published, Pu.1 MO  or Sunitinib , beclomethasone  and β-amino- proprionitrile  were applied directly to the egg water and refreshed every 2 days.Zebrafish and embryos were raised, staged and maintained according to standard procedures in compliance with the local animal welfare regulations. The transgenic lines Tg(fli1:GFP) and Tg(mpx:GFP)in vitro for > 10 passages. Zebrafish fibroblast cell lines ZF4 and PAC2 were cultured as previously described Murine aortic endothelial (MAE) cells and tumourigenic FGF2-over-expressing FGF2-T-MAE cells m above the ventral end of the duct of Cuvier where it opens into the heart. After implantation with mammalian cells, zebrafish embryos (including non-implanted controls) were maintained at 34 °C to compromise between the optimal temperature requirements for fish and mammalian cells Dechorionized 2dpf zebrafish embryos were anaesthetized with 0.003% tricaine (Sigma) and positioned on a 10 cm Petri dish coated with 1% agarose. Mammalian cells were trypsinized into single cell suspensions, resuspended in phosphate-buffered saline , kept at room temperature before implantation and implanted within 3 h. Non-fluorescent cells were labelled with the fluorescent cell tracker CM-DiI (Invitrogen) according to the manufacturer's instructions. The cell suspension was loaded into borosilicate glass capillary needles  and the injections were performed using a Pneumatic Pico pump and a manipulator (WPI). 50–400 cells, manually counted, were injected at approximately 60 µFor live imaging, embryos were anaesthetized using 0.016% tricaine (Sigma) and mounted in 0.6% low-melting agarose. Fixed embryos were imaged in PBST. Fluorescent image acquisition was performed using a Leica MZ16FA stereo microscope, a Leica TCS SPE confocal microscope or a Zeiss LSM exciter on an Axio Observer confocal microscope. Confocal stacks were processed for maximum intensity projections with Zeiss ZEN2009 software or ImageJ software. Images were adjusted for brightness and contrast using ImageJ. Overlays were created using Adobe Photoshop CS4 or ImageJ. 3D reconstructions and movies were assembled using ImageJ.Second-harmonic generation (SHG) was used to image the helical structures of collagen fibres, which are capable of combining two photons into one L-plastin was performed after the myeloperoxidase activity assay and the Mpx−/L-plastin+ cells were counted as macrophages in vivo+ cells from the posterior end of the tail fin in each embryo, which exhibited consistent intensity for at least 15 min, were tracked and analysed using the Manual Tracking plugin of ImageJ.In the Tg(fli1:GFP) line, the neutrophils in fixed embryos were detected using the Peroxidase/ Myeloperoxidase Leukocyte Kit (Sigma), as described previously L-plastin  and then wounded on the ventral side of the tail fin with the tip of a glass capillary needle, as described Tetramethylrhodamine dextran was injected into the sinus venosus of anaesthetized zebrafish embryos, as described t-tests with confidence intervals of 90% or 95%: ns, not significant (p > 0.05); *0.01 < p < 0.05; **0.0001 < p < 0.01; ***p < 0.0001.Statistical analysis was performed using Prism 4 software (GraphPad), two- or one-tailed unpaired In order to develop an experimental metastasis model, as frequently used in mice, we implanted up to 400 fluorescent tumour cells into the blood circulation of zebrafish embryos at 2 dpf. The tumour cells haematogenously disseminated in the embryo immediately after injection into the duct of Cuvier; a wide circulation channel on the embryonic yolk sac connecting the heart to the trunk vasculature A, B. WheTaking advantage of the transparency of zebrafish embryos, we performed high-resolution confocal imaging to characterize the localized tumour growth. Shortly after implantation, the non-disseminated tumour cells accumulated locally in close association with the primitive endothelial cells of the duct of Cuvier A, which de novo vascularization in a zebrafish xenograft system. This phenomenon is fundamentally different from tumour vascularization in a previously described angiogenesis assay, which monitors growth of new vessels from existing vessels towards the tumour implant Notably, only in the embryos where such functional tumour neovasculature had formed did the localized tumour cells proliferate and form a tumour mass of > 0.15 mm diameter by 6dpi. Phosphorylation of histone H3 (pH3), a proliferation marker, was significantly increased in both fluorescence-positive tumour cells and in fluorescence-negative stromal cells of the localized tumour mass after 4dpi H, when tThe occurrence of micrometastasis, independent of the localized tumour growth, was also characterized by high-resolution confocal imaging. Micrometastases originated from tumour cells disseminated haematogenously directly after implantation. Disseminated cells were arrested in the host vasculature in the first hour A, and exThe spatially specific invasion site was validated by other tested tumour cell lines . Such tu+ for neutrophils and Mpx−/L-plastin+ for macrophages Leukocytes are known to contribute to different steps of tumour progression, including the formation of tumour vasculature and metastasis m morpholino; m morpholino; To investigate the role of myeloid cells in tumour progression, we used morpholino oligonucleotides to knock down the transcription factor Pu.1, which controls the development and differentiation of myeloid cells in zebrafish embryos L-plastin+ myeloid cells To further decipher the impact of neutrophil CHT–TF transmigration on the spatially co-localized tumour cell invasion process, two-photon excited confocal microscopy and SHG was performed to visualize the extracellular fibrillar collagen matrix upon neutrophil migration m beclomethasone (m beclomethasone 4 h before implantation (Pharmacological approaches were taken to confirm the functional involvement of neutrophil migration in tumour cell invasion. A prototype glucocorticoid receptor agonist, beclomethasone, previously shown to inhibit neutrophil migration ethasone A, B, D. antation C. In addantation A–C. Notaantation D, indicaantation D and tumantation I. Taken Tumour vascularization is an important process in tumour progression. In our model, the localized tumour growth was determined by successful vascularization . AdminisWe then investigated the contribution of myeloid cells to the tumour cell invasion and micrometastasis promoted by VEGFR inhibition. We found that VEGFR inhibition led to a reduction of macrophage numbers, altered the typical elongated macrophage shape into a rounded morphology and suppressed the migratory activity of macrophages D, 9A–C. The recent discoveries in mice models and clinical studies showed that anti-angiogenic therapies by targeting the VEGF signalling inhibited primary tumour growth but promoted tumour invasion and metastasis. Unfortunately, the mechanism of this very relevant clinical problem is still elusive. Answering this question requires live imaging of tumour-microenvironment interactions at the cellular level—a process severely limited in current animal models.in vivo, including tumour vascularization, localized tumour growth, tumour invasion and micrometastasis formation, we discovered a new mechanism of the metastatic niche formation. In addition to the published roles of macrophages in this process Using a novel zebrafish xenograft model that allows simultaneous single-cell resolution monitoring of multi-step tumourigenesis Recently, the zebrafish myeloid cell population has been studied in detail and similarities with the human myeloid cell functions and embryonic differentiation have been demonstrated The involvement of myeloid cells in critical tumourigenesis, steps such as vascularization and invasion, was revealed by both genetic and pharmacological approaches. We demonstrated that myeloid cells, particularly macrophages, are essential for tumour vascularization. Importantly, we also obtained evidence that the micrometastasis site was determined by the physiological migration of neutrophils. Our results show that yet undefined environmental cues favour neutrophil transmigration at the posterior end of the caudal haematopoietic tissue, which remodels the fibrillar collagen matrix and in turn conditions the premetastatic niche for tumour cells to invade into the tail fin. These findings support the ‘seed and soil’ hypothesis, that tumour metastasis is controlled by both the tumourigenic property of disseminated cells and their microenvironment It is known that tumour-infiltrating immune cells can have both tumour-promoting and tumour-antagonizing roles Our data support the recent notion that inhibition of VEGF signalling may have opposite effects on localized tumour growth and on metastasis. It has been suggested that hypoxia-resistant cells may escape from a starving primary tumour treated with VEGFR inhibitors and then result in elevated malignancy in vivo cooperativeness between VEGF signalling and neutrophils in metastasis. In addition, we observed that macrophages were suppressed by the VEGFR inhibitors, which may indicate an indirect involvement of macrophages in this process. The remaining question is, what are the targets of the VEGFR inhibitors in the neutrophils that cause them to enhance their migration? The VEGFR inhibitors used in this study are sufficiently different in structure to ensure that the observations were not due to an off-target effect. However, we can not be certain that observed phenotypes are restricted to targeting of VEGF receptors, since sunitinib works also on PDGFR family members and affects pericyte coverage of endothelium, promoting extravasation of tumour cells and the formation of micrometastasis Our data provide evidence that the physiological migration of neutrophils is elevated upon inhibition of VEGF signalling, which in turn promotes tumour invasion. This is the first demonstration, to our knowledge, of the in vivo model. In conclusion, we exploited this model here to demonstrate that neutrophil migration affects metastasis and that this is enhanced by VEGFR inhibitors.Our findings implicate myeloid cells as critical mediators of tumour invasiveness elicited by VEGFR inhibitors, independent of inflammation and the adaptive immune system. It provides a new mechanism underlying the recent clinical observations of anti-VEGF therapies promoting tumour invasiveness. We propose that dual targeting of angiogenesis and myeloid cells can be beneficial in future treatments. Of course, it will only be relevant for medical studies if the neutrophil transmigration is not restricted to this model. Currently the role of macrophages in intra- and extravasation of cancer cells is well established


Biological sources of free radicals include mitochondrial electron transport chain, enzymes like cytochrome P450, xanthine oxidase, and phagocytosis. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are well known for playing a dual role as both deleterious and beneficial species, since they can be either harmful or beneficial to living systems [Oxidative stress is an important aspect of toxicology and therefore of considerable interest. It can be traced back to 1954 when Gerschman et al. propounded the free radical theory which for the first time implicated partially reduced forms of oxygen in its toxic mechanism . The disIn addition to disease states, oxidative stress has been implicated in the mechanisms of drug-induced toxicity , chemicaAfaf K. El-AnsaryMalak KotbMaha Zaki RizkNikhat J. Siddiqi


Amphimedon queenslandica, undergoes metamorphosis from a free-swimming larva into a sessile adult that bears no morphological resemblance to other animals. While the genome of A. queenslandica contains an extensive repertoire of genes very similar to that of complex bilaterians, it is as yet unclear how this is drawn upon to coordinate changing morphological features and ecological demands throughout the sponge life cycle.The biphasic life cycle with pelagic larva and benthic adult stages is widely observed in the animal kingdom, including the Porifera (sponges), which are the earliest branching metazoans. The demosponge, A. queenslandica, we compared global gene expression profiles at four key developmental stages by sequencing the poly(A) transcriptome using SOLiD technology. Large-scale changes in transcription were observed as sponge larvae settled on the benthos and began metamorphosis. Although previous systematics suggest that the only clear homology between Porifera and other animals is in the embryonic and larval stages, we observed extensive use of genes involved in metazoan-associated cellular processes throughout the sponge life cycle. Sponge-specific transcripts are not over-represented in the morphologically distinct adult; rather, many genes that encode typical metazoan features, such as cell adhesion and immunity, are upregulated. Our analysis further revealed gene families with candidate roles in competence, settlement, and metamorphosis in the sponge, including transcription factors, G-protein coupled receptors and other signaling molecules.To identify genome-wide events that accompany the pelagobenthic transition in This first genome-wide study of the developmental transcriptome in an early branching metazoan highlights major transcriptional events that accompany the pelagobenthic transition and point to a network of regulatory mechanisms that coordinate changes in morphology with shifting environmental demands. Metazoan developmental and structural gene orthologs are well-integrated into the expression profiles at every stage of sponge development, including the adult. The utilization of genes involved in metazoan-associated processes throughout sponge development emphasizes the potential of the genome of the last common ancestor of animals to generate phenotypic complexity. For many animals, the genome controls the construction and function of two or more discrete life forms, each with its own morphological, physiological, behavioral and ecological characteristics. These life cycles, which typically include larval and adult phases, require the successive unfolding of morphogenetic programs at embryogenesis and metamorphosis. The pelagobenthic life cycle, in which a pelagic, microscopic larva metamorphoses into a large, benthic adult, is one common variation. An inherent feature of the pelagobenthic life cycle is the settlement of the planktonic larva, usually in response to an environmental cue, and its subsequent metamorphosis into the benthic form. This major ecological transition dictates the likelihood of future reproductive success because adults usually are sessile or sedentary, and therefore, larvae must settle at a distance from conspecifics to allow subsequent effective matings. Before settling and metamorphosing, the larvae must first develop the capacity, or competence, to detect and respond to benthic environmental cues typically associated with post-settlement survival and reproduction  .During development, a single gene inventory is drawn upon to implement the vast morphological and ecological changes associated with metamorphosis from one body form into another. Gene expression studies at developmental transitions, therefore, offer a window into the genetic framework that corresponds to distinct morphological features and ecological demands. Genome-wide activity as the animal prepares and moves from the pelagic to the benthic phase has been studied in just a few animals, including ascidians, gastropods and corals -8. TheseAmphimedon queenslandica, the pelagobenthic life cycle starts with the emergence of larvae from brood chambers of the adult . Observed mapping statistics are similar to those reported in previous RNA sequencing studies  following the manufacturer’s protocol. Adult samples were cleaned of macroscopic debris then ground in liquid nitrogen before RNA extraction with Trizol. Contaminating DNA was removed using the DNAfree kit [Ambion]. The poly(A) RNA fraction was enriched using the MicroPoly(A)Purist kit [Ambion] and ribosomal RNA was depleted using the RiboMinus Eukaryote kit [Invitrogen]. RNA quality was monitored using the Agilent Bioanalyzer RNA 6000 Pico Assay.Fragment libraries were prepared as previously described . BrieflyAssembled sponge contigs and gene models (Aqu1) are publicly available . RedundaComparison of sequencing results from different libraries prepared from the same sample or libraries prepared from different biological samples showed that variable sequencing depths did not induce any detectable read count-dependent bias but rather affected read counts by a single scaling factor which is of the order of the difference in the total number of mapped reads. Read counts from different libraries were normalized by multiplying with a proportionality constant reflecting different sequencing depths to obtain read count distributions similar to the precompetent library, which was used as the reference sample. The distribution of reads in each sample after normalization is shown in Additional file t-test on the log of read counts and filtered out transcripts for which expression varied by less than four-fold. This method would filter out any gene that does not exhibit a strong stage-specific expression, particularly genes with dynamic expression across development. Therefore, to identify genes that exhibit differential expression between stages, we tracked the expression trajectory for each expressed gene in pair-wise comparisons between successive stages. Individual genes were categorized as either up or downregulated if their expression changed more than four-fold and were greater than the sampling error on the read count between stages. We also identified genes differentially expressed by two-fold between stages.In technical replicates of libraries derived from the same biological sample, the variation in expression for genes detected above 64 reads did not exceed three-fold. Fold change cutoffs as calculated based on read count error within technical replicates are 1.36, 1.68, and 2.25-fold for false calling rates of 5%, 1%, and 0.1%, respectively. Thus, we selected a four-fold change as a conservative criterion for differential expression. To identify genes with stage-specific expression across the pelagobenthic transition, we compared gene expression at each stage to the mean expression of the other three stages by performing a two-tailed -4. Genes were assigned the names and Gene Ontology (GO) [Sponge gene models were aligned to proteins in the UniProt database  using BLogy (GO)  annotatiPrimers for quantitative RT-PCR were designed to span exon junctions of predicted transcripts whenever possible to ensure specificity for target mRNAs  Distribution of transcript read counts after global normalization for sequencing depth. (B) Comparison of normalized read counts from two sequencing runs of the same library preparation and (C) comparison of normalized read counts from sequencing runs of two independent libraries made from the same RNA sample show that determination of transcript expression is reproducible above a cutoff threshold of 64 reads (red lines).Click here for fileFigure S2. Comparison of expression trends from sequencing and quantitative RT-PCR. The expression of 50 transcripts was determined by quantitative RT-PCR (qPCR) from three separate pools of individuals at each developmental stage. The expression profile for each transcript is shown relative to the precompetent sample . The relative fold change in expression estimated by the two methods showed similar trends across development for 40 of the genes tested (Pearson r ≥ 0.70). Variance in pooled sample replicates was assessed using one-way ANOVA with Bonferroni’s post-test . Error bars indicate the standard deviation.Click here for fileFigure S3. Expression of annotated and non-annotated genes. Sponge genes were aligned to sequences in the UniProt database. Sequences with significant matches  were designated as ‘annotated’ and those without as ‘non-annotated.’ (A) Non-annotated genes (red line) have lower overall expression compared to annotated genes (blue line). (B) Both gene sets exhibit similar patterns of variation across development. Heatmaps show relative expression of annotated and non-annotated genes . The number of genes in each set is indicated to the left of each heatmap.Click here for fileTable S1. Four-fold differentially expressed genes at stage transitions. List of genes that are differentially expressed (>4-fold and greater than sampling noise) at indicated stage transitions grouped by direction of change (up or downregulation). Transcript length, normalized read counts, name and accession number of best sequence match in the UniProt database, Gene Ontology (GO) annotation, PFAM domains, and PANTHER annotation is indicated for each gene.Click here for fileTable S2. Two-fold differentially expressed genes at stage transitions. List of genes that are differentially expressed (>2-fold and greater than sampling noise) at indicated stage transitions grouped by direction of change (up or downregulation). Transcript length, normalized read counts, name and accession number of best sequence match in the UniProt database, Gene Ontology (GO) annotation, PFAM domains, and PANTHER annotation is indicated for each gene.Click here for fileTable S3. Gene ontology (GO) analysis for genes exhibiting greater than two- or four-fold change in expression between successive stages. Selected functional terms enriched in the set of genes that are upregulated or downregulated at specific stage transitions are shown with the corresponding p-values. The number of genes belonging to each functional category in the genome or within each differentially expressed group is indicated.Click here for fileTable S4. PANTHER functional group enrichment analysis for genes that exhibit greater than four-fold change in expression between successive stages. Functional groups enriched in the set of genes that are upregulated or downregulated between stages are shown . The number of genes in the genome or within each differentially expressed group that belong to a category is indicated.Click here for fileFigure S4. Expression of genes in selected functional groups. Genes within each category were retrieved using the Gene Ontology (GO) annotation of their best UniProt sequence match or by the presence of PFAM domains. Transcription factors, G-protein coupled receptors, and kinase genes were obtained from previous studies [ studies ,56,57. SClick here for fileTable S5. Genes exhibiting greater than 100-fold upregulation in relative expression level during sponge development. Maximum upregulation (max. upreg.) is the highest relative expression ratio for the gene, given as the log2 of reads for a gene in one stage minus the average of the log2 of reads in the other three stages.Click here for fileFigure S5. Relative expression heatmaps for genes belonging to developmental signaling pathways, transcription factor families, or kinase classes. (A) Genes in the Wnt, Notch, and TGF-β pathways have homologs in A. queenslandica  and are expressed throughout sponge development . Major receptors and ligands in each pathway are highlighted in red. (B) Relative expression of genes in transcription factor families. (C) Relative expression of genes in kinase classes. Heat maps show the relative expression of genes across stages . Genes were retrieved from previous studies [ studies ,11,23,87Click here for fileTable S6. Quantitative RT-PCR validation cycle numbers, read counts, and primer sequences.Click here for file


The reduction of HIV transmission risk behaviors among those infected with HIV remains a major global health priority. Psychosocial characteristics have proven to be important correlates of sexual transmission risk behaviors in high-income countries, but little attention has focused on the influence of psychosocial and psychological factors on sexual transmission risk behaviors in African cohorts.The CHAT Study enrolled a representative sample of 499 HIV-infected patients in established HIV care and 267 newly diagnosed HIV-infected individuals from the Kilimanjaro Region of Tanzania. Participants completed in-person interviews every 6 months for 3 years. Using logistic random effects models to account for repeated observations, we assessed sociodemographic, physical health, and psychosocial predictors of self-reported unprotected sexual intercourse. Among established patients, the proportion reporting any recent unprotected sex was stable, ranging between 6–13% over 3 years. Among newly diagnosed patients, the proportion reporting any unprotected sex dropped from 43% at baseline to 11–21% at 6–36 months. In multivariable models, higher odds of reported unprotected sex was associated with female gender, younger age, being married, better physical health, and greater post-traumatic stress symptoms. In addition, within-individual changes in post-traumatic stress over time coincided with increases in unprotected sex.Changes in post-traumatic stress symptomatology were associated with changes in sexual transmission risk behaviors in this sample of HIV-infected adults in Tanzania, suggesting the importance of investing in appropriate mental health screening and intervention services for HIV-infected patients, both to improve mental health and to support secondary prevention efforts. The reduction of HIV transmission risk behaviors among those infected with HIV, or secondary HIV prevention, remains a major global health priority. While large investments have been made over the past decade to expand access to HIV treatment and medications,Among the predictors of sexual transmission risk behaviors, psychosocial characteristics such as depression, substance use, stressful and traumatic experiences, and post-traumatic stress have been identified in high-income countries as playing important roles,Accordingly, in the present paper we investigate predictors of sexual risk behaviors over 3 years of longitudinal follow-up among a cohort of over 750 newly diagnosed and established HIV-infected patients in Tanzania, with a particular emphasis on the association of psychological characteristics with sexual risk.The Coping with HIV/AIDS in Tanzania (CHAT) Study is a longitudinal observational cohort study designed to define the determinants of HIV-related behaviors and health outcomes among Tanzanian adults in the Kilimanjaro Region. The CHAT Study includes 5 distinct cohorts: representative samples of HIV-infected patients engaged in routine clinical care at the regional tertiary referral hospital (n = 228) and largest public hospital (n = 271); consecutively recruited individuals newly testing HIV-seropositive at voluntary counseling and testing (VCT) centers (n = 267); individuals testing HIV-seronegative at the same VCT centers (n = 182); and a random sample of community-dwelling adults (n = 249). Study participants were recruited in 2008–2009 and completed in-person interviews in Swahili with trained local interviewers every six months through 2013. The study protocol has been described in greater detail previously.The present analysis focuses on predictors of self-reported sexual transmission risk behaviors, over the first three years of follow-up, among HIV-infected CHAT participants, i.e. those recruited from the two hospitals (“established HIV patients”) and those newly diagnosed at VCT centers (“newly diagnosed HIV patients”).This study and all study activities were specifically approved by the Kilimanjaro Christian Medical Center Institutional Review Board in Tanzania and the Duke University Health System Institutional Review Board in the United States, and written informed consent was obtained from all participants.Sexual transmission risk behaviors were assessed via self-report during each in-person interview (every 6 months). Participants were asked to report the number of sexual partners in the past 6 months. Separately for primary and non-primary partners, participants were asked to report the total number of acts of anal or vaginal intercourse in the past 6 months and the number of those acts in which a condom was used. Measures taken to enhance the accuracy of these self-report items included anchoring (repeatedly orienting the participant to a date or participant-identified event six months ago), asking separately about primary vs. non-primary partners, and interviewer prompts to assist respondents, if necessary, in estimating frequency of intercourse by day, week, or month. The percentage of sexual acts that involved a condom was then calculated and reported back to the participant for confirmation or correction. Participants were also asked whether they had used a condom the last time they had sex with a primary and a non-primary partner, and whether they had exchanged (given or received) sex for gifts, money, shelter, food, or other benefit in the past 6 months .sociodemographic information at baseline including age, gender, marital status, highest level of completed education, religion, household assets, and tribe. Overall health-related physical functioning was assessed every 6 months with the Short Form (SF)-8, a validated shortened version of the extensively used SF-36.Participants self-reported Depressive severity was assessed every 6 months with the Patient Health Questionnaire-9 (PHQ-9), a widely used depression case identification tool which has been validated in African populationsPost-traumatic stress (PTS) symptom severity was assessed every 6 months with the PTSD Symptoms Checklist (PCL) based on DSM-IV criteria that include re-experiencing a traumatic event, numbing/avoiding, and hyperarousal symptoms. This scale has strong reported reliability, and correlates highly with a clinician-administered PTSD measure.The number of categories of lifetime potentially traumatic events, adapted from prior research, included sexual abuse, severe physical trauma, childhood physical and emotional neglect, and other potentially traumatic experiences.Recent stressful life events were measured every 6 months with a modified version of the Life Events Survey (LES)Distributions of variables are summarized with means and standard deviations or percentages. We defined the primary outcome variable as a dichotomous measure of any reported unprotected sexual intercourse with a primary or non-primary partner in the past 6 months. We used random effects logistic regression models with an unstructured correlation matrix to estimate the associations between any reported unprotected sex and predictor variables while accounting for correlations between repeated observations on the same individual. In this model, we included outcomes data from rounds 2–7 in order to focus on the period when all participants were aware they were HIV-infected. The model included random intercepts for each participant to account for the natural heterogeneity among participants. The appropriateness of modeling continuous variables using simple linear terms was confirmed through visual examination of locally weighted scatterplot smoothing (lowess) graphs and comparison of linear to more flexible (e.g. quadratic) modeling choices. The potential for model instability due to collinearity was evaluated by assessing whether standard errors for coefficients changed substantially as potentially correlated variables were added to the model.The multivariable model included predictors that were of interest a priori, with a particular focus on the association between time-varying psychosocial characteristics and reported unprotected sex. Each time-varying predictor  is represented in the models by two terms.Two potential confounders, alcohol use and fertility intentions, were only available from a subset of time points . Therefore these variables were not included in the primary model, but the potential for uncontrolled confounding was assessed by comparing models with and without these variables in the sample of observations for which measures were available. Effect modification by gender and marital status was assessed via likelihood ratio tests comparing models with and without interaction terms between these variables and key psychosocial variables of interest, with a likelihood ratio P value <0.05 considered indicative of effect measure modification.All analyses were conducted using STATA version 11 .Of 766 HIV-infected participants enrolled, 755  provided information on sexual behaviors and are included in the analyses . Follow-At baseline, a majority of unmarried participants reported no sexual partners in the past 6 months, and a majority of married or cohabitating participants reported 1 sexual partner; between 3–14% of participants reported 2 or more sexual partners in the past 6 months . Among nOver time, the proportion of established patients reporting any recent unprotected sex remained fairly stable, ranging between 6% and 13% over 3 years . Among nIn multivariable logistic regression random effects models controlling for correlation between repeated measures on each individual , female When considering time-varying covariates, those with better mean physical health scores were more likely to report unprotected sex , but within-person changes in physical health over time were not associated with changes in sexual risk . Those with higher mean post-traumatic stress symptom severity scores were also more likely to report unprotected sex . Moreover, within-person changes in PTS symptom severity over time were also associated with changes in sexual risk , meaning that if an individual scored 5 points above her norm in PTS symptom severity at a given interview, she had a 13% increased odds of reporting unprotected sex in the 6 months before that interview. Depression and recent stressful events were not associated with overall sexual risk or within-person changes in sexual risk over time.Likelihood ratio tests did not indicate presence of effect measure modification of the above associations by gender or marital status. Comparison of models including and excluding fertility intentions and alcohol use in the subset of observations with information on those variables indicated that neither variable was likely to represent substantial uncontrolled confounding in the final full sample model. Model diagnostics indicated that while several psychosocial predictor variables included in the final model were moderately correlated (ρ 0.4–0.7), collinearity did not affect model stability  or substantive conclusions.In this study, HIV-infected participants reported stable rates of unprotected sexual intercourse over 3 years of follow-up. While nearly half of newly diagnosed patients reported unprotected sex in the 6 months before testing positive for HIV, the proportion dropped to approximately 10–20 percent after diagnosis. Patients with established HIV infection, meanwhile, reported even lower rates. Unprotected sex with a non-primary partner or by unmarried participants was rarely reported, as was transactional sex. Even within marriage, unprotected sex was unusual among patients with established HIV infection. For comparison, in studies of male circumcision as an HIV prevention strategy in Uganda, 85–86% of participants reported some unprotected sex at baseline.We found that individuals with higher levels of post-traumatic stress symptomatology were more likely to report unprotected sex. Further, for a given individual, an increase in PTS symptoms during longitudinal follow-up relative to the individual's overall mean was linked to an increase in the likelihood of reporting unprotected sex. These findings are consistent with other reports that have linked sexual risk behaviors with mental health symptomatology and exposure to traumatic experiences.The finding in this sample that PTS symptom severity was associated with sexual risk, while number of lifetime traumatic experiences was not, is consistent with literature demonstrating marked changes in the brain associated with PTSD. For example, MRI studies of brain volume have found smaller hippocampal volumes, among other structural brain differences, in trauma-exposed individuals with PTSD compared to trauma-exposed individuals without PTSD.In considering the absence of an association between traumatic life events and sexual risk in the present analysis, it is worth noting the very high prevalence of exposure to traumatic life events in the CHAT sample. Nearly all participants in the present analysis (88% of established and 85% of newly diagnosed HIV patients) had experienced at least one traumatic life event, with a mean of 2.3 lifetime experiences. Thus the coefficient on the variable representing lifetime traumatic experiences, modeled continuously, represents less a contrast between those with any vs. no traumatic experiences but rather a contrast between those with more vs. fewer experiences.We also identified gender differences in reported unprotected sex, with women more likely to report unprotected sex than men. This may reflect gender differences in ability to negotiate condom use, as has been described in Uganda.Notable strengths of this study include the large sample size, systematic sampling approach, inclusion of both established and newly diagnosed patients, longitudinal design, and analytic approach that specifically identifies predictors for which changes over time are associated with changes in reported sexual risk. One limitation of this study is that sexual risk behaviors were collected via participant self-report, raising the possibility of social desirability bias. Interviewers were carefully trained not to imply any judgment in administering these questions, and respondents were assured that all information would be kept confidential. Also, these data do not suggest increasing reports of sexual risk over time, which might be expected if participants were more willing to disclose risk behaviors as they developed stronger rapport with interviewers over time. An additional limitation is that the serostatus of the sexual partners was not collected, thus we cannot distinguish unprotected sex with a seronegative partner from unprotected sex between two HIV-infected individuals. However, it is important to note that even between two HIV-infected individuals, use of a condom is recommended to avoid the risk of super-infection with a heterologous HIV strain.As described in detail elsewhere, recruitment strategies for the CHAT cohort were carefully designed to yield a representative sample of HIV-infected patients in established care and individuals newly diagnosed with HIV in the study area.The present sample was similar in physical health measures to some other studies of HIV-infected individuals with comparable measures. Compared to the SF-8 physical composite score mean of 47.5 in the present sample, Fox et al. reported a mean of 48.2 in an oral health study of HIV-infected individuals throughout the US,Other studies have also documented associations between mental health symptomatology and sexual risk behaviors among HIV-infected individuals. While the majority of such research has been conducted in high-income countries,Every HIV sexual transmission event involves one HIV-infected and one HIV-uninfected partner, inextricably linking primary and secondary HIV prevention efforts. The present study highlights the longitudinal association of changes in post-traumatic stress symptomatology and sexual transmission risk behaviors among HIV-infected adults in Tanzania, suggesting the importance of investing in appropriate mental health screening and intervention services for HIV-infected patients, both to improve mental health and to support sexual transmission prevention efforts.


Over the past several years, there has been newly emerging and overlapping fields of research investigating CSF pressure and flow pulsatility (CSFp), and cerebral blood flow (CBF). It is not known whether CSFp is merely a passive epiphenomena or alternatively, plays a physiologically significant role in cerebral compliance and blood flow. Thus, the relationship between CSFp and CBF is not clearly understood. In order to study the effect of CSF pulsatility on blood flow, we have developed a novel method and device for controlling  pulsatility.In 15 canine subjects, a small (0.3-3.0cc), custom-made polyurethane bladder was surgically implanted in either the cranial or spinal epidural space such that it would alter the underlying CSF space. CSFp were controlled via an oscillating air pump gated to the cardiac cycle timed to deflate (ICP reduction) or inflate (ICP augmentation) during systole. Measures for ICP (fiberoptic probes) and CBF  were obtained intra-operatively under different acute and chronic conditions.CSPp reduction or augmentation was successfully achieved via cardiac-gated oscillation of a bladder device. Alteration of ICP-waveform was global and measured remotely on the contralateral side and with device activation in the spinal space. Under specific inflation cycles and physiological conditions, operation of the system increased CBF up to 15 mL/min*100gm, or by as much as 40%. Mean systemic pressure, cardiac output, and mean ICP did not significantly change with system activation.We have developed a method of altering CSF pressure pulsatility using cardiac-gated oscillating bladder which dynamically alters CSF space volume. The ability of the system to increase CBF without affecting cerebral perfusion pressure (MAP or ICP) suggests it may work through alteration of cerebrovascular compliance and impedance. Understanding the effect of CSFp on cranial compliance may ultimately allow a new therapy for increasing blood flow in acute and chronic states, including hydrocephalus, where CBF and CSFp are abnormal.SMD, MGL serve as Scientific Advisors for CSF Therapeutics, Inc.


Stress increases the risk of drug abuse, causes relapse to drug seeking, and potentiates the rewarding properties of both nicotine and cocaine. Understanding the mechanisms by which stress regulates the rewarding properties of drugs of abuse provides valuable insight into potential treatments for drug abuse. Prior reports have demonstrated that stress causes dynorphin release, activating kappa opioid receptors (KOR) in monoamine circuits resulting in both potentiation and reinstatement of cocaine and nicotine conditioned place preference. Here we report that kappa opioid-dependent reinstatement of cocaine and nicotine place preference is reduced when the mice are exposed to a randomized chronic mild stress (CMS) regime prior to training in a conditioned place preference-reinstatement paradigm. The CMS schedule involves seven different stressors  rotated over a 3-week period. This response is KOR-selective, as CMS does not protect against cocaine or nicotine drug-primed reinstatement. This protection from reinstatement is also observed following sub-chronic social defeat stress, where each mouse is placed in an aggressor mouse home cage for a period of 20 min over 5 days. In contrast, a single acute stressor resulted in a potentiation of KOR-induced reinstatement, as previously reported. Prior studies have shown that stress alters sensitivity to opioids and prior stress can influence the pharmacodynamics of the opioid receptor system. Together, these findings suggest that exposure to different forms of stress may cause a dysregulation of kappa opioid circuitry and that changes resulting from mild stress can have protective and adaptive effects against drug relapse. It is well-established that stress increases the risk of drug abuse, relapse to drug seeking, and potentiates the rewarding properties of cocaine . Previoui/o protein-coupled receptor (GPCR). In addition to the classical inhibitor effects on adenylate cyclase activity, KOR also couples to mitogen-activated protein kinase pathways  is a widely adopted animal model for inducing depression- and anxiety-like behaviors because it mimics the unpredictable intermittent stress exposure that humans experience, for complete review see Here we determined how different types of exposure to stress  impact subsequent kappa opioid-mediated reinstatement of cocaine and nicotine place preference. We determined how different forms of stress, including a 3-week CMS, sub-chronic social defeat stress (SDS), and acute forced swim stress (FSS), impact kappa opioid-induced reinstatement. We found that the initial cocaine and nicotine conditioned place preference were unchanged in sub-chronic stress and CMS exposure, however, KOR-induced cocaine and nicotine reinstatement was absent in mice that were pre-exposed to CMS, in contrast to acute stress, which caused potentiated KOR-reinstatement. In addition, we found that cocaine and nicotine drug-primed reinstatement is not affected by pre-exposure to stress, suggesting that this protective ablation of KOR-induced reinstatement by CMS, selectively affects KOR-mediated behavioral responses.ad libitum, and maintained in specific pathogen-free housing. Mice were transferred at least 1 week before testing into a colony room adjacent to the behavioral testing room to acclimatize to the study environment and prevent stress during conditioning phases. Housing rooms were illuminated on a 12-h light/dark cycle with lights on at 7 AM. All animal procedures were approved by the Animal Care and Use Committee of Washington University in St. Louis, in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.Male C57BL/6 wild-type mice, bred locally and maintained in Washington University mouse facility (20–30 g) were used for all experiments. All mice were group-housed within the Animal Core Facility at Washington University in St. Louis, given access to food pellets and water Cocaine HCl and racemic U50,488 methanesulfonate were provided by the National Institute on Drug Abuse and Drug Supply Program and in some instances Sigma Aldrich . Nicotine hydrogen tartrate salt was purchased from Sigma Aldrich , dissolved in phosphate buffered solution. The free base form of nicotine was used for calculating all injection doses. All drugs were dissolved in saline unless otherwise indicated.Mice were trained in an unbiased, balanced three-compartment conditioning apparatus and the reinstatement of cocaine place preference (CPP) paradigm was conducted as previously described . On pre-On reinstatement test day mice were injected with KOR agonist, U50,488 , placed in their home cage for 30 min (cocaine CPP) or 20 min (nicotine CPP) as previously described , after wDuring reinstatement (day 10) mice were subjected to FSS in which they were placed in an 18 cm deep bucket of water at 30°C and allowed to swim for upto 6 min. Mice were removed immediately if they appeared to be at risk of drowning. After a 5 min dry off and recovery period mice were injected with U50, 488  in the home cage for 30 min and then placed in the CPP chambers, with access to all three compartments to access reinstatement to cocaine place preference.Social defeat stress was performed as previously described . From thTable 1) prior to conditioned place preference/reinstatement procedure. During reinstatement phase the mice were injected with U50,488  in the home cage (30 min) and then placed in the CPP chambers to assess reinstatement to cocaine place preference. On the following day, mice were injected with cocaine  or nicotine  priming injection and again allowed free access to all three chambers to determine drug prime-induced reinstatement of place preference.In order to model randomized mild stress exposure, we adapted a previously validated CMS paradigm. Stressors were randomly assigned during the 3-week stress period  throughout the 30 min period using video tracking (Canon) of animal movement and Ethovision 8.5 software analysis (Noldus). During the conditioning period the total distance (cm) on conditioning days are represented. During reinstatement post-test trials distance (cm) is represented as 5 min bins throughout the 30 min test period.t-tests, one-way ANOVA, or two-way ANOVA as appropriate. ANOVA’s were followed by post hoc Bonferroni comparisons if the main effect was significant at p < 0.05. Statistical analyses were conducted using GraphPad Prism 5.0F .Data were expressed as means ± SEM. All raw data were calculated via Ethovision video tracking and then place preference or locomotor data were calculated as described. Data were normally distributed, and differences between groups were determined using Student’s independent Figure 1A). Mice subjected to FSS prior to KOR activation by U50,488 showed significant and robust potentiation of reinstatement to cocaine seeking when compared to mice that were not subjected to FSS but that were injected with only U50,488 to induce reinstatement . Locomotor activity during the reinstatement test was measured and the combination of FSS, followed by KOR-induced reinstatement to cocaine seeking showed a reduction in locomotor activity when compared to both the non-stressed group and the FSS alone group . These data suggest that acute stress exposure induces a potentiation of KOR agonist-induced reinstatement of cocaine place preference.It has previously been shown that FSS is sufficient to induce reinstatement and potentiation to drug seeking in a KOR-dependent manner . FurtherFigure 2A). Prior exposure to sub-chronic SDS resulted in a significant block of U50,488 induced reinstatement of cocaine place preference . This inhibition of reinstatement was selective for activation of KOR because a priming injection of cocaine  still caused significant reinstatement of cocaine CPP following exposure to the sub-chronic SDS paradigm . We also measured locomotor activity during the 30 min reinstatement phase. Both the non-stress + U50,488 group and the SDS + U50,488 group show a similar KOR-mediated reduction in locomotor over the 30 min period, supporting the notion that prevention of reinstatement by pre-exposure to stress, is not due to additional alterations in mouse locomotor activity . In contrast, as shown in previous studies . Taken together, these data suggest that exposure to a sub-chronic repeated SDS causes a significant prevention of kappa agonist-induced reinstatement of cocaine place preference.To determine the effects of multiple rounds of stress exposure on KOR-induced reinstatement, mice were subjected to 20 min of SDS at the end of the day during extinction training and prior to the reinstatement phase. On day 10, the reinstatement test day, a single dose of U50,488  was administered to induce reinstatement of cocaine place preference  influences cocaine and nicotine conditioning, as well as KOR-induced reinstatement of cocaine and nicotine CPP. The CMS paradigm involved a random assignment of seven mild stressors during a 3-week period (see Table 1) prior to the cocaine or nicotine CPP/reinstatement training protocol.Kappa opioid receptor agonists and stress have been shown in numerous models to affect the magnitude of cocaine and nicotine place preference as well as cause reinstatement of cocaine and nicotine preference behavior . HoweverFigures 3B and 4B). Extinction rates of place preference between no stress, SDS-exposed and CMS-exposed groups were not significantly changed. In contrast, following extinction of either cocaine or nicotine place preference in CMS groups, KOR-induced reinstatement of both cocaine and nicotine place preference was significantly reduced  and cocaine . No significant differences in locomotor activity were found between no stress group and the CMS group following cocaine treatment . As predicted, distance traveled significantly increased over the 3-day cocaine conditioning period, as previously shown . However, nicotine-induced elevations in locomotor activity were significantly reduced following CMS on the second and third day of nicotine conditioning . Locomotor activity was also measured during U50-induced reinstatement in both the nicotine and cocaine groups. In the cocaine group only at the 5 min time point was there a significant decrease in locomotor activity in the CMS group compared to the no stress group . The nicotine group showed a similar profile, locomotor activity was also significantly decreased in the CMS group compared to the no stress group at both the 5 and 10 min time point . Together, these results support the conclusion that CMS protects against subsequent KOR-mediated cocaine and nicotine reinstatement and further suggests that CMS may act to regulate KOR system function.There was no significant difference between the no stress and CMS-exposed groups in the magnitude of the initial cocaine  or nicotine  conditioned place preference scores. This finding suggests that exposure to CMS does not effect the subsequent conditioned rewarding effects of cocaine or nicotine . In contrast, repeated SDS or CMS protected against subsequent KOR-mediated reinstatement . We also found that this CMS protective effect not only influenced KOR-induced cocaine reinstatement  but it also blocked KOR-induced reinstatement of nicotine place preference , suggesting a conserved mechanism for multiple drugs of abuse. Interestingly, CMS exposure had no effect on the initial cocaine or nicotine conditioning, nor did CMS exposure impact the magnitude of drug-primed reinstatement. These findings suggest that SDS and CMS induce a type of “tolerance” to subsequent KOR activity, which has also been previously reported when administering repeated doses of KOR agonists  suggesting that SDS acts in specific neural circuits associated with reinstatement but not locomotion. Therefore selective blockade of KORs in specific brain regions during repeated SDS, and CMS and subsequent measures of KOR-mediated behavioral responses will be required in future studies. In addition, utilizing GRK3 knockout mice, or expression of KOR-S369/358 mutants in vivo, and exposure to CMS are interesting extensions of this work as prior work has shown that GRK3 signaling is required for KOR-mediated behavioral effects via phosphorylation, arrestin recruitment, and p38 signaling (The precise neuronal and molecular mechanisms for SDS- and CMS-induced attenuation of KOR-induced reinstatement were not identified in this study but are important and exciting extensions of this work. There are a number of mechanisms that maybe involved in CMS-induced regulation of KOR-reinstatement. KOR regulation following repeated and high agonist-receptor occupancy has been previously reported to be mediated by GRK3 phosphorylation of serine 369 in mouse/rat KOR or serine 358 in the human KOR . In repeignaling . It has ignaling . Togetheignaling  it is poignaling . HoweverThe effects of stress on cocaine and nicotine seeking have been widely reported by numerous groups (for reviews see  In summary, we report that frequency and duration of stress differentially influences KOR-induced reinstatement of cocaine and nicotine preference. The present study shows that repeated stress or CMS prior to reinstatement prevents a KOR-induced reinstatement response, while acute exposure to stress induces potentiation of KOR-reinstatement. The wide array of recent studies investigating the interactions between stress and KOR function on reward, reinstatement, and dysphoria suggest that KOR interacts with multiple neurotransmitter systems and circuits to mediate its complex role in behavioral output. We identified previously unrecognized roles for acute, sub-chronic, and chronic stress exposure on KOR-mediated behavioral function. These findings suggest that understanding the regulation of dynorphin/KOR systems in response to various stress exposures is critical to understanding and identifying KOR as a potentially novel therapeutic target system in drug relapse, anxiety, and depression.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.