N = 12) for Y-tube conduits. Animal groups comprised intact animals (Group 1), those receiving hypoglossal-facial nerve end-to-end coaptation alone , and those receiving hypoglossal-facial nerve reconstruction using a Y-tube . Videotape motion analysis at 4 months showed that HFA-Y-tube group showed a reduced synkinesis of eyelid and whisker movements compared to HFA alone.The facial nerve is the most frequently damaged nerve in head and neck trauma. Patients undergoing facial nerve reconstruction often complain about disturbing abnormal synkinetic movements of the facial muscles  which are thought to result from misguided collateral branching of regenerating motor axons and reinnervation of inappropriate muscles. Here, we examined whether use of an aorta Y-tube conduit during reconstructive surgery after facial nerve injury reduces synkinesis of orbicularis oris (blink reflex) and vibrissal (whisking) musculature. The abdominal aorta plus its bifurcation was harvested ( The facial nerve is the most frequently damaged nerve due to a variety of reasons including radical parotidectomy, petrous bone surgery, removal of the cerebellopontine angle tumors, and iatrogenic injury –5. The rThe most common surgical approach for repairing transected nerves has been direct suture of the two stumps (end-to-end anastomosis)  but funcIn cases where large segments of the intracranial or intratemporal portions of the facial nerve are destroyed, hypoglossal-facial anastomosis (HFA) is a favoured surgical technique aimed at encouraging some innervation  of the facial muscles and minimizing facial palsy , 11, 12.We have previously shown that, following facial nerve injury and end-to-end anastomosis, collateral branching can be reduced by local application of neutralizing antibodies to neurotrophic factors  or by stIn human patients receiving facial nerve reconstruction surgery, the most common synkinesis is eyelid closure during a smile. However, little is known about the effects of different types of surgical reconstruction on the likelihood of synkinesis after nerve injury.Rats provide an ideal model to examine synkinesis because they display spontaneous bursts of activity of the vibrissal muscles (whisking) that can be analyzed in relation to eyelid closure (blink) , 26, 27.We therefore examined the extent of synkinesis after facial nerve reconstruction using HFA alone or HFA using an aorta Y-tube conduit. We undertook two quantitative motion analyses: one on eyelid closure during spontaneous whisking and the other on vibrissal whisking during a blink reflex elicited by an air puff to the cornea and periorbital region. Video recordings taken at the time of data collection for a previously published experiment  were usen = 12 in each) comprising intact controls (Group 1), rats subjected to hypoglossal-facial nerve end-to-end coaptation alone , and those receiving hypoglossal-facial nerve reconstruction using a Y-tube . After 4 months, all animals were subjected to video-based motion analysis of orbicularis oculi and vibrissal motor performance.The data were collected, but not previously reported, from animals used in an earlier study . Forty-eThroughout, allocation was concealed; that is, the person undertaking the surgery did not know to which group the animal would be allocated. Animals were randomised into groups using a randomised number sequence and assessment was blinded.The aortal bifurcation into the right and left common iliac arteries was used as a Y-tube conduit. In brief, once exposed, an artery clamp was applied to the aorta just below the diaphragm to avoid excessive bleeding. The aorta was cut and traced inferiorly taking care to avoid punctures and a 1.5–2.0 cm long segment including the common iliac arteries removed and irrigated with phosphate buffer to remove blood clots. Small branches originating from the aorta were cauterized.Animals were anesthetized with a mixture of ketamine  and xylazine HCl . Hypoglossal-facial anastomosis  was performed unilaterally (right side) as described previously [HFA using a Y-tube has also been described previously , 25. ProRodents make a number of different movements with their whiskers, namely, (i) large-amplitude “explorative” sweeps of the vibrissae (in the frequency range of 5–11 Hz); (ii) low-amplitude “foveal” or “palpating” whisker movements at 15–25 Hz , 29; andVideo-based motion analysis of vibrissal motor performance and eyelid closure has been previously established and examined in a series of recent experiments , 32, 33.Four months after nerve reconstruction, all animals were videotaped for 3–5 min during active whisker exploration using a digital video camera (Sony Handycam DCR-SR70 HDD Camcorder). Care was taken to handle the animals gently to avoid stress on the animal. For calibration, a ruler laid 20 cm below the recorder  was videotaped before each trial. Selected sequences containing the most prominent whisks  on the intact side contralateral to surgery were captured by a 2D/Manual Advanced Video System . Frame rate of the video analysis was 25 frame/second. Eyelid closures were assessed from the same videotape sequences.To assess whisker movements, we used a geometrical model consisting of three reference points: (i) a point in the medial sagittal line close to the end of the nose, (ii) a point corresponding to the medial angle of the left orbita, and (iii) a point corresponding to the medial angle of the right orbita. Using this model we collected and evaluated data on the (i) whisking frequency (cycles of protraction and retraction per second), (ii) angle at maximal protraction , and (iii) amplitude  Figures . The maxTo assess spontaneous eyelid movements, video sequences were inspected on screen. In addition to the reference points used for evaluating whisking, we also added a reference point half-way along the rim of each eyelid to measure the mean distance between both eyelids. Frames were selected when eyelid closure on the intact left side was maximal, that is, when the distance between the eyelids was smallest . The minThe following are formulas used to calculate synkinetic movements:To assess elicited eyelid movements, we used a custom-designed apparatus that delivered a single standardized volume of 20 mL air as a “puff” to the cornea and periorbital region bilaterally at a distance of 3 cm. Ten puffs were delivered sequentially over 120 s. Eyelid closure was evaluated as above.All frames of our video material were evaluated to find the highest amplitude of the vibrissal hairs during blink reflex (contraction of the orbicularis oculi muscle) or vice versa. This is why we just concentrate to find spatial relation between whisker movement and eyelid contraction on the same sequence of the movements (the full length of the movement could be observed in 3–10 consecutive frames). Full length of the video material was examined and intereyelid distance and amplitude (in degree) of the vibrissal hair movement were measured in all sequences of the movements. For instance, when an animal contracted its orbicularis oculi muscle and showed a minimal intereyelid distance, we also measured the angular changes of the vibrissal hair to find the amplitude .It should be noted that there was a negative correlation in calculation paradigms of synkinetic movements. Minimal intereyelid distance (in mm) means more synkinetic movement. In other words during whisking if the animals show full close of its eyelid, we consider that the animal showed 100% synkinesis. However maximal protraction angle value  means more synkinetic movement.Movements of eyelid and vibrissal hairs are reverse in direction. Function of the eyelid is to diminish palpebral rima, but function of the vibrissal muscles is to move the hairs in forward direction, for example, enlarge the amplitude. In intact animals smaller intereyelid distance will mean good function (this means that orbicularis oculi muscle contracted to decrease intereyelid distance), but larger amplitude of the vibrissal hairs will mean good function . In other words if some axons in the buccal branch showed collateral axonal branching and reinnervated both of the whisker pad and orbicularis oculi muscles, smaller intereyelid distance measured during whisking will mean more synkinesis .Data Analysis. Data were presented as mean ± standard deviation and analyzed by ANOVA (post hoc Tukey test). The level of significance was set to P < 0.05. For analysis, GraphPad Prism version 5.0  was used.The formulas which were used to calculate synkinetic movements of the eyelid and vibrissal muscles are shown in .P < 0.05, Synkinetic movements of the eyelid during whisking were analyzed in the frame of first experimental set. The ratio (%) of synkinetic eyelid closure occurred at the same time as whisking movement was significantly lower in the HFA+Y-tube group  compared to the HFA group  in the HFA-Y-tube conduit group compared to those of HFA-coaptation group.Synkinetic movement ratios were 42.4 ± 6.5% and 29.2 ± 3.7% in HFA-coaptation and HFA-Y-tube groups, respectively. There was a significant difference between both groups . SynkineIn this study, we examined the function of vibrissal and orbicularis oculi muscles after two types of hypoglossal-facial reconstruction using an objective and quantitative analysis of simultaneous movements of both muscle groups, that is, synkinesis. We showed that synkinesis was reduced following hypoglossal-facial nerve reconstruction (HFA) using a Y-tube compared to HFA-coaptation, that is, direct anastomosis, alone. Evaluation of synkinesis of eyelid and vibrissal movements may be a useful model with clinical relevance for assessing functional recovery after facial nerve injury.Direct measurement of orbicularis oculi electromyography in conjunction with search coil tracking in a magnetic field has been used previously as an indicator of eyelid motor activation and lid movements, respectively –36. HoweA critical feature of our video-based assessment is to measure the amplitude of natural vibrissal bouts during free exploration. Earlier studies have shown that, during free exploration, rats rhythmically move their heads so as to bring microvibrissae into contact with the surfaces they are exploring. These periodic “microvibrissal placements” are synchronized with macrovibrissal movements not only during free exploration, but also during texture discrimination tasks , 38. As However, if the head is immobilized, the finely-tuned physiological synchrony of micro- and macrovibrissal whisking is impaired thereby introducing a confounder. Thus, in an attempt to compensate for the resulting decrease in sensory stimuli, rats react with significantly larger than normal, and therefore nonphysiological, vibrissal excursions of the order of 90 degrees –43.Immediately after facial nerve lesion, the retractor bulbi extraocular musculature has been shown to support blink response in rabbits  as well Synkinesis following nerve injury refers to involuntary contraction of some muscles during voluntary movement of others, for example, involuntary eye closure at the same time as smiling. This phenomenon is thought to arise from axonal misrouting during regeneration of the facial nerve . TreatmeImproving clinical outcome requires reliable and reproducible experimental models of peripheral nerve injuries and their reconstruction , 58, 59.Another important issue is that we could not understand whether recovery from facial nerve cut causes abnormal whisker movements desynchronization of whisker movement, more side to side asymmetry. Although we tried to measure abnormal coactivation (contraction) of muscles, it is possible that there may be abnormal whisker synchronization. Cortical motor neurons can drive whisker movement but only of a few whiskers , 63, andFacial nerve palsy can be scored clinically using the Sunnybrook, House-Brackmann, and Yanagihara grading systems whose reliability has also been established . ScoringA different scoring system  has beenIn a recent study, Hadlock et al.  describeIn our previous study , we founIn conclusion, the analysis of our video material revealed that reconstruction of the hypoglossal nerve with an injured facial nerve using a Y-tube conduit results in lower synkinetic activities of the orbicularis oculi and vibrissal muscles compared to when reconstruction is undertaken using simple end-to-end anastomosis. Surgical reconstruction that enables transected axons to regenerate in an unforced manner towards their targets via a Y-tube conduit may both reduce collateral axonal branching and synkinetic movement and improve function.


Lung cancer remains the leading cause of cancer-related death in the United States. Cigarette smoking is a well-recognized risk factor for lung cancer, and a sustained elevation of lung cancer risk persists even after smoking cessation. Despite identifiable risk factors, there has been minimal improvement in mortality for patients with lung cancer primarily stemming from diagnosis at a late stage when there are few effective therapeutic options. Early detection of lung cancer and effective screening of high-risk individuals may help improve lung cancer mortality. While low dose computerized tomography (LDCT) screening of high risk smokers has been shown to reduce lung cancer mortality, the high rates of false positives and potential for over-diagnosis have raised questions on how to best implement lung cancer screening. The rapidly evolving field of lung cancer screening and early-detection biomarkers may ultimately improve the ability to diagnose lung cancer in its early stages, identify smokers at highest-risk for this disease, and target chemoprevention strategies. This review aims to provide an overview of the opportunities and challenges related to lung cancer screening, the field of biomarker development for early lung cancer detection, and the future of lung cancer chemoprevention.  Lung cancer is the leading cause of cancer mortality, accounting for almost 27% of all cancer-related deaths  and 20% This review will provide an overview of the progress that has been made in lung cancer screening, early detection, chemoprevention, and biomarker development using surrogate tissues. It will also discuss some of the progress and controversies in these areas, and how evolving technologies may improve our ability to target interventions for the subpopulations at highest risk for lung cancer. The prime focus of this review is on smoking-associated lung cancer since cigarette smoking is a major contributor to development of this disease.Although 80%–85% of patients with lung cancer have a history of smoking, only 10%–15% of patients who smoke will actually develop lung cancer. There is presently no widely-accepted method to accurately predict which current and former smokers will develop lung cancer. The risk of developing lung cancer increases with accumulated exposure to cigarette smoke, which alters antioxidant, xenobiotic, inflammatory-related pathways and oncogenic genes. Identifying the subset of cigarette smokers at highest risk for developing lung cancer would enhance the ability to target lung cancer surveillance, chemoprevention, and early detection strategies. Furthermore, risk modeling can facilitate the design of targeted clinical trials by enrolling higher risk subjects.et al. estimated a lung cancer incidence in never-smokers age 40–79 years ranging from 4.8–13.7 per 100,000 person-years in men and 14.4–20.8 per 100,000 person-years in women [et al. showed that non-smoker’s lung cancer had a greater portion of their genome altered compared to smokers lung cancer [Although cigarette smoking is the major risk factor for lung cancer, lung cancer also occurs at lower rates in nonsmokers . Wakelee in women . Thu et g cancer . These oet al. were the first to apply modern statistical methods to assess 10-year lung cancer risk in heavy smokers by considering age, sex, smoking history, and asbestos exposure [et al. developed another risk assessment model that incorporated clinical information to predict 1-year lung cancer risk in current, former, and never smokers [Bach exposure . This moexposure . Spitz e smokers . The Liv smokers . Incorpo smokers . For exa smokers . These oet al. expanded their clinical risk model for current and former smokers to include two markers of DNA repair with marginal improvement in discriminatory power of the model [Incorporating molecular information with clinical risk features may improve the ability to identify individuals with the highest risk for developing lung cancer. For example, it is well established that smokers who are first-degree relatives of individuals with lung cancer have a two- to three-fold higher risk of developing lung cancer themselves . Polymorhe model . While sThe drastic drop in survival rates with delayed diagnosis of lung cancer demonstrates the pressing need for effective screening strategies. Ideally, a screening tool would accurately identify early stage lung cancer patients in a safe and cost effective manner . FurtherPrevious lung cancer screening trials using chest radiography ,21 and/oComputerized tomography (CT) has also been investigated as a lung cancer screening tool given its improved ability to radiographically evaluate other lung diseases compared to chest radiography. While smaller and observational CT screening studies did not show a mortality benefit, the large randomized National Lung Screening Trial (NLST) did demonstrate a statistically significant benefit to using annual low-dose CT (LDCT) screening in individuals at high risk for lung cancer. This randomized trial involved greater than 53,000 current and former heavy smokers (>30 pack-years) compared to annual low-dose helical CT with chest radiography for lung cancer screening. High-risk individuals were defined as men and women aged 55 to 74 who were active or former (<15 years since quitting) smokers with a cumulative exposure to cigarettes of at least 30 pack-years. A significant improvement in mortality from lung cancer and all causes was observed for LDCT screening (20% and 6.7% respectively), with a number needed to screen of 320 . Based oDespite the improvement in mortality with LDCT screening, a high proportion of false positives is observed with this technique. This raises questions on how best to implement LDCT screening in clinical practice, and additional trials are aimed at further evaluating the role of LDCT in lung cancer screening. The randomized NELSON trial in The Netherlands and Belgium is comparing LDCT to no intervention to determine whether LDCT improves lung cancer mortality . This tret al. stratified participants in the NLST into five groups based on their risk of developing lung cancer based on clinical factors. Individuals with the highest lung cancer risk benefited most from LDCT screening, suggesting that targeting highest-risk individuals and incorporating lung cancer risk models into CT-screening programs may ultimately improve the benefit and reduce the potential harm and cost from screening [Despite the mortality benefit for high-risk smokers screened with LDCT, the debate over safety and cost effectiveness of this screening protocol is ongoing. Most notably, 23.3% of LDCT screening tests performed in the NLST had false positive results . The potcreening . Another potential harm of annual LDCT screening is the inevitable exposure to radiation. This is particularly important to consider given the high proportion of patients that will require additional follow up imaging for indeterminate nodules, further increasing cumulative radiation exposure . It has The financial implications of annual LDCT screening are unclear, and this will likely be closely monitored as additional data becomes available. The rate of over diagnosis and false positives may strongly influence the ultimate economic burden of any large-scale lung cancer-screening program, particularly when compared to the cost effectiveness of smoking cessation . FurtherWhile the overall 5-year survival for non-small cell lung cancer (NSCLC) is 14%, survival for Stage I disease is 83% . This hiet al. identified loss of heterozygosity in bronchial brushings obtained from patients with lung cancer [Bronchial airway biomarkers for early lung cancer detection are based on the concept of “field cancerization” which stems from the observation that some alterations found in the lung tumor are also present throughout uninvolved areas of the respiratory tract and lung. This field cancerization effect, measurable throughout the respiratory tract, is thought to stem from: (1) an airway-wide response to the toxins in cigarette smoke that reflect an individual’s risk for lung cancer; (2) response of the lung and respiratory tract to the presence of tumor; and/or (3) clonal-expansion of the tumor . Prior sg cancer , suggestSubsequent studies have leveraged this field cancerization concept to develop clinical-useful lung cancer diagnostics. Using cytologically normal large-airway epithelial cells obtained at bronchoscopy from active and former smokers undergoing clinical evaluation for suspected lung cancer, Spira and colleagues developed an 80-gene expression biomarker that distinguis0hed individuals with and without lung cancer. This airway gene-expression biomarker had an accuracy of 83%  in an independent test set, and ~90% sensitivity for Stage I lung cancer across all subjects, suggesting utility as an early diagnostic tool . FurtherObservations that cigarette smoke-induced alterations in the bronchial airway epithelium ,51 are sin situ hybridization (FISH) can be used to detect genetic changes in sputum and microRNA panels, identified by profiling primary lung tumors and adjacent noncancerous lung tissue, and have been measured in sputum as a potentially useful tool for early lung cancer detection [Another potential surrogate tissue for developing early diagnostic biomarkers for lung cancer is sputum. Despite sputum cytology not being an effective screening method to achieve early lung cancer diagnosis , subsequetection . et al., for example, have shown that hypermethylation of p16 and MGMT are detectable in the sputum of patients with squamous cell lung cancer, and could potentially serve as an early diagnostic for lung cancer [Furthermore, DNA methylation, an epigenetic event that affects cell function by altering gene expression, has been shown to be altered in sputum samples from patients with lung cancer. Belinsky g cancer . In addig cancer . Some ofg cancer .et al. identified a group of miRNA in serum samples that might serve as an early diagnostic biomarker in asymptomatic patients [Whole blood, serum and plasma are other easily obtainable surrogate tissues that could be used to develop biomarkers for early lung cancer detection. The source of these biomarkers could be from the tumor itself, or from the host response to the presence of a lung tumor. Serum measurement of the protein pentaxin 3 has been described as a potential early lung cancer diagnostic . Anotherpatients . Other spatients ,65.et al. developed a series of potential biomarkers for early lung cancer diagnosis using a proteomic profiling of serum samples [Ostroff  samples . Other g samples . Recentl samples  added di samples . Importa samples . TogetheBefore the manufacturing of cigarettes became widespread in the early 20th century, lung cancer was a rare disease. Today, cigarette smoking remains a leading cause of lung cancer, with 85% of lung cancer cases attributable to this exposure . 20% of Smoking prevention and cessation are the foundation of any strategy for preventing lung cancer . HoweverDespite numerous trials, no chemoprevention studies to date have demonstrated an improvement in lung cancer mortality, and few studies have demonstrated an effect on intermediate endpoints such as genomic markers of epithelial cell damage and sputum atypia. Several publications review the results of randomized trials and the rationale for evaluating new chemopreventive agents ,79,80,81Based on preclinical studies providing a rationale to study α-tocopherol (vitamin E) and β-carotene, one of the earliest chemoprevention trials evaluated these agents for the primary prevention of lung cancer in smokers as part of the α-tocopherol, β-carotene (ATBC) Cancer Prevention Study. This trial showed an 18% increased incidence of lung cancers and an 8% increased overall mortality for those on β-carotene at a median follow-up of 6 years . In a suSelenium, a component of the antioxidant enzymes glutathione peroxidase and thioredoxin reductase, is thought to improve cellular defense against oxidative stress and has also been studied as a potential primary chemoprevention for lung cancer. The results of studies investigating selenium as a potential agent for lung cancer chemoprevention have been conflicting. In a secondary analysis of The Nutritional Prevention of Cancer Trial, a randomized, double-blind, placebo-controlled trial of over 1300 participants with a history of skin cancer , the incCyclooxygenase-2 (COX-2), an enzyme in the arachidonic acid cascade, is up-regulated in many tumors, including lung cancer. Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit COX enzymes, and have been investigated as potential agents in the primary chemoprevention of lung cancer. One meta-analysis that included 19 studies of NSAIDs and lung-cancer risk suggests an overall benefit of regularly taking aspirin (but not other NSAIDs) for those at high risk of lung cancer , and a sProstacyclin analogs, in addition to their anti-inflammatory effects, selectively increase peroxisome proliferator-activated receptor gamma (PPARγ) activity in non-small cell lung cancer . In NSCLSeveral randomized trials studied the effects of retinoids given alone ,101,102 The selective cyclooxygenase-2 (COX-2) inhibitor celecoxib was studied in two randomized trials in smokers, in which cellular proliferation and COX-2 expression levels were study endpoints ,106. In cis-1,2,3,5-trans-4,6-cyclohexanehexol) is a well-tolerated inhibitor of the PI3K pathway. A clinical study of 10 smokers with bronchial dysplasia demonstrated a significant rate of regression of the dysplastic lesions following treatment with myo-inositol [Phosphatidylinositol 3-kinases (PI3Ks) regulate cell growth and survival through activation of protein kinase B (PKB or AKT) in the PI3K/AKT/mTOR pathway. PI3K is a well-recognized oncogenic pathway, with higher levels of pathway activity associated with several malignancies including lung cancer. Myo-inositol  was observed in current smokers, suggesting that lung cancer chemopreventive approaches may have the greatest likelihood of success in the absence of ongoing tobacco-driven carcinogenesis . A multi-faceted approach combining smoking cessation and prevention, early lung cancer detection, and targeted chemoprevention is urgently needed to improve mortality from lung cancer. Since there are approximately 45 million former smokers in the United States and more than 1.2 billion smokers worldwide, tobacco use must be the first target, since smoking prevention and cessation are the most effective ways to prevent lung cancer. Identifying which former and current smokers are at highest risk for developing lung cancer is the next most important step in any screening, early detection, or chemoprevention program. Several groups have used genomic studies in an attempt to identify a subset of smokers who are at highest risk of developing lung cancer. Ideally, these approaches must be integrated with clinical early detection and screening strategies such as low-dose computer tomography (LDCT) scanning to effectively differentiate benign from malignant lung nodules, and to establish therapeutically exploitable differences between normal, precancerous, and malignant cells. A combined approach that integrates molecular biomarkers with CT characteristics  may further help distinguish biologically aggressive lung cancers from indolent lesions. Since molecular, genetic, and epigenetic abnormalities precede morphological changes in the bronchi and alveoli, molecular biomarkers may help identify a group of high-risk patients who would most benefit from LDCT screening. Genetic abnormalities can be detected from respiratory cells from sputum, bronchial airway samples, and blood. Gene expression profiles generated from those specimens offer a wide area of investigation for biomarker development, though to be clinically applicable, biomarkers must be specific, cost-effective, and efficient.The availability of agents that might be effective in reversing the pre-malignant changes in airway epithelial cells has driven the search for biomarkers that identify individuals at highest risk for developing lung cancer. Chemopreventive agents are increasingly selected for further development based on the biologic mechanisms underlying lung carcinogenesis. Given the long latency between progression from pre-malignancy to overt lung cancer, consensus groups in the United State and Europe have suggested that it is essential to identify earlier end-points in the study and development of chemopreventive agents ,119. Serial tissue sampling could be used to measure biomarkers that reflect the clinical effectiveness of chemopreventative agents. As an example of this approach, Mascaux and colleagues used biospecimens from the iloprost lung cancer chemoprevention trial to investigate whether miRNAs could serve as predictive biomarkers or intermediate endpoints of response to iloprost . The expWhile chemoprevention for lung cancer is not currently recommended, it remains a promising strategy to potentially reduce lung cancer mortality. Studies using biomarker and pathway-driven approaches aimed at identifying the subsets of patients most likely to respond to specific agents can serve as models for assessing novel molecular targets and chemopreventive agents. It is also possible that combination-targeted therapy directed at multiple oncogenic pathways may ultimately prove more effective than single agents alone. Integrating clinical markers of lung cancer risk with molecular biomarkers may also help improve early lung cancer detection through the development of comprehensive risk-assessment and early-detection models. Incorporating these biomarkers and integrating clinico-genomic models using surrogate tissue sampling into large-scale clinical studies promises to improve our ability to diagnose, treat, and prevent lung cancer and thereby improve mortality from this disease.


Functional imaging studies in human reliably identify a trio of scene-selective regions, one on each of the lateral [occipital place area (OPA)], ventral [parahippocampal place area (PPA)], and medial [retrosplenial complex (RSC)] cortical surfaces. Recently, we demonstrated differential retinotopic biases for the contralateral lower and upper visual fields within OPA and PPA, respectively. Here, using functional magnetic resonance imaging, we combine detailed mapping of both population receptive fields (pRF) and category-selectivity, with independently acquired resting-state functional connectivity analyses, to examine scene and retinotopic processing within medial parietal cortex. We identified a medial scene-selective region, which was contained largely within the posterior and ventral bank of the parieto-occipital sulcus (POS). While this region is typically referred to as RSC, the spatial extent of our scene-selective region typically did not extend into retrosplenial cortex, and thus we adopt the term medial place area (MPA) to refer to this visually defined scene-selective region. Intriguingly MPA co-localized with a region identified solely on the basis of retinotopic sensitivity using pRF analyses. We found that MPA demonstrates a significant contralateral visual field bias, coupled with large pRF sizes. Unlike OPA and PPA, MPA did not show a consistent bias to a single visual quadrant. MPA also co-localized with a region identified by strong differential functional connectivity with PPA and the human face-selective fusiform face area (FFA), commensurate with its functional selectivity. Functional connectivity with OPA was much weaker than with PPA, and similar to that with face-selective occipital face area (OFA), suggesting a closer link with ventral than lateral cortex. Consistent with prior research, we also observed differential functional connectivity in medial parietal cortex for anterior over posterior PPA, as well as a region on the lateral surface, the caudal inferior parietal lobule (cIPL). However, the differential connectivity in medial parietal cortex was found principally anterior of MPA. We suggest that there is posterior–anterior gradient within medial parietal cortex, with posterior regions in the POS showing retinotopically based scene-selectivity and more anterior regions showing connectivity that may be more reflective of abstract, navigationally pertinent and possibly mnemonic representations. Functional magnetic resonance imaging (fMRI) studies investigating scene-perception in human reliably and repeatedly identify a trio of spatially distinct scene-selective regions, one on each of the lateral [occipital place area (OPA) or transverse occipital sulcus (TOS)] , ventralDespite their shared category-selectivity, a division of labor between these regions has been suggested recently, with OPA and PPA reportedly more involved in the visual representation of scenes  and landper se  and scene-selectivity with independently acquired resting-state functional connectivity analyses to explore scene and retinotopic processing in medial parietal cortex. Across participants, medial scene-selectivity was identified reliably within the posterior bank of ventral parieto-occipital sulcus (POS) and did not extend into retrosplenial cortex . We propSixteen participants  completed retinotopic mapping (eight runs) and functional localizer (two runs) scans. Ten of these participants  also completed an additional event-related experiment with scenes presented in the four quadrants of the visual field. A separate group of 48 participants  completed resting-state functional connectivity sessions. All participants had normal or corrected-to-normal vision and gave written informed consent in accordance with the declaration of Helsinki. The National Institutes of Health Institutional Review Board approved the consent and protocol. This work was supported by the Intramural Research program of the National Institutes of Health – National Institute of Mental Health Clinical Study Protocol 93-M-0170, NCT00001360.Participants were scanned on either a research-dedicated GE 3 Tesla Signa scanner or a research-dedicated Siemens 7 Tesla Magnetom scanner in the Clinical Research Centre on the National Institutes of Health campus . Across scanners, oblique slices were oriented approximately parallel to the base of the temporal lobe and extended posteriorly through all of visual cortex. All participants completed at least eight runs of pRF mapping as well as two runs of an additional category-selective functional localizer. Participants in the event-related sessions completed six event-related runs as well as two runs of an additional category-selective functional localizer.Partial volumes of the occipital and temporal cortices were acquired using an eight-channel head coil .Partial volumes of the occipital and temporal cortices were acquired using a 32-channel head coil .Population receptive field mapping sessions were conducted at either 3T (11 participants) or 7T (5 participants) field strengths. During pRF mapping sessions a bar aperture traversed gradually through the visual field, whilst revealing random scene fragments. During each 36 s sweep the aperture made 18 evenly spaced steps, one every 2 s (1TR), to traverse the entire screen. During each bar position (1TR) five scene fragments were displayed in rapid succession (400 ms per image). Across the 18 aperture positions all 90 possible scene images were displayed once. Thus, in any single sweep, each scene occurred only once, reducing the likelihood that participants mentally ‘fill-in’ in the underlying image, a problem that can arise if a single background image is revealed gradually. A total of eight sweeps were performed in each run . Specifically, the bar aperture progressed in the following order for all (8) runs: Left–Right, Bottom Right–Top Left, Top–Bottom, Bottom Left–Top Right, Right–Left, Top Left–Bottom Right, Bottom–Top, and Top Right–Bottom Left. The bar stimuli covered a circular aperture  or 7T (5 participants) field strengths. All participants completed two runs in order to localize scene- and face-selective areas. These scans employed an on/off design (scenes/faces) with 16 alternating blocks (16 s) of 20 stimuli (5° × 5°) presented while participants performed a one-back task. Within a block, 20 stimuli were presented for 300 ms each, separated by a 500 ms inter-stimulus interval. Additionally five participants completed independent localizer scans employing a multiblocked design. In these sessions, images from eight categories including faces, buildings, scenes , and man-made and natural objects, were presented in 16-s blocks with an 8 s blank fixation period separating blocks. Each category was presented twice per run, and the order of presentation was counterbalanced across participants and runs.n = 24) appeared at each location (n = 4) for 400 ms, with a jittered (4–12 s) interstimulus fixation period; thus, each run contained 96 trials. The order of presentations and fixation arm extensions was randomized within each run. Participants completed six runs of the event-related experiment.Event-related scanning sessions were conducted at either 3T (six participants) or 7T (four participants) field strengths. Participants performed an attention-demanding task at fixation whilst whole-scene images were presented randomly in one of the four quadrants of the visual field . Stimuli subtended 6.5° × 6.5° and were centered 6.5° from the central fixation cross into one of the quadrants of the visual field. Participants maintained fixation throughout. Scene stimuli were scaled to subtend the same visual angle of the screen during both 3T and 7T scans. As a scene was presented, one arm of the fixation cross, either the horizontal or the vertical, increased in length. Participants were required to identify, via button response, the longer fixation arm. Stimulus presentation and fixation cross changes occurred simultaneously. Within each run, each scene  software package1 . All funp < 0.0001 (uncorrected). Significance maps were then projected onto surface reconstructions of the gray–white matter boundary of individual hemispheres. Only contiguous clusters of nodes exceeding the above threshold were defined as scene- or face-selective. The anatomical locations of these clusters were then inspected in order to define ROIs consistent with previously published work (To identify scene- and face-selective regions of interest (ROIs), significance maps of the brain were computed in each participant by performing a correlation analysis between the assumed hemodynamic response function and the activation time courses thresholded at hed work .t-tests between each condition  and baseline generated significance maps. The β-values for each condition were extracted from voxels within each ROI and averaged.For event-related runs, performing R2, which corresponds to the explained variance of the fit and can be used to statistically threshold these data.Population Receptive Field analyses of unsmoothed data were conducted in AFNI, using a pRF implementation for the AFNI distribution . The modVisual field coverage plots, which are built by combining the best Gaussian receptive field model for each voxel within an ROI, can be computed from these data. The coverage plots for a given ROI are an aggregation of these Gaussians. Assuming a strong central tendency in the centers of the receptive fields, a linear aggregation  will result in a coverage plot appearing as a single large Gaussian. In our analyses a max operator is used. This creates a coverage plot that reflects, at each point, the maximum pRF value from all of the receptive field models within an ROI . Thus, t2. Retinotopically organized maps were visible and present in all tested hemispheres. Notwithstanding subtle inter-participant variability, the main features of the maps, in particular the reversals in visual field representation at the vertical and horizontal meridians were consistent across participants. In accordance with previous reports  were acquired.Preprocessing of the fMRI data was performed using AFNI. Each echo was processed independently prior to optimal combination of the data from each TE (see below). Slice-time correction was applied (3dTShift) and signal outliers were attenuated [3dDespike ]. Motion∗. Because T2∗ varies across the brain, single echo images are not optimal to see this variation. By acquiring multiple echoes, this enables the calculation of the “optimal” T2∗ weighted average of the echoes, which allows one to recover signals in dropout areas and improves contrast-to-noise ratio . Using the contrasts described above, these ROIs  were restricted to the top 300 category selective nodes in each region to account for large differences in ROI size. This thresholding procedure revealed anterior and posterior components of PPA, which were considered separately (aPPA and pPPA) in some analyses, resulting in the following ROI set .Category-selective ROIs for resting state analysis were derived from the group data . The locations of our OPA, PPA and RSC ROIs  are entirely consistent with previous literature , ventral (PPA), and medial (RSC) cortical surfaces were identified, where possible, in each participant  using the contrast of scenes > faces . At lower thresholds, a small peak of activation does emerge on the cingulate gyrus, potentially corresponding to BA29/30, but it is dorsal to the isthmus and only extends into the callosal sulcus, where most of retrosplenial cortex is contained, at very low thresholds . To investigate individual variability in the spatial extent of the MPA, we calculated in how many participants the scene-selective activation extended onto the cingulate gyrus and into the commonly drawn BA 29/30. In the left hemisphere, activation in BA29/30 was only present in 2/13 participants and in 1/15 participants in the right hemisphere. Finally, to investigate to what extent the specific contrast we employed (scenes vs. faces) impacts the spatial extent of the medial parietal scene-selectivity, we compared the extent of scene-selective activation in a subset of our participants (n = 5) who participated in a separate experiment allowing us to define scene-selective activation by the contrast scenes vs. objects. This alternative contrast identified a similarly located region within the POS , shows that scene features, without detailed context, are sufficient to drive this region. Second, in both single participant and group time-series, eight peaks of activity are clearly evident, which reflect the eight sweeps our pRF mapping stimulus made through the visual field and thus through the pRF’s of MPA voxels . Third, the stimuli at the top of Figure 3A, shows position in the visual field corresponding to the peak of our group level time-series during each sweep . The region of visual space over which these bar positions overlap maximally, reflecting the positions in the visual field that right MPA is most sensitive to are shown in . Importantly, although this region covers portions of the visual field both above and below the horizontal meridian, they are restricted exclusively to the left  visual field. Finally, the width of each peak of activity is wide, spanning much of the width of each sweep , which is suggestive of large receptive fields within MPA . Crucially, if MPA were retinotopically insensitive, such a systematic pattern of activity would be unlikely to occur.Inspection of Figure 4). These plots were computed from pRF values derived from all nodes within the functionally localized MPA ROIs, without thresholding of the pRF values themselves. Both left and right MPA exhibit clear biases for the contralateral visual field, with more pRF’s centered within, and more representation of, the contralateral visual field in both hemispheres . Such contralaterally biased representations mirror those present in both left and right OPA and PPA, respectively . We tested for any biases for a single quadrant of the visual field using repeated-measures ANOVA with factors Hemisphere , Visual Field  and Vertical Position . There was a main effect of Hemisphere , reflecting on average stronger responses in the right than left hemisphere. The main effect of Visual Field was also significant , reflecting on average stronger responses in the contralateral visual field within both regions. Further, there was a significant Hemisphere by Visual Field interaction , reflecting on average a stronger contralateral bias in the right than left hemisphere. However, there was no main effect of Vertical Position  and no interactions involving Vertical Position reflecting on average the largely equal representations within the upper and lower visual fields within both regions. These data demonstrate that both left and right MPA exhibit a contralateral bias, but no bias for a specific quadrant of the visual field.Next, we sought to characterize MPA’s receptive field profile in a number of ways that mirror our previous characterizations of retinotopy within both OPA and PPA , 2016. Iectively . Unlike minus ipsilateral pRF value) and elevation  bias measurements in each participant and ROI. Consistent with our previous characterizations of OPA and PPA  in both hemispheres . In contrast however, the elevation biases within MPA were not significant in either hemisphere .To further quantify these biases, we calculated contralateral . In agreement with our pRF mapping, univariate analyses  demonstrated that both left and right MPA exhibited stronger responses for stimuli presented in the contralateral over ipsilateral visual fields  to stimuli in the contralateral visual field, confirming this organizational principle within MPA. Elevation biases in MPA, however, were not significant for either hemisphere .To confirm these retinotopic biases within MPA, independently, we measured responses in MPA during a condition-rich event-related experiment. Participants viewed 24 whole scene images presented randomly into one of the four quadrants of the visual field, whilst performing an orthogonal task at fixation . Indeed, this region either encompassed entirely or overlapped substantially with our MPA ROIs in many participants, but importantly, was separated spatially from both antecedent visual areas (V1 and V2d) and retinotopic maps found in more dorsal regions of POS, such as V6, V6Ad, and V6Av  could localize this region. Importantly, across participants we observed reliable and statistically significant pRF responses within the posterior and ventral bank of POS in both hemispheres  and ventral surfaces (PPA), we analyzed functional imaging data collected from a separate group of 48 participants whilst at rest.ventral surface contrast) and (ii) OPA vs. OFA . A region overlapping our MPA ROI was clearly identifiable on the basis of differential connectivity between PPA and FFA in both hemispheres , but not, between OPA and OFA , suggesting that MPA is more strongly associated with PPA than OPA.Initially, we examined whether MPA could be identified on the basis of differential connectivity with scene- vs. face-selective regions on both the ventral and lateral surfaces, considered independently. Our principal localization of MPA was based on the direct contrast of scenes > faces, for consistency therefore, we computed differential connectivity measurements for (i) PPA vs. FFA . Second, highly differential aPPA-pPPA connectivity was also found on the lateral surface, overlapping considerably with the caudal inferior parietal lobule (cIPL) ROI, taken from the Eickhoff–Zilles PGP probabilistic cytoarchitectonic map  consistent with prior work . The spatial adjacency but limited overlap between scene-selective MPA in the posterior bank of POS and the more anterior precuneus region showing significant association with aPPA, suggests a posterior–anterior gradient exists within medial parietal cortex and the POS.Previous work has suggested that medial scene-selective regions show stronger functional connectivity with anterior PPA (aPPA) than posterior PPA pPPA; . Given tior work . In contTo investigate the functional connectivity in medial parietal cortex further, we created a connectivity-defined ROI (CON) based on the differential connectivity between aPPA vs. pPPA. Importantly, we defined this connectivity ROI on the basis of differential connectivity data acquired during one rest period and subsequently applied this ROI to independent data acquired during a separate rest period within each participant .Figure 9A). Consistent with our pRF analyses, MPA exhibited stronger connectivity than CON with visual cortical areas, in particular pPPA and OPA, which have been shown previously to evidence strong retinotopy . Stronger CON connectivity was also present within anterior temporal, as well as, orbitofrontal and superior frontal cortical locations .Next, to compare the connectivity profiles of the selectivity-defined MPA with the more anterior CON, we directly contrasted the connectivity patterns for these two regions in both hemispheres . In general, there are positive correlations between all these ROIs in both hemispheres. However, CON shows very low correlation with both pPPA and OPA . Next we analyzed the relative strength of connectivity of MPA and CON by computing differential correlations between our medial parietal ROIs (MPA and CON) and scene-related regions on the (i) ventral surface , and (ii) lateral surface (OPA and cIPL) in both hemispheres. For each surface we conducted separate two-way repeated-measures ANOVAs with Hemisphere  and Seed ROI .To characterize the functional connectivity profiles of CON and MPA further, we focused on all scene-related ROIs  and first computed all pair-wise time course correlations  = 21.53, p =< 0.0001], reflecting on average greater differential connectivity with aPPA and pPPA in the left compared to right hemisphere. The main effect of Seed was also significant , reflecting on average greater differential connectivity for CON than MPA. Further, there was a significant Hemisphere by Seed interaction , indicating a stronger difference between MPA and CON in the right than left hemisphere .For the ventral surface we observed a significant main effect of Hemisphere . The main effect of Seed was significant , reflecting on average greater differential connectivity with OPA and cIPL for CON over MPA. There was also a significant Hemisphere by Seed interaction , again indicating a stronger difference between MPA and CON in the right than left hemisphere . Thus, within the scene-related regions, CON shows a relatively stronger association than MPA with aPPA and cIPL.For the lateral surface, there was no significant effect of Hemisphere  . Finally, we calculated the contralateral bias exhibited by both MPA (defined by its scene-selectivity) and CON. Again, MPA exhibited a significantly greater contralateral bias , although of note, CON also exhibited a numerical bias to the contralateral visual field . These data support the conclusion that MPA is more sensitive than CON to both retinotopic location and visual scene information and suggest a gradient of visual responsiveness across the medial parietal surface.Finally, we compared these two regions within medial parietal cortex in terms of both scene-selectivity and retinotopy. Initially, we examined the relationship between these two functional properties within the MPA alone. Within our group-based MPA ROIs we calculated the correlation between each nodes scene-selectivity index and the explained variance of the pRF model  7.01° (0.52°)]. Our resting state analyses highlight differential patterns of connectivity between MPA and PPA over OPA. Moreover, a region directly anterior of MPA exhibited significant differential connectivity with anterior over posterior PPA divisions, and strong connectivity with cIPL, suggesting a posterior–anterior gradient exists within medial parietal cortex from retinotopic and scene-related representations more posteriorly to more abstract, navigationally relevant and potential mnemonic representations more anteriorly.Although originally labeled ‘RSC’ , we, aloThe precise boundaries of human retrosplenial cortex are actively debated in the literature, with conflicting opinions as to its spatial extent with respect to the cingulate gyrus . RetrospImportantly, the location of scene-selectivity we report here is located largely within the ventral and posterior bank of the POS – it is not only dorsal of the anterior tip of the calcarine sulcus, but also, dorsal and posterior to the isthmus of the cingulate gyrus , suggestWe demonstrate a posterior–anterior gradient across medial parietal cortex in terms of both retinotopic properties and functional connectivity  presented scenes have been reported previously within this region , a systeClear and orderly progressions of the visual field within MPA were not visible consistently in our data, despite the strong retinotopic sensitivity shown by MPA as a whole. MPA’s large receptive fields may prohibit accurate estimates of polar angle in this region under the current pRF paradigm as voxels will respond to a number of visual field positions. Additionally, we do not observe clear clustering within MPA for different quadrants of the visual field . Again, the large receptive fields within MPA could underpin this. Importantly, however, these data cannot rule out the possibility that a map(s) of the visual field within MPA could be delineated with more sophisticated mapping paradigms at higher resolution. MPA exhibits large pRF sizes are suggestive of more global computations allowing neurons within MPA to pool visual scene information across large areas of the contralateral visual field. The large size of MPA pRF’s likely underpin the more complete hemifield representation exhibited by MPA, compared to the largely quadrant representations within OPA  and PPA , respectively . AlthougTogether, our data highlight that scene-selective MPA, in the posterior and ventral banks of POS, evidences strongly retinotopic voxels, contains large receptive fields and represents predominantly the contralateral visual field. MPA is also differentially connected to ventral (PPA) over lateral (OPA) scene-selective regions. Dividing PPA into posterior and anterior portions revealed a region of medial parietal cortex directly adjacent and anterior to MPA that exhibited highly significant aPPA associations. The spatial adjacency within medial parietal cortex, exhibited by this region and our scene- and retinotopically sensitive MPA, suggests that scene-related visual information undergoes a transformation from strongly retinotopic and visually grounded in posterior and ventral portions of POS, to more abstract, navigationally- and memory-relevant more anteriorly.ES jointly designed the study, collected and analyzed the retinotopic, event-related and functional localizer fMRI data, and wrote the manuscript. AS collected and analyzed the resting-state functional connectivity data and wrote the manuscript. CB jointly designed the study and wrote the manuscript.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.


Cerebral toxoplasmosis is a leading cause of central nervous system (CNS) lesions in severely immunocompromised human immunodeficiency virus+ (HIV+) patients who are seropositive for the parasite and have not received effective chemoprophylaxis. A 48-year-old HIV+ woman presented to our emergency room with seizure and decreased level of consciousness. She had developed fever, productive cough, and malaise 1 week prior to her current admission. Multifocal sensory and motor deficits in her extremities were revealed on neurologic examination. The brain imaging of our patient showed a distinctive ring-enhancing lesion that narrowed our differential diagnosis considerably.The patient was admitted on January 20, 2009 with decreased level of consciousness, urinary incontinence, upward eye deviation, right arm tonic flexion and right leg clonic jerks while the left extremities were tonically extended. Her seizure was controlled with intravenous diazepam and phenytoin, and she was fully conscious for history taking 5 h after presentation.Rosa canina, Urtica dioica, and Tanacetumv ulgare in addition to selenium, flavonoids, and carotenes. Ten years ago, after a bout of jaundice, she had been diagnosed with acute hepatitis B. The resolution of which was documented with hepatitis B surface antigen clearance and hepatitis B surface antibody seroconversion at the time.The patient had a positive history for HIV infection and reported prolonged close contact with a known tuberculosis (TB) case 2 years ago. She had developed fever, productive cough and malaise 1 week prior to her current admission; CD4 count of 18 cells/μL, skin purified protein derivative reactivity of 5 mm and a negative sputum smear for acid-fast bacilli had been reported at the time waiting for culture results. She also complained of headaches and weight loss during the past month. Oral co-amoxiclav and co-trimoxazole were administered for her recent symptoms. For her HIV, she had received IMOD intermittently  during the year prior to admission – Setarud (IMOD) is a herbal medicine prepared from a mixture of extracts of Neurologic examination revealed multifocal sensory and motor deficits involving her left ulnar nerve distribution, right median nerve distribution, proximal and distal right lower extremity weakness and atrophy, and symmetric sensory deficits in both feet. The patient’s lab data during the past year revealed a steady decline in CD4 counts from 361 cells/μL in August 2007 and variable peripheral eosinophilia. The patient’s lab results during her admission are shown in Staphylococcus, Streptococcus, Salmonella, Aspergillus, Nocardia, Rhodococcus, Listeria, Cryptococcus, Treponemapallidum, and cytomegalovirus (CMV).In developing countries, the differential diagnosis for a severely immunocompromised HIV+ patient with a solitary ring-enhancing brain lesion should include primary CNS lymphoma (PCNSL), TB, toxoplasmosis, neurocysticercosis, and much less commonly infections caused by 1 Single and multiple lesions are equally common in patients with PCNSL and because of the inadequacy of neuroradiological features to distinguish between TE and PCNSL, a high suspicion for the latter should be maintained throughout the follow-up of a presumed TE patient with a solitary CNS lesion – especially for lesions that are larger than 4 cm. The “eccentric target sign” in post-contrast T1-weighted magnetic resonance imaging (MRI) has been proposed as a highly characteristic feature found in 30% of biopsy proven cerebral toxoplasmosis cases.2 This imaging feature is more commonly found in cortical-based toxoplasmosis abscesses organizing around a cerebral sulcus with the eccentric core representing the inflamed cerebral vessels traversing that sulcus.3 Neurocysticercosis can produce a similar ring-enhancing lesion with an eccentric hyperdense nodule representing the scolex of the live tapeworm, but the parasite cysts in this stage of development can usually be differentiated from an abscess by the absence of diffusion restriction within the cyst in diffusion-weighted imaging and minimal parenchymal edema surrounding the lesion;4 in addition, Taenia solium infections are extremely rare in Iran.The typical magnetic resonance finding in toxoplasmic encephalitis (TE) is that of multiple ring-enhancing lesions with a predilection for basal ganglia; solitary lesions are reported in approximately 30% of cases.Pneumocystis jiroveci pneumonia and Mycobacterium avium complex prophylaxis. HIV infection by itself was identified as the probable cause of the patient’s eosinophilia.5Empiric treatment for TE as the most probable cause of the patient’s CNS lesion was began with pyrimethamine, sulfadiazine, and leucovorin. Highly active antiretroviral therapy for her HIV infection and isoniazid for her TB infection were also initiated as well as co-trimoxazole and azithromycin for Despite being rare, CMV infection can present as ring-enhancing lesions in severely immunocompro-mised HIV patients and because of the presence of mononeuropathy multiplex in this patient was considered a possibility. Since PCNSL, TB, and CMV could not be definitely ruled out, a follow-up MRI after 2 weeks of empirical therapy was planned. Our patient responded favorably to the initial empiric therapy thus consolidating a diagnosis of cerebral toxoplasmosis.The most probable diagnosis of cerebral lesion in HIV positive patients is cerebral toxoplasmosis and in resource poor settings for cerebral biopsy, the patients can be treated according to radiographic and laboratory findings.


Senescent cells secrete senescence-associated secretory phenotype (SASP) proteins to carry out several functions, such as sensitizing surrounding cells to senesce; immunomodulation; impairing or fostering cancer growth; and promoting tissue development. Identifying secreted factors that achieve such tasks is a challenging issue since the profile of secreted proteins depends on genotoxic stress and cell type. Currently, researchers are trying to identify common markers for SASP. The present investigation compared the secretome composition of five different senescent phenotypes in two different cell types: bone marrow and adipose mesenchymal stromal cells (MSC). We induced MSC senescence by oxidative stress, doxorubicin treatment, X-ray irradiation, and replicative exhaustion. We took advantage of LC-MS/MS proteome identification and subsequent gene ontology (GO) evaluation to perform an unbiased analysis (hypothesis free manner) of senescent secretomes. GO analysis allowed us to distribute SASP components into four classes: extracellular matrix/cytoskeleton/cell junctions; metabolic processes; ox-redox factors; and regulators of gene expression.We used Ingenuity Pathway Analysis (IPA) to determine common pathways among the different senescent phenotypes. This investigation, along with identification of eleven proteins that were exclusively expressed in all the analyzed senescent phenotypes, permitted the identification of three key signaling paths: MMP2 - TIMP2; IGFBP3 - PAI-1; and Peroxiredoxin 6 - ERP46 - PARK7 - Cathepsin D - Major vault protein. We suggest that these paths could be involved in the paracrine circuit that induces senescence in neighboring cells and may confer apoptosis resistance to senescent cells. Several genomic stressor events, such as telomere shortening, non-telomeric DNA damage, extreme mitogenic signals, and alteration of chromatin organization, may induce cellular senescence, which arrests cell division and induces loss of cell's functions. Senescence can counteract cancer growth since it blocks the proliferation of transformed cells, but it also contributes to organismal aging. Recently, evidence has emerged that senescence is involved in other biological processes such as tissue repair and development , 2.Senescent cells present several attributes including flattened and enlarged morphology; increase in β-galactosidase activity; telomere-dysfunction-induced foci; senescence-associated heterochromatin foci; DNA scars ; altered gene expression; and a senescence-associated secretory phenotype (SASP).Collectively, this SASP accomplishes several tasks. It can sensitize surrounding cells to senesce to avoid situations where normal neighboring cells, some of them with minimal DNA damage, fail to enter senescence. SASP can attract and activate immune system cells to dispose senescent cells. SASP also produces detrimental physiological consequences. It can undermine tissue and organ functionality and contribute to organismal aging. Moreover, tumor cells can misuse SASP for their own growth. Actually, the inflammatory cytokines, growth factors, and proteases present in SASP allow the modification of tissue micro-environment that support tumor growth by promoting angiogenesis, epithelial–mesenchymal transition, and cancer cell proliferation -6. All tIn the current research, we decided to study the SASP produced by bone marrow and adipose mesenchymal stromal cells (MSC), which contains a subpopulation of stem cells able to differentiate in mesodermal derivatives  and can also contribute to the homeostatic maintenance of several organs . FurtherMSCs accomplish their functions through the secretion of cytokines and growth factors, which exert paracrine and autocrine functions . SenesceOn these premises, the study of MSC SASP in different experimental conditions may illuminate the key functions exerted by senescent secretomes under different genotoxic stresses and the impairment of MSC autocrine and paracrine activity that may occur in senescence.Senescent phenotypes can be classified according to the stressor type into acute or chronic senescent cells.Exogenous stressors may trigger acute senescence to arrest the damaged cell growth or become part of physiological phenomena (embryonic development and tissue repair). Continuous proliferation is associated with the DNA replication process and consequent accumulation of genomic damage. This enduring stress induces senescence called chronic senescence or, alternatively, replicative senescence .We induced senescence on bone marrow and adipose mesenchymal stromal cells (BM MSC and A MSC), which share several biological features but also demonstrate peculiarities in their differentiation potential, transcriptome, proteome, and immuno-modulation function . In a prTo identify protein secretome composition, we conducted LC-MS/MS analyses on peptides from the tryptic digestion of secretome samples. Using high-resolution MS in a search of the Protein Metrics database, several hundred proteins were identified in all senescence conditions, ranging from 279 in BM MSC DOXO to 876 in A MSC DOXO, as shown in Figure High-throughput experiments, such as proteome analysis, enable the interrogation of thousands of data points simultaneously. These data have to be systematically organized in order to obtain biological information.Gene Ontology (GO) performs an enrichment analysis to evaluate the relative frequency of biological functions, named ontology terms, in the proteomic profile of interest. GO is based on a database of three non-overlapping controlled vocabularies that describe molecular functions, biological processes, and cellular components.We used PANTHER gene ontology enrichment analysis to match our experimental data onto reference ontology terms. This allowed the identification of ontology terms that occurred more frequently in our datasets when compared with a reference protein set. This experimental approach enabled the identification of protein sets of interest in a hypothesis free manner , 16.We focused our GO analysis looking for two ontological terms: biological functions and molecular processes. For each of the ten different experimental conditions, we identified dozens of ontologies, as shown in GO scrutiny is useful for an overall description of the key functionalities in the samples under analysis, but the enriched ontology terms, found in experimental datasets, cannot directly pinpoint the most important proteins in the analyzed proteomes.In order to reach a deeper insight on the most meaningful proteins in proteome samples, we used IPA to identify the set of interactions for each protein in our datasets and compared this with a reference interactome dataset . The keyFor every senescent phenotype, the proteins present in the secretome could be attributed to hundreds of canonical pathways, as shown in A closer look at the list of the proteins belonging to overlapping canonical pathways evidenced the presence of several structural proteins ; extracellular remodelers ; and proteins of misfolding and degradation signaling .We then focused on the identification of proteins exclusively expressed in all the SASPs and absent in the secretomes of healthy functional MSCs. We collected the conditional media from early passage BM MSC and MSC, then performed the LC-MS/MS analyses on peptides from the tryptic digestion of secretome samples. Using high-resolution MS in a search of the Protein Metrics database, we found 372 proteins in early passage BM MSC and 254 in A MSC, as shown in Currently, researchers are trying to identify the common features for SASPs. The present investigation compared the secretome composition of five different senescent phenotypes in two different cell types (BM MSC and A MSC). These cells were selected because their activities are accomplished by producing factors acting in both paracrine and autocrine signaling processes. Changes in the secretome profile as it passes from a healthy to a senescent state greatly affects the MSC physiology.Many researchers have focused their analysis of senescent secretomes on specific proteins, such as the released factors that may modify extracellular environment , proteins that promote inflammation , as well as those that affect the behavior of cancer cells (growth factors) . We tookOur results are in good agreement with the hypothesis that senescent program includes two distinct events: cell cycle arrest and geroconversion. Cells may arrest and preserve their ability to re-enter cell cycle o, alternatively, they may undergo geroconversion. This latter phenomenon leads to permanent cell cycle arrest, hypertrophy and hypersecretion (SASP) , 18.GO analysis allowed us to distribute SASP components across four classes: extracellular matrix/cytoskeleton/cell junctions; metabolic processes; ox-redox factors; and regulators of gene expression. These classes increase the number of SASP categories that have been identified so far  and demoWe used IPA analysis to identify common canonical pathways among the different senescent phenotypes. The list of the proteins belonging to overlapping canonical pathways demonstrated the presence of several structural proteins, extracellular remodelers, and proteins of misfolding and degradation signaling. The examination of these proteins, along with the 11 proteins that were exclusively expressed in all the analyzed senescent phenotypes, allowed us to hypothesize that these factors are part of some key circuits that mediate paracrine interactions between senescent cells and surrounding environment. As shown in Figure Matrix metalloproteinases (MMPs) play a key role in physiological and pathological remodeling of the ECM during embryogenesis, angiogenesis, wound healing, metastasis, and inflammatory diseases -24. The MMP2 is found in most tissues and cells, and besides its activity as an ECM degrading enzyme, it also acts on several nonmatrix substrates, such as stromal cell-derived factor 1, big endothelin-1, interleukin 1β precursor, and monocyte chemoattractant protein-3. This suggests that MMP2 plays a key role in promoting and auto-regulating the inflammation process -28. TIMPFactors regulating the IGF signaling pathways are expressed in senescent secretomes . Our preReactive Oxygen Species (ROS) play a central role as mediators of senescence regardless of the genotoxic stress that induces senescence. ROS concentration has to be strictly controlled to avoid a huge increase in oxidative phenomenatriggering apoptosis. It is well known that senescent cells are resistant to apoptosis, but there is no clear indication how this is accomplished . Our finCathepsin D is an acid protease active in intracellular protein breakdown. Several findings demonstrated that this protein participates in signaling that regulates cell death. Indeed, Byun and colleagues found that the Cathepsin D level may play part of the process governing cell decision about apoptosis and senescence following DNA damage stimulus . They foin vivo and in vitro. MVP upregulation facilitates apoptosis resistance of senescent cells [Major vault protein (MVP) is the main component of large ribonucleoparticles called vaults. The exact function of vaults and their components is still unknown. There are reports showing that the MVP gene is p53 responsive and the corresponding protein increases with age nt cells -43.We propose that PRDX6, ERP46, PARK7, Cathepsin D, and MVP could be part of the common circuit that allows senescent cell survival following stress. We also speculate that the presence of these proteins (singularly or in combination) may be part of the mechanism that regulates cell response to stress: either senescence or apoptosis.5 cells/cm2 in alpha-MEM containing 10% FBS and bFGF. After 72 hours, non-adherent cells were discarded and adherent cells were cultivated to confluency. Cells were then further propagated for the assays reported below.Bone marrow was obtained from three male healthy donors (range: 6-10 years old) who provided informed consent. We separated cells on a Ficoll density gradient , and the mononuclear cell fraction was collected and washed in PBS. We seeded 1-2.5 × 10Lipoaspirates were obtained from three male healthy donors (range 20 - 30 years old) undergoing plastic surgery after they provided their informed consent. The dispersion of adipose tissue was achieved via collagenase digestion, after which the lipid-filled adipocytes' ability to float caused them to separate from the stromal vascular fraction by way of centrifugation. Stromal pellets were washed with phosphate-buffered saline (PBS) and further purified on a density gradient . Mononuclear cells fractions were collected and cultivated in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. These cells were further amplified to conduct experiments.in vitro cultivation for 30 days (replicative senescence) as previously described [For the induction of acute senescence, we used three different stressors: irradiation, doxorubicin, and peroxide hydrogen treatments. Chronic senescent MSCs were obtained by extensive escribed .Exponentially growing cells (passage 3) were irradiated with 40 and 2000 mGy X-rays at room temperature. X-rays were administered via a Mevatron machine  operating at 6 MeV. Following irradiation, cells were cultivated for 48 hours before carrying out further experiments.Cells were incubated with 1 μM doxorubicin in complete culture medium for 24 hours, then the medium was discarded, and the cells were incubated for 24 hours in a fresh medium before further analysis.2O2 for 30 minutes in complete medium, then the medium was discarded, and the cells were incubated for 48 hours in a fresh medium before further analysis.Cells were incubated with 300 μM HThe percentage of senescent cells was calculated by the number of blue beta-galactosidase-positive cells out of at least 500 cells in different microscope fields, as previously reported .Following genotoxic stress, we induced as reported in previous paragraphs, cells were incubated in serum free media for 24 hours to obtain conditioned media (secretomes). We did not observe increase in apoptosis after incubation in serum free media in all the experimental conditions.Without disturbing the attached cells, 5 mL of MSC secretomes were collected from culture dishes and culture debris removed by centrifugation at 10,000 g. Supernatants were used for protein pooling with resin  using dried beads mixed with 1× Laemmli gel loading buffer and run on a gradient gel 4-15% SDS-PAGE . Following electrophoresis at 100 V, the gels were stained with Coomassie Brilliant Blue and gel lanes of interest were excised for in-gel digestion, as previously described .After digestion, peptides were eluted from the gel matrix by immersing the reaction tube in an ultrasonic bath for 5 min with a sequential elution of 0.4% formic acid in 3% ACN, 0.4% formic acid in 50% ACN, and 100% ACN. The supernatant containing the peptides was centrifuged, transferred to low binding tubes, and desalted using pipette tips . Following that, the extracted peptides were dried and stored at −80°C until LC-MS/MS analysis was performed. A more detailed protocol of CM preparation appears in ® TF v.1.6 (AB SCIEX).Tandem mass spectrometric analysis was carried out using AB SCIEX TripleTOF 5600+ instrument  coupled to an Eksigent expert nano-LC 400 system (AB SCIEX). MS and MS/MS data was acquired using AnalystMass spectrometry data was analyzed by using ProteinPilot 4.5 Beta (AB SCIEX) for the peptide identifications. The detailed protocol is described in http://www.pantherdb.org) and Ingenuity Pathway Analysis (IPA) (http://www.ingenuity.com/products/ipa).Proteins expressed in secretomes were analyzed with PANTHER  to compare classifications of multiple clusters of lists with a reference list to statistically identify the over- or under-representation of PANTHER ontologies. Significance was set to a p-value of .05.Differentially expressed proteins were imported into IPA to identify canonical pathways present in senescent secretomes. Fischer's exact test was used to calculate a p-value that would determine the probability that the association between genes in the dataset and canonical pathway could be explained by chance alone. Significance was set to a p-value of .05.Statistical significance was evaluated using ANOVA analysis followed by Student's t and Bonferroni's tests. We used mixed-model variance analysis for data with continuous outcomes. All data were analyzed with a GraphPad Prism version 5.01 statistical software package .We identified four classes of SASP components: extracellular matrix/cytoskeleton/cell junctions; metabolic processes; ox-redox factors; and regulators of gene expression. These classes are common among senescent phenotypes obtained with very different stressors, suggesting that they may represent a common feature of senescent secretome.In addition, the 11 proteins we identified exclusively in all the analyzed senescent phenotypes need further investigations, since the fact that they can be found in very different experimental conditions suggests that they may represent a common signature of senescence phenomenon. Further investigations are needed to evaluate the role of the pathways we identified with our study. This could contribute to understand how senescence may be triggered by paracrine signaling pathways. The importance of these further investigations resides also on the consideration that senescence may affect the therapeutic performance of MSCs.


The use of non-caloric sweeteners can interfere in the regulation of appetite, promoting greater food intake and weight gain. In previous data, our results showed that animals who consumed yogurt with saccharin and aspartame had a increase in weight compared to the group using sucrose. However, as the total calorie intake was similar between the groups, we speculated that weight gain might be associated with decreased energy expenditure induced by artificial sweetener.Determine the caloric expenditure at rest in rats receiving saccharin or sucrose for 12 weeks.We conducted a controlled experiment with adult male Wistar rats randomly divided into 3 groups: non-caloric sweetener (saccharin-SAC); calorie sweetener (sucrose-SUC) or control ( non sweetened yogurt -CON) given daily over a period of 12 weeks with free chow and water. Weight gain, food intake and water control were determined weekly, basal oxygen consumption was measured at 0, 5 and 12 weeks. We used one-way ANOVA with Dunnett's test and ANOVA by repeated measures and mixed model assessment.The SAC group promoted greater weight gain than control (p=0.031). All groups had similar total caloric intake. The maximal oxygen consumption was not diferent between groups during the whole experiment, respectivelly: SAC , SUC  and CONT .The cumulative weight gain in the animals fed with saccharin can not be attributed to a reduction in energy expenditure. Further studies are necessary to determine metabolic causes for weight gain induced by saccharin in rats.