This is the first pilot study to demonstrate the temporal and spatial separation of a pain-induced blood flow and an itch-induced blood flow in human cortex during information processing.Pain and itch are closely related sensations, yet qualitatively quite distinct. Despite recent advances in brain imaging techniques, identifying the differences between pain and itch signals in the brain cortex is difficult due to continuous temporal and spatial changes in the signals. The high spatial resolution of positron emission tomography (PET) and functional magnetic resonance imaging (fMRI) has substantially advanced research of pain and itch, but these are uncomfortable because of expensiveness, importability and the limited operation in the shielded room. Here, we used near infrared spectroscopy (NIRS), which has more conventional usability. NIRS can be used to visualize dynamic changes in oxygenated hemoglobin and deoxyhemoglobin concentrations in the capillary networks near activated neural circuits in real-time as well as fMRI. We observed distinct activation patterns in the frontal cortex for acute pain and histamine-induced itch. The prefrontal cortex exhibited a pain-related and itch-related activation pattern of blood flow in each subject. Although it looked as though that activation pattern for pain and itching was different in each subject, further cross correlation analysis of NIRS signals between each channels showed an overall agreement with regard to prefrontal area involvement. As a result, pain-related and itch-related blood flow responses  were found to be clearly different between pain ( Despite great strides in understanding the neuronal processing of pain over the last several years, the underlying mechanism of dynamic processing in the brain cortex remains a fundamental question Distinct from pain, itch is an unpleasant perception that evokes the desire to scratch. Although there are dissimilarities between their processing, pain and itch are thought to be closely related in that weak activation of nociceptors mediates itch, while strong activation of the same receptors results in weak pain In the 1990’s, functional human brain imaging utilizing positron emission tomography (PET) and functional magnetic resonance imaging (fMRI) was developed based on measurements of the dynamic changes in cerebral blood flow. These studies demonstrated that neural processing of nociception and pain signals occur in several brain regions operating in parallel Near infrared spectroscopic (NIRS) topographic imaging, based on the measurement of oxygenated hemoglobin (HbO2) and deoxygenated hemoglobin (HbR) concentrations, was recently developed to obtain dynamic brain images that overcome the limited operation in the shielded room of PET and fMRI In the present study, we applied NIRS imaging to compare the dynamic activation pattern of cortical areas between pain- and itchy-related information processing. For pain stimulation, NIRS images suggested on first appraisal that the activated area was changing rapidly in the frontal cortex and the activation pattern was largely different among each subjects. However, on closer inspection, there was a statistical difference found in time-dependent cross correlation of NIRS signals between the central region and peripheral region of the prefrontal cortex (p<0.05 by cross correlation method). Although itch-activated NIRS signals were well correlated between those regions, there was no or a miniscule time-lag between them. This suggested differences in how the prefrontal cortex processed information from pain and itch signals.Seven healthy human subjects  were enrolled in the study upon providing written consent. Subjects with past history of atopic diathesis, including allergic rhinitis, asthma, atopic dermatitis, and allergic conjunctivitis were excluded. In addition, subjects with past history of contact hypersensitivity and urticaria were both excluded. All subjects were right-handed. Subjects with excessive Holmes and Rahe Stress Scale (>100) were excluded Brain imaging was performed using an optical topography system ETG-4000  with 22 channels for the frontal area and 24 channels for the parietal area. The operator checked if the NIR sensors were closely applied to the scalp of the subjects using an established standardized process according to the manufacturer’s instructions. The NIRS system generates NIR at 780 and 830 nm, which is transmitted via optical fiber bundles to the brain through the cranium via an emitter  and Y(t). The cross-correlation function between the trains X(t) and Y(t) is calculated as follows:[HbO2] NIRS trains from two channels are represented as the time series T is a number of measure points in the time series and (XY)τ is a time-lag between X(t) and Y(t). The value of XYC((XY)τ) represents the variation of the [HbO2] NIRS signal from channel Y, conditioned on the fact that the [HbO2] NIRS signal from channel X changes (XY)τ second earlier. In the present study, to calculate the cross-correlation function of channel Y(ChY) against channel X (ChX), waveform of ChY was slid against ChX from −60 sec to +60 sec at every 100 msec and the correlation coefficient was calculated for each sliding time. A delay time that showed a maximum correlation coefficient was defined as a time lag (tau) for ChY against ChX. To remove physiological fluctuation, the raw data of [HbO2], [HbR], and [HbT] from individual channels were smoothed by moving average (average from −0.2 sec to +0.2 sec).Where p<0.05.Significance was assessed using the Kruskal-Wallis test followed by the Mann-Whitney’s U test (α = 0.05). The significance level was set at In a typical response to pain, the [HbO2] exhibited a transient increase immediately after stimulation, a gradual increase to a peak at approximately 20 seconds after pain stimulation, and then an exponential reduction to baseline  of stimulation resulted in gradually decaying reported perceived pain and NIRS response in channel 10 in the frontal area, which may be due to adaptation or habituation . StimulaWe examined the correlation of NIRS signals between frontal and parietal areas after 7 pain stimulations of different intensities. The pain-stimulation-dependent change in [HbO2] was obtained from three subjects on the same day, each given a different random order of pain stimulation intensities. Each subject showed a different response pattern in the frontal area to the set of stimulations , rangingτ = +19 sec) and that of Ch2 and Ch6 were middle . No delay was observed at both Ch3 and Ch8. Averages of delay time for each channel from the four subjects are summarized in τ = +19.2 sec). These results suggest a direction of pain signal information flowing from center of prefrontal area (Ch7) to peripheral area (Ch1). Therefore, by correlating responses from channels in prefrontal cortex, we could identify possible time-dependent connections between regions.In order to decide the most effective method analyzing moving and changing pattern with time, we observed time-dependent NIRS pattern at prefrontal cortex in pain stimulation. As shown in After determining the pain signal processing pattern in the frontal area, we next examined whether distinctive signals between itch and pain stimulation could be detected using NIRS topography . BecauseAs described in the pain response analysis section, cross correlation analysis was applied to [HbO2] NIRS signals for itch stimulation. The correlation between Ch7 and channels 1, 2, 3, 4, 5, 6 and 10 were significant  law By smoothing the traces using the moving average, the present study found the oscillation frequency of both [HbO2] and [HbT] to be approximately 0.08 Hz. These low frequency oscillations were apparent in the frontal and parietal areas. Changes in the signals detected by NIRS topography fluctuate due to breathing (∼0.16 Hz), heart beat (∼1 Hz), and vasomotor responses (∼0.1 Hz) According to the lateral views of the cortical maps proposed by Campbell The neuro-imaging work during the last three decades have shown the SII and insular areas to be key pain integration areas The patterns of activation following pain and itchy stimulation are similar, but distinct and quite different delay time in information processing in prefrontal cortex. It is plausible that pain and itchy are different sensation systems processed by distinct sets of neurons NIRS can simultaneously measure dynamic changes of [HbO2] and [HbR] in the cortex with a temporal resolution of 1 second and spatial resolution of 3 cm. The change in [HbO2] represents neural activity in the human brain. In the present study, we proposed that analyzing methods for NIRS signal could be used to determine a pain-related activation pattern in the prefrontal cortex. This was found feasible as evidenced by a highly reproducible characteristic NIRS pattern for pain and itchy stimulation among each subject. Agreement was excellent between subjective human perception and objective [HbO2] levels measured using NIRS. Non-invasive NIRS can potentially be used measure pain and itchy signal processing in awake, responsive human subjects, though large scale experiments are needed to further confirm its reliability.Figure S1Dynamic frontal cortex responses and imaging after pain stimulation. Twenty sequential NIRS images were displayed every 10 seconds. Red color indicates the region that [HbO2] NIRS signal was above baseline (increase) and blue color express the region that [HbO2] NIRS signal was under baseline (decrease).(TIF)Click here for additional data file.Figure S2Direct comparison of NIRS brain images of pain and itch stimulation. NIRS images from subjects I, II, and III when the subjects felt the most pain (A) and the most itch (B). I, II, and III corresponds to subjects F-2, F-3, and F-1, respectively.(TIF)Click here for additional data file.Figure S3The distribution of VAS scores.(TIF)Click here for additional data file.Text S1NIRS responses in frontal area after pain stimulation.(DOC)Click here for additional data file.Text S2Distinctive topographic imaging of pain compared with that of itch.(DOC)Click here for additional data file.


Recurrent myelitis is one of the predominant characteristics in patients with neuromyelitis optica (NMO). While paresis, visual loss, sensory deficits, and bladder dysfunction are well known symptoms in NMO patients, pain has been recognized only recently as another key symptom of the disease. Although spinal cord inflammation is a defining aspect of neuromyelitis, there is an almost complete lack of data on altered somatosensory function, including pain. Therefore, eleven consecutive patients with NMO were investigated regarding the presence and clinical characteristics of pain. All patients were examined clinically as well as by Quantitative Sensory Testing (QST) following the protocol of the German Research Network on Neuropathic Pain (DFNS). Additionally, plasma endocannabinoid levels and signs of chronic stress and depression were determined. Almost all patients (10/11) suffered from NMO-associated neuropathic pain for the last three months, and 8 out of 11 patients indicated relevant pain at the time of examination. Symptoms of neuropathic pain were reported in the vast majority of patients with NMO. Psychological testing revealed signs of marked depression. Compared to age and gender-matched healthy controls, QST revealed pronounced mechanical and thermal sensory loss, strongly correlated to ongoing pain suggesting the presence of deafferentation-induced neuropathic pain. Thermal hyperalgesia correlated to MRI-verified signs of spinal cord lesion. Heat hyperalgesia was highly correlated to the time since last relapse of NMO. Patients with NMO exhibited significant mechanical and thermal dysesthesia, namely dynamic mechanical allodynia and paradoxical heat sensation. Moreover, they presented frequently with either abnormal mechanical hypoalgesia or hyperalgesia, which depended significantly on plasma levels of the endogenous cannabinoid 2-arachidonoylglycerole (2-AG). These data emphasize the high prevalence of neuropathic pain and hyperalgesia in patients with NMO. The degree of mechanical hyperalgesia reflecting central sensitization of nociceptive pathways seems to be controlled by the major brain endocannabinoid 2-AG. Neuromyelitis optica  is a demyelinating inflammatory autoimmune disease of the central nervous system leading to recurrent optic neuritis (ON) and episodic myelitis Apart from the deterioration of visual and motor function, the presence of severe pain in a substantial proportion of NMO patients with a high impact on their health-related quality of life was only recently reported Although spinal cord inflammation is a defining aspect of NMO, there is still no knowledge of the pattern of somatosensory changes accompanying the disease, nor the degree of on going stress and depression frequently observed in NMO patients suffering from chronic pain. Thus, it is essential to characterise the complete somatosensory phenotype of patients with NMO, since this may contribute to a better understanding of the underlying pathophysiological mechanisms of pain generation in NMO, which may enable potential treatment options Eleven consecutive Caucasian patients with NMO seen at the Institute for Clinical Neuroimmunology were included into the study. All of them fulfilled diagnostic criteria of definite AQP4-antibody positive NMO After given written informed consent nine female and two male patients with a median age of 51 years ranging from 24 to 69 were included . Median time since onset of disease was 8 years ranging from 3 months to 24 years . MedicalThe patients were interviewed for the presence, intensity, location, and characteristics of pain. Neuropathic pain was considered to be present if pain was (a) located in the area corresponding to CNS lesion, (b) could not be attributed to any other condition and (c) had specific characteristics of neuropathic pain according to the DN4 neuropathic pain scale Venous blood samples for endocannabinoid measurements were taken peripherally and drawn into Lithium-Heparin containing tubes , immediately centrifuged and then stored at –80°C to minimize ex-vivo synthesis of endocannabinoids from nucleated blood cells as has been described earlier QST was conducted under minimal distraction in a silent, air-conditioned room, with an ambient temperature of 24–25°Celsius. QST followed the standardised protocol as described by the German Network on Neuropathic Pain (DFNS) Thermal testing included Warm Detection Threshold (WDT), Cold Detection Threshold (CDT), Thermal Sensory Limen (TSL), Cold Pain Threshold (CPT), Thermal Sensory Limen (TSL), Cold Pain Threshold (CPT) and Heat Pain Threshold (HPT). Furthermore, Paradoxical Heat Sensations (PHS), that means identification of a cold stimulus as either hot or burning pain, were recorded. For mechanical testing we determined Mechanical Detection Threshold (MDT), Vibration Detection Threshold (VDT), Mechanical Pain Threshold (MPT), Mechanical Pain Sensitivity for pinprick stimuli (MPS), Wind-up Ratio (WUR), Pressure Pain Threshold (PPT), and Dynamic Mechanical Allodynia (DMA). For an elaborate discussion see The presence and severity of depression was assessed by the German version of the Center for Epidemiological Studies Depression Test (CES-D). Presence of stress symptoms such as sleep disturbances, nightmares and generalized irritability were measured using the German Version of the Post-Traumatic Stress Symptom 10-Questionnaire (PTSS-10) Overall health-related quality of life (HRQL) was assessed by the German version of the Short Form 36 (SF-36). reference data base)/SD reference data base). The sign of the respective z-score was adjusted in a way that a gain of function was indicated by a positive sign, whereas a negative sign indicated a loss of function http://home.clara.net/sisa/binomial.htm) All QST data  were transformed into decadic logarithms to achieve secondary normal distributions Data of SF36 were normalized as described above to a representative sample of the US-General population (n = 2393). In addition, correlations between QST results, endocannabinoid levels and the time since last relapse of NMO were calculated.Clinical and MRI characteristics of NMO patients are shown in Eight out of the eleven consecutive NMO patients (72%) reported on going pain , in the arms or shoulder (n = 5), in thoracic or abdominal dermatomes (n = 8) or combined (n = 7). Five patients had spontaneous on going pain as well as painful paroxysms; two patients complained of either solely attacks or constant pain, respectively .Pain was treated continuously with analgesic or co-analgesic drugs in 7/11 patients (63%), all of whom needed more than one analgesic substance. One patient only took medication on demand during pain attacks and two more patients received antiepileptic drugs during previous relapses that where associated with pain but had recovered completely. Three patients administered a combination of sodium channel blocker to treat electric shooting pain attacks and a calcium channel blocker to reduce constant burning pain .Mean number of traumatic memories was 0.91; PTSS-10 score was 26.1±7.9. Three patients indicated no traumatic memories (PTSS-10∶18.3±4.2), 6 patients had one traumatic memory (27.8±7.9), while 2 patients displayed two traumatic memories (32.5±0.7). However, no patient displayed a PTSSD score>35, which would be firmly indicating comorbid posttraumatic stress disorder. Mean CES-D score was 34±8 indicating the presence of clinical relevant depression. Patients with NMO showed a significant decrease in three of the eight dimensions of SF 36, namely physical functioning, general health and noteworthy bodily pain .10mean ± SEM: −0.017±0.032) and −1.79°C in the feet (log10mean ± SEM: 0.253±0.065), NMO patients detected cooling not until at a much more larger temperature change of −3.04°C (log10mean ± SEM: 0.483±0.107) and −7.85°C (log10mean ± SEM: 0.895±0.106), respectively (both p<0.001).Thermal detection  was significantly impaired in NMO patients in upper and lower extremities as compared to healthy controls (10mean ± SEM: −0.231±0.036) in the hands and +4.31°C (log10mean ± SEM: 0.635±0.050) in the feet, while NMO patients detected warming only at a temperature change of +4.16°C  and +7.51°C , respectively. Likewise, combined alternating warm and cold stimuli (TSL) was significantly increased in the hands and feet of patients with NMO . Warming was detected at a stimulus temperature difference of +1.70°C (logDetection of vibration (VDT) assessed by an 8/8 calibrated Rydel-Seiffer tuning fork in NMO patients was strongly reduced in the hands , where only one NMO patients exhibited vibration sensitivity in the normal range of bedside testing (>6/8) and none compared to a reference data base Cold pain thresholds (CPT) did not differ significantly between NMO patients and healthy controls in hands and feet (both p>0.30). In contrast, heat pain threshold (HPT) was significantly decreased in the hand , indicating significant heat hyperalgesia. Heat pain thresholds in the feet, however, were unaltered  excluding those without cervical lesions . While patients without cervical spinal lesion did not differ from healthy controls at all regarding their heat pain threshold , patients with structural cervical lesions displayed a highly significantly reduction of heat pain threshold , as compared to control group at the upper extremities . Consequently, there was a highly significant correlation of heat pain thresholds with time since last relapse of NMO for both hand and feet , which reached normal thresholds not until one year after the last NMO relapse .PHS was a very frequent finding in TSL trials with NMO patients at either the examined hands or feet. Overall, PHS was detected in 8/11 patients with NMO, and 33 of 66 trials (50%) at the feet, but only in 8 of 66 trials at the feet of healthy controls . Moreover, whereas none of the healthy controls showed PHS in any trial at the dorsum of their hand, PHS regularly occurred in 8/9 patients with NMO at the hands, and was experienced in 26 of 66 trials, .Concerning the mechanical pain thresholds, QST testing revealed no signs of mechanical hyperalgesia. Neither mechanical pain thresholds (MPT), pressure pain threshold (PPT) nor wind-up ratio (WUR) following pin-prick stimulation differed significantly in any extremity of NMO patients compared to healthy controls . In addition, pain sensitivity for pinprick stimuli (MPS) was significantly reduced in the hand of NMO patients (p<0.05), accompanied by a strong trend towards MPS reduction in the feet . Mechanical detection thresholds were abnormal in 50% of patient test sites, but remained relatively unchanged overall, since abnormal sensitivity encompassed a similar proportion of abnormal gains or losses . AdditioSecondly, patients with NMO exhibited thermal hyperalgesia (to painful heat and cold stimuli), when they exhibited signs of cervical spinal lesion as a marker of inflammatory spinal cord damage. The amount of hyperalgesia was strongly correlated to the time since the last relapse of NMO. In contrast, mechanical pain parameters did not reveal clear signs of hyperalgesia.Thirdly, NMO patients displayed distinct signs of dysesthesia in both hands and feet, namely the frequent occurrence of paradoxical heat sensations following alternating warm/cold stimulation, and significant pain to light touch.Finally, the magnitude of on going pain at the time of QST assessment was strongly negatively correlated to any of the QST pain parameters with no difference between thermal and mechanical pain modalities  suggesting a deafferentation-type of central neuropathic pain.The two major chemically related cannabinoid lipids anandamide and 2-arachidonoylglycerol (2-AG) were elevated in patients’ plasma compared to age and gender-matched healthy controls. 2-AG plasma levels were doubled , while anandamide exhibited only a trend  . While pBoth, 2-AG and anandamide were not significantly related to the time since last relapse . In contrast, there were significant relationships of both lipid signalling molecules to the patients’ somatosensory status. Anandamide levels were positively, but weakly correlated to pain sensitivity as calculated by normalized pain thresholds  is a frequent, but often neglected phenomenon in various forms of CNS diseases such as NMO. Notwithstanding, mechanisms underlying this type of pain are poorly understood. Accordingly, pain has recently been described as another key symptom of NMO, but the predominant quality of pain has not yet been characterized As almost all patients reported pain at the time or in the three months preceding the examination, our data emphasize the high incidence of pain also in Caucasian NMO patients compared to patients with multiple sclerosis (28%−47%) or spinal cord injury (30−40%)The pain characteristics in NMO patients were burning or tingling, sensory descriptions discriminating neuropathic from non-neuropathic pain states Similar to previous findings, we consistently found significantly reduced scores in three of the eight dimensions of the SF 36 reflecting impaired Health Related Quality of Life, Consequently, we strongly recommend the routine use of supplementary questionnaires amending the EDSS, in order to achieve timely recognition and treatment of pain as well as depression in NMO.As the pattern of pain suggested a neuropathic origin of the ongoing pain in patients with NMO, in the next step we used QST in order to delineate the accompanying somatosensory changes and to get insights into the pathophysiological background of neuropathic pain in NMO as recently published for a n>1200 cohort of neuropathic pain patients In NMO patients, the ability to detect either warm or cold was considerably impaired, indicated by the threefold increase in CDT, WDT and TSL. The occurrence of negative thermal sensory signs is characteristic for patients with all kinds of neuropathic pain disorders, regardless of a peripheral or central site of lesion Similarly, a pronounced impairment of vibration detection was observed, which is a characteristic of somatosensory changes in patients with neuropathic pain as well There was apparently no overall mechanical hyperalgesia to pinprick or blunt pressure. However, approximately 20% of the patients nevertheless displayed abnormal mechanical hyperalgesia, yet balanced by patients with mechanical hypoalgesia . ImportaThe most conspicuous positive mechanical sign was dynamic mechanical allodynia (DMA), i.e. pain to stroking light touch stimuli, which marks a crosstalk of tactile inputs into pain pathways, and is considered as a hallmark sign of central sensitization However, the low incidence of abnormal pain sensation (wind up pain sensation), despite clear signs of central sensitization is not contradicting the finding of central nociceptive sensitization, as central sensitization and wind-up represent independent phenomena Hyperalgesia to noxious heat or cold is usually attributed to peripheral sensitization of primary afferent C fibers, a hallmark sign of inflammatory processes In animal models of neuropathic pain, gliopathy, involving microglia in the early stages and astrocytes in the later stages, parallels persistent pain in spinal cord injury Currently, there is no comprehensive characterization of the interaction between analgesic medication and somatosensory testing and we cannot rule out interference with analgesic treatment. However, no influence of antidepressant medication on thermal thresholds could be found in patients suffering from major depression , for which we propose a double inhibitory role on both mechanical hyperalgesia and potentially also on neuroinflammation.In aggregate, patients with NMO are displaying both the clinical as well as the typical somatosensory signs of neuropathic pain. The “unhappy” trias of central lesions, accompanied by central sensitization of pain pathways and contemporaneous CNS inflammation in NMO accounts for the high incidence of neuropathic pain. Additionally, the pain phenotype appears to be modulated by 2-AG, the major endogenous cannabinoid of the brain


The drug development process is widely hampered by the lack of human models that recapitulate liver functionality and efficiently predict toxicity of new chemical compounds. Moreover, liver failure is a global medical problem, with transplantation being the only effective treatment currently available. The bipotent liver progenitor cell line HepaRG can be differentiated into cholangiocyte and hepatocyte-like cells that express major functions of mature hepatocytes, representing a valuable tool to model hepatic function . Current2. Spinner vessels with ball impeller (Wheaton) were inoculated with inoculums ranging from 5 to 8 × 105 cell/mL and an agitation ranging from 35 to 45 rpm to attain the desired aggregation conditions. Aggregate size was determined by measuring Ferret's diameter using the Image J software (NIH). After 3 days of aggregation, spheroids were encapsulated in 1.1% and 2% (w/v) of Ultra Pure MVG alginate  in NaCl 0.9% (w/v) solution. Encapsulation was performed in an electrostatically driven microencapsulation unit VarV1 (Nisco) and cultures were maintained for 14 days in stirred culture conditions. Viability was determined by the double stain viability test - alginate beads were collected from stirred cultures, incubated with fluorescein diacetate (10 μg/mL) and TO-PRO3 ® (1 μM) and observed on a fluorescence microscope (Leica DMI6000) - and by the Trypan blue exclusion method - alginate beads were dissociated with a solution of Sodium citrate 50 mM, Sodium chloride 104 mM and spheroids were dissociated by incubation with Trypsin 0.05%-EDTA (Gibco) and counted trypan blue exclusion dye. For characterization of the cultures, encapsulated spheroids were fixed as previously described [HepaRG cells were routinely propagated in static conditions as previously described . BrieflyIn 2D cultures, HepaRG cells proliferate until confluence is reached and the cell-cell interactions established associated with the spatial constriction are postulated to trigger the differentiation program and maintain the differentiated state ,4. MoreoIn both culture conditions, the viability was maintained above 85%, showing that the alginate concentration does not affect diffusion of nutrients or oxygen to supply effectively the cell spheroids Figure . MoreoveThe structural organization of the cell spheroids in both stiffness environments was characterized by the arrangement of actin filaments, which is associated to the tight junctions in highly polarized epithelial cells. As shown in Figure In the current work, the encapsulation of liver spheroids with different stiffness conditions was evaluated as a strategy to culture HepaRG cells. It was observed that the encapsulation with different alginate concentrations is compatible with maintenance of highly viable cultures of liver spheroids, with growth arrest and cell polarization promoted by spatial constriction and the enhanced cell-cell interactions in 3D.


Enterobacteriaceae represents a major clinical and public health challenge, especially concerning those organisms that produce Klebsiella pneumoniae carbapenemase (KPC)–type b-lactamase.Â In this study, we examined the molecular epidemiology of KPC-producing Klebsiella pneumoniae strains, isolated during sporadic or epidemic cases in different hospitals of North-Western Tuscany (NWT).The emergence and spread of carbapenem-resistant Klebsiella pneumoniae, occurred in the UniversityÂ Hospital of Pisa and in other hospitals from the surrounding area; during this period we isolated 57 Klebsiella pneumoniae strains in our hospital and 23 from other health-care facilities; all strains, isolated mostly from different patients hospitalized over protracted periods of time, showed reduced susceptibility to carbapenems and were assayed for KPC detection.Starting from April 2010, an outbreak of clinical infection caused by multiresistant 60 out of 61 tested strains produced KPC type 2; molecular typing results indicated the spread of a prevalent clone during the outbreak in Pisa. The PFGE method allowed to identify 7 variants clonally related (pt A1-A7), sharing the same MLST sequence type (ST 258).Molecular characterization confirmed the higher discriminatory power of PFGE compared to MLST and it may be useful in local and short-time outbreaks; however MLST, providing unequivocal and comparable data, remains the gold standard for molecular typing.None declared.


Bric-a-brac,Tramtrack, Broad) domain proteins such as Fruitless are known to play key roles in the neural differentiation underlying such responses. We previously identified a gene, which we have termed jim lovell (lov), encoding a BTB protein with a role in gravity responses. To understand more fully the behavioral roles of this gene we have investigated its function through several approaches. Transcript and protein expression patterns have been examined and behavioral phenotypes of new lov mutations have been characterized. Lov is a nuclear protein, suggesting a role as a transcriptional regulator, as for other BTB proteins. In late embryogenesis, Lov is expressed in many CNS and PNS neurons. An examination of the PNS expression indicates that lov functions in the late specification of several classes of sensory neurons. In particular, only two of the five abdominal lateral chordotonal neurons express Lov, predicting functional variation within this highly similar group. Surprisingly, Lov is also expressed very early in embryogenesis in ways that suggests roles in morphogenetic movements, amnioserosa function and head neurogenesis. The phenotypes of two new lov mutations that delete adjacent non-coding DNA regions are strikingly different suggesting removal of different regulatory elements. In 47lov, Lov expression is lost in many embryonic neurons including the two lateral chordotonal neurons. 47lov mutant larvae show feeding and locomotor defects including spontaneous backward movement. Adult 47lov males perform aberrant courtship behavior distinguished by courtship displays that are not directed at the female. 47lov adults also show more defective negative gravitaxis than the previously isolated 91Ylov mutant. In contrast, 66lov produces largely normal behavior but severe female sterility associated with ectopic lov expression in the ovary. We propose a negative regulatory role for the DNA deleted in 66lov.Innate behaviors have their origins in the specification of neural fates during development. Within Drosophila, BTB ( Understanding the molecular origins of behavioral responses is a central goal of neuroscience. Innate behaviors are a particular focus since these reflexive activities originate in neural circuitry “hard wired” into the organism during development to give robust, invariant responses. As the most intensely studied complex multi-cellular organism, Drosophila offers the best possibilities for understanding the processes by which neural circuitry is laid down in development.Within Drosophila, the relatively simple larval nervous system, which is formed during embryogenesis, is providing many insights into neurogenesis and neural differentiation. The critical role of transcription factor cascades has emerged from studies of the peripheral nervous system (PNS), where major components of the transcriptional hierarchies that give rise to various classes of sensory neurons have been identified (reviewed in jim lovell (lov) in honor of this astronaut’s pioneering work in microgravity. lov encodes a putative transcription factor of the BTB/POZ domain family. These proteins contain a ∼120 amino acid domain, initially identified in the Drosophila proteins Bric-a-brac, Tramtrack and Broad Poxvirus zinc finger proteins lov in transcription regulation mediated via protein-protein interactions. Based on a partial cDNA sequence for CG16778, the gene was initially proposed to belong to the tyrosine kinase family and was named Tyrosine-kinase related (Tkr) Through a screen for defective responses to gravity, we previously identified CG16778 as a neurally expressed gene that influences behavioral responses lov function based on analysis of its expression and on study of new lov mutants derived from our initial gravitactic mutant, 91Ylov. These studies show that lov is widely expressed in the embryonic nervous system and indicate a role in the late development of some sensory neurons, potentially producing distinctions in the properties of closely related neurons within a given class. We also demonstrate that lov is essential for proper execution of several innate behaviors - ranging from coordinated locomotor responses in larvae to negative gravitactic climbing and male courtship behavior in adults. We further provide evidence that lov has non-neural roles in early embryonic pattern formation and in the testis.We present here an investigation of 38lov, 47lov and 66lov were generated by mobilizing the + P{GawBw} 91Ylov−w viable mutations. Each deletion was initially identified by genomic PCR and the precise endpoints for each deletion were determined by sequencing appropriate PCR fragments.Deletion mutations SB1 Kr10), which removes the entire lov locus 38lov, 47lov, 66lov, 91Ylov and deficiency SB1 were all isogenized by six rounds of outcrossing to a +w; Sco/CyO stock. SB1 and 66lov, which cannot be maintained as homozygous stocks, are carried over the CyO chromosome from this +; Sco/CyO stockw. Stock 5702  from BDSC was used to generate 47/CyO-GFPlov and 66lov/CyO-GFP lines for larval and embryo analysis, respectively. For ectopic expression of lov, the following GAL4 drivers were obtained from BDSC: MVD1(GAL4::VP16-nos.UTR) CG6325 (stock 4937), P{GawB}c204 (stock 3751), P{GawB}c355 (stock 3750) and P{GawB}c532 (stock 30841).Deficiency lov transcripts, the primer 5′ TCAACTTCTGGTGGGCGTCCTTTA 3′, which is the reverse complement of residues 41–64 of the first common exon of the four lov transcripts, was paired with primers specific for the immediate upstream 5′UTR exon of each transcript (see below). PCR (30 cycles) at appropriate annealing temperatures was then used to generate fragments specific for each lov mRNA. Primers for transcripts from the ubiquitously expressed Actin gene at 57B were used to standardize output for analysis. Fragments were detected after agarose gel electrophoresis and ethidium bromide staining. Primer sequences were as follows.RNA was prepared from various tissues or life cycle stages using Trizol lysis as described in ATCCGAGTGTCATCTTCAACGCGA 3′.Transcript A - 5′ ACATACGCTCATTCGTTACCCGCT 3′.Transcript B - 5′ TGTCTTGAACAGAACTATATTGTG 3′.Transcript C - 5′ GTTTCCAAAGAAGCAATCAAACGGC 3′.Transcript D - 5′ TTCCAAGCCGTACACACCGTAACT 3′.Actin 57B forward –5′ TCATCACCGACGTACGAGTCCTTCT 3′.Actin 57B reverse –5′ lov coding sequence with no detectable similarity to any other protein sequence in the Drosophila genome was chosen for antibody generation. This region lies within the first protein coding exon , 948 residues downstream of the ATG start site and 270 residues downstream of the BTB domain (CTGGAAGTTCTGTTCCAGGGGCCCCTGGGATCC 3′ and 5′ GAATTCCCGGGTC GACTCGAGCGGCCGCAT 3′) that add an upstream BamH I site and downstream EcoR I site were used to generate the required PCR fragment from lov cDNA clone GH08221 (Berkeley Drosophila Genome Project) for CG16778. After initial cloning into pCR4-TOPO (Invitrogen), the coding region was transferred to expression vector pGEX-6P-1 as a BamH I/EcoR I fragment, to generate a GST-lov fused coding sequence. After induction, fusion protein was collected on a glutathione column. The Lov region was released by cleavage with PreScission Protease (GE Life Sciences) and used to raise antibodies in a rabbit and a guinea pig . The recombinant Lov protein fragment cross-linked to NHS-activated Sepharose resin (GE Life Sciences) was used for affinity purification.A 218 amino acid region of the B domain . Primerslov protein coding sequence in cDNA clone GH08221 was amplified using primers (5′ GTTGAATTCCTGGATACGAGAATTGAAGCACGC 3′ and 5′ GTTAGATCTGTTTCAATCATGCCCGGTC 3′) that add an upstream EcoR I site and a downstream Xho I site. The resulting PCR fragment was cloned into these two sites in the pUAST vector lov on the second chromosome was used here.The entire 66lov/66lov embryos, a 66lov/CyO-GFP stock was used. Embryos were co-stained with anti-GFP peptide antibody  and anti-Lov antibody to identify 66lov homozygous embryos.Standard procedures were used to collect embryos and then to dechorionate and fix them in 3% paraformaldehyde and to remove their vitelline membranes. Embryos were stored at −20°C in methanol prior to staining. After rehydration in phosphate buffered saline (PBS), embryos were blocked with 5% goat serum in PBS and then stained with primary antibodies. Antibodies used were as follows: guinea pig anti-Lov 1∶500, rabbit anti-Lov 1∶200, mouse 22C10  1∶200. For single labeling, biotin-labeled secondary antibodies  were used, followed by streptavidin-horse radish peroxidase (Pierce) and detection via metal-enhanced 3,3′-Diaminobenzidine (DAB) treatment (Pierce). For double labeling with anti-Lov and 22C10, alkaline phosphatase (AP) labeled anti-mouse secondary antibodies  were also applied and detected with an X-phosphate nitroblue tetrazolium detection kit (Vector labs). For examination of Males and virgin females 3–6 days of age were set up in egg lay dishes over small Petri dishes containing grape juice agar smeared with yeast paste. 12–15 male-female pairs were used per egg lay dish. After discarding the initial plate, 100 eggs were collected from each of three successive overnight collections. The eggs were arrayed in a 10×10 grid on a new plate and after 36 hours, the number of unhatched eggs was counted. For each male/female combination tested, at least two separate sets of mating pairs were set up to give at least six estimates of egg hatch rate. For eggshell analysis, after preparing a 10×10 grid of eggs, eggshells and dorsal appendages were scored and classified as described in the text.47lov, only forward strides were seen. But in 47lov, trains of backward strides were also detected. To quantitate this effect, any larva showing three or more consecutive backward locomotor strides was scored as showing spontaneous backward movement. For food shoveling assays, newly hatched first instar larvae were collected from egg lay plates, and placed on 2% agar plates for one hour prior to assay. Individual larvae were then transferred to a 2% agar plate coated with a layer of 2% w/v yeast paste. After a minute of acclimatization, the number of mouth hook contractions (shovels) in one minute was scored. 66lov homozygous larvae were collected from the 66lov/CyO-GFP stock based on lack of GFP fluorescence. At least 100 larvae of each genotype were tested in each assay.Assays were modified from the protocols described by Neckameyer Newly eclosed males were placed in individual food vials and aged for two days. Each was then transferred to an empty food vial with a circumferential marking line 5 cm from the base of the vial. The fly was then tapped gently to the bottom of the vial and the time to climb to the 5 cm mark was recorded. Each fly was tested 10 times with a one-minute rest period between tests. Any trial in which a fly failed to reach the 5 cm line in one minute was scored as a climb failure. At least 50 adults of each genotype were tested.Courtship assays were modified from the protocol described previously lov locus as predicted by Flybase in 2008 is shown in lov transcripts all generate the same protein encoded by three common 3′ exons of the gene. However, three different promoters are used and although transcripts B and D share a promoter, transcript D contains 5′ UTR sequences not present in transcript B. Thus unique 5′ non-coding sequences are present in each of the mRNAs. The transcript B/D promoter and C promoters are close to the protein coding exons, whereas the promoter for transcript A is remote, lying ∼ 43 kb upstream of the first protein coding exon. We used Semi-quantitative (Semi-Q) RT-PCR to investigate the expression patterns of the transcripts. A primer from the first protein coding exon was paired with transcript-specific primers derived from the non-coding 5′exons of the individual mRNAs so as to generate PCR fragments for each of the final four mRNAs. The structure of the lov transcription is detected and Lov protein expression in the nervous system fades away. But as the remodeling of the pupal stages begins, transcript B expression is again expressed  results in loss of elements of the antibody immunostaining from components of the nervous system (see below).To examine Lov protein expression we generated polyclonal antibodies against a unique protein-coding region  in rabbilov mRNA expression, from transcript C to transcripts B and D, as embryogenesis progresses (see above). These changes are associated with two distinct phases of Lov expression: an early non-neural phase and a later phase associated with differentiation of the CNS and PNS. The early pattern is shown in lov mRNA in the ovary  neurons, chordotonal (ch) neurons and three classes of multiple dendritic neurons – the dendritic arborization (da), bipolar dendritic (bp) and tracheal dendrite (td) classes. With the possible exception of one da class cell  A, Lov iAt stage 15, the nuclei of lch2 and 4 together with the two td neurons of the ventral’ cluster show the strongest Lov staining. In the final stages of embryogenesis and immediately before cuticle formation, these four nuclei are often the only peripheral neurons retaining Lov expression, creating a distinctive pattern of four dots in each abdominal segment.lov locus (91Ylov) as part of a screen for mutations that affect gravity responses in Drosophila P{GawB} element associated with 91Ylov is inserted close to the transcription start sites for the B, C and D lov mRNAs and within the large intron of the A transcript. Imprecise excision of the 91Ylov transposon generated three mutations that delete genomic DNA from the locus  is indistinguishable from wild type (data not shown) and they show a normal preference for fructose (a strongly attractant stimulus) versus lactose  suggesting, as for 47lov larvae, that many sensory pathways are intact. However, two major behavioral defects were detected. When assayed in a negative gravitactic climb test, 47lov homozygous and hemizygous adults showed two defects. For flies that completed climb assays, the climb rate was slower than for controls, with 47lov hemizygotes taking three times as long to complete the 5 cm climb, as a result of pausing and non-upward movement . 38lov adults show poor climbing ability although the defects are not as marked as those of 47lov . Eggs from homozygous or hemizygous 66lov mothers have a low hatch rate (∼20%) irrespective of mating partner genotype  and after mating, 66lov females carry stored sperm in their seminal receptacles as frequently as controls (Table S1).In genotype Figure 766lov homozygous or hemizygous mothers show a range of defects. Some aberrant eggs . However, abnormalities consistent with the defects seen in laid eggs were detected during the vitellogenic and later stages, including malformed DAs and incomplete nurse cell dumping associated with premature formation of a chorion-like structure between the nurse cells and oocyte proper. Overall, these findings show that 66lov affects late events in egg morphogenesis including aberrant activity of the somatic cells that form the chorion and associated structures.An analysis of oogenesis established that defects in egg chamber formation for lov transcripts  CG6325MVD166lov phenotype. Although a lower fraction of the eggs were abnormal  have largely normal egg cases and show normal hatch rates. This finding, which may indicate a transvection effect at the locus . The resultant larvae grew as well as controls when selected from the balanced CyO-GFP stock and grown as homozygotes in yeast paste. These larvae also showed none of the larval behavioral defects associated with the 47lov mutation  but the number of emergent 66lov homozygotes dropped considerably in crowded conditions, suggesting some growth disadvantage in the presence of heterozygous siblings. The lower expression of lov transcript B during larval and early pupal life , with the exception of a statistically significantly slight reduction in response to sucrose as a tastant  pnr and the Dorsocross genes are both required for formation of the amniserosa and are regulated by decapentaplegic (dpp) and zernknullt (zen), which act early in the formation of the dorsal embryonic tissues. Both the Zen protein dpp mRNA pattern fushi tarazi (ftz) and its ubiquitously expressed cofactor, ftz-F1lov in these early genetic hierarchies.By comparing the Lov expression pattern to that of other early acting genes, we have identified loci that may be upstream regulators of lov acts late in neural development to direct final neuronal differentiation of subsets of neurons. Given the expression pattern of Lov within the various classes of sensory neurons, we can predict some elements of the transcriptional hierarchies that regulate lov in the PNS. Thus the lov expression in most eso neurons indicates that lov is activated downstream of achaete and scute, the proneural genes of the achaete-scute complex (ASC) that direct formation of the eso mother cells cut, which maintains the “eso” identity in the mother cell lineage lov expression in these neurons. Similarly the absence of Lov in the bp multiple dendritic class suggests suppression of lov by amos, the proneural regulator of this class The timing and expression pattern of Lov in the PNS indicates that lov regulation. Limitation of Lov expression to two neurons of the ch class  is a striking finding. To our knowledge this is a unique observation that suggests for the first time functional differences amongst the five chordotonal organs (CHOs) at this site. Developmentally, these CHOs are already known to originate via two distinct pathways atonalrhomboid expression in these precursors leads to EGF receptor signaling, induction of argos in adjacent cells, and formation of two further chordotonal precursors. It is not known precisely which of the final five lateral CHOs correspond to the two derived from the second phase of development but one possibility is that they are the two Lov expressing neurons. Lov expression would thus be downstream of both atonal and EGF signaling in these neurons.The expression pattern of Lov within the chordotonal lineages indicates considerable complexity in One of the two CHOs of the ventral cluster (VchA) is also induced by the later wave of EGF signaling activity lov mRNA expression in the midline lineages performed previously lov within the CNS is also dynamic and complex and that here too lov has roles in the final differentiation of particular neuronal sub-types. The single cell per segment that first expresses Lov protein in the CNS  cell and a subset of ventral unpaired median motorneurons (VUMs) to give a final expression pattern at stage 17 in just three neurons: a single cell of the MNB progeny and the sister interneuron and motor neuron derived from division of midline precursor neuron 6 (MP6). Notch and lethal-of-scute act at various points to regulate these expression patterns The analyses of  the CNS  has been47lov and 66lov mutations produce strikingly different phenotypes, with 47lov mutants showing multiple behavioral defects and 66lov mutants proving behaviorally unexceptional but strongly female sterile. These differences indicate that the mutually exclusive, non-coding, DNA sequences deleted in the two mutations have differing regulatory roles for the individual lov transcripts. Some of the effects on individual transcripts produced by each mutation generate the differing phenotypes detected here but other effects are without consequence, at least in terms of the traits we assayed. Thus the DNA deleted in 47lov has a strong positive role in production of lov transcript D, a lesser positive role in production of transcript A and a negative role in production of transcript C  within the CNS coordinates the rhythmic locomotor contraction waves of the larval body wall and prior work has shown that loss of sensory input from the periphery disrupts CPG performance, producing both decreased forward, and spontaneous backward, contraction waves 47lov could diminish sensory feedback to the CPG and contribute to abnormal backward movement.The larval phenotypes identified for 47lov adults. The defective 47lov gravitactic climb responses cannot be considered a consequence of general sluggishness since 47lov adults show enhanced locomotor activity when alone in courtship chambers. They appear to represent a more severe form of the diminished negative gravitaxis produced by the original 91Ylov mutation. Thus the original 91Ylov mutant has normal climb behavior , another proprioceptor organ with a demonstrated role in gravity responses in other insects  leading to abdomen curling and copulation. 47lov males showed a marked inability to pursue females continuously but still performed non-directed wing extension, and occasionally abdomen curling, often towards the courtship chamber walls. As noted earlier, Lov belongs to a class of putative transcription factors that includes Fruitless (Fru), the master regulator of male courtship behavior. Given that this class of transcriptional regulators is known to form homo- and hetero-dimers, it is tempting to speculate that Lov might affect courtship responses through protein-protein interaction with Fru. We have not yet attempted a detailed analysis of Lov protein expression in the adult head but preliminary experiments indicate that Lov is expressed in a very large number of adult CNS neurons, a subset of which also express Fru (data not shown).The larval defects of 66lov induced female sterility appears to represent a neomorphic phenotype resulting, at least in part, from ectopic expression of lov in the ovarian germline. However, some aspects of our data for this phenotype are puzzling. First, although 66lov hemizygotes show the same phenotype as homozygotes, 66lov heterozygotes are essentially wild type both with respect to eggshell features and egg hatch rate  correlates well with the fraction of unhatched eggs (20%), for 66lov mothers, the fraction of unhatched eggs (∼80%) is significantly higher than the fraction of detectably abnormal eggs (∼45%). Given that the unhatched eggs from 66lov mutant mothers appear undeveloped and unfertilized, one possible explanation is that 66lov can disrupt egg maturation and fertilization through a second route, involving other elements of the female reproductive system. Adult females lacking the midline CNS neuron population (which includes lov expressing neurons) show sterility thought to reflect loss of innervation of the female genitalia, perhaps producing failed fertilization 66lov causes loss of elements of lov CNS expression in addition to producing ectopic ovarian expression.A further puzzling comparison involves the hatch rates and eggshell defects associated with lov in the ovary must be presumed to produce interference with the action of one or more factors active in eggshell synthesis and morphogenesis. Making the simplest assumption that a single factor is affected by Lov to produce both phenotypes, we sought to identify regulators that affect both eggshell synthesis and eggshell morphogenesis and for which action in the follicle cells is controlled by signals from the germline. Two transcriptional regulators, Broad broad and dominant interference with ecdysone signaling produce eggshell phenotypes similar to those seen with 66lov. In particular i) the fragile chorions and short/malformed/branched DAs from mothers mutant for the rbp class of broad mutations 66lov mothers and ii) the short eggs with stubby DAs and cupshaped dorsal anteriors produced by dominant negative inhibition of ecdysone receptor function in the follicle cells, or loss of ecdysone production, are like the Class 3 eggs identified for 66lov66lov phenotype in detail, we have determined that, in ovaries from 66lov mothers, effects on broad are not limited to effects at the level of protein/protein interaction. broad transcription is depressed in 66lov ovaries in addition to disruption of Broad protein localization , which was not studied here and which contains additional 3′ protein coding sequences.In addition to lacking transcript A (RA), the Flybase page for CG16778 now shows a new minor Figure S147lov larvae show normal taste responses to sugars. Petri dishes filled with two adjacent semicircles of 1% agarose gel, one with 10% fructose (strongly attractant) and red food coloring and the other with 10% lactose (non-stimulating) and blue food coloring, were used to test larval sugar preference. Twenty-five larvae, collected 65 hours after egg laying, were placed on the boundary between the two sugars and the number of larvae at the boundary and on each of the two sugar semicircles was counted after 5, 10 and 15 minutes. The experiment was repeated eight times  and mean values with standard error for all eight repeats are shown. A. Control Canton-S larvae. B. 47lov larvae. Although 47lov larvae take longer to move to the compartment of their choice, their preference for fructose at 15 minutes is indistinguishable from that of the controls. Error bars represent standard error. Assay modified from Xu et al., Nature Neuroscience 11, 676–682, 2008 and Schipanski et al., Chem. Senses 33, 563–573, 2008.(DOCX)Click here for additional data file.Figure S2lov mutants show normal responses in a fast phototaxis assay. The various lov mutants were tested for their ability to detect and respond to light. Flies were placed in a t-test apparatus in which one tube was exposed to a light source and one tube remained in the dark. The flies were allowed one minute to choose which tube to enter and then the number of flies in each tube was counted. For this assay, vials of up to 25 newly eclosed males were collected and allowed to age for three to five days before testing. The assay was repeated up to six times. Wild type flies show positive phototaxis. The responses of all lov mutants are indistinguishable from the control line .(DOCX)Click here for additional data file.Figure S366lov has a decreased response to sucrose. The proboscis extension response (PER) assay of Gordesky-Gold  was used to investigate adult taste responses. Newly eclosed males were starved overnight in food vials with paper tissue moistened with water. Flies were then mounted on toothpicks with Tissue Tack and allowed to recover for three hours. Prior to testing, flies were satiated with water to ensure that tastant, and not water, responses, were assayed. Attractive (1% or 4% sucrose) or repellent (4% caffeine +4% sucrose) tastants were touched to the front legs and the proboscis response noted. Flies were tested three times, and given water between testings. A score of one (“tasting”) was given if a fly extended its proboscis all three times. Two additional trials were performed for flies with mixed responses in the first three trials. After five trials, any fly that extended its proboscis three or more times was scored as “tasting”. Approximately 50 flies from each line were tested. A chi square test was used to determine significance relative to the control, Ore R flies. The reduced response of 66lov to sucrose and stronger response of 91Ylov to sucrose+caffeine are statistically significant. , 66/CyOlov n = 44 (χ2 = 56.7); 1% Sucrose: df 5; Ore R n = 50, 91Ylov n = 50, 38lov n = 50, 47lov n = 48, 66lov n = 47 (χ2 = 50.2), 66/CyOlov n = 44 (χ2 = 50.2); 4% Sucrose and 4% Caffeine: df 5; Ore R n = 50, 91Ylov n = 50 (χ2 = 13.5), 38lov n = 50, 47lov n = 50, 66lov n = 51, 66/CyOlov n = 51).(DOCX)Click here for additional data file.Figure S4Olfactory responses to a repellent odorant for lov mutants. Newly eclosed males were collected, aged for four to six days, then placed in empty food vials in groups of five. A Q-tip pre-soaked with 100 µl of repellent odorant  was then placed in the vial so that the tip was in the middle of the vial. For one minute, the number of flies at the far end of the vial (opposite the plug) was counted every five seconds. The number of flies at the far end was averaged to give an Olfactory Response Index (ORI). The assay was repeated ten times for each genotype. An ORI above 3 (above the red line) means flies are repelled by the odorant. A one-way ANOVA was performed to determine if there were differences among the lines. A Dunnett’s Test was used to compare the results of the mutant lines to the control, Ore R. Error bars represent standard error. Responses of the lov mutants to 1% benzaldehyde are indistinguishable from the control, Ore R, response . The slightly stronger responses of 91Ylov, 38lov, 66lov and 66lov/CyO to 0.1% benzaldehyde as compared to the control are statistically significant. . Assay modified from Anholt and Mackay, Behav. Genet. 31,17–27, 2001.(DOCX)Click here for additional data file.Figure S5Olfactory responses to a neutral odorant for lov mutants. Newly eclosed males were collected, aged four to six days and then placed in an empty food vials in groups of five. A Q-tip pre-soaked with 100 µl of neutral odorant (water) was then placed in the vial so that the tip was in the middle of the vial. For one minute, the number of flies at the far end of the vial (opposite the plug) was counted every five seconds. The number of flies at the far end was averaged to give an Olfactory Response Index (ORI). The assay was repeated at least ten times so that a minimum of 50 flies were tested. An ORI between 2 and 3 (between the green and red lines) means flies are neutral to the odorant. A one-way ANOVA was performed to determine if there were differences among the lines. A Dunnett’s Test was used to compare the results of the mutant lines to the control, Canton S. Error bars represent standard error. The slight attraction to water shown by the control and the slight repulsion to water shown by 47lov are statistically significant. . Assay modified from Anholt and Mackay, Behav. Genet. 31,17–27, 2001.(DOCX)Click here for additional data file.Figure S666lov decreased fertility is attributed to the females. Average total progeny number for the crosses shown above was determined as follows. Single male/single virgin female mating pairs were set up in vials and transferred on day 8 and then day 16 to new vials. On day 24 they were removed from the third vial and discarded. Adult offspring were then collected from each vial for eight days after eclosion of the first adult. A minimum of 10 mating pairs were scored for each line. Female 66lov mutants have significantly decreased fertility when compared to 66lov males in terms of producing viable progeny when mated to control flies. A one-way ANOVA was performed to determined to assess significance of differences between crosses. A Dunnett’s Test was performed with 1118w as the control. Error bars represent standard error. *** = p<0.001 relative to the 1118w control; M = males; F = females.(DOCX)Click here for additional data file.Figure S766lov affects Broad expression in the ovary. In stage 10 of oogenesis, two patches of somatic follicle cells on the dorsal surface of the oocyte compartment show elevated nuclear expression of Broad . This enhanced Broad expression commits the affected cells to dorsal appendage formation. A. The Broad patches (arrowheads) on a stage 10 wild type Canton-S egg chamber. Broad staining of the nurse cell nuclei (n) and oocyte nucleus (o) is artifactual and was intermittently seen with the antibody used. B. A normal size stage 10 egg chamber from a 66lov mother. Although the overall pattern of Broad expression is similar to wild type, enhanced Broad expression at the position of the two presumptive patches of dorsal appendage cells is barely detectable. C. A short stage 10 egg chamber from a 66lov mother. Again, overall patterning of Broad expression is relatively normal but expression within the patches is highly aberrant. D. Semi-Q RT-PCR to probe for all broad transcripts within ovaries from one-day-old control (1118w) and 66lov homozygous females shows that Broad expression is depressed in 66lov ovaries. Methods. A-C. Ovaries from Canton-S or 66lov homozygous mothers were fixed in 4% paraformaldehyde and stained with the Broad-core monoclonal antibody 25E9-D7 (1∶250 dilution) from the Developmental Studies Hybridoma Bank and an Alexafluor-488 labelled goat anti-mouse secondary antibody . The Broad-core antibody recognizes sequences common to all isoforms of Broad. D. Semi-Q RT-PCR on ovarian RNA was performed as described in TGCAGGATGTCAACTTCATGGACC 3′) and Broad R (5′TATCTGAGCCAGATGGCTGTGTGT 3′), which span an exon-exon junction within the shared protein coding sequences of all broad transcripts, were used to probe for all processed broad transcripts. Actin 57B primers (see main text (DOCX)Click here for additional data file.Table S1Sperm transfer and storage is not impaired in 66lov mutants. A third chromosome don juan-GFP construct , which generates GFP-expressing sperm, was used to monitor sperm storage in the seminal vesicles and spermathecae of mated females. Males and virgin females were aged for 3–6 days after eclosion and set up in matings of the genotypes indicated. The sperm storage organs of females were dissected out after confirmed mating and were examined for GFP fluorescing sperm. There is no significant difference in the ability of 66lov males to transfer sperm or 66lov females to store sperm, relative to the 1118w and 66lov/CyO controls. χ2 = 1.04.(DOCX)Click here for additional data file.


Reduction or depletion of mitochondrial DNA (mtDNA) has been associated with cancer progression. Although imbalanced mtDNA content is known to occur in prostate cancer, differences in mtDNA content between African American (AA) and Caucasian American (CA) men are not defined. We provide the first evidence that tumors in AA men possess reduced level of mtDNA compared to CA men. The median tumor mtDNA content was reduced in AA men. mtDNA content was also reduced in normal prostate tissues of AA men compared to CA men, suggesting a possible predisposition to cancer in AA men. mtDNA content was also reduced in benign prostatic hyperplasia (BPH) tissue from AA men. Tumor and BPH tissues from patients ≥60 years of age possess reduced mtDNA content compared to patients <60 years of age. In addition, mtDNA content was higher in normal tissues from patients with malignant T3 stage disease compared to patients with T2 stage disease. mtDNA levels in matched normal prostate tissues were nearly doubled in Gleason grade of >7 compared to ≤7, whereas reduced mtDNA content was observed in tumors of Gleason grade >7 compared to ≤7. Together, our data suggest that AA men possess lower mtDNA levels in normal and tumor tissues compared to CA men, which could contribute to higher risk and more aggressive prostate cancer in AA men. In vitro and in vivo analyses demonstrate that reduction of mtDNA content is associated with acquisition of an androgen-independent phenotype and leads to prostate cancer progression In terms of diagnosis and mortality, prostate cancer is one of the leading cancers among American men. Increased prostate cancer-related diagnoses and mortality are commonly associated with African American (AA) men compared to Caucasian American (CA) men How mtDNA regulates the tumorigenesis process is not clearly defined, but current evidence suggests that mutation, reduction, or deletion of mtDNA lead to defective oxidative phosphorylation (OXPHOS), increased reactive oxygen species (ROS) production, induction of the glycolytic pathway, increased expression of prosurvival proteins, which ultimately results in cancer proliferation and tumorigenesis OXPHOS defects are one of the hallmarks in multiple types of cancer. The OXPHOS system consists of five multisubunit complexes, and mtDNA encodes 7 subunits of complex I, 1 subunit of complex III, 3 subunits of complex IV, and 2 subunits of complex V Age comparison of mtDNA content in prostate cancer patients demonstrated that in normal prostate epithelial tissues, median mtDNA content increased ∼229% in patients ≥60 years compared to patients <60 years of age. Similarly, mean values were also increased in patients ≥60 years of age . In contProstate cancer is highly aggressive in AA men compared to CA men; however, the underlying mechanisms are not yet defined. Decreased mtDNA content is one of the key factors in cancer progression, including in prostate cancer To further establish and evaluate the importance of mtDNA in cancer progression, we compared different prostate cancer stages in both groups of combined populations. Surprisingly, we observed higher mtDNA content in matched normal tissues at the T3 stage  compared to the T2 stage . Because T3 stage tumors have worse prognoses compared to the T2 stage, mtDNA in normal tissues from T3 tumors needs further investigation. Although the reason for increased mtDNA in normal tissues from tumors of higher grade and stage is not known, one plausible explanation might be due to accumulation of an increased number of altered or defective mitochondria in normal cells that were not transformed, thus causing increased amount of mtDNA in normal tissues at higher grade and stage. Interestingly, mtDNA content was not affected in tumor tissues or in BPH samples. Importantly, increased mtDNA levels in WBCs at the T3 stage suggest that mtDNA content could also be a possible marker for staging and predicting aggressiveness of prostate cancer .Analyses of PSA levels and mtDNA content demonstrated that median mtDNA content was positively associated with PSA levels in normal tissues, whereas it was decreased in tumors from patients with PSA levels >10. No specific trend was observed in BPH samples .With regard to Gleason grade, mtDNA content in normal prostate epithelial tissues was nearly doubled in Gleason grade of >7 compared with Gleason grade ≤7. In contrast, prostate tumors showed reduced median values of mtDNA in samples with Gleason grade ≥7; however, no difference in mtDNA content between different Gleason grades was observed in BPH samples .When we compared median values of mtDNA content in tumors between Gleason grade ≤3 and ≥4, we observed ∼30% reduction in mtDNA in Gleason minor grade ≥4 compared to grade ≤3, whereas no change in median mtDNA content was observed in tumors of Gleason major. Increased mtDNA levels were observed in Gleason grade ≥4 in matched normal prostate epithelial tissues of Gleason major; however, the median mtDNA levels were decreased in matched normal tissues of ≥4 in Gleason minor  and 6.In order to establish the importance of mtDNA in dissecting prostate cancer health disparities, it is essential to measure the levels of mtDNA in normal noncancerous prostate epithelial tissues as well as tumor tissues obtained from AA and CA prostate cancer patients. We observed that mtDNA content was reduced in tumor tissues compared to matched normal prostate tissues in combined data from both AA and CA patients (p = 0.053). In CA prostate cancer patients, mtDNA content was 42% lower in tumor tissues compared to matched normal prostate epithelial tissues (p = 0.068). In AA prostate cancer patients, the mtDNA levels in tumors showed limited changes compared to matched normal tissues when we compared the median values. However, the mean mtDNA content was reduced in tumor tissues compared to matched normal prostate tissues. Nonetheless, mtDNA content in tumors from AA men was lower than those from CA men .Analyses of mtDNA content in combined populations of prostate cancer patients demonstrated significantly lower levels of mtDNA in BPH and WBCs compared with normal prostate tissues (p = 0.001). Similarly, mtDNA content was reduced in BPH samples and WBCs in comparison with normal tissues in AA and CA patients individually . CompariWhen we compared mtDNA content in tumors obtained from AA and CA patients after adjusting for age or Gleason grade, or PSA, although not statistically significant, we observed a ∼7–8, ∼5, or 4–5 fold decrease in mtDNA, respectively, in AA patients. Age, Gleason grade, and PSA adjustment of BPH demonstrated no changes in the mtDNA levels in BPH from AA patients compared to CA patients . SimilarOur study provides the first evidence that prostate tumor tissues from AA men harbor reduced mtDNA levels compared to CA men. Normal prostate epithelial tissues from AA men also show decreased mtDNA content as compared to CA men. Although the exact significance of these results requires further investigation, our data suggest that reduced mtDNA levels in AA men may be associated with the higher incidence and aggressive nature of prostate cancer in AA men. Future studies with increased numbers of DNA samples from AA and CA patients may provide greater understanding regarding the impact of reduced mtDNA content on prostate cancer health disparities. It is unknown how mtDNA content decreases in prostate tumors from AA men compared to CA men, but it is possible that mtDNA polymerase γ (pol γ), the only DNA polymerase in mitochondria that plays an essential role in mtDNA replication and repair kB. Therefore, reduced mtDNA in normal cells from AA men suggests that the AA population is susceptible to a higher incidence of prostate cancer. Indeed, the incidence of prostate cancer in AA men is ∼1.6 times greater than the CA population Because the occurrence of prostate cancer in AA men is higher than in CA men, as well as prostate cancer is more aggressive in AA men 0F1-ATP synthase. These effects may also be due to differences in cancer cell types. The relatively higher mtDNA content in CA patients may lead to enhanced mitochondrial function and respiration, thereby causing enhanced apoptotic cell death in CA patients compared to AA patients. In contrast, higher mtDNA content in tumors from CA prostate cancer patients may lead to proliferation of mitochondria due to induction of genes involved in mitochondrial biogenesis. This suggests that the relatively higher mtDNA content in CA prostate cancer patients could cause accumulation of abundant but morphologically altered mitochondria, which may induce apoptosis or alternate forms of programmed cancer cell death in prostate tumors. These findings are consistent with earlier reports that increased mtDNA mutations in thyroid tumors induce accumulation of defective and altered mitochondria Reduction or depletion of mtDNA and abrogation of mitochondrial respiration inhibits apoptosis induced by multiple anticancer agents How is the reduction in mtDNA associated with aggressive nature of prostate cancer in AA men? Depletion of mtDNA induces prostate cancer progression Together, our findings provide the first evidence that mtDNA content is decreased in normal prostate epithelial and cancer tissues from AA men compared to CA men. These findings may have significance in predicting the differential outcomes of prostate cancer progression and treatment in American men. Because both normal prostate tissues and tumor tissues of CA men harbor higher mtDNA levels compared to AA men, we anticipate that CA men will be more likely to respond to anticancer therapy and be more susceptible to the cytotoxic effect of anticancer agents compared to AA men. Therefore, a clear understanding of the mechanisms of mtDNA imbalance in American men may provide new and efficient approaches for prostate cancer prevention and therapy.http://www.louisianacancercenter.org/collaborative-initiatives/shared-resources/biospecimen-core).Sample collection was performed after obtaining written informed consent from patients for the use of their samples for diagnostic and scientific purposes. All clinical samples were provided based on a unique and random alphanumerical coded system without referring to the patients by name or using any other personal identifier term. During data processing and analyses, samples were assigned a different random number. Ethical approval was obtained from the Institutional Review Boards (IRBs) at Louisiana State University Health Sciences Center (LSUHSC), School of Medicine, New Orleans, LA, and Roswell Park Cancer Institute, Buffalo, NY. The study conformed to the principles outlined in the Declaration of Helsinki. All tissue biospecimens were obtained from the biospecimen core facilities at the Louisiana Cancer Research Consortium (LCRC) affiliated with Tulane Medical School and School of Medicine, New Orleans and LSUHSC, New Orleans  in this study are defined as men with no evidence of prostate cancer. White blood cells (WBCs) were obtained from patients diagnosed with primary prostate cancer. Fresh frozen matched normal and malignant prostate tissues were obtained from African American and Caucasian American patients diagnosed with primary tumors. In this study, normal prostate tissues are considered as matched normal prostate specimens taken from patients with primary prostate cancer. Only malignant tissues with 70% or higher tumor content were considered in the study. Genomic DNAs were extracted using the QIAGEN DNeasy Blood & Tissue Kit, as recommended by the manufacturer. Genomic DNA samples were quantitated using a NanoDrop 8000 Spectrophotometer (Thermo Scientific).β-actin and cytochrome c oxidase subunit II (COX II) were used for amplification of nuclear and mtDNA, respectively. Primers for β-actin and COX II were used as described previously β-actin (forward): 5′-TCAC CCACACTGTGCCCATCTACGA-3′, β-actin (reverse): 5′-CAGCGGAACCGCTCATTGCCAATGG-3′. COX II (forward): 5′-CCCCACATTAGGCTTAAAAACAGAT-3′, COX II (reverse): 5′ TATACCCCCGGTCGTGTAGCG GT-3′. Real-time PCR reactions were performed in a total reaction volume of 10 µl containing 5 µl 2X iTaq SYBR Green Supermix with ROX , 10 ng of template DNA, 500 nM each of forward and reverse primers, and nuclease-free water. Melting curve analyses were performed at the end of the amplification to verify the absence of nonspecific amplification or primer dimer formation. The threshold cycle number (Ct) values for each reaction were calculated using the 7300 system SDS software. Standard curves were generated using 10 ng to 10 pg of DNA prepared from untreated LNCaP cells and provided PCR efficiency based on the equation E = 10∧(−1/slope) −1 COX II (mtDNA-specific) and β-actin (nDNA-specific). mtDNA content was determined as 2∧ΔCt, or fold difference of mtDNA from nDNA mtDNA content was determined as described previously mtDNA content was reported using the means, medians, and standard deviations. Other clinical and demographic characteristics were reported using frequencies and relative frequencies. Graphical analyses (QQ plots) found the mtDNA content to be non-normally distributed and therefore, non-parametric approaches were considered for analyses of this variable. For each group , the mtDNA content was compared between age, race, tumor stage, prostate specific antigen (PSA), Gleason sum, Gleason minor and Gleason major using Wilcoxon rank-sum or Kruskal-Wallis exact tests, as appropriate. Additional analyses within each group included comparisons of mtDNA content between racial groups while adjusting for age, Gleason sum and PSA using two-way analysis of variance (ANOVA). Because mtDNA content is non-normally distributed, appropriate p-values were obtained using standard bootstrap methodologies. mtDNA content was compared in a pairwise fashion between groups in the overall sample and within each race using the Wilcoxon signed-rank  or Wilcoxon rank-sum  exact tests. All data analyses were conducted in SAS v9.3  at a significance level of 0.05.


Melanoma cutis is virtually incurable when it has metastasized to distant organs. Unfortunately, there is no safe and effective adjuvant treatment available for early stage patients that may prevent the development of metastasis. Since early melanoma development is accompanied by impaired immune effector functions in the sentinel lymph node (SLN), there is a strong rationale for therapeutic immune modulation of the SLN aimed at strengthening cellular immune functions. In two placebo controlled trials, the immunological effects of i.d. administration of CpG type-B  ± GM-CSF  at the tumor site, one week before SLN excision, were investigated. The trials showed that this treatment is clinically safe and flow cytometric and functional analyses of viable immune effector cells isolated from the SLN showed increased frequencies and activation state of LN resident CD141+ cDC subsets as well as activation of pDC and CD1a+ skin derived cDC subsets in the treated groups. This DC activation was accompanied by an increased Th1 cytokine response profile and melanoma-specific CD8+ T cell reactivity. Interestingly, when combining the treated groups of both studies (n=31), we found remarkable clinical differences compared to the combined placebo groups (n=22). Important predictors for recurrence and SLN tumor positivity are Breslow depth and ulceration of the primary tumor. These two clinical parameters were higher  in the treated group. Nevertheless, the number of tumor positive SLN in this group was significantly lower (3/31 vs. 8/22 p=0.02). At a median follow-up of 71 months we also found a longer recurrence free survival in the treated group (p=0.14). We conclude that local administration of CpG ± GM-CSF is safe, shows very promising immunological efficacy with evidence for clinical impact on lymphatic spread and recurrence free survival and could potentially be used as adjuvant treatment combined with primary excision of suspected melanoma lesions.


Decisions guiding environmental management need to be based on a broad and comprehensive understanding of the biodiversity and functional capability within ecosystems. Microbes are of particular importance since they drive biogeochemical cycles, being both producers and decomposers. Their quick and direct responses to changes in environmental conditions modulate the ecosystem accordingly, thus providing a sensitive readout. Here we have used direct sequencing of total DNA from water samples to compare the microbial communities of two distinct coastal regions exposed to different anthropogenic pressures: the highly polluted Port of Genoa and the protected area of Montecristo Island in the Mediterranean Sea. Analysis of the metagenomes revealed significant differences in both microbial diversity and abundance between the two areas, reflecting their distinct ecological habitats and anthropogenic stress conditions. Our results indicate that the combination of next generation sequencing (NGS) technologies and bioinformatics tools presents a new approach to monitor the diversity and the ecological status of aquatic ecosystems. Integration of metagenomics into environmental monitoring campaigns should enable the impact of the anthropogenic pressure on microbial biodiversity in various ecosystems to be better assessed and also predicted. Natural microbial diversity encompasses a broad spectrum of microorganisms  that exert a strong influence on global processes such as the carbon, nitrogen and sulphur biogeochemical cycles. Quick responsiveness to environmental changes and the rapid reproductive capacity of microorganisms allow for changes in both the qualitative and quantitative composition of particular habitats. Indices of microbial diversity are considered a sensitive measure of the state of the environment and the health of a given habitat or ecosystem. Assessment of biodiversity, therefore, represents a keystone in understanding the complex processes within ecosystems and needs to be taken into account in decisions concerning environmental resource management and conservation priorities In view of the intrinsic connection between environmental quality and human health Water quality assessment represents an important aspect of environmental monitoring but is commonly restricted to chemical monitoring, despite numerous studies indicating that biodiversity in marine ecosystems is consistently reduced because of anthropogenic contamination For a long time microbiological monitoring has been restricted to the detection of microbes affecting human health, excluding the majority of microbial species mainly because of technical limitations In the present study we demonstrate the applicability of metagenomic profiling to assessing the extent of anthropogenic impact on two different marine ecosystems, complementing traditional monitoring measurements such as chemical analyses. Water samples were collected from two coastal regions of the Mediterranean Sea, the Port of Genoa, in 2009 and the protected area around Montecristo Island in 2010, two areas distinguished by high and low anthropogenic impact, respectively. Sequencing of total community DNA allowed us to generate metagenomic profiles and compare the microbial diversity caused by the anthropogenic stress response in these two distinct coastal marine ecosystems.For sampling in the port of Genoa, no specific permit was required for the described field studies since the area is not privately-owned or protected in any way. We confirm that the field studies did not involve endangered or protected species.For the sampling in the protected area Montecristo Island, all necessary permits were obtained for the described field studies. A permission was requested to the regulatory body Ministero dell’Ambiente e della Tutela del Mare - Parco Nazionale Arcipelago Toscano, which authorized the sampling with the authorization n. L. 394191; D.P.R. 22/07/96.Two coastal marine environments in the Mediterranean Sea were sampled using a sterile Ruttner sampler . During Each sediment sample (0.2 g) was analyzed by ICP-MS using an APEX system  following a microwave (Milestone ETHOS 900) assisted digestion 2PO4 buffer overnight at 4°C for better recovery of cells. Subsequently, they were treated with 5 U µl−1 of lyticase , 4470 U µl−1 of lysozyme  and finally 2.5 µl of ß-mercaptoethanol . DNA was extracted using the DNeasy Blood and Tissue kit  according to the manufacturer’s instructions. DNA was quantified on 1% agarose gel using a MassRuler™ High Range DNA Ladder .Total community DNA was recovered from the shallow and homogeneous mixed water column by pooling these samples together. All samples collected from the PolS were pooled together (depth 0 to 8 m), while only samples from the upper layers (depth 0 to 7 m) of PriS were pooled together excluding the deepest samples (14 m). Prior to DNA extraction each sample of 20 liters was split into aliquots of approximately 250 ml to avoid clogging of the filters. Aliquots were then directly filtered on separate 0.22 µm pore size membrane filters . According to the Rapid Library Kit manufacturer's instructions (Roche), ∼500 ng of sampled DNA should be used for the preparation of pyrosequencing samples. Depending on the plankton density in the pooled samples a variable number of filters corresponding to different sample volumes were therefore needed in order to extract the desired amount (500 ng) of community DNA. As a consequence the total volume filtered for the March 12 and September 18, 2009 PolS samples was 5.05 and 5.625 liters, respectively, and volumes of 10 liters were filtered for each of the PriS sample. The filters were stored at –20°C. Before DNA extraction, the thawed filters were incubated and shaken (160 rpm) in 5 ml of 50 mM K3 particles of the internal standard were used .To determine bacterial abundance, samples fixed in 1% paraformaldehyde were sonicated for 5 min at 4°C , stained with SybrGreen I  and analyzed by flow cytometry . For quantification, ∼50×10http://metagenomics.anl.gov/metagenomics.cgi?page=MetagenomeSelect) with ID numbers 4449589.3 (PolS1), 4449685.3 (PolS2), 4451102.3 (PriS1), 4451593.3 (PriS2).Total community DNA was directly sequenced using the equipment and tools available at the Zürich Functional Genomics Centre according to the manufacturer's instructions: a GS Rapid Library Kit (Roche), Roche 454 Genome Sequencer FLX with the GS Titanium Sequencing Kit XLR70, GS Titanium PicoTiterPlate Kit (70×75) and gsRunProcessor from GS FLX SW v2.3. Two samples were run in parallel on one plate yielding 200–300 Mbp of sequence information per sample. The metagenomic data is available through the MG-RAST server (http://weizhong-lab.ucsd.edu/rammcap) implemeted in CAMERA  and the MG-RAST server (http://metagenomics.anl.gov/). In RAMMCAP, exact read duplicates were removed using CD-HIT http://greengenes.lbl.gov/cgi-bin/nph-index.cgi) applying a 70% sequence identity, 100 bp alignment length and 1.0e-05 e-value threshold.Raw reads were processed using the Rapid Analysis of Multiple Metagenomes with a Clustering and Annotation Pipeline (RAMMCAP http://tools.metagenomics.anl.gov/m5nr/) applying an e-value threshold 1.0e-05. Functional annotation and classification relied on the KEGG Orthology (http://www.genome.jp/kegg/ko.html) or SEED subsystem (http://www.theseed.org/wiki/Home_of_the_SEED) databases applying an e-value threshold of 1.0e-05. Functional annotation with PFAM (http://pfam.sanger.ac.uk/) was performed using the CoMet server (http://comet.gobics.de). Annotation against the Clusters of Orthologous Groups (COG) database In the MG-RAST analysis, the optional initial quality control (QC) filter was applied to the raw sequence data combined with their associated quality scores (FASTQ format) to remove duplicate and low quality reads. Organisms were classified in MG-RAST using the M5NR protein database (http://www.r-project.org/). Functional annotation profiles obtained from RAMMCAP, MG-RAST or COMET were reformatted for ShotgunFunctionalizeR analysis using custom-generated Perl scripts (see Data S2). Prior to ShotgunFunctionalizeR analysis, all counts associated with an individual sample were normalized taking into account the number of total reads post MG-RAST QC.Gene family and category enrichment were analyzed on the metagenomic profiles using the ShotgunFunctionalizeR tool Samples were collected at two environmentally distinct sites in the Ligurian Sea during 2009 and 2010 . The Por−1) than in PriS (median 0.10 µg Chla l−1) (p<0.001), consistent with the expected high level of eutrophication at the polluted site. The same trend was observed for the total number of bacteria (TNB), which was significantly higher at PolS  than PriS (median 0.41×106 cells ml−1). Taken together, the Chla and TNB values indicate a global shift in biodiversity towards planktonic organisms such as bacteria and algae consistent with a higher level of nutrients at the polluted site.In order to determine general environmental parameters characterizing the two sites, samples were collected as two replicates at different depths within a column of homogeneously mixed water mass . Salinit−1, median in PriS 0.955 µg Mn l−1, p = 0.048, see Data S3). Differences between the polluted and pristine sites were more pronounced in the sediment samples with several metals showing a statistically significant enrichment in PolS as compared to PriS: Cd , Co , Fe  and Mn  (see Data S3).The concentration of trace metals in the water column as revealed by mass spectrometry did not differ between sites, with the exception of Mn, which was about three times higher at the polluted site  were generated from the samples , a small fraction of Eukaryotes (2–3%) and generally less than 1% of Archaea, viruses and other unclassified organisms , values Annotation quality was consistently high across all M5NR database sources with more than 80% of similarities falling below the e-value cutoff value of 1.0e-05 subsequently applied during functional characterization of the metagenomic profiles. A more detailed comparison of organism abundance at the phylum and class levels correctly grouped the samples according to their origin, with some intra-group variations recognizable especially between the polluted samples PolS1 and PolS2 . Both inPredicted protein features were annotated using a panel of complementary tools: RAMMCAP for COG protein families The enrichment analysis highlighted a large number of COG families with significant differences between the two sample groups . PFAM anBecause ShotgunFunctionalizeR also allows groups of functionally connected genes, e.g. whole pathways, to be examined concurrently, the analysis was extended to COG gene categories and enzymatic pathways. Several COG categories differed significantly between the sample groups (see Data S6) with “Signal transduction” and “Carbohydrate transport and metabolism”  appearinThe biodiversity distribution within the four metagenomic profiles had indicated a substantial difference between the two polluted samples, in particular at the OTU level. Therefore, functional protein annotation was also compared separately within each group, again using ShotgunFunctionalizeR. For the two pristine samples very few COG families showed a significant difference (Benjamini-Hochberg (BH) adjusted p-value <1.0E-05), confirming their high similarity. In contrast, the abundances of many more COGs differed between the two polluted samples (Data S6). However, importantly, only a small number of these COG families had also been found in the between-groups ShotgunFunctionalzie comparison (see Data S7). A small bias due to the more heterogeneous composition of the polluted samples can therefore not be excluded; nevertheless, the major conclusions from the group-wise analysis should be considered valid.Present practice in environmental monitoring lacks tools that are adequate for detecting and analyzing the impact of complex factors on aquatic and terrestrial ecosystems. In particular, monitoring the health of aquatic environments typically focuses on chemical pollutants In the present study two coastal marine systems exposed to rather different anthropogenic pressures have been examined: the Port of Genoa, located in a heavily urbanized and industrialized area, and the uninhabited Montecristo Island resource. In both cases, samples were collected before and at the end of the summer period of 2009 (Port of Genoa) or 2010 (Montecristo). A more synchronized schedule might have been preferred at first glance, but one aim of the present study was to gain insight into how much sample variation should be expected when sampling a large complex ecosystem. In routine monitoring practice, water samples would in general be taken only from “suspected” sites and not necessarily always at the same time point during the year. Knowledge about the potential global variations in the polluted samples will be important for comparing them optimally to a “healthy” reference sample (or reference profile). We have therefore chosen, as a first test case, to examine four water samples collected at different time points from two environmentally very distinct sites located in distant areas  of the Mediterranean coastal sea.Analysis of the metagenomic profiles proved consistent with the expected sample characteristics. The functional protein annotation recapitulated the results from phylogenetic analysis well, showing clear differences between the two sample groups exposed to distinct anthropogenic pressures. In the pristine sample site, the high abundance of Actinobacteria and the associated enzymatic functions and pathways detected are consistent with an environment containing oligotrophic organisms adapted to low levels of nutrients and characterized by a high potential for utilizing low concentrations of organic substrates through energy-efficient ABC transporter systems In contrast, polluted environments are generally characterized by high levels of numerous stress factors and high level of nutrients.Organisms adapted to such environments are in general copiotrophs, e.g. Gammaproteobacteria, which are the “feast and famine” strategists optimizing rapid growth in the presence of labile nutrients. Reflecting this characteristic, the microbial community at the polluted site had significantly higher levels of signal transduction regulator proteins involved in a variety of cellular responses to environmental stimuli Despite being collected at different seasonal time points during the year, the metagenomic profiles from the two pristine samples closely resembled each other. Generation of a “healthy” reference profile therefore seems feasible and further refinement through incorporation of additional samples should allow the profile to be consolidated. In contrast, the metagenomic profiles of the two polluted samples exhibited much larger variation, probably reflecting fluctuations of the harsher environmental conditions caused by quantitative and qualitative variations of pollutants. Consequently, a much larger sampling study will be required to identify robust generally-applicable indicators of pollution. Importantly, in order to extend the applicability of metagenomic monitoring to potentially much less polluted samples , a large number of samples with a broad distribution of pollution levels will have to be examined.Compared to more traditional methods, PCR-based direct sequencing of total community DNA mostly avoids several of the methodological problems that potentially introduce various biases Although it is still at an early stage, metagenomic analysis of total community DNA using direct sequencing without cloning and with long sequence fragments (∼400 bp or more) opens up new perspectives in environmental monitoring by providing a relatively simple, robust and reproducible approach to studying samples with unknown diversity and exposed to unknown anthropogenic pressures. Until very recently 454 pyrosequencing had the advantage to produce longer average read length compared to the rather short reads (<100 bp) obtained by Illumina, the main alternative and cheaper technology. Since the average gene length in bacteria is about 1 kB Finally, it should not be neglected that microorganisms are known to make up the bulk of the biota in both natural and managed ecosystems. Proposals to restore the function and integrity of ecosystems are increasingly being put forward, and any initiative would be incomplete if considerations regarding the complexity and integrity of the underlying microbial systems are not included.Data S1Minimal Information about Metagenomics Sequences (MIMS).(XLS)Click here for additional data file.Data S2Description of the analysis workflow together with a collection of custom-written Perl scripts and example data files.(ZIP)Click here for additional data file.Data S3Table describing sediment metal concentrations.(XLS)Click here for additional data file.Data S4Complete GreenGenes OTU classification table.(XLS)Click here for additional data file.Data S5Complete table describing sample differences by functional annotation against COG, Pfam, SEED and KEGG .(XLS)Click here for additional data file.Data S6Complete table describing sample differences by pathway annotation against COG, SEED and KEGG .(XLS)Click here for additional data file.Data S7ShotgunFunctionalizeR analysis comparing the COG functional annotation of two pristine or the two polluted samples.(XLS)Click here for additional data file.