Behaviour and distribution of striped marlin within the southwest Pacific Ocean were investigated using electronic tagging data collected from 2005–2008. A continuous-time correlated random-walk Kalman filter was used to integrate double-tagging data exhibiting variable error structures into movement trajectories composed of regular time-steps. This state-space trajectory integration approach improved longitude and latitude error distributions by 38.5 km and 22.2 km respectively. Using these trajectories as inputs, a behavioural classification model was developed to infer when, and where, ‘transiting’ and ‘area-restricted’ (ARB) pseudo-behavioural states occurred. ARB tended to occur at shallower depths (108±49 m) than did transiting behaviours (127±57 m). A 16 day post-release period of diminished ARB activity suggests that patterns of behaviour were affected by the capture and/or tagging events, implying that tagged animals may exhibit atypical behaviour upon release. The striped marlin in this study dove deeper and spent greater time at ≥200 m depth than those in the central and eastern Pacific Ocean. As marlin reached tropical latitudes (20–21°S) they consistently reversed directions, increased swimming speed and shifted to transiting behaviour. Reversals in the tropics also coincided with increases in swimming depth, including increased time ≥250 m. Our research provides enhanced understanding of the behavioural ecology of striped marlin. This has implications for the effectiveness of spatially explicit population models and we demonstrate the need to consider geographic variation when standardizing CPUE by depth, and provide data to inform natural and recreational fishing mortality parameters. Kajikia audax) Striped marlin  data and efforts to mitigate bycatch. Although known to spend the majority of their time near the surface, less is known about their utilization of other water column strata. Utilization of the water column by pelagic fishes tends to be shallower in the eastern tropical Pacific (ETP) than in most of the rest of the Pacific because of a sharp vertical oxycline and shallow thermocline The geographic distribution of Pacific striped marlin has been inferred from commercial longline CPUE and conventional tagging data Double-tagging data are used here to investigate the movement patterns of striped marlin within the southwest Pacific Ocean. A model is developed to classify pseudo-behavioural states and then to infer the frequency and duration of area-restricted and transiting behaviours. As a result, a basis for investigating their response to the capture/tagging process is also established. We show how different behavioural modes are related to swimming depths. Relationships among the behaviour of individuals and the distribution of the population are illustrated for the first time. This study provides the detailed analysis of long-term behaviour reported thus far, describing new insights into the biology and ecology of striped marlin. We discuss how this research can inform population dynamics models including spatio-temporal stratification, depth standardized CPUE, recreational fishing mortality, and natural mortality.This research was approved under permit AEC-R431, issued by the Animal Ethics Committee of University of Auckland School of Biological Sciences.et alStriped marlin were caught between 2005–2008 from recreational fishing vessels as described in Holdsworth Rpackage)’. Unless stated otherwise, averages are noted as mean ± standard deviation.All data analyses were conducted using the statistical environment uKFSSTSLRT locations were computed directly by Argos satellites and properties of these positions  have been assessed extensively crawlMost telemetry studies record observations at irregular intervals, and it is common to use inferential models which require regular time-steps to analyze latent pseudo-behaviours A movement model was formulated to infer four discrete behavioural states; “slow-transiting“, “fast-transiting“, “slow-ARB“, “fast-ARB“ (ARB is short for area restricted behaviour). Trajectory segments with high turning angles have been referred to as ‘area-restricted search’ (ARS) previously fields) Comparisons were made among the behaviours inferred by the model and distributions of maximum depths reported by PSAT tags. Profiles of depth and temperature (PDT)s, were transmitted by PSAT tags at 8 discrete depths (and mean temperature at depth) distributed between the minimum and maximum during pre-programmed summary periods. Maximum depths were chosen for analysis because they are the most commonly observed discrete measure of position in the water column across all animals. Maximum depths were non-normally distributed, so non-parametric tests for asymptotic distributions  were used to compare maximum depths with transiting and ARB behaviours modes (without respect to speed). Probability-density plots of time spent within discrete depth bins summarized over the same temporal periods described above were used to illustrate changes in depth distributions over time. Gaps in the depth data bins were interpolated using the function loess The probability of occurrence of ARB with respect to the number of days at liberty was estimated by fitting a proportional hazards model to produce a Kaplan-Meier survival curve using the function Surv(n = 3), 2006 (n = 15), 2007 (n = 5), and 2008 (n = 2) were included in the analysis . The highest quality location data generally occurred within the first 4–8 weeks of the tracks when SLRT locations were available. Exceptions to this included STM06.8 and STM07.2, which had gaps of 14 and 20 days at the beginning of their tracks. STM07.1 and STM08.1 provided exceptionally good SLRT data for 103 and 115 days (max gaps 6.7 and 6.2 days) respectively . Root meAll four behavioural modes were predicted, but not all were observed in each individual . The numResults for at least one marlin from each tagging season (2005–2008) are discussed. Two archival datasets were recovered from fish tagged in 2006 , and anac. 75–150 m. During the ensuing two weeks STM05.4 spent the majority of its time at or near the surface, with only brief forays below 50 m  its northerly progress slowed, with the final SLRT location observed on 2 June 2007.STM07.1 was tagged on 19 February 2007 at Waihau Bay in the eastern Bay of Plenty . Upon deSTM08.1 was tagged on 6 March 2008 at Waihau Bay . It depap≤0.001, θ = 11.8). Relative turning angles (angles between consecutive locations) were significantly lower during the first 10 days at liberty than all subsequent periods . Trajectories were aligned significantly more northwards during the first 10 days at liberty than all subsequent days . In addition to the strong tendency to move away from tagging locations, a seasonal trend was observed in the likelihood of marlin returning to their capture vicinity within the same tagging year. Of the marlin tagged before 1 March, 5/14 circled back to within 500 km of their capture location  until after 16 days post-release . Proportlocation . None talocation .p≤0.001) for transiting behaviours (127±57 m) than for ARB (108±49 m). Across all modes, they spent 51.4% of their time between within 1 m of the surface during the day, and 64.0% of their time in this range at night , initially tagged and captured in different months (February–April) and different locations  moved towards the tropics and suddenly reversed directions at 20–21°S latitude upon arrival during the months April–August . These dDealing with error-prone geolocations can be challenging, but Kalman filters enable the errors of marine telemetry techniques to be quantified and minimized STM06.1 made rapid and uncharacteristic west-to-east movements immediately upon release . The magIn spite of the improved accuracy of our longitudes derived from light level geolocation, significant error remains for latitude estimates, which may lead to erroneous predictions of fast-ARB. Fast-ARB may not represent a real biological state, but rather a temporally autocorrelated series of location estimates of higher-than-average spatial error. Biologically relevant signals almost certainly lie within these segments, but the magnitude of measurement error precluded making biological inferences. Recently developed methods can reduce, or even eliminate bias in light level latitudes Dermochelys coriacea) using switching state-space models (SSM) ARB is representative of all behaviours such as foraging, resting and breeding that occur in spatially confined areas et al.The segmentation model we used calculates the likelihood of a trajectory segment belonging to a specific behavioural mode, but does not estimate a posterior probability as do other approaches to classification ie. sardines and other planktivorous fish) may sometimes persist for days Transiting behaviour tended to correspond with changes from surface to deeper waters, while ARB mode generally was associated with shallower depths. Opportunities to feed can be sparsely distributed through space and time for pelagic predators, so foraging frequencies observed here probably reflect these conditions. Shoals of prey  which have lower nutritive value, while marlin feed on less abundant but higher nutritive value targets like saury (Family: Scomberesocidae) and mackerels (Family: Scombridae). Accordingly, models designed to identify analogous behaviours (ie. foraging) which driven by different strategies (ie. predatory vs. grazing) probably require unique configurations.Jonsen et al.Thunnus maccoyii) fed on 76% of days, with periods between confirmed feeding events ranging from 3.9–24.4 days (mean 11 days). The ability to detect feeding through such physiological data improves confidence in estimates of foraging success. The intervals of feeding success observed from endothermic animals such as tunas are probably quite reliable, and provide some indication of reasonable feeding/fasting intervals in other highly migratory pelagic fishes.Across all striped marlin, a mean of nearly 3 weeks separated periods of ARB activity. Although up to 50 days separated these periods it should not be assumed that this is truly reflective of foraging frequency given the range of prey types commonly pursued. From visceral warming in archival tagging data, Bestley et al.New Zealand's recreational catch rates of striped marlin peak around March and begin to diminish around April Lampris guttatus) by a warm bodied shark (ie. a lamnid shark) Movement out of range of domestic fishing vessels may also contribute to the particularly low recapture rates (0.52%) of striped marlin in the New Zealand cooperative gamefish tagging program et al.Kajikia albidus) Makaira indica) Makaira nigricans) Time-series analysis indicates that the probability of occurrence of ARB and transiting behaviours was not equal (50∶50) until 16 days after tagging. Bestley We hypothesize that capture and/or tagging triggered migration in at least half of striped marlin sooner than would be expected if they were not captured and tagged. Since it is not possible to know the long-term movements of individual untagged fish, the primary difficulty in assessing the effects of capture and tagging is the challenge of gathering behavioural control data. An approach to obtaining such data is free-tagging uncaptured fish . If substantial differences in behaviour are observed between captured and uncaptured striped marlin, this would suggest that the capture process contributes to departures. Two striped marlin have been free-tagged with a PSAT by blue-water spear fishers to investigate movements of uncaptured fish. One provided short-term data which are suggestive of different movement patterns see , and datet al.A comprehensive investigation into the effects of exhaustive exercise on post-release survivorship and blood-borne stress indicators on sharks, tunas and marlin found compelling evidence that recreationally caught gamefish species are physiologically compromised after release Because the effects of capture and tagging on animals behaviour and fitness are not well understood, further investigation is merited. Furthermore, capture effects that trigger early departure from the tagging region could bias spatio-temporal data used in management and stock assessment.Utilization of the water column by striped marlin is mediated heavily by two primary oceanographic conditions, oxygen saturation and temperature relative to the surface. The maximum depth reported from PSAT tagging in the ETP was 192 m, with only 9% (4/45) of individuals reported to exceed 150 m depth Among the uncertainties of the first southwest-Pacific stock assessment was vertical distribution which effects vulnerability to fishing activity Gear depth is a consideration in recreational catch rates as well. Recreational billfishers usually troll for billfish at the surface, but we show how fishing activity targeted there would probably fail to attract the attention of marlin when they move deeper. This is a likely explanation for the marlin bite turning on and off, and innovations to recreational fishing techniques and gear might provide new opportunities to increase catches at depth.ie. increased mixed layer depth and temperature, decline in oxygen saturation, changing upwelling/downwelling conditions, etc.) might cue these behaviour changes. The probable influence of oceanography on population structure and stratification demonstrates how environmental factors can be important inputs in population dynamics models A 10–20° latitudinal band north and south of the equator is commonly recognized as a break in the geographic distribution of adult striped marlin in the central and western Pacific Figure S1Joint probability densities of errors for CTCRW regularized uKFSST geolocations from transmitted PSAT data.(TIF)Click here for additional data file.Figure S2Effects of CTCRW regularization on double-tagged striped marlin STM06.14. The black line and points are raw location data, with triangles representing uKFSST location estimates and circles representing Argos locations from the SLRT tag. Red represents the smoothed CTCRW pathway.(TIF)Click here for additional data file.Figure S3Striped marlin trajectories by season 2005–2008 after CTCRW regularization.(TIF)Click here for additional data file.Figure S4Controlling for striped marlin capture effects. Red vector (red inset box): Travel distance and direction over 8 day PSAT deployment from speargun. Black vectors: Travel distance and direction over initial 8 days at liberty for six individuals captured with standard recreational fishing methods.(TIFF)Click here for additional data file.Table S1Multiplier function K values used as error model parameters used in CTCRW regularization.(XLS)Click here for additional data file.Table S2Estimates of t-distribution parameters from non-linear minimization for uKFSST longitude and latitude errors.(XLS)Click here for additional data file.Table S3Density function parameters estimated by maximum likelihood for each individual.(XLS)Click here for additional data file.Appendix S1Temporal regularization and behavioural classification of striped marlin trajectories.(DOC)Click here for additional data file.Appendix S2Comparison of free-tagged to recreationally captured and tagged striped marlin movements.(DOC)Click here for additional data file.


Cetacea, odontoceti) use biosonar to navigate their environment and to find and catch prey. All studied toothed whale species have evolved highly directional, high-amplitude ultrasonic clicks suited for long-range echolocation of prey in open water. Little is known about the biosonar signals of toothed whale species inhabiting freshwater habitats such as endangered river dolphins. To address the evolutionary pressures shaping the echolocation signal parameters of non-marine toothed whales, we investigated the biosonar source parameters of Ganges river dolphins (Platanista gangetica gangetica) and Irrawaddy dolphins (Orcaella brevirostris) within the river systems of the Sundarban mangrove forest. Both Ganges and Irrawaddy dolphins produced echolocation clicks with a high repetition rate and low source level compared to marine species. Irrawaddy dolphins, inhabiting coastal and riverine habitats, produced a mean source level of 195 dB (max 203 dB) re 1 µPapp whereas Ganges river dolphins, living exclusively upriver, produced a mean source level of 184 dB (max 191) re 1 µPapp. These source levels are 1–2 orders of magnitude lower than those of similar sized marine delphinids and may reflect an adaptation to a shallow, acoustically complex freshwater habitat with high reverberation and acoustic clutter. The centroid frequency of Ganges river dolphin clicks are an octave lower than predicted from scaling, but with an estimated beamwidth comparable to that of porpoises. The unique bony maxillary crests found in the Platanista forehead may help achieve a higher directionality than expected using clicks nearly an octave lower than similar sized odontocetes.Toothed whales ( Bats and toothed whales have independently evolved a sophisticated biosonar system pp re 1 µPa @1 m Kogia sp.), and the Franciscana dolphin (Pontoporia franciscana) all use Narrow Band High Frequency (NBHF) clicks where energy is concentrated in a narrow frequency band around 130 kHz Toothed whale biosonar signals have been studied in captivity over the last 60 years and increasingly also in the wild. Studies of captive animals have contributed greatly towards our understanding of the biosonar performance Despite the many studies quantifying sonar parameters for free-living, marine toothed whales, much less variation in signal type or biosonar parameters has been found compared to bats, especially among delphinids. However, most of the delphinids studied to date forage in habitats that may differ less acoustically than is the case for the different bat guilds. Instead it seems that an inverse scaling of frequency with body mass to achieve a similar directionality may be a major driving force across the toothed whale suborder Orcaella brevirostris) are freshwater cetaceans living in shallow coastal waters, generally associated with freshwater inputs, as well as far upstream in three large, Indo-Pacific river systems. The extent of their inland range in the Sundarbans varies with seasonal freshwater regimes Platanista gangetica gangetica) are obligate freshwater dolphins found in the Ganges, Brahmaputra and Karnaphuli river systems where they exhibit a peculiar, side-swimming form of locomotion To address this question, we studied two species of toothed whales that co-occur in waterways of the Sundarbans mangrove forest of Bangladesh . IrrawadIn this study, we quantify the biosonar source parameters of Ganges river dolphins and Irrawaddy dolphins to test this hypothesis. We show that these animals use consistently lower source levels and higher repetition rates than oceanic delphinids, possibly limited by high amounts of clutter and reverberation. We demonstrate that Ganges river dolphins have a slightly broader beamwidth than other toothed whales due to their very low centroid frequency but that they achieve a higher directionality than expected from a direct scaling with centroid frequency and size, possibly by using a novel set of bony plates in the forehead. We conclude this study by discussing means to use acoustics to help better understand the conservation needs of these highly endangered freshwater toothed whales.th and16th of February 2010 from a 12 m long, wooden research boat. All research was conducted under a research permit issued to the Bangladesh Cetacean Diversity Project of the Wildlife Conservation Society by the Ministry of Environment and Forest, Government of Bangladesh.Recordings were obtained in the waterways of the Bangladesh part of the Sundarban mangrove forest  where redB re µPa (peak), and the frequency response of the recording chain was flat (±2 dB) from 2–180 kHz.A vertical array of four Reson TC4034 spherical hydrophones  was formed by mounting hydrophones in a Perspex rod  with 0.75 m spacing. The first hydrophone was positioned at 2 m depth while the last hydrophone was at 4.25 m depth. A buoy was attached to the top of the array, and a 4 kg weight was fixed to the bottom to help maintain the array vertical in the water. Signals were amplified 60 dB by a custom-made amplifier and filter box (1 kHz 1-pole high-pass and 200 kHz 4-pole low-pass filter), then digitized by two synchronized National Instruments USB-6251 A/D converters  at a sampling rate of 500 kHz per channel and a resolution of 16 bits. The calibrated clip level of the recording chain was 174Ganges river dolphins were recorded while foraging or resting at the convergences of channels. Irrawaddy dolphins were recorded during different behaviors . The boat engine was turned off and the array was lowered into the water once the animals were within about 100 m of the vessel. Data acquisition was initiated and terminated manually and files were stored approximately every minute. Start and end time, position and depth were recorded for every recording event, as well as group composition and behavior.Signal analysis was carried out with custom-written routines in Matlab 7.5 . Each click series  was examined visually and discarded if more than one animal was present to avoid underestimating interclick intervals. Echolocation clicks were then located on the third hydrophone using an automated click detector with a variable detection threshold chosen during visual inspection of waveforms to exceed the background noise level and detect individual click series. Each click was further analyzed only if detected on all four channels.Source location relative to the hydrophones was obtained through acoustic localization techniques based on time-of-arrival differences of the same click on the four receivers Acoustic localization with this array was calibrated in Aarhus Harbour, Denmark, using artificial clicks (2 cycles at 70 kHz) generated by an omnidirectional HS70 hydrophone (Sonar Products) connected to a waveform generator . Pulses were emitted from a depth of 2 m and at distances from 5 m to 40 m from the array. Speed of sound during this calibration was calculated using the Leroy equation The interclick interval (ICI) was defined as the time between each click and the previous Off-axis signals are subjected to distortion To evaluate the use of sound source parameters for passive acoustic monitoring studies without the potential for identifying on-axis clicks, a set of click series with only one clicking animal was identified. Each of these click series was passed through an automatic click detector (described above) to find accurate inter-click intervals for the two species. Subsequently, the power spectrum of each click was analyzed to find the centroid frequency.Irrawaddy dolphins were recorded on 16 different occasions during a total of 9 hours, 58 minutes of recordings. The median group size encountered during recordings of Irrawaddy dolphins was 3 animals. During recordings, this species was observed while foraging and travelling. Ganges river dolphins  were recorded in two different occasions and a total of 57 minutes of recordings were obtained from these encounters. In both recording occasions, the Ganges river dolphins were located in channel convergences.The hydrophone localization calibration indicated that clicks within 40 m were localized with a resulting error in the transmission loss estimates of less than 3 dB , which wA total of 15 Irrawaddy dolphin and 29 Ganges river dolphin clicks met the on-axis criteria and were recorded within the localization range of 40 meters. Only one click from each scan was used for analysis, and all recording areas were well separated to prevent recording the same groups of animals repeatedly. Clicks for both species were broadband transients  similar Tursiops aduncus) recorded in a 5–8 m shallow bay   . Peak-toolphins) . The cenInterclick intervals were measured for both species for all on-axis clicks. The ICI values for on-axis clicks were higher than ICI values measured across entire click series. Interclick intervals (mean±SD) for Irrawaddy dolphin on-axis clicks was 44.8±24.6 ms and for Ganges river dolphin on-axis clicks it was 35.0±18.4 ms . In addiNeophocaena phocaenoides asiaeorientialis) was calculated using peak frequency (comparable to centroid frequency for narrowband high frequency species) and standard deviations from Li et al. To test the potential for species discrimination in passive acoustic monitoring, probability density functions for Ganges river dolphin and Irrawaddy dolphin centroid frequencies were calculated using means and standard deviations from this paper, and assuming a normal distribution. In addition, a normalized probability density function for the Yangtze finless porpoise species  Echolocating toothed whales normally wait until the echo from a potential target has been received before producing a new click, meaning that the interclick interval between clicks exceeds the two-way travel time plus a processing lag time Feresa attenuataTursiops sp.Lagenorhynchus albirostrisStenella longirostris) and spotted dolphins (Stenella attenuata) Lagenorhynchus obscurus, max 210 dB pp Concurrent with the higher repetition rates, the two species also produced echolocation signals with much lower source level compared to similar sized marine delphinids. Irrawaddy dolphins (mean source levels ± SD of 194.7±4 dB re 1 µPa pp at 1 m) and Ganges river dolphins (183.6±3.5 dB re 1 µPa pp at 1 m) echolocate at more than 10 dB to 20 dB (respectively) lower source levels than other small, oceanic delphinids such as free-ranging pygmy killer whales (EFD)for harbor porpoises (Phocoena phocoena)  with somewhat intermediate source levels  with source levels as low as Ganges river dolphin (mean SLEFD: 125 dB re 1 µPa2*s Orcinus orca) odontocetiThe overall body size of many oceanic delphinids is larger than the animals studied here, and it is possible that this size difference could account for the lower source levels of our animals. Indeed, large echolocating animals tend to produce echolocation clicks at high source levels  and scalOne important challenge that these animals face is the task of locating and catching food in an acoustic habitat with high reverberation and clutter levels. Several studies have shown how close proximity to clutter Tursiops aduncus) in a shallow coastal habitat, compared to deep-water common bottlenose dolphins (Tursiops truncatus) Macrophyllum macrophyllum) gradually decreased the source levels of its echolocation calls when operating in three increasingly cluttered environments. Clutter-imposed constraints from such habitats may have resulted in microchiropteran bats having specialized into guilds inhabiting different foraging niches If reverberation can play an important role in shaping the source levels of echolocating toothed whales, this might also explain the lower source levels found for the Indo-pacific bottlenose dolphins (Delphinapterus leucas) Pseudorca crassidens) A central component in the high source levels of toothed whales is the production of a narrow echolocation beam through partial collimation of the acoustic energy Platanista gangetica gangetica, Orcaella brevirostris and Neophocaena phocaenoides. The on-axis biosonar centroid frequencies of these species are well separated, and spectral parameters may be a promising way of both detecting and discriminating these animals acoustically  Acoustic monitoring has proven to be a powerful method for determining range, seasonality, and abundance of animals pp and Ganges river dolphins, living exclusively in a shallow river habitat, produce even lower source levels of 183.6 dB (max 191) re 1 µPapp. The ultimate cause of these low source levels may be a relaxed selection for long-range echolocation inhabiting restricted, shallow, geomorphically complex river systems, with limits on echolocation range imposed by reverberation and clutter. Interestingly, the centroid frequency of the clicks used by Ganges river dolphins is almost an octave lower than expected from their size. The unusual, air-filled bony maxillary crests found in this species may compensate in part for this lower frequency by providing a larger effective baffle and hence a more directional sound beam than the biosonar frequency and head size would predict. The beamwidth of Ganges river dolphins is still wider than most other toothed whales, and it is possible that this may facilitate capture of highly maneuverable prey items in shallow water. Acoustic discrimination between freshwater odontocetes may facilitate acoustic monitoring efforts and may help prevent a continued decline of these two threatened freshwater species.Irrawaddy dolphins and Ganges river dolphins within the river systems of the Sundarban mangrove forest use high repetition rate, low source level echolocation clicks compared to marine species of similar size. Whereas obligate marine delphinids use high source level echolocation signals, Irrawaddy dolphins, inhabiting coastal and upriver habitats, produce lower source levels, with mean source levels of 194.7 dB (max 203 dB) re 1 µPa


The vast majority of glomerular filtrated phosphate is reabsorbed in the proximal tubule. Posttransplant phosphaturia is common and aggravated by sirolimus immunosuppression. The cause of sirolimus induced phosphaturia however remains elusive. Male Wistar rats received sirolimus or vehicle for 2 or 7 days (1.5mg/kg). The urine phosphate/creatinine ratio was higher and serum phosphate was lower in sirolimus treated rats, fractional excretion of phosphate was elevated and renal tubular phosphate reabsorption was reduced suggesting a renal cause for hypophosphatemia. PTH was lower in sirolimus treated rats. FGF 23 levels were unchanged at day 2 but lower in sirolimus treated rats after 7 days. Brush border membrane vesicle phosphate uptake was not altered in sirolimus treated groups or by direct incubation with sirolimus. mRNA, protein abundance, and subcellular transporter distribution of NaPi-IIa, Pit-2 and NHE3 were not different between groups but NaPi-IIc mRNA expression was lower at day 7. Transcriptome analyses revealed candidate genes that could be involved in the phosphaturic response. Sirolimus caused a selective renal phosphate leakage, which was not mediated by NaPi-IIa or NaPi-IIc regulation or localization. We hypothesize that another mechanism such as a basolateral phosphate transporter may be responsible for the sirolimus induced phosphaturia. The kidney is the major regulator of extracellular phosphate homeostasis. Phosphate is freely filtered in the glomerulus and mostly reabsorbed along the proximal tubule (PT) according to the organisms' needs to maintain a balanced serum phosphate. To date, three distinct sodium dependent phosphate cotransporter with a pivotal role for phosphate reabsorption in the kidney have been identified in the brush border membrane (BBM) of proximal tubule cells: NaPi-IIa (SLC34A1), NaPi-IIc (SLC343) and Pit-2 (SLC20A2) Sirolimus is a potent inhibitor of the mammalian target of rapamycin (mTOR) and routinely used after solid organ transplantation to prevent rejection. mTOR has long been known for its pivotal role in regulating cell proliferation and cell growth. More recently it has been shown in the heterologous Xenopus oocyte expression system to be involved in the regulation of various solute carrier such as the creatinine transporter SLC6A8 and the renal and intestinal sodium dependent phosphate cotransporters NaPi-IIa (SLC334A1) and NaPi-IIb (SLC34A2) Male Wistar rats  were randomly divided into four groups: groups 1 and 3 received vehicle, groups 2 and 4 sirolimus. Each group consisted of six animals and we used samples from each rat for all experiments. Daily subcutaneous injections of sirolimus (1.5 mg/kg body weight) were given for either 2 days (group 2) or seven days (group 4), whereas control animals received daily injections of vehicle for either two days (group 1) or seven days (group 3). Sirolimus  was dissolved in polyethylene glycol 400, 10% polysorbat 80 and 20% dimethylacetamid. Vehicle consisted of the three solvents. Rats were maintained on a standard rodent chow containing 0.8% phosphate  and had access to drinking water ad libitum. All animals were placed in individual metabolic cages for three and eight days, respectively, allowing a 24 hour adaption period to the metabolic cage environment. Food and water consumption, body weight, stool and urine output were monitored daily. Urine samples were collected daily under mineral oil. All animal experiments were performed according to national and international guidelines and laws of animal welfare. Protocols were approved by the local veterinary authorities .2 and calculated HCO3− measurement urine was aspirated from the collectors  into syringes and injected into the blood gas analyzer. Urinary creatinine was analyzed using the Jaffé method +, Na+, Cl,− Ca2+ and Mg2+ concentrations were measured using a chemistry analyzer  by examination of freezing-point depression. A test for low molecular weight proteinuria was performed. Urine samples were normalized for creatinine and samples containing 10 mg creatinine were then solubilized in Laemmli sample buffer and SDS-PAGE was performed on a 15% polyacrylamid gel. Colloidal coomassie blue staining was performed for 1h at room temperature. After destaining for 2h, the gel was photographed and dried. Five micrograms of BSA served as a positive control.At the end of the experiments rats were anesthetized by inhalation of Isoflurane/air and heparinized arterial blood was collected from the tail artery and immediately analyzed for pH, blood gases, and electrolytes on a Radiometer ABL 800 Flex blood gas analyzer . Serum creatinine, serum phosphate and serum glucose concentrations were determined using the clinical chemistry analyzer Piccolo Xpress . Vitamin D plasma levels were determined using the 1,25 Dihydroxy Vitamin D RIA  according to the manufacture's protocol. The levels of intact fibroblast growth factor 23  intact parathyroid hormone  and soluble klotho  were measured by a two-site enzyme-linked immunosorbent assay in rodent serum samples, according to the manufacturer's protocol. Inter-assay and intra-assay coefficients of variation were below 5% for all laboratory analyses. Creatinine clearance was calculated from the measured values. Sirolimus blood levels were measured using HPLC-mass spectrometry [Ct(HPRT) _ Ct(gene)], where Ct is the cycle number at which the fluorescence intensity is above background levels (threshold).Total RNA from kidneys was extracted using the Qiagen RNeasy Mini Kit . Snap-frozen kidneys were homogenized in RLT buffer (Qiagen) supplemented with ß-mercaptoethanol to a final concentration of 1%. Subsequently, 200 µl of each homogenate were used for total RNA isolation according to the manufacturer's protocol. DNAse digestion was performed using the RNase-free DNAase set . Quality and concentration of the isolated RNA preparations were analyzed spectroscopically using the 2100 Bioanalyzer (Agilent Technologies) and the NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies). RNA samples were diluted to a final concentration of 100 ng/µl and 3 µl were used for cDNA preparation using the TaqMan Reverse Transcriptase Reagent Kit . Primers and probes  of all genes of interest were designed using Primer Express Software , and primers were tested by PCR with kidney cDNA and always resulted in a single product of the expected size (data not shown). Sequences of primers and probes are listed in 2+ precipitation technique as previously described Frozen kidneys were used to prepare total membrane proteins and brush border membrane vesicle (BBMV) using the MgTen micrograms of renal BBM protein or 35µg of total membrane protein was solubilized in Laemmli sample buffer and SDS-PAGE was performed on 8% polyacrylamid gels. Proteins were transferred electrophoretically from gels to polyvinylidene difluoride membranes . After blocking with 5% milk powder in tris-buffered saline containing 0.1% tween 20 for 60min, the blots were incubated with the respective primary antibodies: rabbit polyclonal anti-NaPi-IIa  2 and adjusted to 300 mosmol/l with sucrose) and 4% hydroxyethyl starch in saline . After ten minutes the fixative was washed out by perfusion with PBS. Kidneys were then removed and stored in PBS overnight at 4°C. Coronal slices of fixed kidneys were then frozen in liquid propane and cooled with liquid nitrogen and stored −80°C. Serial sections, 5 μm thick, were cut at −20°C on a cryomicrotome  mounted on thermo scientific superfrost plus glass slides , thawed, and kept in cold PBS until further processing for staining. Before immunofluorescence staining, sections were pretreated with blocking solution  for 60 min at room temperature. After blocking sections were incubated with the primary antibody overnight at 4°C either with a rabbit anti-rat antiserum against the NaPi-IIa protein Anesthetized rats were fixed by vascular perfusion through the left ventricle. The thorax was opened and the fixative was manually injected through a 50 ml syringe. The fixative consisted of 3% paraformaldehyde, 0.05% picric acid in 0.1M cacodylate buffer .+, Cl−, Ca2+ showed no significant changes, however, serum K+ was significantly lowered in sirolimus treated rats after two days . Urinary Na+/creatinine ratios ((mmol/l)/(mmol/l)) were significantly higher after two and seven days treatment . Cl−/Creatinine ratio ((mmol/l)/(mmol/l)) was significantly higher after seven days  but was not different after two days. Mg2+/Creatinine ratios and Ca2+/Creatinine ratios ((mmol/l)/(mmol/l)) were significantly higher after two and seven days . Only very weak overall correlations of urinary phosphate with magnesium, sodium and chloride, calcium and potassium were found . Urinary glucose/creatinine ratio ((mmol/l)/(mmol/l)) was significantly higher only after seven days .Mean body weight was similar in all groups at the beginning of the experiment and after two days of treatment with sirolimus but was significantly lower in sirolimus treated animals after seven days of treatment  . Sirolimre found . Whereas3 serum levels were not significantly affected by sirolimus treatment (32Pi uptakes in the absence and presence of phosphonoformic acid (PFA) were similar in sirolimus and vehicle injected rats after two and seven days and revealed no different characteristics in phosphate influx  mRNA and protein expression of NaPi-IIa and Pit-2 were unaffected by sirolimus treatment. Solely mRNA expression of NaPi-IIc was significantly down regulated after two and seven days. However, sirolimus did not alter protein expression of NaPi-IIc iii) the subcellular localization of these three transporters with predominant localization in the BBM of the early PT was not altered, iv) microarray analysis of renal transcripts did not detect changes in any mRNA related to the known phosphate transporters or proteins known to be involved in their expression or activity, and v) hormonal changes are consistent with compensatory adaptations but not with causing phosphaturia.Several lines of evidence support the conclusion that sirolimus did not induce phosphaturia by primarily acting on BBM phosphate transporters despite a significantly lower mRNA abundance of NaPi-IIc. i) the rates of NaOur results are in apparent contradiction with two recent reports suggesting that the mTOR pathway may regulate the renal and intestinal phosphate cotransporters NaPi-IIa and NaPi-IIb It has been previously shown that klotho may exert some phosphaturic effects independently from FGF23 We also performed whole kidney transcript anaylasis using microarrays to detect candidate targets of sirolimus that may participate in the induction of phosphaturia. However, the analysis of significantly altered transcripts did not reveal any genes with a clear relation to renal phosphate transporters or their regulation. Microarray data indicated downregulation of SLC17A4. This transporter belongs to the SLC17 superfamily of transporters with subgroups of urate and vesicular gluatamte transporters The direct target(s) of sirolimus causing phosphaturia remain(s) elusive. Our results suggest that direct dysregulation of BBM phosphate transporters can be excluded and that other mechanisms must be considered. Two alternative targets might be either basolateral PT phosphate exit pathways or phosphate transport mechanisms located in the more distal nephron. Earlier studies suggested the presence of phosphate absorbing mechanisms in the distal tubule, however, their functional significance, regulation, molecular identity, or even existence have remained elusive to date Sirolimus induces renal phosphate wasting and the absence of changes in the expression of all known renal phosphate transporters and normal BBM phosphate transport rates suggest an alternative mechansim. Moreover, the regulation of the major known phosphate regulating hormones, PTH, 1,25 Dihydroxycholecalciferol, FGF23 in response to hypophosphatemia was intact and suggests rather compensatory adaptation. Sirolimus might affect other mechanisms that could contribute to overall renal phosphate handling such as either basolateral exit pathways for phosphate in the PT or other elusive transport pathways in downstream nephron segments. Clearly, further studies are needed to unravel the molecular mechanisms causing sirolimus induced phosphaturia.Data S1Supporting Information(DOCX)Click here for additional data file.


Loa loa (Ll), Onchocerca volvulus (Ov), and Mansonella perstans (Mp).The current antibody tests used for monitoring in lymphatic filariasis (LF) elimination programs suffer from poor specificity because of the considerable geographical overlap with other filarial infections such as W. bancrofti (Wb), these were next assessed for homology to known proteins and nucleotides and to similar assemblies of L3 larval ESTs of B. malayi (Bm – n = 5068), Ov (n = 4166), and Ll (n = 3315). Nineteen potential L3- and Wb- and/or Bm-specific antigens were identified. Sixteen of the 19 antigens could be expressed as fusion proteins with Renilla luciferase (Ruc); these were used in a rapid Luciferase Immunopreciptation System (LIPS) assay.Using bioinformatics to assemble into contigs 2048 expressed sequence tags (ESTs) from the L3 infective larvae of One of the 16 expressed antigens (Wb123) was both highly immunogenic and specific for Wb. Using Wb123-based IgG and IgG4 LIPS assays on well-defined sera from normal North Americans and those infected exclusively with intestinal helminths, we could detect all of the Wb-infected individuals (from diverse geographic regions) with 100% sensitivity and 100% specificity. Using sera from exclusively Ll-infected, Ov-infected Mp-infected or Bm-infected subjects as the negative comparator, the sensitivities were between 98–100% and the specificities ranged between 84–100% (for IgG anti-Wb123) and between 98–100% (for IgG4 anti-Wb123). Blinded assessments using panels of sera from various Wb-, Bm- or non-Wb helminth-infected subjects demonstrated equally high degrees of sensitivity and specificity.Wb infections and as a sensitive method for early detection of recrudescent infections in areas of control and for mapping new areas of Wb transmission.We have identified a Wb-encoded antigen that can be used both as a rapid, high throughput tool to diagnose individual  Wuchereria bancrofti (Wb), a causative agent of lymphatic filariasis, following mass drug administration (MDA) control measures, we used a novel bioinformatic approach to identify Wb-specific antigens expressed primarily by the infective stage larvae of Wb that could be used as the basis for a rapid, high throughput antibody assay. From 19 potential candidates, we identified one, termed Wb123, that could be used as the basis for the rapid detection of IgG and IgG4 reactivity in a luciferase immunoprecipitation system (LIPS) assay. These anti-Wb123 IgG and IgG4 antibodies were only present in those with Wb infection and not in those infected with closely related filarial parasites  that share the same geographical niches with Wb. Our data suggest that this rapid Wb123 assay has the potential to be used in for large scale detection of recrudescent Wb infection and thereby may provide a new tool for the monitoring of transmission control measures.To address an unmet need for a surveillance tool to detect transmission of  Wuchereria bancrofti (Wb), Brugia malayi (Bm) and Brugia timori can lead to disfiguring and disabling lymphedema and elephantiasis. Past and ongoing control measures, aimed at interrupting transmission by eliminating the reservoir of infection  has led to substantial decreases in the prevalence of infection and the risk of disease Loa loa [Ll], is co-endemic As one of the neglected tropical diseases (NTDs), lymphatic filariasis (LF) caused by the filarial parasites O. volvulus (Ov) L3-expressed antigen, Ov16 MDA programs are in progress in more than 50 of the 72 LF-endemic countries, with 13 having stopped following at least 5 annual MDA treatments Mansonella spp.) often co-endemic with Wb in Africa and parts of the Americas will likely limit their utility in these regions.A number of immunoassays using a variety of different filarial Ags have been proposed for use as surveillance tools in LF; these include Bm14/BmSXP-1 In the present study, we utilized a bioinformatics approach to identify potential antigens that are expressed in post-parasitic L3s, that would be Wb- (and/or Bm-specific), and that in turn could be used in a rapid, high throughput format to provide a sensitive method for early detection of recrudescent infections in areas of control, for mapping new areas of transmission of Wb infection, and potentially for certifying regions/countries as free of transmission.All samples used were acquired under a number of registered protocols that were approved by the Institutional Review Board of NIAID with the majority being collected under either NCT00001230, NCT00342576. Written informed consent was obtained from all subjects prior to collection of the samples.Brugia malayi L3 ESTs and Wuchereria bancrofti L3 ESTs  were assembled into contigs using the Desktop cDNA Annotation System (dCAS 1.4.3) software package Wuchereria bancrofti (Wb) and/or Brugia malayi (Bm) and that were without significant homology to the related filariae (Loa loa [Ll] and Onchocerca volvulus [Ov]). Contigs were selected for further evaluation as candidate assay targets based on: 1) length of at least 200 bp with a predicted open reading frame (ORF); and 2) lack of sequence homology to the non-redundant protein database (nr) and other stages  of Bm or Wb  was synthesized commercially  with codon usage optimized for expression in mammalian cells. Using insert specific primers containing BamH1 and Xho1 modifications, each of these 19 DNA inserts was amplified and cloned into the BamH1/Xho1 site of pREN2, a mammalian Ruc expression vector described previously Serum samples  from pat2, 1% Triton X-100) in a 96-well polypropylene microtiter plate  and was added to 50 µl of 1×107 LU of Ruc-antigen in polypropylene plates. The plate was incubated for 5 minutes at room temperature after which the material was added to 7 µl of a 30% suspension of protein A/G beads in PBS  in a 96-well filter HTS plate . After 5 minutes, the filter plate containing the mixture was then applied to a vacuum manifold and washed twice in assay buffer and 8 times with PBS. After the final wash, all plates were processed on a Berthold LB 960 Centro microplate luminometer using a colenterazine substrate mix . All data were the average of duplicates corrected for background reactivity (no serum added).The LIPS assay detects IgG or IgG4 antibodies to an antigen (Wb123) and was performed exactly as described previously For anti-IgG4 antibody determinations, the same protocol was utilized with anti-IgG4 antibody beads substituted for protein A/G beads. The anti-IgG4 antibody beads were generated by combining 10 mg of an anti-IgG4 monoclonal antibody  with Ultralink pre-activated beads (Pierce Biotechnology), as described by the manufacturer. The coupling efficiency was greater than 90%.The Wb123 amino acid sequence was run against databases for other filariae using the basic alignment search tool (BLAST). This sequence was then to aligned to the related filarial sequences using ClustalW U test was used for comparison of antibody levels in different groups. The Spearman rank correlation was used for correlation analyses. All statistical analyses were performed using GraphPad (v5).Geometric means were used as measurements of central tendency. For determining the cut-off limits receiver operating characteristic (ROC) curves were used. ROC analysis originates from signal detection theory as a model of how well a receiver is able to detect a signal in the presence of noise. Its key feature is the distinction between a true positive rate (sensitivity) and a false positive rate (specificity). The non-parametric Mann-Whitney W. bancrofti (n = 2048), B. malayi (n = 5068), L. loa (n = 3351) and O. volvulus (n = 4168) were each assembled into contigs by dCAS analysis . From these, 19 potential candidates were identified by virtue of having limited similarity to all publicly available nematode ESTs, to the non-redundant protein database (nr) and to clustered ESTs derived from filarial microfilariae and adult parasites. Each candidate transcript included a start codon and stop codon separated by at least 200 base pairs, indicating a potential open reading frame (ORF). The 19 candidates (and their annotations) are shown in L3-derived ESTs from W. bancrofti-specific in preliminary testing.Sixteen of these 19 were successfully cloned in pREN2 and used to transfect COS1 cells. The lysates were used in a standard LIPS format, and only 1, termed Wb123 (Genbank Accession number HQ438580), was shown to be both immunogenic and Wb123 contains 372 amino acids. The annotation of its sequence suggested a “hypothetical protein,” but it likely belongs to the serpin family of proteins. These are serine protease inhibitors that have been shown to be secreted proteins in filarial species other than Wb, often L3 enriched, and have also been shown to be highly immunogenic in humans. Although there are human serpins, these share little to no homology to Wb123 (data not shown). Amino acid sequence alignment between Wb123 and homologous sequences in Bm (82% identity), Ov (35% identity), and Ll (61% identity) is shown in To assess the diagnostic utility of Wb123, sera from 52 uninfected normal North Americans were compared to sera from 42 Wb-infected individuals using assays that detected either IgG or IgG4 antibody against Wb123. By ROC curve analysis , a cutofMansonella perstans (Mp)- and other non-filarial helminth-infected individuals . There was, however, a small degree of IgG cross-reactivity (4/38 samples for Ov and 5/62 samples for Ll). In contrast, the IgG4 Wb123 assay  IgG reactivity in the Wb123 LIPS despite the relatively high degree of conservation between Wb123 and the Bm123 homologue  and followed for up to 17 years with neither having returned to a Wb endemic country. As can be seen in W. bancrofti following mass drug administration (MDA) in areas of the world  where the geographical overlap of Ov, Ll, and Mp infections has caused significant cross reactions to antigens currently being used in antibody-based assays. Wb123, a serpin-like Wb-encoded protein, appears to be a good candidate for immunoassays based on the extraordinarily good sensitivity and specificity observed with this antigen in the LIPS format. Not only can Wb123-based assays distinguish Wb-infected individuals from uninfected individuals, but they can also be used to distinguish Wb infection from infections with the closely related filarial parasites, Bm, Ll, Ov, and Mp. Furthermore, the Wb123 LIPS assay is a convenient diagnostic for field use as it provides a rapid, high throughput format that requires minimal serum volume (1 uL per test) and can be used with serum, whole blood, filter spotted material, or saliva (data not shown).The purpose of this study was to identify a Wb- and L3-specific antigen that could potentially be used to monitor changes in transmission of et al). Thus, the rapid-format Wb123 LIPS can provide early identification of new infections and, by inference, ongoing transmission. The Wb123 LIPS test could be used for rapid screening of populations and thereby provide a means by which control programs could be more accurately monitored.Unlike PCR, microscopy, and antigen detection assays, antibody detection assays in LF cannot definitively identify active infection, nor can they distinguish between past and current infection. However, antibody tests can be used for surveillance activities to estimate infection prevalence rates W. bancrofti-endemic countries that are also endemic for other filarial infections, particularly Ll, Ov, and Mp. Although there have been developed many sensitive antibody assays One of the greatest needs of the GPELF is for a highly sensitive and specific surveillance tool to monitor, in sentinel populations Wb infection by antibody profiling with high diagnostic sensitivity and specificity. The high-throughput, rapid  formats used here make these approaches highly feasible for screening large numbers of sera samples and/or for rapid large scale field-testing. Similar to our previous published results with Ss This study also demonstrates that Wb123 LIPS can detect W. bancroftibefore the implementation of MDA programs. Therefore, the target population for this field (or region)-applicable Wb123 LIPS (and other immunoassay formats such as ELISA and/or Luminex using IgG4 detection strategies) would be school age children (6–7 year olds), a population that would provide an efficient target for the detection of recent infection and (by inference) recent or ongoing transmission. Prospective testing of this concept is underway in Mali and American Samoa as are efforts to re-format the Wb123 LIPS assay into a true point of care diagnostic.Because levels of antibody to Wb123 slowly decrease after definitive treatment see , the Wb1Table S1Annotated Excel table ouput from dCAS of 19 antigens used as potential targets for Wuchereria bancrofti and/or Brugia malayi-specific antibody assays.(XLSX)Click here for additional data file.Table S2Composition of serum samples used for blinded analyses of IgG anti-Wb123 LIPS assay.(DOCX)Click here for additional data file.


In most cases of cervical cancers, HPV DNA is integrated into the genome of carcinoma cells. This mutational insertion constitutes a highly specific molecular marker of tumor DNA for every patient. Circulating tumor DNA (ctDNA) is an emerging marker of tumor dynamics which detection requires specific molecular motif. To determine whether the sequence of the cell-viral junction could be used in clinical practice as a specific marker of ctDNA, we analyzed a series of cervical cancer patient serums.Serum specimens of 16 patients diagnosed with HPV16/18-associated cervical cancer, and for which the viral integration locus had been previously localized, were analyzed. Sequential serum specimens, taken at different times during the course of the disease, were also available for two of these cases. ctDNA was found in 11 out of 13 patients with tumor size greater than 20 mm at diagnosis, and analysis of sequential serum specimens showed that ctDNA concentration in patients serum was related to tumor dynamics.We report that HPV mutational insertion constitutes a highly specific molecular marker of ctDNA in HPV-associated tumor patients. Using this original approach, ctDNA was detected in most cervical cancer patients over stage I and ctDNA concentration was found to reflect tumor burden. In addition to its potential prognostic and predictive value, HPV mutation insertion is likely to constitute a new molecular surrogate of minimal residual disease and of subclinical relapse in HPV-associated tumor. This is of major importance in the perspective of specific anti-HPV therapy. Specific types of human papillomaviruses (HPV) are recognized as the major etiological factor in cervical neoplasia The characteristic clonality, specificity and stability over time of HPV DNA insertion into the genome of carcinoma cells constitute a highly specific genetic marker of tumor DNA. Recent data have shown that mutant DNA could be detected in the blood of patients with tumors and that the amount of circulating tumor DNA (ctDNA) was related to tumor dynamics KLK3 gene status was taken as a two copies reference.From a series of HPV16 (14 cases) or HPV18 (2 cases) cervical carcinoma accumulated between 2001 and 2011, we determined the viral insertion locus using the DIPS-PCR method 2 concentrations are given in the DNA was isolated from 200 µl of serum with QIAamp Min Elute Virus Spin Kit  following the manufacturer's instructions. The elution step was performed with 25 µl of supplied buffer. Isolated DNA concentrations were measured using a Real-Time quantitative PCR (RT-qPCR) system based on Long Interspaced Nuclear Elements (LINE) sequence detection 6 to 24 106 HPV16 copies/µg DNA). The concentration of circulating host DNA ranged from 6 101 to 77 103 copies/ml serum (3 copies/ml serum (3 copies/ml) corresponded to cases with high viral load in tumor biopsy specimens, suggesting that DNA derived from episomal molecules was also detected by the assay.Clinically, FIGO stages were from Ib to IVa and one case was a pelvic relapse of cervical SCC Table 1.ctDNA dynamics were analyzed on sequential serum specimens in two patients. The first patient received combined chemoradiotherapy followed by utero-vaginal brachytherapy and surgery Click here for additional data file.


The maintenance of genomic integrity requires the precise identification and repair of DNA damage. Since DNA is packaged and condensed into higher order chromatin, the events associated with DNA damage recognition and repair are orchestrated within the layers of chromatin. Very similar to transcription, during DNA repair, chromatin remodelling events and histone modifications act in concert to ‘open’ and relax chromatin structure so that repair proteins can gain access to DNA damage sites. One such histone mark critical for maintaining chromatin structure is acetylated lysine 16 of histone H4 (AcH4K16), a modification that can disrupt higher order chromatin organization and convert it into a more ‘relaxed’ configuration. We have recently shown that impaired H4K16 acetylation delays the accumulation of repair proteins to double strand break (DSB) sites which results in defective genome maintenance and accelerated aging in a laminopathy-based premature aging mouse model. These results support the idea that epigenetic factors may directly contribute to genomic instability and aging by regulating the efficiency of DSB repair. In this article, the interplay between epigenetic misregulation, defective DNA repair and aging is discussed. The genomes of organisms are organized in the form of a fundamental structure called chromatin in which the repeating nucleosomes form the basic unit. The nucleosome consists of 147 bp of DNA wound 1.7 times around an octamer composed of the four core histones, H2A, H2B, H3 and H4. Multiple nucleosomes are further linked by DNA stretches that are occupied by linker histone H1, to form the 10-nm fibre or ‘beads on a string’ type of arrangement. Chromatin fibres undergo compaction through intramolecular nucleosome-nucleosome interactions to form the 30 nM chromatin fibres. At the next level of organization, chromatin is further stacked and folded to give rise to 100-400 nm interphase chromatin fibres. The DNA that is eventually folded into chromosomes has already undergone compaction by about 10,000 fold. The packaging of DNA into condensed and often inaccessible chromatin imposes a significant constraint for the efficient repair of DNA double strand breaks (DSBs). Recent efforts have been directed towards understanding how the DNA repair proteins gain access to and repair, when DNA damage is embedded within chromatin fibres.Cells utilize two distinct mechanisms to modulate chromatin dynamics during DNA repair. These mechanisms include the post translational modifications of histones and ATP-dependent chromatin remodelling. According to the ‘histone code hypothesis’, the biological outcome of histone modifications is manifested by providing a signalling platform for the recruitment of downstream effector and reader protein or by the physical modulation of chromatin structure . AccordiIt is well known that histone acetylation is associated with a more ‘open’ configuration of DNA, because histone acetylation imparts a negative charge that causes the charge repulsion of negatively charged DNA. The acetylation of H2AX at K5 position by Tip60 regulates the ubiquitination of H2AX at K119 and enhances chromatin dynamics . Since tThe direct recruitment of ATP-dependent chromatin remodelling complexes that enzymatically modulate chromatin structure is another important mechanism in DNA repair. Chromatin remodelling complexes use ATP hydrolysis to increase accessibility of nucleosomal DNA by repositioning nucleosomes or by altering nucleosomal composition . There aIn other examples involving chromatin structure and DNA repair, DSB repair in the heterochromatin requires specialized mechanisms to promote access to repair proteins because heterochromatin is structurally inhibitory to DNA repair owing to its condensed structure. ATM-dependent phosphorylation of the co-repressor Kap1 was shown to allow localized and transient chromatin relaxation at regions of heterochromatin and promote the recruitment of DNA repair proteins . LikewisAmongst the most prominent phenotypes associated with defective DSB repair in both humans and mice, is the onset of accelerated aging (progeria). In several correlative studies, γ -H2AX foci containing senescent cells increase with age in humans, mice and primates, leading to the model that inefficient DSB processing and repair can activate cellular senescence pathways and initiate premature aging -31. Thisde novo point mutation of nuclear protein lamin A gene at position 1824 (C to T) in exon 11, was found to be predominantly responsible for this syndrome. Lamin A is synthesized as the precursor protein, prelamin A which is modified at its carboxyl-terminal through a series of post translational modifications. The modifications include farnesylation of the cysteine in the C-terminus CAAX -motif, followed by proteolytic cleavage of the AAX-peptide, and methylation of the farnesylated cysteine. The metalloproteinase, Zmpste24, is responsible for the sequential proteolytic cleavage of prelamin A into functional and mature lamin A. The point mutation identified from HGPS patients results in the activation of an aberrant cryptic splice site causing the deletion of a 50 amino acid region from the C-terminal end of prelamin A [In our earlier study involving human Hutchinson Gilford progeria syndrome (HGPS) patient fibroblasts, defective DNA repair and genomic instability was demonstrated . Clinicaelamin A . Since tZmpste24−/− lmna+/−) largely ameliorated the progeria-like phenotypes in Zmpste24-deficient mice, suggesting that reducing prelamin A concentration by 50% was enough to reduce accelerated aging phenotypes [Remarkably, human HGPS premature aging phenotypes can be recapitulated in Zmpste24-null mice indicating that the presence of 18 extra amino acid tail of prelamin A is responsible for premature aging . At the enotypes .The observation that Zmpste24-null and human HGPS patient cells have genomic instability was important in understanding the mechanisms contributing to premature aging . FibroblLamin A is a structural constituent of a subnuclear compartment called as the nuclear matrix . The nucIn a recent study, we have investigated the mechanism underlying defective DNA repair and premature aging in Zmpste24-null cells and identified a role for histone H4K16 hypoacetylation in aging . We hypoUsing a combination of imaging techniques and biochemical fractionation, it was found that Mof, the principle histone H4K16 acetyltransferase localizes to the nuclear matrix in wild-type cells. However, in prelamin A-expressing cells, the nuclear matrix association of Mof was found severely reduced. At the molecular level, lamin A co-immunoprecipitates with Mof, whereas prelamin A had several folds lower binding with Mof. These observations implied that the 18 amino acid extra C-terminal tail of unprocessable prelamin A that is lacking in mature lamin A, prevented Mof binding and resultantly, the association of Mof with the nuclear matrix was reduced causing histone acetylation defects.How do reduced global H4K16 acetylation levels affect chromatin structure? In elegant work by Shogren-Knaak et al., the incorporation of acetylated K16 of H4 into nucleosomal arrays was demonstrated to impede the ability of chromatin to form cross-fibre interactions and this converted chromatin into an ‘open’ conformation . In crysAs an extension of this idea, it was rationalised that global reduction of H4K16 acetylation may result in genome-wide chromatin compaction in Zmpste24-null cells, which has to be relieved first before repair proteins such as 53BP1 gain access to DNA damage sites. This model was tested by the re-introduction of Mof, which elevated H4K16 acetylation levels and dramatically promoted the recruitment of 53BP1 to sites of DSBs in Zmpste24-null cells. Consistently, as predicted, Mof knockdown had an opposite effect in that cellular senescence and accumulation of irreparable DNA damage exacerbated even further. Thus, an inverse correlation could be drawn between Mof function, DNA repair and premature senescence /M cell cycle arrest, massive chromosome aberrations, and defects in ionizing radiation-induced DNA repair were observed in Mof-null cells [The relationship between Mof, chromatin structure and DNA repair has been further expounded in three recent independent studies. Owing to global compaction of the genome, the chromatin structure of Mof-null MEFs was altered in a way such that the cells became refractory to DNA damage signalling and repair protein recruitment. In the study by Li ll cells . The recll cells . Importall cells . A purkill cells . TogetheInterestingly, upon careful analysis it is apparent that H4K16 acetylation levels begin to increase only at about 60 minutes post-irradiation (our unpublished observations), while it is known that the recruitment of repair proteins such as 53BP1 and MDC1 occurs temporally at earlier time points after irradiation. Since the recruitment of DNA repair proteins is found defective in Mof-null cells even at early time points after irradiation, it indicates that both basal as well as the DNA damage inducible-H4K16 acetylation levels are important to create the chromatin structure conducive for DNA damage recognition, DSB repair protein recruitment and further signalling.A simplified view of the above model is that promoting chromatin accessibility by increasing histone acetylation levels can improve DNA repair by increasing the recruitment of DNA repair proteins. HDAC inhibitors prove useful for this purpose, since they are well-established as anti-cancer agents and several pharmacological agents are validated to increase histone acetylation levels . Based oZmpste24-null mice or human HGPS cells have been considered as segmental progeroid syndromes, in that they only partially recapitulate the phenotypes associated with normal aging. Hence, it was believed that experiments conducted on premature aging syndromes may not accurately reflect physiological aging. This view is gradually changing owing to efforts made to detect progerin or prelamin A accumulation during normal aging. In the first such report, rare fibroblasts cultured from elderly individuals were found to exhibit nuclear abnormalities similar to HGPS cells . Progeriet al, genomic instability associated with normal aging was found associated with delayed recruitment of repair proteins to sites of DSBs [The above studies raise the exciting possibility that Zmpste24-null/HGPS fibroblasts may offer a simple model to decode the molecular defects contributing to normal aging. Interestingly, in a study by Sedelnikova  of DSBs . Since t of DSBs . An age- of DSBs . Put toget al, ‘old’ yeast cells were found to have increased amounts of H4K16 acetylation, due to decreased abundance of Sir2 deacetylase [In contrast to the above model, global H4K16 acetylation levels were found to increase in a Sir2-dependent manner during yeast replicative aging. In a study by Dang cetylase . The inccetylase . Also, ocetylase , and somcetylase , it is pA fundamental manner in which epigenetic misregulation in the form of DNA methylation and histone modification alterations can contribute to aging is by altering gene expression patterns. Since this has been the topic of discussion elsewhere , here weS.cerevisiae,C.elegans, mice and human cells, alterations in histone modifications and chromatin remodelling factors during aging have been studied. For example, S.cerevisiae cells undergo changes in histone levels and histone modifications with an increase in replicative age. As examples, total histone H3, H4 and H2A protein levels are greatly reduced in aged yeast cells relative to young cells [S.cerevisiae cells [C. elegans, members of the ASH-2 trithorax complex,ASH-2 itself, WDR-5 and the H3K4 methyltransferase SET-2 were found detrimental for longevity [C.elegans, and longevity could be extended with an HDAC inhibitor [Ink4a/Arf locus, it is now come to light that Bmi1 can also maintain genomic integrity by regulating H2A ubiquitination in response to DNA damage [Through the use of model systems such as ng cells . Anotherae cells , 74. In ongevity . In anotnhibitor . A globanhibitor . H4K20 tnhibitor . Recentlnhibitor . In a genhibitor . In an inhibitor . Anothernhibitor , 61. In nhibitor . The losnhibitor .Lastly, A damage . In summA damage . AnotherA damage . Thus, tAlthough it is known that the loss of genomic integrity is an important hallmark of aging, the molecular mechanisms are only beginning to be understood. Through the use of Zmpste24-knock mice as a model to understand premature aging, interesting insights have been obtained on how genomic integrity can be regulated by epigenetic mechanisms such as histone acetylation. H4K16 hypoacetylation affects global chromatin structure to impair DNA damage recognition and repair and this, in turn contributes to genomic instability during aging. Recent findings have indicated that the study of Zmpste24-null mice may have broader relevance in understanding physiological aging. Therefore, it would be interesting to find out if histone acetylation levels can also affect genomic integrity during normal aging. Given the inherent reversibility of epigenetic modifications, it would then be possible to manipulate histone acetylation levels and relax chromosome structure to promote DNA repair during normal aging and restrain some age-associated pathologies.In conclusion, a complete understanding on how exactly chromatin modifiers modulate DNA repair and aging at the molecular level is far from complete. Indeed, we are only beginning to see the ‘tip of the iceberg’, as far as chromatin, DNA repair and aging are concerned. It is anticipated that through the use of modern genome-wide technologies and mouse genetics, the study of epigenetics and aging will gain greater momentum.


A2BP1 gene at 16p13.3 and the MACROD2 gene at 20p12.1. However, neither RNA expression nor functional studies support a tumor suppressor role for either gene. Further analyses suggest instead that these are sites of increased genomic instability and that they resemble common fragile sites (CFS). Genome-wide analysis revealed properties of CFS-like recurrent deletions that distinguish them from deletions affecting tumor suppressor genes, including their isolation at specific loci away from other genomic deletion sites, a considerably smaller deletion size, and dispersal throughout the affected locus rather than assembly at a common site of overlap. Additionally, CFS-like deletions have less impact on gene expression and are enriched in cell lines compared to primary tumors. We show that loci affected by CFS-like deletions are often distinct from known common fragile sites. Indeed, we find that each tumor tissue type has its own spectrum of CFS-like deletions, and that colon cancers have many more CFS-like deletions than other tumor types. We present simple rules that can pinpoint focal deletions that are not CFS-like and more likely to affect functional tumor suppressors.One of the key questions about genomic alterations in cancer is whether they are functional in the sense of contributing to the selective advantage of tumor cells. The frequency with which an alteration occurs might reflect its ability to increase cancer cell growth, or alternatively, enhanced instability of a locus may increase the frequency with which it is found to be aberrant in tumors, regardless of oncogenic impact. Here we’ve addressed this on a genome-wide scale for cancer-associated focal deletions, which are known to pinpoint both tumor suppressor genes (tumor suppressors) and unstable loci. Based on DNA copy number analysis of over one-thousand human cancers representing ten different tumor types, we observed five loci with focal deletion frequencies above 5%, including the  KRAS or TP53, contribute to the selective advantage of tumor cells. However the case is less clear with DNA copy number alterations, where some frequent alterations such as amplification of the ERBB2/HER2 locus clearly provide a selective advantage, whereas others like the frequent deletions of DNA at the telomeric ends of chromosomes likely do not. This means that alteration frequency alone is not sufficient to determine whether or not a given DNA copy number alteration directly impacts oncogenicity. Nowhere has this been more difficult to tease out than for candidate tumor suppressor genes located within common fragile sites. Common fragile sites are found throughout the human genome and are prone to DNA breaks when the cell is exposed to partial replication stress WWOX, FHIT, and PARK2, and these functions include growth suppressive effects of restoring expression in deficient cell lines and loss-of-function mutations leading to enhancement of carcinogen-induced or genetically-engineered cancer in mice In human cancer it is generally the case that highly recurrent point mutations, such as those occurring in A2BP1 and MACROD2. In contrast to the results obtained by screening focal deletions in liver cancer, we did not find that these genes were tumor suppressors, which prompted us to take a genome-wide look at the properties of focally deleted genes in a large dataset of DNA copy number alterations affecting over 1000 cancer samples. This led to the discovery of two classes of focal deletions in human cancer, one that resembles deletions affecting common fragile sites and the other that resembles deletions affecting CDKN2A/B. Since then, a genome-wide examination of small homozygous deletions in 270 human cancer cell lines has been reported that also finds two classes of focal deletions Previously we discovered and validated 26 oncogenes by functionally screening sets of genes that are focally amplified in human cancer and similarly have validated 10 tumor suppressor genes that were found in focal deletions affecting liver cancer CDKN2A/B, FHIT and WWOX loci, known or suspected tumor suppressor genes, and the MACROD2 and A2BP1 loci  were very frequent in colorectal cancer (21%) but considerably less frequent or absent in other tumor types . Similarly, deletions affecting MACROD2 were most frequent in colorectal cancer (17%) but considerably less frequent or absent in other tumor types . Frequent deletions affecting A2BP1 and MACROD2 in colon cancer have been observed by others Our dataset was generated by array CGH analysis of 850 primary tumors and 304 cancer cell lines or xenografts of diverse tissue types including brain, breast, colon, liver, lung, ovarian, pancreatic, prostate, and skin (melanoma) . FollowiBP1 loci . DeletioA2BP1 and MACROD2 deletions on underlying gene expression in colon cancers and normal colon tissues. RNA expression of A2BP1 could not be detected by real-time RT-PCR in any of the normal colon tissues, tumors, or cancer cell lines that we examined . Additionally, the deletions affecting the A2BP1 and MACROD2 loci were more separated and the majority did not converge upon a single common site of overlap, in contrast to CDKN2A/B and PTEN loci .We noted several differences in the types and patterns of deletions affecting TEN loci . By examTEN loci . We measA2BP1 and MACROD2 loci on the one hand and CDKN2A/B and PTEN loci on the other hand held true when comparing deletions affecting known common fragile sites and recessive tumor suppressor genes from the Cancer Gene Census CDKN2A/B have been suggested to arise from aberrant recombination or DNA repair by nonhomologous end-joining We then wanted to determine if these two distinctions between deletions affecting MACROD2 was a more generalizable observation that could be used to distinguish common fragile site-like genes from tumor suppressor genes. We determined the correlation of RNA expression and DNA copy number for 115 cancer samples where we had both gene expression profiling and ROMA aCGH data. Compared to the correlations of the tumor suppressor group, there was very little effect of DNA copy number on gene expression in the common fragile site group  or in only one out of 255 primary tumors (MACROD2)  . FHIT waMACROD2 were relatively isolated along the genome and that the frequency count fell precipitously to the genes immediately left and right of MACROD2 . We first explored whether any of the six properties were redundant by determining if they were highly correlated with any of the other five. Although deletion size was moderately correlated with deletion separation (r = 0.39), all of the other pairwise correlations were negligible (ranging from −0.13 to 0.18), and we proceeded to include all six properties for unsupervised analysis. We then transformed the six properties using principal component analysis and plotted the 4,823 genes using the first three principal components. The resultant graph showed that the majority of genes were most similar to recessive tumor suppressor genes . With onMACROD2 or A2BP1, which, together with the known common fragile site gene gene FHIT, are the three genes most frequently affected by focal deletions in colon cancer. We designed a custom tiling array for these three genes and then performed aCGH, comparing the original cell line DNA to DNA from eight different clones isolated after brief induction of replicative stress. Four out of eight clones showed deletions within MACROD2, and two out of eight clones showed deletions within FHIT  under culture conditions, and the same may hold true for the increased incidence of deletions in other CFS-like genes when comparing cell lines to primary tumors.The frequency and spectrum of deletions in CFS-like genes was different for all tumor types when comparing cell lines to primary tumors . In coloy tumors . It seemCDKN2A/B and MAP2K4MEN1), and four are candidate tumor suppressors based on either mutational analysis  repository, accessions GSE31586 and GSE22916.We analyzed 850 tumor samples for this study . Many of200 ng of genomic DNA was used to make representations for whole-genome copy number analysis by ROMA array comparative genomic hybridization as described Common germline copy number polymorphisms (>5% frequency) were removed from the CBS segmented data by previously described methods The set of 10,835 focal deletions was used to develop metrics that were based on 2 Mb intervals (windows). For genes that were affected by 8 or more deletions, we constructed a set of windows that contained deletions based on the deletion start and stop positions, and assigned to the gene the values of the closest possible window (center of gene to center of the window). “Deletion Size” is the median length of all deletions that overlap the window. “Deletion Separation” is calculated by determining for each deletion how many other deletions in the window are separate (non-overlapping), then taking the average of these values, and then standardizing this score so that is independent of the number of deletions.Other metrics were calculated on the level of individual genes. “Deletion Isolation” was developed to quantify whether deletions also affected nearby genes. For each gene, a gene-size neutral metric was determined by summing the fraction of the gene deleted in all samples. Deletion Isolation was then determined by subtracting the average of the same metric for both the nearest upstream and nearest downstream neighboring genes. “Cell Line Proportion” was determined from the fraction of deletions in a given gene that occurred in cell lines relative to the total number of deletions in all sample types. “RNA/DNA Correlation” for each gene was calculated from the Pearson correlation coefficient of the log2-transformed segmented DNA copy number value with the log2-transformed relative RNA expression level. For genes with more than one probe on the Nimblegen expression array (see Supplementary Methods), the average value was used.http://www.R-project.org). The algorithm was trained on a set of 24 known tumor suppressors and 11 known common fragile site genes (http://cran.r-project.org/). We used the Gaussian kernel, and set the kernel function parameter to 0.01 and the soft margin parameter C to 10, based on a grid search for the best (three-fold) cross-validation error. The final cross-validation error was 3.2% and the training error was 2.9%. To assign class probabilities, we used the ‘prob.model’ option  and Support Vector Machine (SVM), to perform genome-wide classification of deleted genes with respect to their tumor suppressor-like or CFS-like properties. We used the Random Forest algorithm as implemented in the randomForest R software package  and . Only samples that contained at least 1 deletion in the set of 35 genes were used in this analysis.For a subset of the cancer samples, we performed whole-genome expression profiling using Nimblegen’s recommended single-color hybridization protocol and their 385,000 probe gene expression array (design ID #1877). Gene calls were generated using the Robust Multichip Average (RMA) algorithm provided with the NimbleScan software package. Subsequently, RMA expression values for a given tumor type were quantile normalized using R. Before computing the correlation between DNA copy number and expression, the normalized RMA expression values were log2-transformed and standardized with values obtained with Stratagene’s universal reference RNA.MACROD2, A2BP1, and FHIT loci with Agilent’s eArray tool (https://earray.chem.agilent.com/earray) using an 8×15K format. Arrays were hybridized and scanned as recommended by the manufacturer and following normalization the fluorescent ratios representing DNA copy number measurements segmented using the CBS segmenter.Induction of focal deletions by replicative stress was performed by treatment of Caco-2 cells with a subtoxic dose (0.3 µM) of aphidicolin  for 5 days followed by 24 hours recovery. Treated cells were trypsinized and 150 cells were plated onto 10 cm plates. After two weeks, clones were selected and expanded. Genomic DNA was isolated from each clone using the Gentra Puregene Core kit (Qiagen) and used to generate probes for array CGH analysis. Microarrays were custom designed to tile the A2BP1 expression vectors (pMSCV-A2BP1 and pMSCV-FlagA2BP1) by using high-fidelity PCR to amplify the coding-sequence insert from cDNA clone AF107203.1  with primers encoding a wild-type N-terminus (for pMSCV-A2BP1) or using the insert from cDNA clone NM_018723.2  using primers encoding a Flag-tagged N-terminus. We chose these two isoforms for functional studies as they were the most abundant based on annotation at http://genome.ucsc.edu. After validation by DNA sequencing, these plasmids were transfected into cancer cell lines and assayed for effects on tumorigenicity in nude mice as described Taqman probes were designed with ABI software and used as described for both real-time PCR analyais of DNA and RNA Figure S1Average DNA segment sizes. Plotted as a function of position on chromosomes 2 through 13.(EPS)Click here for additional data file.Table S1Tumor samples, cancer cell lines, and xenografts used in this study. This table contains four columns describing for each sample the tumor type, sample ID, array CGH ID, and sample type.(XLS)Click here for additional data file.Table S2Properties of the 10,835 focal deletions. This table contains ten columns describing for each focal deletion the tumor type, chromosome (23 = X), chromosomal start position (hg18), chromosomal stop position, the start and stop position on an absolute genome scale, the segmented DNA copy number value, the array CGH ID, and the sample type.(XLS)Click here for additional data file.Table S3Number of focal deletion involving individual telomeres. Focal deletions  that effect individual telomeres are shown.(XLSX)Click here for additional data file.Table S4The known common fragile site genes and tumor suppressor genes affected by ≥15 focal deletions. This set of genes was used for exploring properties that distinguished the two classes and for the training sets for the machine-learning tools.(XLS)Click here for additional data file.Table S5Differential frequency of deletions in CFS genes between breast cancer cell lines and primary breast tumors. This table show the numbers of breast cancer samples affected by focal deletions in the indicated genes, broken out by sample type (tumor or cell line). The average number of deletions affecting RefSeq genes is shown in the last row.(XLSX)Click here for additional data file.Table S6The importance of individual predictors in the RF classifier. The mean decrease in the Gini index was used to calculate the relative important of the seven individual properties in the RF classifier.(XLSX)Click here for additional data file.Table S7Validation of focal deletion calls by TaqMan real-time PCR. TaqMan probes were designed to nine different locations within the A2BP1 locus and used to determine DNA copy number in four different colon cancer cell lines. Shown for each cell line are two rows comparing the DNA copy number estimates based on aCGH versus real-time PCR. Note that there is good agreement except for the boundary of the hemizygous deletion in the cell line LIM2045. Real-time PCR has a greater dynamic range and can more accurately distinguish homozygous from heterozygous deletions than aCGH.(XLSX)Click here for additional data file.Table S8Functional classification of 4,823 genes for their TSG or CFS probabilities. This table contains fourteen columns describing for each gene with >15 focal deletions the chromosome, chromosomal start position, chromosomal stop position, the seven properties that distinguish TSG from CFS genes (mean-centered and variance-adjusted), the probability of being a TSG based on Random Forest classification (using either 7 or 3 properties), and the probability of being a TSG based on SVM classification. The probability of being a CFS is 1– TSG probability.(XLS)Click here for additional data file.Table S9DNAse I hypersensitivity for six common fragile site or CFS-like genes in four different cell types. The average DNAse I hypersensitivity for the indicated genes was taken from ENCODE data from the University of Washington (http://genome.ucsc.edu/cgi-bin/hgFileUi?db=hg19&g=wgEncodeUwDnase). Higher numbers indicate greater sensitivity and more open chromatin structure.(DOCX)Click here for additional data file.