To test the “tinnitus gap-filling” hypothesis in an animal psychoacoustic paradigm, rats were tested using a go/no-go operant gap detection task in which silent intervals of various durations were embedded within a continuous noise. Gap detection thresholds were measured before and after treatment with a dose of sodium salicylate (200 mg/kg) that reliably induces tinnitus in rats. Noise-burst detection thresholds were also measured to document the amount of hearing loss and aid in interpreting the gap detection results. As in the previous human psychophysical experiments, salicylate had little or no effect on gap thresholds measured in broadband noise presented at high-stimulus levels (30–60 dB SPL); gap detection thresholds were always 10 ms or less. Salicylate also did not affect gap thresholds presented in narrowband noise at 60 dB SPL. Therefore, rats treated with a dose of salicylate that reliably induces tinnitus have no difficulty detecting silent gaps as long as the noise in which they are embedded is clearly audible. The ototoxic effects of high doses of salicylate, the active ingredient in aspirin, are well-established –4. A recIn addition to electrophysiological studies, some of the perceptual effects of salicylate have been cataloged. Depending upon the dose, humans and rats typically develop hearing loss, with temporary threshold shifts of ~5–20 dB SPL, following salicylate administration , 12. In In addition to inducing temporary hearing loss and temporal processing deficits, salicylate also reliably induces tinnitus, i.e., a phantom auditory perception, in humans . Given iIn a typical Gap-PPI paradigm, a 50 ms silent interval embedded in 60 dB SPL continuous noise is presented ~100 ms before a high-intensity noise burst used to evoke the startle reflex motor response , 27. Norper se, but rather to tinnitus interfering with the processing of the silent gap  known to induce tinnitus  and obtaSix female Sprague-Dawley rats were used in the gap detection experiments, and three of these six rats were used to obtain noise thresholds . The rats began training at ~4 months of age and they were tested until they were ~14–15 months of age. The rats were obtained from Charles River Laboratories and all procedures were approved by the University at Buffalo, SUNY’s Institutional Animal Care and Use Committee. All rats were housed separately and were kept on a 12 h day/night cycle . Rats were food restricted and kept at ~85% of their free-feeding weight during the course of the experiment. Test sessions lasted ~1 h, and the rats were run once per day, 6–7 days/week.Rats were tested in an acoustically transparent acrylic cage (28 cm × 30 cm × 38 cm) located inside a sound attenuating chamber (76 cm × 71 cm × 71 cm) lined with 5 cm thick sound attenuating foam . The behavior of the animals during test sessions was monitored by a digital camera . The test cage was equipped with a speaker , feeder , and nose-poke hole equipped with infrared sensors .The experiment was run using custom behavioral software running on a personal computer (Microsoft Windows XP) similar to that described previously . The cusThe stimuli used in the gap detection experiments were a continuous broadband noise (BBN) containing frequencies up to 42 kHz, and 3 continuous NBNs: 10–20 kHz , 16–20 kHz (4 kHz bandwidth), and 15.3–16.7 kHz (1/8th octave bandwidth centered at 16 kHz). The BBN was presented at 20, 30, 40, and 60 dB SPL, and the NBNs were presented at 60 dB SPL. The rats were trained using a go/no-go operant conditioning procedure to detect a silent gap embedded within the continuous noise.nose-poke hole, which initiated a variable waiting interval ranging from 1 to 4 s. During this time, the rat had to maintain its position in the nose-poke hole; failure to do so resulted in an aborted trial. After the waiting interval, a single silent gap was presented in the noise. In the go condition, the target stimulus was the silent gap. In this trial type, a hit was recorded if the rat correctly responded to the gap within 2 s by removing its nose from the nose-poke hole and receiving a food pellet . A miss was recorded if the rat failed to remove its nose from the nose-poke.The rat began a trial by nose poking through the catch trials. These constituted the no-go part of the procedure and the silent gap was absent during these trials. False alarm (FA) rates and response biases were calculated from these catch trials. If the rat removed its nose during a catch trial, a false alarm was recorded and the rat received a 4 s timeout, during which the house light was turned off and the rat could not initiate another trial. However, if the subject continued to nose-poke, a correct rejection was recorded. No reinforcement was given for a correct rejection, but the next trial began immediately. Chance performance was represented by the animal’s false alarm rate. Sessions were excluded from analysis if the percentage of FAs was >20%. Less than 1% of all sessions were excluded for this reason.Approximately 30% of all trials were catch trials. During testing, the target gaps were chosen so that the smallest one or two gaps were rarely detected by the rats, whereas the largest gaps were well above threshold and nearly always detected. The gap durations used for testing were adjusted depending on each rat’s performance.The target stimuli were presented according to the psychophysical method of constant stimuli (MOCS). Within each 10-trial block, 7 predetermined targets were presented randomly along with 3 After baseline gap detection thresholds were collected, the rats were tested once per week with either a single injection of sodium salicylate (200 mg/kg IP) dissolved in saline (50 mg/ml), or an equivalent volume of saline (control). The injections were administered 2 h before testing. The 200 mg/kg dose of sodium salicylate has previously been shown to reliably induce tinnitus-like behavior in rats , 33.hit and false alarm rates were used to calculate all gap thresholds using signal detection theory and a threshold criterion of d′ = 1.5. This d′ value is comparable to that used by others . Each noise stimulus was 300 ms in duration with cosine rise/fall times of 5 ms. Rats were trained using a go/no-go operant conditioning procedure to detect a noise burst in an otherwise quiet chamber.go condition for this experiment were noise bursts embedded in a quiet background. Approximately 30% of all trials were catch trials, and no stimulus was presented on no-go trials. Noise-burst threshold testing began when the animals finished the gap detection experiment. As in the gap detection experiment, noise bursts were presented using the MOCS. The target intensities were chosen so that only the lowest one or two intensities were not detected whereas the highest sound levels were well above threshold.The procedure for obtaining the noise-burst thresholds was nearly identical to the procedures used in the gap detection experiment, but the target stimuli in the After baseline noise-burst thresholds were collected, the rats were tested once per week with either a single injection of sodium salicylate (200 mg/kg IP) dissolved in saline (50 mg/ml) or an equivalent volume of saline (control). As in the gap detection experiments, the injections were administered 2 h before testing.The data analysis for this study was the same as that used in the gap detection experiment.Table F = 15.77, 3 df; p < 0.001) and a significant interaction between intensity and baseline and saline conditions . A Holm–Sidak multiple comparison of the baseline condition revealed a significant difference in gap thresholds between 20 and 60 dB and 20 and 40 dB (p < 0.01); a similar analysis of the saline condition revealed a significant difference between 20 and 30 dB, 20 and 40 dB, and 20 and 60 dB (p < 0.05).Figure Mean gap thresholds during salicylate treatment were similar to baseline and saline at 60 dB SPL Figure . At 40 dF = 14.77, 2 df, p < 0.001), intensity , and a significant interaction between condition and sound level . A Holm–Sidak post hoc analysis revealed significant differences between the salicylate and saline conditions (p < 0.05) at 30 dB SPL and between salicylate and baseline conditions (p < 0.05) at 30 dB SPL, but not at 40 and 60 dB SPL (p > 0.05). Within the saline treatment group, the Holm–Sidak post hoc analysis revealed a significant difference between 30 and 60 dB SPL (p < 0.05) and 30 and 40 dB SPL (p < 0.05). No significant differences were found between the baseline and saline conditions for any of the sound levels (p > 0.05).A two-way repeated measures ANOVA with three sound levels  and three conditions  as factors revealed a significant effect of condition  in Figure Gap detection thresholds were also obtained at 60 dB SPL for three NBNs  located near the most sensitive region of the rat’s audiogram and in the vicinity of the presumed tinnitus pitch , 39. TheF = 0.42, p > 0.05], and there was no significant interaction between condition and bandwidth .In a two-way repeated measures ANOVA with three conditions  and four noise bandwidths  as factors, the gap threshold differences among the three conditions were not great enough to exclude the possibility that the differences were due to chance  and noise bandwidth , but there was no significant interaction between condition and bandwidth . Holm–Sidak post hoc tests found significant differences between the salicylate and saline conditions (p < 0.05), and the salicylate and baseline conditions (p < 0.05) for all four noises, with the salicylate condition having significantly higher thresholds than the baseline and saline conditions. No significant differences were found between the baseline and saline conditions for any of the noises (p > 0.05). The post hoc analyses also revealed significant differences between BBN and the 16–20 and 15–17 kHz NBNs (p < 0.05), but not the 10–20 kHz NBN (p > 0.05). Overall, the 16–20 and 15–17 kHz NBNs consistently produced higher thresholds than the BBN and 10–20 kHz NBN.Mean thresholds were the lowest for BBN and the 10–20 kHz NBN and 7–9 dB higher for the 10–20 and 15–17 kHz NBN. Noise thresholds for BBN, 10–20 kHz NBN, 16–20 kHz NBN, and 15–17 NBN during the salicylate condition increased ~18, 19, 16, and 20 dB, respectively, compared to the saline control condition. A two-way repeated measures ANOVA with three conditions  and four noise bandwidths  as factors revealed significant main effects of condition [Most gap detection studies employ BBN stimuli to minimize problems associated with spectral splatter and off-frequency listening , 41. OurOur NBN gap thresholds measured at 60 dB SPL were larger than those obtained in BBN Figure ; Table 2The main goal of the current project was to determine if a dose of salicylate sufficient to induce hearing loss and tinnitus would significantly impair gap detection performance in rats. However, since previous research in normal-hearing humans  and animBecause the 200 mg/kg dose of salicylate increased the BBN threshold from 2 to 19 dB SPL Table , it was Gap detection thresholds at 40 and 60 dB SPL were largely unaffected by salicylate treatment. Likewise, NBN gap detection thresholds measured at 60 dB SPL were largely unaffected by salicylate treatment; mean gap thresholds increased by <1.08 ms post-salicylate when compared to saline values Figure , and salEven under the most difficult listening conditions post-salicylate (30 dB SPL BBN), gap thresholds never exceeded 18 ms in any of the animals tested Table . The dosIn addition to providing important animal psychoacoustic data, our results also have implications for tinnitus research. One of the most widely used animal behavioral models of tinnitus, Gap-PPI, operates on the hypothesis that tinnitus interferes with processing silent gaps in noise , 29, 31.conscious perception of silent gaps in noise. Campolo et al. (However, this “tinnitus gap-filling” hypothesis has been evaluated in only three human studies to date. Two of these human studies used psychoacoustic procedures to determine whether tinnitus interferes with the o et al.  found tho et al. . More reo et al.  found tho et al. .The third recent human study used an eye-blink Gap-PPI paradigm to test gap detection in tinnitus patients . FournieOur current psychophysical results provide the first test of the “tinnitus gap-filling” hypothesis in an animal. The results of our gap detection experiment mirror the results found in the human psychophysical gap detection tests , 30, 54,One of the difficulties in developing an animal behavioral model of tinnitus is controlling for the confounding effects of the tinnitus inducer, i.e., salicylate or noise exposure , 56. In Researchers using Gap-PPI, including our lab, have attempted to account for salicylate-induced hearing loss and gap detection deficits by using background noises of at least 60 dB SPL and 50 ms silent gaps –21, 24. One explanation for the changes in Gap-PPI following salicylate or noise exposure could be related to hyperacusis. Briefly, hyperacusis is defined as abnormal sound level tolerance where moderate-level sounds are perceived as uncomfortably loud , 59. Botfacilitated the ASR, (Another explanation for changes in Gap-PPI following salicylate are alterations in temporal processing. Sun et al.  found ththe ASR,  determinthe ASR, , and sinthe ASR, , it is pthe ASR,  results,Although the Gap-PPI paradigm has many advantages over other animal models of tinnitus, it is not yet clear that tinnitus alone can account for changes in Gap-PPI when both salicylate and noise exposure appear to induce hearing loss, temporal processing deficits, and hyperacusis as well , 32.In summary, the gap detection thresholds for rats in the current study were unchanged with salicylate for BBN and NBN presented at high-sound levels, suggesting that salicylate-induced tinnitus was not “filling in” the conscious perception of the silent gaps. Furthermore, given the threshold shifts found in the noise detection experiments, the effects of salicylate on gap detection at low-sound levels are most parsimoniously explained by hearing loss alone and not caused by tinnitus. However, the possibility remains that tinnitus interferes with peripheral, pre-attentive filtering of sensory stimuli involved in sensorimotor gating. As such, the conscious perception of silent gaps in background noise may involve different neural processes than those underlying prepulse inhibition of the acoustic startle reflex; however, hearing loss, temporal processing deficits, and hyperacusis remain confounding variables when using traditional gap detection paradigms to measure tinnitus in animals.KR, DS, and RS conceptualized and designed the experiment. KR, MU, and RB collected and analyzed the data. KR and RS interpreted the data and wrote the manuscript. KR, DS, MU, RB, and RS revised the manuscript and approved the final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


P < 0.05). Long‐term imaging showed oscillations in the accumulation of FBA over periods of hours. Overall, the studies suggest that high accumulation of FBA can indicate the sensitivity of cultured hepatocytes to hydrophobic bile acids and other toxins.Primary hepatocytes undergo phenotypic dedifferentiation upon isolation from liver that typically includes down regulation of uptake transporters and up regulation of efflux transporters. Culturing cells between layers of collagen in a three‐dimensional (3D) “sandwich” is reported to restore hepatic phenotype. This report examines how 3D culturing affects accumulation of fluorophores, the cytotoxic response to bile acids and drugs, and whether cell to cell differences in fluorescent anion accumulation correlate with differences in cytotoxicity. Hepatocytes were found to accumulate fluorescent bile acid (FBA) at significantly higher levels than the related fluorophores, carboxyfluorescein diacetate, (4.4‐fold), carboxyfluorescein succinimidyl ester (4.8‐fold), and fluorescein (30‐fold). In 2D culture, FBA accumulation decreased to background levels by 32 h, Hoechst nuclear accumulation strongly decreased, and nuclear diameter increased, indicative of an efflux phenotype. In 3D culture, FBA accumulation was maintained through 168 h but at 1/3 the original intensity. Cell to cell differences in accumulated FBA did not correlate with levels of liver zonal markers L‐FBAP (zone 1) or glutamine synthetase (zone 3). Cytotoxic response to hydrophobic bile acids, acetaminophen, and phalloidin was maintained in 3D culture, and cells with higher FBA accumulation showed 12–18% higher toxicity than the total population toward hydrophobic bile acids ( These studies use automated image analysis and fluorescent dye accumulation to demonstrate that 3D culturing enhances organic anion accumulation and cytotoxic response in long‐term hepatocyte cultures. The level of anion accumulation was found to vary through days in culture and also between single cells, and higher fluorescent bile acid accumulation correlated with higher toxic response to hydrophobic bile acids. Primary hepatocytes undergo phenotypic change, or dedifferentiation, upon isolation from liver and subsequent laboratory culturing. This dedifferentiation response, which can vary depending on culture conditions and animal species, involves an inflammatory response arising from ischemia, tissue disruption, and partial digestion of plasma membrane protein that occurs during the isolation procedure , chenodeoxycholylglycylamidofluorescein  as a marker of hepatocyte phenotype. CDCGamF and other FBAs are substrates for bile acid transporters . Cells were plated at 1200 cells/mm2 on coverslip‐bottomed chambers or 96‐well plates that had been coated by 5 min exposure to 0.1 mg/mL rat tail collagen . To create 3D cultures, medium including 0.15 mg/mL rat tail collagen on day 0, and 0.06 mg/mL thereafter was allowed to gel. This minimal concentration of collagen and promoted hepatocyte phenotype, but could be removed without damaging the cells. The collagen overlay was found to delay the cellular accumulation of fluorescent dyes and was therefore removed during the wash procedures, prior to application of the dyes, for all applicable experiments. The medium was changed on day 0, day 1, and every 2 days thereafter (but not immediately prior to experiments). Washes and incubations were in serum‐free medium  unless otherwise noted.Reagents were from Sigma‐Aldrich  unless noted. Fluorescent bile acid, CDCGamF   Figs. . At variμg/mL Hoechst and the exact field positions were re‐imaged. Images were scored automatically by identifying individual cells via Hoechst fluorescence, segmenting the nuclei, and measuring cell fluorescence within a cellular ROI, consisting of the nuclear border to 3 microns beyond the nuclear border.Experiments were performed similarly to those described in Murray et al.  Fig. 4)4). Rat h3, 20 mmol/L Glucose, 10 mmol/L Hepes, 4.8 mmol/L KCl, 1.2 mmol/L MgSO4, 1.2 mmol/L KH2PO4, 1.2 mmol/L CaCl2) followed by 400 μL of 125 μmol/L CDCGamF, and then 300 μL of 1 mmol/L Hoechst . The fields were then re‐imaged once every 10 min for 30 h at 37°C to observe cell death. Individual cells were identified by Hoechst nuclear stain, and FBA and propidium iodide intensity were measured in the cellular ROI. Cell death within the first 30 h was measured by an increase in propidium iodide fluorescence. FBA fluorescence was measured in frame 1, it decreased with addition of the bile acids.Hepatocytes were plated on collagen‐coated dishes in cell culture media supplemented with 100 mmol/L Hepes and diluted with Hure Fig. . 100 nmohttp://rsb.info.nih). For Fig. 2 for nuclear area, or if circularity was <0.6. Nuclear diameter was the maximum Feret diameter. Anion fluorescent intensities were subtracted from control . For Hoechst, Lysotracker, and propidium iodide, background intensity (image mode) was subtracted. For Fig. 2) with propidium iodide pixel intensity <200 in nuclear ROI, and Hoechst standard deviation <mean ×2 and >mean ÷2, and circularity exclusion as above. Image processing for Fig. 2 and circularity <0.05. More strenuous outlier removal was not needed for day 0 cells. Cells were determined to be nonviable in the first 30 h if propidium iodide cell fluorescence exceeded 100 units above background. Cell fluorescence was expressed as a ratio to Hoechst fluorescence.Images were quantified using ImageJ  on an Olympus iX71 with an automated X‐Y‐Z stage  and 60× 1.4 NA oil, 20×, 0.75 NA, or 20× or 10× long‐working distance lenses. Epifluorescence imaging employed a DG‐4 xenon lamp  with Dapi, Cy2, Cy3, Cy5 fluorescence and bright‐field channels and a cooled CCD camera . Spinning disk confocal utilized PhotoFluor metal halide white light excitation  with similar channel capture using a CARV II spinning‐disk unit  and an iXon 897 EMCCD camera .Typically, primary hepatocytes will dedifferentiate from their absorptive, secretory epithelial phenotype when cultured on a two‐dimensional substrate such as plastic or collagen‐coated glass. As part of this dedifferentiation, hepatocytes lose their ability to take up and secrete bile acids. However, culturing between layers of collagen in a three‐dimensional matrix, termed a collagen “sandwich” . The panels  show representative fields of cells in the FBA channel with the cytosolic ‘region of interest’ outlined. Cells appear round at early time points. Some pixel intensities may appear saturated, but this is due to image scaling. For comparison, we assayed for the accumulation of three other fluorescein‐containing anions: fluorescein (FL), carboxyfluorescein diacetate (CFDA), and carboxyfluorescein succinimidyl ester (CFSE). All these have been shown to be taken up into hepatocytes , yet at 7 h there was 4–5‐fold greater accumulation of FBA than CFDA and CFSE for both 3D and 2D culturing . Fluctuations through time in culture are also evident in accumulation of CFSE as well as other fluorophores as well as in 2D culturing, although the lower overall signal makes them more difficult to discern. The fluctuations may reflect nutrient levels in the culture media, such as glucose, which can affect nuclear receptor transcription factors such as FxR, HNFalpha and SHP , lithocholic acid, and chenodeoxycholic (CDCA) acid, can induce hepatocyte cell death both within the liver and in cultured cells , whereas by day 1, hepatocytes in 2D culture had reduced responsiveness to APAP, GCDCA, and phalloidin. By day 2, the 2D‐cultured cells were refractory to all of the toxins. Under 3D culture there was a loss of response to APAP and phalloidin on day 1, but by day 2, hepatocytes regained significant toxic response to all of the agents, although the response appears somewhat diminished. Overall, the studies indicate that 3D culturing increases the level of anion accumulation . L‐FABP localizes to the periportal region (zone 1) (Kazantzis and Seelaender n = 1150). These studies therefore imply that variability of FBA accumulation is not strongly related to the zone from which the hepatocytes were derived.Several studies have noted that the level of fluorescent bile acid accumulation in hepatocytes varies significantly from cell to cell, and that this is especially apparent in primary cultures . Cell parameters from each experimental condition were sorted according to initial FBA accumulation and divided into three groups of equal numbers of cells, referred to as high, medium, and low FBA accumulation groups. P < 0.05 for each compared to the whole population). The high FBA accumulating cells showed a 13.3 and 18% greater cell death than the whole population for GCDCA and TLCA treatments. In control cells, high FBA accumulating cells were not more likely to undergo cell death compared to the whole population , indicating that high accumulation of FBA itself did not result in toxicity. Sorting the data another way, it was found that the cells that that underwent cell death had 1.2‐, 1.4‐, 2.2‐, and 1.8‐fold higher initial FBA fluorescence in control, APAP‐, GCDCA‐, and TLCA‐treated cells as compared to cells that survived . Interestingly, the low uptake cells for all experiments showed a lower death rate, suggesting that cells with low FBA accumulation may be protected from cell death, a possibility that requires further investigation.To examine for consequences of different levels of accumulated fluorescent bile acids within individual hepatocytes, we performed cytoxicity correlation analysis of individual cells using live microscopy. We reasoned that cells with high FBA accumulation should also accumulate high amounts of hydrophobic bile acid, and that the ensuing cellular damage would also be higher in these cells. To accomplish this, primary rat hepatocytes were treated with FBA, propidium iodide, and Hoechst (not shown), imaged, and then treated with bile acids or APAP and imaged over 30 h Fig. . IndividThe studies of Fig. These studies were initiated to further understand the effects of culturing rat hepatocytes between layers of collagen in the sandwich configuration, and to determine whether the fluorescent bile acid, CDCGamF , can serve as a marker of hepatocyte phenotype in automated image analysis. In the 3D configuration, the top layer of collagen can form a barrier to solute diffusion. This was overcome by the use of low concentrations of collagen (0.15 mg/mL) and removal of the top layer prior to experimental manipulation. As a technical note, imaging of hepatocytes in the presence of vital dyes and markers of cell death provided an important tool to inspect for cellular damage that can occur during uptake assays. At least three levels of variability, or heterogeneity, of fluorescent anion accumulation are observed in these studies; 1) acinar zonal variability, which here did not play a dominant role Fig. ; 2) pop acinar z pop acinBile acid and drug‐induced toxicities were maintained in 3D culture and can also be analyzed by automated imaging Figs. . This prNone declared.


Our objective was to estimate the incidence of influenza‐associated hospitalizations and in‐hospital deaths in Central American Region.We used hospital discharge records, influenza surveillance virology data, and population projections collected from Costa Rica, El Salvador, Guatemala, Honduras, and Nicaragua to estimate influenza‐associated hospitalizations and in‐hospital deaths. We performed a meta‐analysis of influenza‐associated hospitalizations and in‐hospital deaths.The highest annual incidence was observed among children aged <5 years .Annually, 7 625–11 289 influenza‐associated hospitalizations and 352–594 deaths occurred in the subregion.Our results suggest that a substantive number of persons are annually hospitalized because of influenza. Health officials should estimate how many illnesses could be averted through increased influenza vaccination. Influenza is a vaccine preventable disease that annually causes approximately 1 million influenza‐associated hospitalizations among children younger than 5 years and 500 000 deaths among all age‐groups.Few studies have estimated the burden of influenza illness in Central America, a tropical region comprised of lower to upper‐middle‐income countries.Annual influenza vaccination remains a key strategy for the prevention of influenza‐related hospitalizations and deaths. In addition since 2004, countries in the Americas have expanded the use of influenza vaccine among subpopulations at increased risk of complications following influenza infections.Our objective was to estimate the incidence of influenza‐associated hospitalizations and in‐hospital deaths in five Central American countries.We used hospital discharge records, influenza surveillance virology data and population projections collected during January 1, 2009, to December 31, 2012, from Costa Rica, El Salvador, Guatemala, Honduras, and Nicaragua to estimate influenza‐associated hospitalizations and in‐hospital deaths.In all countries except Guatemala, we obtained public hospital discharge records and in‐patient survival data from national Health Information Systems available from the Ministries of Health. In Guatemala, we recruited a survey team to collect the information at all public hospitals. For each month during 2009–2012, we determined the number of patients hospitalized with pneumonia or influenza diagnoses corresponding to ICD‐10 codes J09–J18, the WHO proxies for severe acute respiratory infections (SARI),Influenza surveillance among SARI case‐patients was conducted in the study countries by sentinel hospitals and National Influenza Centers according to PAHO‐CDC 2006 generic protocol for influenza surveillance.We assumed that every inhabitant was at risk of developing severe influenza illness and requiring admission to a public hospital. Thus, we defined the population at risk per country as the most recent population estimate based on census projections, stratified by year and age‐group. National Statistical Offices provided the data for each country. As a sensitivity analysis, we also assumed that in each country, only the proportion of the population typically seeking care at public hospitals network was at risk of developing severe influenza and being identified in national data sets. Public hospitals cover 87% of the population in Costa Rica, 80% in El Salvador, 70% in Guatemala, 60% in Honduras, and 61% in Nicaragua.We estimated incidence of influenza‐associated hospitalizations and in‐hospital deaths for each year, age‐group, and country by multiplying the age‐stratified monthly number of patients with pneumonia or influenza clinical diagnoses by the age‐stratified percentage of samples from SARI surveillance case‐patients that tested positive for influenza. We then divided the estimated annual number of influenza‐associated hospitalizations and deaths by the population at risk. The formula is presented in the Data S1. We calculated 95% confidence intervals (95% CI) assuming a Poisson distribution. We performed a meta‐analysis of individual country rates by pooling the information (2009–2012) from the five countries, using a random‐effects model, and assuming significant heterogeneity in the data. We conducted a second sensitivity analysis stratifying influenza rates into pandemic (2009–2010) and seasonal influenza years (2011–2012).The project was reviewed by Institutional Review Committees of the Universidad del Valle de Guatemala and the countries' Ministries of Health and judged to be a public health evaluation. Only records without personal identifiers were used for the analysis.During 2012, Costa Rica, El Salvador, Guatemala, Honduras, and Nicaragua had 40 325 186 inhabitants: 4 951 130 (12%) children aged <5 years, 33 299 080 (83%) persons aged 5–64 years, and 2 074 976 (5%) adults aged >64 years. During 2009–2012, there were 313 453 hospitalizations with pneumonia and influenza diagnoses, of which 13 218 (4%) resulted in death during hospitalization (Tables S1–S5).P‐value<0·001).Children aged <5 years comprised the greatest proportion (226 026 [72%]) of the 313 453 pneumonia and influenza hospitalizations. Older adults aged >64 years comprised the greatest proportion (5532 [42%]) of the 13 218 in‐hospital deaths, followed by children aged <5 years (4092 [31%]). The age‐stratified percentage of hospitalizations and in‐hospital deaths varied significantly between countries (Data S1) : 9% among children aged <5 years, 32% among persons aged 5–64 years, and 13% among adults aged >64 years. In 2009 and 2011 influenza A (H1N1) pdm09 was the most commonly identified subtype among SARI case‐patients samples   were hospitalized in the five countries with influenza‐associated SARI. Among them, 5595 (59%) were children aged <5 years, 2997 (31%) persons aged 5–64 years, and 913 (10%) adults aged >64 years. The highest incidence of influenza‐associated hospitalizations occurred among children aged <5 years (113 [95% CI: 91–134] per 100 000 children) which was 12‐fold higher than among persons aged 5–64 years (P < 0·001)  died during their influenza‐associated hospitalization in the five countries. A total of 64 (17%) in‐hospital deaths occurred among children aged <5 years, 166 (43%) among persons aged 5–64 years, and 151 (40%) among adults aged >64 years. The highest influenza‐associated mortality occurred among adults aged >64 years (7·3 [95% CI: 5·5–9·1] per 100 000 persons) and was 14‐fold higher than among persons aged 5–64 years .Our results suggest that influenza, a vaccine preventable illness, is associated with substantial morbidity and mortality in Central America. In the five countries studied, 7563–11 397 influenza‐associated hospitalizations and 292–473 in‐hospital deaths occurred annually among 40 million inhabitants.Very young children were most commonly hospitalized as a result of influenza‐associated hospitalizations and older adults most commonly died as a result of influenza. While persons aged 5–64 years were more commonly hospitalized during 2009 because of influenza A (H1N1) pdm09 influenza pandemic,Influenza‐associated hospitalizations rates in this study were similar to those published in a recent global meta‐analysisOur estimated influenza‐associated in‐hospital mortality was similar to that reported previously in the region, except for the lower mortality rates we estimated among adults aged >64 years.We extrapolated influenza laboratory results from SARI case‐patients identified through surveillance to all untested hospitalized patients with SARI proxy discharge diagnoses. We were unable to assess differences between SARI case‐patients identified through surveillance and the SARI proxies. In addition, surveillance systems and medical practices vary from country to country; we therefore used random‐effects models because we assumed that influenza‐associated hospitalization rates incidence varied by country but followed same distribution. Our results might underestimate the burden of influenza‐associated hospitalizations and mortality because underreporting of deaths is common in developing countries; a study estimated ~50% underreporting in 2003 for some countries in the region.Influenza is among the leading causes of hospitalizations and deaths.This work was supported by cooperative agreement between Centers for Disease Control and Prevention and Universidad del Valle de Guatemala “Strengthening Infectious Disease Research Capacity for Public Health in Guatemala and Central America” No. 1U01GH000028‐04.MAD had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. MAD, WC, JJ, and EAB contributed to study concept and design. GG, RM, JA, BL, CS, AA, RC, MC, and JM contributed to acquisition of data. MAD, WC, GG, RM, JA, BL, CS, AA, RC, NEO, JM, MC, RP, JJ, and EAB contributed to analysis and interpretation of data. MAD drafted the manuscript. MAD, WC, GG, RM, JA, BL, CS, AA, RC, NEO, JM, MC, RP, JJ, and EAB critically revised the manuscript for important intellectual content. MAD performed statistical analysis. MAD, WC, and JJ contributed to study supervision.None of the authors have any conflicting interests to declare.Data S1. Mathematical formulaTable S1. Influenza‐associated hospitalizations and deaths in Costa Rica during 2009–2012.Table S2. Influenza‐associated hospitalizations and deaths in El Salvador during 2009–2012.Table S3. Influenza‐associated hospitalizations and deaths in Guatemala during 2009–2012.Table S4. Influenza‐associated hospitalizations and deaths in Honduras during 2009–2012.Table S5. Influenza‐associated hospitalizations and deaths in Nicaragua during 2009–2012.Table S6. Sensitivity analysis of influenza‐associated hospitalizations and deaths.Click here for additional data file.


Mitochondrial disorders belong to the most prevalent inherited metabolic diseases with the m.3243A > G mutation reflecting being one of the most common mutations in mitochondrial DNA. Previous studies showed little relationship between mitochondrial genetics and disease manifestation. Relationship between genotype and disease manifestation with patient reported quality of life and other patient reported outcomes is still unexplored.Seventy-two out of the 122 invited adult patients with m.3243A > G mutation completed online standardized questionnaires on quality of life, functional impairment, fatigue and mental health as assessed by the RAND-SF36, the Sickness Impact Profile (SIP), the Checklist Individual Strength (CIS) and the Hospital Anxiety and Depression scale (HADS). Data were related to clinical manifestation reflected by the Newcastle Mitochondrial Disease Adult Scale (NMDAS) score and heteroplasmy levels of the mutation in urine epithelial cells.r = <.30) and weak (.30 < r < .50) to moderate (.50 < r < .70) relationship with clinical manifestation.Patients reported impaired quality of life. Sixty percent showed severe levels of fatigue, and 37 % showed clinical relevant mental health problems, which was significantly more than healthy norms. These patient reported health outcomes showed negligible relationship with levels of heteroplasmy (Patient reported outcomes on quality of life, fatigue and mental health problems, are only partly reflected by clinical assessments. In order to support patients more effectively, integration of patient reported outcomes, alongside symptoms of their disease, in clinical practice is warranted. Mitochondrial disorders, as a group, belong to the most prevalent inherited metabolic diseases. The incidence of congenital mitochondrial disorders based on oxidative phosphorylation (OXPHOS) defects is at least 1:8500 of all life births –5    and three months (T3). Newcastle Mitochondrial Disorder Assessment Scale  . Scores on different scales range from 0 (maximum limitations) to 100 . As reference group we used a Dutch norm group for general population of men and women age 18 to 75 years [75 years  . Th. ThFunctle , was used as indication for physical quality of life. Total scores were used for SIP, CIS and HADS as indication of functional impairment, subjective fatigue and mental health. Correlations lower than .30 were considered negligible, between .30 and .50 low, between .50 and .70 moderate and between .70 and .90 strong  (Hincle p = .482). The same was true for NMDAS subscales 1 and 2. Non response group showed significantly more problems on the NMDAS 3 subscale  and nearly significant more problems on total NMDAS scale . Age , BMI  and height  did not differ, however, non responders had a lower weight .A total of 122 patients with the m.3243A > G mutation was invited to participate. Seventy-two patients completed the questionnaires, a response rate of 59 %. Demographic characteristics of the patients are summarized in Table Response rate on repeated assessment at T2 and T3 was 50 (69 %) and 48 (67 %) respectively. Correlations on outcomes measures between T1 and T2 assessments varied from .837  to .922 . Correlations between T1 and T3 assessments varied from .761  to .858 .Levels of heteroplasmy were assessed in Leucocytes ), urinary epithelial cells  and buccal mucosa .Mean NMDAS score was 17 (SD = 15) varying from 1 to 98, median 14.5. Twenty-one percent of the patients showed mild symptoms, 50 % moderate and 29 % severe. There were no asymptomatic patients in the sample.p < .001) to −2.98 ).Quality of life of patients with m.3243A > G mutation is presented in Table p < .001). Most limitations were experienced in putting great physical efforts, 61 % of the patients experienced serious impairments. About one third of the patients experienced serious impairments with climbing stairs and walking more than one kilometer. One out of five indicated serious problems with putting moderate physical efforts, carrying groceries or stooping. Only a one out of about ten patients or less indicated severe impairments with walking more than 500 m, climbing one stair or washing and dressing.Scores on the RAND-36 subscale physical functioning indicated significantly more problems  compared to norm group of comparable age and sex : Mean scores on home management were 86.89 , on work 30.89 , and on recreation 76.54  reflecting more functional impairments compared to healthy controls  to recreation t = 8.016; p < .001).p = .004; ambulation t = 5.405; p < .001). Thirty-nine percent % of the patients experienced no impairments in alertness. Mean alertness score was 127,10 , this was more than healthy controls .74 % of the patients experienced no impairment in mobility, 57 % no impairment in ambulation. Mean mobility score was 39.5 , mean ambulation score was 60.0 . Patients show more impairments than healthy comparison group . The mean CIS fatigue score was 37.4  but less than patients with chronic fatigue syndrome . Age and gender did not differ between fatigued patients and non-fatigued patients.The majority of the patients with a m.3243A > G mutation reported abnormal levels of fatigue (78 %), 60 % reported severe levels of fatigue (p = .003) as well as total symptom scores in the patient group . Scores on anxiety were comparable to healthy control and better than a comparison group of general medical patients  [Mental functioning was assessed with the HADS. Results indicated that 26,4 and 31,6 % of the patients indicated clinical relevant symptoms of respectively depression and anxiety. 36,9 % scored above the cutoff of clinical relevant levels of general distress. Comparing the patient groups with healthy Dutch controls shows higher levels of depression  .To assess to what extend patient reported outcomes were related to disease manifestation as indicated by NMDAS, ANOVA’s were performed with three NMDAS categories as factor and set of patient reported outcomes as dependent variables. ANOVA’s indicated that scores on physical functioning, functional impairment, fatigue and mental health differed significantly between NMDAS severity groups, with patients with worst clinical manifestation indicating most physical problems, most functional impairment, most fatigue and most mental health problems Table .Table 4PTo assess the strength of the relationship between disease presentation and patient reported outcomes, spearman correlations between NMDAS subscale scores and different patient reported outcomes were calculated. Correlation of physical functioning, functional impairment, fatigue, and mental health with three subscales and total scale on NMDAS varies from strong (r > =.70) to negligible (r < .30). Perceived physical functioning was most strongly related with NMDAS scores with strong correlations with the NMDAS 1 (rho = −.727) and total score (rho = −.714), and moderate correlation with NMDAS 2 (rho = −.528) and 3 (rho = −.574). Functional impairment was most strongly related to NMDAS1 (rho = .665) and NMDAS3 (rho = .382). Fatigue is moderately correlated with NMDAS 1 (rho = .564) and total NMDAS (rho = .546), and weak with NMDAS 2 (rho = .446) and 3 (rho = .344). Mental health was only weakly correlated with NMDAS 1 (rho = .376), 2 (rho = .454) and total score (rho = .425). The correlation between mental health and NMDAS 3 was negligible.To explore which factors predicted patient perceived health status, four aspects of perceived health were taken into account as outcomes in different regression analyses: physical functioning, impairments in daily functioning, fatigue and mental health. Hierarchical regression analyses were performed in which mitochondrial genetics and clinical manifestation were entered in the first steps, and subsequently physical functioning, functional impairment, fatigue and mental health in the next steps. The correlations are presented in Table  p < .001; R2 = 0.468). Heteroplasmy did not significantly explained variance in physical function, after controlling for genotype, NMDAS significantly added 45.0 % explained variance. In the total model, NMDAS  was a significant predictor of physical functioning.A first hierarchical multiple regression analysis was performed to predict physical functioning. Next to genotype, clinical manifestation was entered as predictor. The total model significantly predicted physical functioning  = 28.174; p < .001; R2 = 0.519). NMDAS  and physical functioning (15.9 %), significantly added explained variance. In the total model, only physical functioning  = 22.617; p < .001), with an R2 of .655. Genotype explained 7.3 % variance, after controlling for genotype, NMDAS added another 9.0 %; physical functioning another 32.7 %, and, after controlling for previous factors, functional impairment added another 16.5 % of the variance.A third hierarchical linear regression was calculated to predict perceived fatigue based on genotype, clinical functioning, physical functioning, and functional impairment. A significant regression equation of the total model was found  = 29.461, p = .053), NMDAS , physical functioning , and functional impairment  significantly predicted perceived fatigue.In the total model, heteroplasmy , with an R2 of .531. Heteroplasmy explained 6 % of the variance. After controlling for heteroplasmy, NMDAS additionally explained 9.1 % of the variance, physical functioning also 11.6 %, functional impairment 19.6 % and fatigue 5.8 %. In the total model, functional impairment  and fatigue  significantly predicted mental health.A last hierarchical linear regression analysis was calculated to predict mental health, also based on genotype, clinical manifestation, physical functioning, functional impairment, and fatigue. A significant regression equation of the total model was found  = 13.250; Patients with the m.3243A > G mutation, experience important impairments in several domains of their health. Sixty percent showed severe levels of fatigue, and 37 % percent showed clinical relevant mental health problems. The variation in quality of life, functional impairment, fatigue and mental health could not be explained by level of heteroplasmy in body fluids and only partly by disease manifestation as indicated by NMDAS. This means that patients with a comparable clinical presentation can have a substantial variation in the impact of the disease in their daily life. In order to tailor treatment to complaints experienced by patients, integrating the perspective of the atient in clinical assessment is warranted  . From sThis study included a broad range of patient reported outcomes based on standardised measures also shown to be sensitive for investigating these outcomes in other patient groups. In addition, also genotype and disease manifestation were taken into account reflecting the broad range of disease manifestation from genotype, to clinical phenotype, to daily functioning and perceived health. Quality of life and patient reported outcomes are only sporadically integrated in studies concerning patients with mitochondrial diseases. Mitochondrial diseases are characterised by a great variability in expression. This even more supports the need for systematic assessment of patient reported outcomes. This study focused on a relatively homogeneous group of patients carrying the same mutation. Including also patients with different diagnoses even increase this variability. This makes it even more important to built agreement on the conceptualisation and subsequent assessment of patient reported outcomes in patients with mitochondrial disease. Especially in small patient groups such as patients with the m.3243A > G mutation, agreement in outcome assessments could importantly increase gain in knowledge on variability in patient reported outcomes, and, even more important, its possible predictors.This study also revealed interesting information regarding outcomes of clinical trials predominantly focusing on medical outcomes. This study shows that, next to survival rates and clinical manifestation, also patient perceived functioning on different health domains is important to explain the relevance of trial outcomes for patients’ daily functioning. It could also explain the possible role of psychological and behavioral mediators explaining the relationship between disease manifestation and perceived functioning , 31, 32 Longitudinal research should shed more light on this issue. It could explain the role of these mediators in changes in perceived health. It could be highly assumed, for instance, that coping and cognitions regarding somatic complaints are important mediators able to explain variation in the relationship between disease activity and patients’ complaints. Future studies should also include more objective assessment of daily functioning e.g. exercise tests and by using registrations of daily movements. However, previous studies revealed that ambulation subscale of the SIP, as used in the present study, highly correlates with physical fitness and objective physical activity  . Instruments assessing general quality of life are needed to compare outcomes of patients with mitochondrial disorders with patients with other, more or less comparable medical conditions. Disease specific measures could be used to compare the impact of the disease of a specific patient in relation to the whole patient group. These measures are also useful as outcomes for clinical trials because of their frequently reported higher sensitivity for chance.Developing these measures for the field of mitochondrial medicine asks for integrating instruments available in other patient groups with information from interviews with patients to be able to point to specific quality of life aspects for patients with a mitochondrial disease. One could think about the specific burden of the inherited aspect of the disease, the lack of tangible symptoms of the disease in communicating about impairments in the social environment, the unpredictable course, the possible lethal character and the lack of control patients could have on the symptoms they experience. These specific characteristics need to be integrated in a mitochondrial disease specific instrument to assess quality of life, or burden of the disease.This study showed that patients reported considerable impairments in their quality of life and daily functioning. In addition, it indicated the complexity of the relationship between patient reported outcomes, clinical manifestation as well as genotype in patients with m.3243A > G mutation. This point to the importance of integrating patient reported outcomes in clinical assessment as well as clinical trials to gain more understanding in the complexity of factors explaining outcomes in these patients, as well as to provide starting points for interventions to support patients in the management of their disease.


Valsa mali var. mali (Vmm), a Vmm–apple callus interaction system was developed to evaluate the induced resistance of an attenuated Vmm strain LXS081501 against further infection by a virulent Vmm strain LXS080601. The infection index was up to 97.32 for apple calli inoculated with LXS080601 alone at 15 days after inoculation whereas it was only 41.84 for calli pretreated with LXS081501 followed by LXS080601 inoculation. In addition, the maximum levels of free proline, soluble sugar, and protein in calli treated with LXS081501 plus LXS080601 were 2.14 to 3.47 times higher than controls and 1.42 to 1.75 times higher than LXS080601 treatment. The activities of defense-related enzymes such as phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO), peroxidase (POD), and catalase (CAT) as well as β-1,3-glucanase and chitinase in apple calli inoculated with LXS080601 alone or LXS081501 plus LXS080601 increased significantly 24 hai and peaked from 48 to 120 hpi. However, in the latter treatment, the maximum enzyme activities were much higher and the activities always maintained much higher levels than control during the experimental period. These results suggested the roles of osmotic adjustment substances and defense-related enzymes in induced resistance.To study the induced resistance in apple against  Valsa mali var. mali (Vmm), is a destructive disease that is prevalent in main apple orchards in China. Cankers usually occur on branches and tree trunk, eventually leading to the death of the whole tree and sometimes the failure of the whole orchard [ Vmm invades apple tree by secreting cell wall degrading enzymes and toxins to collapse and kill host cells [ Vmm at satisfactory levels [Apple tree canker disease, caused by orchard . Officia orchard . In addi orchard , 4. As ast cells –8. Presey levels , 10. To  Colletotrichum fragariae that was able to induce defense responses against anthracnose caused by a virulent isolate of C. acutatum in strawberry plants. In this case, protection effects were accompanied by a rapid accumulation of the reactive oxygen species including hydrogen peroxide and superoxide anion in leaves [ Fusarium oxysporum as a biological agent to control Fusarium wilt disease on tomato.As an alternative to traditional control method, it is of great interest to employ induced resistance in plants to combat economically important plant diseases , 12. It n leaves . Similarn leaves  utilized Vmm-apple callus interaction system for disease evaluation in laboratory. In this system, the induction of resistance in apple calli mediated by the attenuated Vmm strain LXS081501 against a virulent Vmm strain LXS080601 was verified. To gain an understanding of the underlying mechanisms, osmotic adjustment substance contents and elicitation of defense-related enzymes were evaluated in apple callus challenged with attenuated strain LXS081501, virulent strain LXS080601, or both. This would be the first report to describe induced resistance in apple mediated by attenuated pathogenic fungi.Apple canker disease usually displays symptoms on branches and trunk. However, there are several shortcomings when utilizing branches as experimental materials for research, due to variations in branch ages and water content as well as the sampling times. To circumvent these disadvantages, we established a stableMalus domestica Borkh.), were used in the experiments. “Fuji” and “Gala” are main varieties grown in China but are susceptible to Vmm. Five-year-old apple trees were grown in a greenhouse under natural daylight conditions.Two apple cultivars, “Fuji” and “Gala” (M. spectabilis). Strains were single-hyphen propagated to obtain pure cultures on PDA medium (potato dextrose agar) and maintained on PDA slants at 4°C [A virulent strain LXS080601 was obtained from infected “Fuji” trees and an attenuated strain LXS081501 from diseased Chinese flowering crabapple  standards established by Liang et al.  with somSamples were designated as follows: (i) callus inoculated with LXS081501 48 h prior to LXS080601 inoculation; (ii) callus inoculated with LXS081501; (iii) callus inoculated with LXS080601; (iv) callus inoculated with PDA as control. For each treatment, calli were sampled randomly at different time points . Each treatment contained three replicates with each replicate having five calli. The whole experiment was repeated twice. Calli were arranged in sealed Petri dishes at 95% relative humidity and 25°C for 144 h. At the established time intervals, tissue cylinders (6 mm) from each callus were excised from the inoculation sites. In the case of inoculation with both LXS081501 and LXS080601, samples were only taken from LXS080601-inoculated sites. The excised tissues were rapidly frozen in liquid nitrogen and stored at −86°C until use for osmotic adjustment substance contents analysis and enzyme activity assays.μg/g FW).Free proline was extracted according to Abrahám et al.  with somμg/g FW.The method to determine soluble sugar was modified from a procedure by Brugnoli . 1 g calβ-mercaptoethanol (w/v), pH 7.6). The mixture was kept in agitation on ice for 30 min and then centrifuged at 10000 g for 10 min at 4°C with the supernatant being saved. The quantification of the soluble protein was determined at 595 nm with Coomassie Brilliant Blue G-250, and bovine serum albumin  was used to prepare standard curve [The determination of soluble protein was carried out with 1 g callus sample, which was homogenized in 5 mL extraction buffer , 10% glycerin (w/v), and 0.5% rd curve .Phenylalanine ammonia lyase (PAL), peroxidase (POD), polyphenol oxidase (PPO), and catalase (CAT) were extracted according to the method of Moerschbacher et al.  with som2O2 (24 mM) was added.PAL activity was analyzed with modified method of Youssef et al. . The rea2O2 according to the method of Cakmak and Marschner [2O2. The change in the absorbance at 240 nm was assayed. The activity of PAL, POD, PPO, and CAT was expressed as units per milligram of total protein per minute or hour (U/mg protein/min or U/mg protein/h), where one unit was defined as the change in absorbance per minute or per hour.PPO activity was determined by adding 0.5 mL enzyme extract to 2.5 mL catechol substrate . The increase in the absorbance at 405 nm was measured . CAT actarschner . The reaβ-1,3-glucanase and chitinase activity assay, 1 g tissue sample was homogenized on ice in 3 mL of 50 mM sodium acetate buffer (pH 5.0). The homogenate was centrifuged at 12000 g for 20 min at 4°C, and the supernatant was used for the enzyme activity assays.For β-1,3-Glucanase activity was determined using the method described by Wang et al. [μmol of N-acetyl-glucosamine per hour for β-1,3-glucanase (U/mg protein/min) and chitinase (U/mg protein/h), respectively.g et al.  with modg et al. . Final aP < 0.05).All statistical analysis was carried out with SPSS software . Analysis of variance (ANOVA) was performed to determine the statistical significance by Duncan's multiple range tests . Similar trends were observed for the free proline levels in calli pretreated with LXS081501 followed by LXS080601 infection. However, in the latter case, free proline slightly decreased after 96 hpi but still remained at high levels at 144 hpi (45.27 μg/g FW). During early time points until 72 hours, the free proline of LXS080601 treated calli increased slowly, which declined to control level at 120 hpi. Overall, LXS080601-inoculated calli produced much less free proline compared with the other treatments. The control exhibited no significant change overtime , and then the soluble protein content decreased to control level at 120 hpi. Calli inoculated with either LXS081501 or LXS081501 plus LXS080601 experienced continuous rise in soluble protein content, with the later treatment increasing more dramatically, reaching 0.86 mg/g FW at 120 hpi. It was noticeable that, until 72 hpi, the soluble protein levels were similar for LXS081501 and LXS080601 treatment; however, after that time point, soluble protein content for calli inoculated with LXS081501 kept accumulating while the other diminished to control level.Four enzymes, including PAL, POD, PPO and CAT, were selected to determine their activities for different treatments. All these enzymes remained unchanged activities for PDA inoculated calli, which served as control. The other three treatments  showed iFor PAL, its activity started to increase at an early time point (24 hpi) for all pathogen treated calli. Specifically, LXS080601 treated calli showed enhanced PAL activates until 72 hpi (2.61 times higher than control), since when the enzyme activity declined dramatically to control level at 144 hpi (1.39 U/mg protein/min). In contrast, when the calli were inoculated with LXS081501, PAL activity increased remarkably and peaked at 72 hpi (3.29 times higher than control). PAL then showed decreased activity but still remained highly active at 144 hpi compared with control or LXS080601 treatment. Substantial rise in PAL activity was observed for LXS081501 plus LXS080601 treatment (5.29 U/mg protein/min at 96 hpi).POD activity for three treatments showed similar changing patterns, with activity peaking at different time points with varying levels . In detaThe changing patterns for PPO activity were similar to those of POD activity. As shown in β-1,3-glucanase and chitinase activities, all treatments except control displayed various changing patterns. As shown in β-1,3-glucanase and chitinase activities at 24 and 48 hpi, and for the other following time points their activities were similar to those of control treatment. After LXS081501 inoculation, β-1,3-glucanase and chitinase activities increased significantly from 48 hpi and peaked at 96 hpi which was 2.08 and 2.40 times higher than control, respectively  and at 72 hpi for chitinase (3.05 times higher than control).For ectively . CompareValsa canker disease is a typical apple trunk disease and the research is hindered due to the experimental variations caused by the branch ages, water content, positions on the tree, and the sampling times. Wei et al. [ Vmm-apple callus interaction system to investigate Valsa canker disease. With culturing conditions amendable to manipulation, calli have good uniformity in growing status and can be propagated at any time, which minimizes variations and provides enough experimental materials. In order to evaluate the stability and reliability of Vmm-apple callus interaction system, we examined the pathogenicity of 28 Vmm strains using “Fuji” calli, detached branches, and younger trees as experimental materials. Interestingly, the results were consistent in the three independent experiments. In addition, this interaction system allows for evaluating the resistant apple germplasm resources such as M. hupehensis Rehd., M. baccata Borkh., and M. sieboldii Rehd., and the results were in line with the identification in field. Furthermore, the calli can be used to estimate the effects of cell wall degradation enzymes and toxic compounds produced by Vmm [ Vmm pathogenesis.i et al.  developed by Vmm , 8, whic Vmm strain LXS081501 [ Vmm strain LXS080601 infection (see Supplement Table 1 available online at http://dx.doi.org/10.1155/2014/201382). In this study, when “Fuji” and “Gala” calli were pretreated with LXS081501, the disease symptoms and infection index produced by LXS080601 decreased significantly  on plant cells [ Rhizoctonia treatment increased POD and PAL activities and the elevated levels were positively correlated with the disease resistance in rice. In this study, LXS081501 in combination with LXS080601 also markedly induced POD and CAT activities . When ‘Fuji' and ‘Gala' younger trees were inoculated with LXS080601 alone, the lesion sizes were 5.31 cm2 and 3.93 cm2 at 15 dpi, respectively. In contrast, pretreatment of ‘Fuji' and ‘Gala' younger trees with LXS081501 effectively mitigated the disease development caused by LXS080601 since the lesion sizes were only 1.11 cm2 and 1.15 cm2, respectively, much smaller than when inoculated with LXS080601 alone.To evaluate the induced resistance of the attenuated


Fragile X Syndrome is the leading monogenetic cause of autism and most common form of intellectual disability. Previous studies have implicated changes in dendritic spine architecture as the primary result of loss of Fragile X Mental Retardation Protein (FMRP), but recent work has shown that neural proliferation is decreased and cell death is increased with either loss of FMRP or overexpression of FMRP. The purpose of this study was to investigate the effects of loss of FMRP on behavior and cellular activity.Xenopus laevis tadpoles and performed a series of behavioral and electrophysiological assays. We investigated visually guided collision avoidance, schooling, and seizure propensity. Using single cell electrophysiology, we assessed intrinsic excitability and synaptic connectivity of tectal neurons.We knocked down FMRP expression using morpholino oligos in the optic tectum of We found that FMRP knockdown results in decreased swimming speed, reduced schooling behavior and decreased seizure severity. In single cells, we found increased inhibition relative to excitation in response to sensory input.Our results indicate that the electrophysiological development of single cells in the absence of FMRP is largely unaffected despite the large neural proliferation defect. The changes in behavior are consistent with an increase in inhibition, which could be due to either changes in cell number or altered inhibitory drive, and indicate that FMRP can play a significant role in neural development much earlier than previously thought. Fragile X Syndrome (FXS) is the leading monogenetic cause of autism and most common form of inherited intellectual disability –3. FXS iXenopus laevis tadpoles were used to assess the early effects of FMRP upregulation and knockdown in the optic tectum. Tadpoles express FMRP throughout central nervous system (CNS) development . Brains were then pinned to a submerged block of Sylgard in a recording chamber and maintained at room temperature (24 °C). To access tectal cells, the ventricular membrane surrounding the tectum was carefully removed using a broken glass pipette. For evoked synaptic response experiments, a bipolar stimulating electrode (FHC) was placed on the optic chiasm to activate retinal ganglion cell (RGC) axons.For whole-brain recordings, tadpole brains were prepared as described by  and 22]22]. In bA antagonist picrotoxin (100 μM) was added to the external saline in a subset of experiments. Spontaneous synaptic events were collected and quantified using a variable amplitude template . Recordings were restricted consistently to retinorecipient neurons in the middle one-third of the tectum, thus avoiding any developmental variability existing along the rostrocaudal axis , 23, 24.template . Voltagestudy by , by measBehavioral and electrophysiological experiments were performed on stage 49 tadpoles in which FMRP expression was knocked down using a morpholino-antisense oligomer  during cXenopus laevis tadpoles, and across species that relies heavily on the optic tectum for sensory integration and perception. Organisms engage in this behavior to maneuver away from impending predators or objects. We observed that FMRP KD tadpoles have significantly decreased background swimming speed prior to stimulus presentation , indicating an overall decrease in activity levels. However, we found that FMRP KD tadpole collision escape responses were not significantly different from controls in measures of escape speed  and collision escape distance . These data indicate that while FMRP KD tadpoles are overall slower swimmers, the basic visual function and escape responses are largely unchanged by FMRP KD.We performed a series of behavioral experiments designed to test various functional aspects of neural circuit development . Visual KS < 0.05; N = 51 for FMRP and control tadpoles; see Fig. KS < 10−20; N = 51 for both FMRP KD and control tadpoles). Both of these measures indicate that FMRP KD tadpoles show decreased schooling behavior.Without major effects to the basic escape behavior, we wanted to identify if behaviors particularly relevant to FXS were affected in our FMRP KD tadpoles. A common behavioral marker of FXS and autism spectrum disorders (ASDs) is social interaction deficits. Tadpoles normally engage in a social aggregation behavior, called schooling . Schoolit = 0.038 1.06 ± 0.16 events per minute for controls and 0.94 ± 0.23 events per minute for FMRP; N = 22 for controls and FMRP KD tadpoles) and significantly longer seizures . The decreased seizure frequency and increased seizure length  and potassium currents , and found no significant difference. We also did not find any significant differences in spike output evoked by direct depolarization over a range of current injections . These results indicate that despite altered arborization, FMRP KD does not affect normal development of intrinsic excitability in tectal neurons.To begin to examine the underlying causes of the behavioral phenotypes, we first investigated the intrinsic properties of the tectal neurons . We founons Fig. –e and thMW > 0.67, 4.73 ± 2.95 events per second, n = 28 for controls, 5.18 ± 3.24 events per second, n = 28 for FMRP KD) and amplitude , as did the inhibitory post-synaptic currents (sIPSC) . Spontaneous recurrent activity is measured as the occurrence of barrages of activity of at least 200 ms duration with a 10 pA change in holding current and indicates the amount of recurrent circuitry present in the network. It is a measure of how interconnected the tectal network is. We found no difference in the frequency of spontaneous recurrent activity . These results indicate that the synaptic connectivity of the cells present in the network appears similar, and that a given cell has roughly the same number, frequency and type of synapses.We next examined the connectivity of the tectal network. Given the known cell proliferation defect , we knowt = 0.78, 2.3 ± 1.0, n = 13 for controls, 3.4 ± 2.2, n = 8 for FMRP KD). Furthermore, paired pulse facilitation, a presynaptic measure of synaptic efficacy [t > 0.22, 1.68 ± 0.60, n = 12 for control, 2.10 ± 0.86, n = 9 for FMRP KD). In the tectum, afferent visual input recruits a large amount of recurrent excitation [t = 0.84, 1620.9 ± 1342.9 pA.ms, n = 18 for controls, 1716.7 ± 1395.7 pA.ms, n = 15 for FMRP KD; 0-300 ms: Pt = 0.91, 7126.6 ± 6365.6 pA.ms, n = 18 for controls, 7339.9 ± 4784.7 pA.ms, n = 15 for FMRP KD), indicating an overall normal network level responses to visual inputs. We also measured the ratio of charge evoked by the monosynaptic afferent input, to the total amount of network activity. This provides us with a measure which we term monosynapticity index, which is a measure of how much network activity is evoked by visual input [MW = 0.44, 1.0 ± 0.64, n = 18 for controls, 0.77 ± 0.51, n = 15 for FMRP KD), indicating that excitatory network connectivity is unaffected. Together, these data show that excitatory synaptic activity and recurrent excitation are not changed by FMRP knockdown by FMR1 morpholino despite decreased proliferation and dendritic arborization.While spontaneous synaptic transmission might be indicative of the total input a tectal neuron receives, it does not tell us anything specific about the state of visual inputs to the tectum. To that end, we investigated responses to evoked visual stimulation. Evoked synaptic activity provides a measure of synaptic strength and network connectivity. First, we assayed basic synaptic properties. The AMPA to NMDA ratio is a measure of synaptic maturation, as cells incorporate additional AMPA receptors as they mature; therefore more mature cells have a larger AMPA:NMDA ratio . We founefficacy , was notcitation , 31. Firal input . We founMW = 0.048, 1714 ± 1363 pA.ms, n = 11 for controls, 3421 ± 2490 pA.ms, n = 17 for FMRP KD). However, the excitation is not affected by FMRP KD , which yields a significantly smaller excitation to inhibition ratio in FMRP KD tadpoles . We also looked at the overall time course of recurrent inhibition, and found that inhibitory activity remained elevated for a longer time following synaptic stimulation in the FMRP KD tadpoles , indicating altered dynamics of inhibitory circuits.It has been noted in other studies that a discoordination of excitation and inhibition may underlie many neurodevelopmental disorders , 33. To Our findings indicate that FMRP knockdown by FMR1 morpholino in the developing optic tectum has behavioral and electrophysiological consequences. Behaviorally, FMRP KD animals showed slightly reduced baseline swimming activity, decreased schooling behavior and decreased seizure susceptibility, but normal visual avoidance. These behavioral findings are consistent with decreased excitation within the tectum. Electrophysiologically we confirmed that neuronal intrinsic excitability and development of synaptic connectivity appear to be normal, however we found a significantly larger amount and longer lasting evoked network inhibition within the tectum, suggesting that this imbalance in the excitation to inhibition ratio may be responsible for the behavioral phenotypes.These findings are unexpected since Faulkner et al.  identifiDisruptions in the normal balance of excitation and inhibition during development have been implicated in a number of neurodevelopmental disorders, ranging from schizophrenia to autism , 35–37. To follow up on this study, it will be important to investigate the network properties of the optic tectum. The interaction of the decreased neural proliferation found by Faulkner et al.  and the Fragile X Syndrome and other neurodevelopmental disorders affect people in many ways, but have proven difficult to treat clinically. With our work, we have shown an explanation of why that may occur. It is clear that one particular insult does not result in a single, robust phenotype. Our research shows the opposite: small changes at the cellular level, combined with a neural proliferation defect, gives rise to the behavioral phenotype.P value for Kolmogorov-Smirnov test; Pmw, P value for Man-Whitney u-test; Pt, P value for t-test; PTZ, pentylenetetrazol; RGC, retinal ganglion cell; RNA, ribonucleic acid; sEPSC, spontaneous excitatory post-synaptic current; sIPSC, spontaneous inhibitory post-synaptic currentASD, autism spectrum disorders; CNS, central nervous system; FMR1, fragile X mental retardation 1 gene; FMRP, fragile X mental retardation protein; FXS, Fragile X Syndrome; K, Potassium; KD, knock down; MO, morpholino; Na, sodium; Pks,


Castanopsis tibetana, Machilus thunbergii and Schima superba. Contrary to a chorus of previous phylogeographic studies and the results of species distribution modelling (SDM) of this study , the three species displayed three different phylogeographic patterns that conform to either an in situ survival model or an expansion-contraction model. These results are partially congruent with the inference of PBR that EBLF was absent to the north of 24° N at the LGM. This study suggests that the constituents of EBLF could have responded idiosyncratically to climate changes during the Late Quaternary. The community assemblages of EBLF could have been changing over time, resulting in no palaeo-analogs to modern-day EBLF, which may be the main reason responsible for the failure of PBR to detect the occurrence of EBLF north of 24° N at the LGM.Subtropical evergreen broad-leaved forest (EBLF) is one of the most important vegetation types in China. Inferences from palaeo-biome reconstruction (PBR) and phylogeography regarding range shift history of EBLF during the late Quaternary are controversial and should be reconciled. We compared phylogeographic patterns of three EBLF constituents in China,  Machilus thunbergii displays the lowest nucleotide diversity, while C. tibetana has the highest. The average population genetic diversity values are all considerably low in C. tibetana, M. thunbergii, and S. superba .Estimates of haplotype diversity . As shown in M. thunbergii (P < 0.001), but not in C. tibetana or S. superba , indicating phylogeographic structure of haplotype distribution, but this was not significant in either C. tibetana  or M. thunbergii .In the multiple regression analyses of genetic variation, haplotype diversity was correlated negatively with latitude in  superba . NonetheC. tibetana, the neutrality tests showed that both Tajima’s D  and FS  were negative. The mismatch distribution for haplotypes was unimodal  years bp, although this estimate should be treated with caution given the wide confidence interval  and FS , unimodal mismatch distribution and positive values of SSD and HRag (P > 0.05) consistently suggested a past expansion event  and a significant positive FS . Despite a good statistical fit of the expansion model   Curve’ (AUC) all have values > 0.95, indicating good predictive model performance. In general, the predicted distributions of the three species under present conditions are similar to their actual distributions in mainland China, each with a potentially patchy range in mountainous areas of subtropical China and a slightly more continuous range for tability . Therefotability . ENMTOOLsuperba) .C. tibetana, M. thunbergii and S. superba, have different range shift trajectories during the late Quaternary, which cannot be predicted purely from either the ISS or EI model.Recently, phylogeographic patterns of subtropical EBLF constituents in China have received increasing attention . These s910Castanopsis tibetana (Fagaceae), displays a pattern of multiple northern refugia  and PBR (24°N). These results suggest that the inference of previous phylogeographic studies could be biased by stochastic variance among species and phylogenetic niche conservatism within Fagaceae. More phylogeographic surveys, particularly CP studies with the same sampling strategy and molecular markers, may provide an opportunity to reconcile the discrepancy between PBR and phylogeography.Although there are contradictory results in a few cases42M. thunbergii retreated to the south during the LGM, the complete absence of EBLF to the north of 24°N at that time suggested by PBR is still contrary to the phylogeographic patterns of most plant species characteristic of this vegetation. Although additional paleopalynological investigations are needed to verify the conclusion of previous PBR, the absence of EBLF to the north of 24°N during the LGM cannot be simply ascribed to the sparse pollen record. In fact, by comparing fossil records of individual forest plants and palaeo-biome maps in North America, Williams et al.C. tibetana and S. superba) as well as with a different species composition (as suggested by the evidence that M. thunbergii retreated to the south). This kind of community dynamics makes it likely that PBR would omit some EBLF components in the fossil record or misidentify the “EBLF” of the LGM as other types of vegetation because the LGM “EBLF” has no counterpart in the present vegetationDespite the fact that some species in EBLF such as in situ during the LGM C. tibetana and S. superba has been repeatedly revealed in the past 37Comparative phylogeography is a powerful tool for addressing community dynamics in distribution and abundance at the regional scale. However, the conclusions of this study might be subject to two limitations. First, because of financial constraints, this study was limited to two cpDNA markers to allow for a large number of individuals and populations to be surveyed. However, a given spatial genetic structure inferred by cpDNA reflects the history of maternal lineages, as recorded by seed movement, whereas pollen flow, as measured by the nuclear genome, may be more extensive than seed movement, and stochastic processes may lead to a wide range of genetic patternsC. tibetana, 392/46 of M. thunbergii, and 515/54 of S. superba, were sampled  from many localities to sufficiently cover the entire geographic range of each species: 332 individuals/45 populations of  sampled ; Fig. 1.ca. 20 mg of silica-dried leaf material using a modified cetyltrimethyl ammonium bromide (CTAB) protocolpsbA–trnH and rpl32–trnL(UAG) intergenic spacer (IGS) regions for the full phylogeographic survey because they could be successfully amplified in all three species and contained relatively rich polymorphic sites.Total genomic DNA was extracted from Taq polymerase/μl, 0.5 mmol/L dNTP each, 20 mmol/L Tris-HCL (pH 8.3), 100 mmol/L KCl, and 3 mmol/L MgCl2; Tiangen]. PCR amplifications were conducted under the following conditions: 5 min at 80 °C, 31–35 cycles of 1 min at 95 °C, 1 min of annealing at 50–58 °C, followed by a ramp rate of 0.3 °C/s to 65 °C, and extension at 65 °C for 4 min, with a final 5-min extension at 65 °C. PCR products were purified using TIANgel Midi Purification Kit  prior to sequencing. Sequencing was carried out from both directions using the amplification primers. Sequences containing microsatellites and singletons in some individuals were amplified and sequenced twice to exclude the possibility of PCR errors.PCRs were performed in 20-μl volume containing 50–100 ng genomic DNA, 0.5 μmol/L of each primer, and 10 μl 2 × Taq PCR MasterMix [0.1 U sequencher  and were aligned using clustal_x 1.81network 4.1.0.8 (http://www.fluxus-engineering.com).All sequences for each region were edited with haplonsthT and hS), as well as population differentiation based on ordered (NST) and unordered (GST) haplotypes. The values of NST and GST were then compared using U-statistics to test for the presence of phylogeographic structure. To test the EI model for each species, we calculated the correlation of genetic variation with latitude by performing a multiple regression analysis in spss version 13.0 . Spatial genetic structure of each species was analyzed by the program samova K) of populations that are geographically homogeneous and maximally differentiated from each other.We used the program DFSarlequin (ver. 3.1)SDD) between observed and expected mismatch distributions, and raggedness index (HRag), using 1000 parametric bootstrap replicates. The expansion time (in generations) for expanding species or groups was calculated using the formula T = τ/2 μkgμ is the substitution rate in substitution/site/year, k is the average sequence length used for analysis , and g is the generation time in years. The μ of C. tibetana was set as 0.71 × 10−9 substitutions per site year−1 (s/s/y), which is the mean substitution rate of two chloroplast intergenic spacers (ndhJ-trnF and atpI-atpH) in a closely related genus Fagus we previously studiedμ of three chloroplast intergenic spacers  in another Fagaceae member, Quercus glauca (0.96 × 10−9 or 0.69 × 10−9 s/s/y)M. thunbergii,a phylogenetic tree of Lauraceae was reconstructed based on the chloroplast sequences  of species in et al.beast v.1.7.1 using the same settings as that study used. We chose the local substitution rate (4 × 10−9 s/s/y) of clade D of et al.psbA–trnH and rpl32–trnL(UAG) in M. thunbergii, because substitution rates varied among clades and clade D is a minimally monophyletic group that includes M. thunbergii when only chloroplast sequences were used for phylogenetic reconstruction. Similarly, we reconstructed the phylogenetic tree of Theaceae in et al.beast v.1.7.1 to approximate the substitution rate of chloroplast genome in S. superba. The local substitution rate (0.6 × 10−9 s/s/y) of the clade C1 [see et al.Schima was embedded, was adopted in this study. There is no accurate record for the first reproduction age of the three species. For C. tibetana, 25-year was used based on the generation time reported in another Castanopsis speciesOcotea tenera takes at least 5 years to reach its reproductive maturitySassafras was reported as 10 yearsM. thunbergii. We used 8 years to approximate the generation time of S. superba based on observations on age of first reproduction of cultivated trees at the arboretum of Jiangxi Agricultural University .Tajima’s beast v. 1.7.1 for estimating fluctuations in the effective population size of each species using the above substitution rates accordingly. MCMC chains were run for 20,000,000 generations for C. tibetana, M. thunbergii and S. superba under the GTR, GTR + G and HKY model chosen by jModelTest 2.1.5 respectivelyWe used the Bayesian skyline plots (BSPs) methodmaxent)http://www.worldclim.org)r ≤ 0.70  were retained for subsequent analyses.We employed the maximum entropy approach  data are available. Each model was run 10 times using the default parameters  and the following user-selected features: application of a random seed, duplicate presence records removal and logistic probabilities used for the output. 80% of species records were used to train the model and 20% to test the model. Model accuracy was measured by the area under the ‘Receiver Operating Characteristic (ROC) Curve’ (AUC)71SDMs were constructed using 205/127/169 presence records of et al.enmtools, version 1.3, was used to calculate Schoener’s DWe then measured niche differences between each species’ ENM following the suggestions of Warren How to cite this article: Fan, D. et al. Idiosyncratic responses of evergreen broad-leaved forest constituents in China to the late Quaternary climate changes. Sci. Rep.6, 31044; doi: 10.1038/srep31044 (2016).


Carbon storage at Wanang was somewhat lower than in other lowland tropical forests, whereas local variation among 1-ha subplots and the contribution of small trees to total AGLB were substantially higher. We speculate that these differences may be attributed to the dynamics of Wanang forest where erosion of a recently uplifted and unstable terrain appears to be a major source of natural disturbance. These findings emphasize the need for locally calibrated forest carbon estimates if accurate landscape level valuation and monetization of carbon is to be achieved. Such estimates aim to situate PNG forests in the global carbon context and provide baseline information needed to improve the accuracy of PNG carbon monitoring schemes.Efforts to incentivize the reduction of carbon emissions from deforestation and forest degradation require accurate carbon accounting. The extensive tropical forest of Papua New Guinea (PNG) is a target for such efforts and yet local carbon estimates are few. Previous estimates, based on models of neotropical vegetation applied to PNG forest plots, did not consider such factors as the unique species composition of New Guinea vegetation, local variation in forest biomass, or the contribution of small trees. We analysed all trees >1 cm in diameter at breast height (DBH) in Melanesia's largest forest plot (Wanang) to assess local spatial variation and the role of small trees in carbon storage. Above-ground living biomass (AGLB) of trees averaged 210.72 Mg  ha The Uniet al. et al. et al. −1  forest plot with measurements of all trees over 1 cm DBH to examine local spatial variation and the contribution of small trees to above-ground forest carbon. The large size of the plot provides a carbon estimate that is robust to local spatial variation and which is useful for interpreting differences among previous estimates based on smaller plots. Extensive measurement of small trees (<10 cm) further enabled the discovery of a substantially greater contribution of small trees to overall forest carbon than was known from forests elsewhere. Incorporating this new information improves the overall accuracy of estimates for PNG.The Wanang Forest Dynamics Plot (FDP) was established in 2009 in lowland rainforest in the Middle Ramu region of Madang Province, Papua New Guinea (PNG). The plot is gridded to 20 m by 20 m quadrats according to protocol developed by the Center for Tropical Forest Science Condit . TopograDuring 2009 through 2013, every woody stem >1 cm DBH in the Wanang FDP was tagged, DBH measured, mapped and identified to species. The 50-ha plot included 288 204 stems. In total, we recorded 536 taxa including 33 unnamed morphospecies and 503 named species. Morphospecies refer to undescribed taxa for which there may be uncertainty in identification beyond the genus level. The dataset also included 15 067 trees that could not be assigned to a morphospecies or species.et al. Aglaia brownii did not have a wood specific gravity value in our data set, so we assumed phylogenetic conservatism and used the average Aglaia wood specific gravity estimate. Likewise, if no genus level data were available, a family level average was used. If no family level data were available, a community-wide average for Wanang forest species was used. Wood specific gravity values were assigned as specifically as compiled information and taxonomic identifications allowed, assuming phylogenetic conservatism of wood specific gravity. We assigned wood specific gravity values to 289 species, 194 taxa were assigned a genus average value, 26 taxa were assigned a family average value, and the community average wood specific gravity was used for 27 taxa lacking taxonomic information.Wood specific gravity was obtained either from a destructively sampled forest plot at Wanang for 208 species Whitfeld  or publiet al. (ρ(−1.239 + 1.980 ln(D) + 0.207(ln(D))2 − 0.0281(ln(D))3), where ρ is wood specific gravity (g/cm2) and D is diameter (cm). Averages and 95% confidence intervals (CI) for AGLB and stem density were produced by 1000 bootstraps on 1-ha sub-plots to provide information on spatial variation in biomass . A two-tailed t-test compared per-hectare small tree biomass (<10 cm) based on measurements of individuals >1 cm to estimates based on the common assumption that small trees constitute 5% of large tree biomass.The relationship between AGLB and plot topography was explored using ordinary least squares regression. Topographic variables were calculated from plot survey data using the CTFS R package. Relationships between elevation, slope, convexity of quadrats and AGLB were explored in separate regression models. AGLB was log-transformed for normality and homoscedasticity. Significance was evaluated for each linear model at α = 0.05 and the explanatory value of models was assessed based on evaluation of the portion of variance explained  in trees >1 cm DBH  as well as estimates for lowland rainforests of the Americas (287.9 Mg  ha−1), Asia (393.24), Africa (393.3)   Mg  ha−1 AGLB (95% CI 14.8–15.6), or 7.2% of total AGLB in the plot as a whole , making up 72.6% of total biomass (Table −1 (95% CI 28.9–46.3) and 17.92% of total AGLB  averaging 5240 stems per hectare (95% CI 5100–5385) and accounting for 15.19 Mg  ha−1 . Few studies have reported comparable biomass estimates including trees as small as 1 cm DBH , or being much higher than ours, greater than the American estimate, and nearer to the global mean , perhaps the divergent estimates of different authors might be explained by a bias toward locating the plot in more massive forests. Re-analysing data from the 1-ha destructively sampled plot (Whitfeld et al. et al. (−1. This figure is much larger than our average per hectare AGLB estimate of 210.7, but does fall within the range of 96.5–347.8 Mg  ha−1 within the 50 ha FDP. This large value relative to our plot average illustrates the possible bias introduced by limited spatial sampling to characterize forest biomass.This study, in agreement with Fox et al.  and Brya (et al. , found t (et al. . The que (et al.  closely  (et al.  and Brya (et al.  and is s (et al. , who inc (et al.  was obta. et al.  located . et al.  was subs. et al.  producedThe lesser biomass of PNG forests compared to the global average could be due to their dynamic nature (Swaine & Whitmore By including measurements of small trees, species-specific wood density measures and local spatial variation our estimate provides a unique perspective into above-ground carbon storage in PNG. Although our estimate applies only to lowland primary rainforest, this forest type covers a vast area of 18.65 million hectares in PNG (Shearman & Bryan et al. et al. et al. Forest succession theory predicts that a more dynamic forest will contain a higher proportion of biomass in small trees Chazdon . Shaped −1. This error may seem small but extrapolation could propagate a minor discrepancy across millions of hectares and grossly underestimate forest carbon at larger scales. On the other hand, our assumptions about wood density may incorrectly estimated small tree biomass. Wood density is known to vary within trees (e.g. Wiemann & Williamson et al. Plot-based vegetation methods inevitably set a minimum diameter for stem tree measurements and, commonly, only trees > 10 cm DBH are measured. The popular assumption that biomass of trees < 10 cm DBH is equal to 5% of large tree biomass underestimated Wanang tree biomass by 4.4 Mg  haet al. et al. et al. et al. et al. Variation observed among subplots within 50 ha of contiguous forest Fig.  supportset al. Our study examined some of the methodological limitations of prior work including plot size, lack of small tree measurements and wood density assumptions. Further limitations that also apply to our own estimates include failure to incorporate height measurements or to validate the assumption that carbon represents 50% of AGLB (Martin & Thomas et al. (et al. (We extrapolated our Wanang estimate and AGLB estimates from Fox et al.  and Brya (et al.  for the  (et al. . LikewisAs with any extrapolation, scaling estimates of forest biomass per hectare to a landscape level will propagate errors. The extrapolations required by schemes to monetize carbon and incentivize forest preservation for the purposes of reducing global atmospheric carbon concentrations should be regarded with great caution. Our results show substantial biomass variation in a contiguous forest that many would assume to be homogenous. Although large plots like Wanang are impractical for REDD+ implementation, we suggest that greater consideration be given to local spatial heterogeneity. Should projects such as REDD+ move past the pilot phase and into wider implementation it will be important to consider how errors associated with carbon estimates may impact economic reality.Carbon estimates are ideally generalizable across the landscape. The largest and most detailed measurement of a continuous forest in Melanesia suggests that PNG lowland rainforests contain less biomass per hectare than lowland tropical rainforests on average. This pattern could be due to the elevated disturbance and dynamism of lowland PNG forests, an explanation that has been suggested in the literature but remains untested. A recensus of the Wanang plot is needed to examine this possibility.et al. et al. et al. We also found a higher proportion of biomass in small trees than is typically assumed for lowland tropical rainforests. Our findings suggest that the carbon estimates reported by (Bryan