Identification of tomato varieties able to exhibit higher accumulation of primary and secondary metabolites in their fruits is currently a main objective in tomato breeding. One tool to improve fruit quality is to cultivate the plants under salt stress, although improvement of fruit quality is generally accompanied by productivity losses. However, it is very interesting to implement strategies aiming at enhancing fruit quality of tomato by means of growing plants in moderate salt stress that allows for a sustainable fruit yield. The traditional tomato varieties adapted to the Mediterranean environmental constraints may be very attractive plant materials to achieve this goal, given the wide range of fruit quality traits because of their genetic diversity. Here, agronomic responses and fruit quality traits, including primary and secondary metabolites, were analyzed in fruits of two Mediterranean traditional tomato varieties named “Tomate Pimiento” (“TP”) and “Muchamiel Aperado” (“MA”) because of the pepper and pear shape of their fruits, using as reference the commercial cultivar “Moneymaker” (“MM”). Plants were grown without salt (control) and with moderate salt stress (50 mM NaCl), which did not affect fruit yield in any variety. “TP” is of great interest because of its high soluble solids content (SSC) in control, which is even higher in salt, whereas “MA” is very attractive because of its high Brix yield index (SSC × fruit yield), used as overall fruit quality measure. Similitude between both traditional varieties were found for primary metabolism, as they significantly increased sucrose contents compared with “MM” in red ripe fruits from plants in control and, especially, salt stress conditions. The most remarkable difference was the high constitutive levels of total amino acids in “TP” fruits, including the three major free amino acids found in tomato fruit, GABA, glutamate, and glutamine, which even increased under salinity. Regarding secondary metabolites, the most interesting change induced by salinity was the increase in α-tocopherol found in red ripe fruits of both “TP” and “MA.” These results reveal the interest of traditional varieties as sources of genetic variation in breeding because of their improvement of tomato fruit quality without production losses under moderate salt stress. Solanum lycopersicum) is one of the most important horticultural crops worldwide and ranks second after potato in terms of global production and first in terms of yield , and seeds were stored and registered in a seed bank maintained by the Agricultural and Forestry Experimentation Service of the University of Murcia .v/v peat/perlite mixture inside a growth chamber under controlled conditions of 28°C and 90% relative humidity (RH). After emergence, seedlings were grown in the same growth chamber under environmental conditions of 18–25°C temperature, 50–70% RH, and a photoperiod of 16 h light/8 h darkness. A photosynthetic photon flux (400–700 nm) of 345 μmol m2 s–1 was provided at plant level by fluorescent tubes . During this period, plants were irrigated daily with half-strength Hoagland solution  solution was prepared in 2000-L tanks with local irrigation water [electrical conductivity (EC) = 0.9 dS m–1], with pH and EC regularly monitored. Plant fertigation was achieved using a drip irrigation system with 3 L h–1 drippers. Salt level (50 mM NaCl) was selected from preliminary assays using irrigation waters with increasing NaCl concentrations from 30 to 60 mM, being this salt level where plants were able to maintain fruit production. At the six-leaf stage, when time elapsed from sowing was 45 days, fertigation solution supplemented with 50 mM NaCl was applied to seven plants of each tomato variety for 80 days, while the other seven plants of each variety were irrigated without salt (control condition). Temperature and RH were recorded daily and showed daily fluctuations from 30 to 15°C and 40 to 60% (day/night), respectively. A completely randomized design with seven plants per variety was used for each treatment (0 and 50 mM NaCl).A spring–summer culture was maintained in a greenhouse located on the campus of the University of Murcia  that offered controlled culture conditions. At the four-leaf developmental stage (30 days after sowing), plants were moved to the greenhouse and transplanted; 14 plants per variety were grown in plastic pots containing 18 L of a 2:1 At the start of salt treatment, plant height, number of leaves, and chlorophyll content and fluorescence were determined to verify the homogeneity of the plants. The latter two physiologic parameters were measured as previously described .To determine fruit yield (based on fruit number and fruit weight), ripe fruits from the first to third truss of each plant were collected, weighed, and counted. At the end of the experiment, the shoot of plants was weighed, and the harvest index was estimated as the ratio of fruit yield to total plant biomass (vegetative and reproductive).2 and samples stored at −80°C until analyses were completed. Frozen samples were homogenized with a mortar and pestle to proceed with the metabolic analyses. A portion of frozen material was lyophilized for water content analysis, weighting it before and after the freeze-drying process. For each variety, fruit stage, and treatment, three biological replicates of 10 fruits each were analyzed.To determine fruit quality standard parameters [soluble solids content (SSC) and titratable acidity (TA)], fruits at the red ripe (RR) stage were harvested. Mature-green (MG) fruits that had reached their final size were also harvested for mineral and metabolite analyses. Pericarp of MG and RR fruits was chopped into pieces and immediately frozen in liquid N2O, followed by pH titration with 0.1 M NaOH up to pH 8.1; the result is expressed as grams of citric acid per 100 g of fresh weight (%). Fruit quality indices such as maturation index (SSC/TA ratio) and Brix yield  were calculated.For SSC analysis, an aliquot of frozen tomato fruit sample was thawed and filtered through a nylon membrane filter. The supernatant was collected and SSC was measured using a refractometer with automatic temperature compensation ; the result is expressed as Brix at 20°C. TA was determined in triplicate from the juice by taking 5 g of the thawed fruit pericarp homogenate and adding 45 ml of distilled H3:HClO4 (2:1 v/v) solution. Na+, K+, Ca2+, and Mg2+ contents were determined by inductively coupled plasma optical emission spectrometry (ICP-OES) on an ICAP 6500 DUO/IRIS Intrepid II XLD system . ICP-OES analysis was carried out at the Ionomics Platform of Centro de Edafología y Biología Aplicada del Segura (CEBAS)–Consejo Superior de Investigaciones Científicas (CSIC) .Fruit samples (pericarp) were lyophilized, milled to powder, and digested for 24 h in a concentrated HNOv/v) H2O:MeOH was added to 50 mg of lyophilized fruit sample (pericarp) and vortexed for 1 min, followed by sonication for 1 min and centrifugation . The supernatant was collected in a 2-ml microtube and dried in a rotary vacuum evaporator. The dried extract was reconstituted in 800 μl of D2O phosphate buffer [100 mM KH2PO4 (pH 6.0)] containing 0.01% trimethyl silyl propionic acid (TSP)  as an internal standard and vortexed for 1 min. The mixture was centrifuged  and 600 μl of the supernatant was transferred to nuclear magnetic resonance (NMR) tube for analysis.Primary metabolites were analyzed as previously described , 2006, w1H NMR spectra were recorded at 298 K on a Bruker AVIII HD 500 NMR spectrometer (500.13 MHz for 1H) equipped with a 5-mm CryoProbe Prodigy Broadband Observe cryogenic probe . The 1H spectra were referenced to the TSP signal (δ = 0.00 ppm), whereas 13C spectra were referenced to CH-1 resonance of α-D-glucose (δ = 93.10 ppm). For each sample, 32 scans were recorded with the following parameters: 0.126 Hz/point, pulse width = 4.0 μs (30°), and relaxation delay = 1.0 s. Free induction decay was Fourier-transformed with line broadening = 0.5 Hz, Gaussian broadening = 0, and peak-picking sensitivity = 1.0, and peak integral was used for quantitative analysis. Whole-peak intensities in every 0.02 ppm in 1H NMR spectra in the range of δ 0.30–12.0 were used as variables. 1H NMR spectra were manually corrected for phase and baseline distortions using TOPSPIN v3.2 (Bruker). Peak-fitting on the resultant spectra was performed using an algorithm in Chenomx NMR Suite v8.1 software  to obtain the concentrations of primary metabolites detected in plant material. The region δ = 4.67–5.15 was discarded to eliminate the effects of imperfect water presaturation. Spectral areas of all buckets were normalized to the weight of extracts used for measurements. The intensities of the selected 1H resonances attributable to hydroalcoholic metabolites were measured with respect to the intensity of the TSP signal used as the internal standard at a concentration of 0.58 mM. The metabolite analysis was performed at the Metabolomics Platform of CEBAS–CSIC.All w/v NaCl solution and 200 μl of dichloromethane, and vortexed for 1 min. Next, 500 μl of hexane/ether (1:1 v/v) was added to the mixture, which was stirred for 1 min and centrifuged at 13,000 × g at 4°C for 5 min. The supernatant was collected in a 2-ml microtube. This procedure was repeated three times and the organic phases from the repeats were combined. The remaining hexane phase was evaporated under an N2 atmosphere. The dried carotenoid extract was reconstituted in 300 μl of the injection solvent composed of acetonitrile (ACN)/MeOH (7:3 v/v)/acetone (6.7:3.3 v/v) for LC analysis. All sample solutions were filtered through a Millex 0.2-μm nylon membrane syringe filter  before injection into an Acquity I Class Ultra Performance LC system connected to a tunable ultraviolet (TUV) detector measuring absorbance at 286 and 450 nm . Chromatographic separation was performed on a reversed-phase Acquity UPLC ethylene-bridged hybrid (BEH) C18 column  (Waters) as previously described . The chloroform phase was recovered and the methanol phase  was re-extracted with 1 ml of chloroform as described previously. The chloroform phases were pooled and adjusted to a final volume of 2 ml and evaporated under an N2 atmosphere. The dried α-tocopherol extract was reconstituted in 0.2 ml of injection solvent (2:1 v/v CH2Cl2/MeOH) for LC analysis. All sample solutions were filtered through a Millex 0.2-μm nylon membrane syringe filter before injection into the UHPLC instrument.The analysis of α-tocopherol was carried out by ultra-high–performance liquid chromatography (UHPLC). The extraction procedure was performed as previously described . First, v/v ACN/MeOH. The column temperature was set at 30°C, the flow rate was 0.5 ml min–1, and the total runtime including column equilibration was 5.0 min. Eluting compounds were detected at 292 nm. α-Tocopherol was identified and quantified by comparing the retention time and peak area with those of a standard (Sigma-Aldrich). The stock solution of α-tocopherol was prepared by dissolving 2.54 mg in 1.0 ml HPLC-grade EtOH. Before use, aliquots were diluted in 3:2 v/v ACN/MeOH and calibration was performed with dose–response curves generated using the standard solutions. The analysis was carried out at the Metabolomics Platform of CEBAS–CSIC.The α-tocopherol content was determined using a Waters I-Class HPLC system coupled with a TUV detector. Separation was carried out on a normal-phase BEH C18 column  using an isocratic solvent system (mobile phase) consisting of 3:2 a and b contents in tomato fruit were determined as previously described  at 663 and 645 nm was measured using a spectrophotometer. Chlorophyll a and b contents were calculated according to the following equations.Chlorophyll escribed  using frp < 0.05) were evaluated by one-way ANOVA, Student’s t-test, or Tukey’s test. Statistical analyses were performed using Minitab v19.0 software . A principal component analysis (PCA) biplot and heatmap were generated from data matrices and used to ascertain the overall variability across cultivars and treatments for each fruit stage (MG and RR). Multivariate analysis was performed using the Metaboanalyst 4.0 server  were estimated for the first three trusses . AlthougRegarding fruit quality parameters, SSC was significantly higher in RR fruits of “TP” but significantly lower in RR fruits of “MA” compared with “MM” under control treatment . Salt stGiven that physiologic and metabolic changes can vary during ripening, fruits were analyzed at two developmental stages, namely MG and RR. Notably, water content was lower under salt stress in all genotypes at both fruit stages . “MA” sh+ contents were similar between the two landraces and “MM” at both stages of ripening and did not change with salt stress ], there is an inverse relationship among these metabolites, which is evident in the PCA biplots of MG and RR fruits , althougd plants . RR fruid plants . Total cd plants .There was a very small separation between control and salt treatments for “MA” fruits at the RR stage whereas for “MM” and “TP,” salt stress had a significant effect on the metabolite composition of RR fruits, similar to the changes observed in MG fruits . This seThe application of controlled abiotic stress can improve the sensory and nutritional value of fruits . As chan2+/Mg2+ratio, which is often correlated with organoleptic quality traits such as SSC and TA (2+/Mg2+ ratio and MI (SSC/TA ratio) in RR fruits  produced by the methylerythritol 4-phosphate pathway. Because of this metabolic cross-talk, changes in one of these compounds can affect the biosynthesis of the others . We obseThe mechanism of salt tolerance in plants subjected to salinity depends on the level of salt stress , which aIn conclusion, the two traditional varieties used in this study are attractive sources of genetic variation in breeding because of their improvement of tomato fruit quality under moderate salt stress. These traditional varieties were collected in the semiarid area of Spanish Southeast, featured by its very hot summers and the use of saline waters for irrigation due to the scarcity of hydric sources. Therefore, they constitute a rich reservoir of valuable tomato traditional varieties for breeding focused in improvement of fruit quality of tomato growing in a scenario of climate change. It is interesting to remark that development of new cultivars on the basis of their contents in health-promoting compounds is still very limited. Finally, the fact that α-tocopherol level in fruit nearly doubled in “TP” and “MA” plants relative to “MM” upon irrigation with 50 mM NaCl while fruit yield was unaffected demonstrates that cultivating these landraces under moderate salt stress is an effective agronomic strategy for improving the nutritional value of tomato fruit.The original contributions presented in the study are included in the article/SLRM, IE, and BM performed the experiments. SLRM, IE, and ILM collaborated in the design of the experiments and data analyses. EP, JME-F, MCB, and FBF conceived the project and research, and supervised the experiments. MCB and FBF wrote the manuscript. All authors contributed to the article and approved the submitted 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.


Two-photon imaging studies in mouse primary visual cortex (V1) consistently report that around half of the neurons respond to oriented grating stimuli. However, in cats and primates, nearly all neurons respond to such stimuli. Here we show that mouse V1 responsiveness and selectivity strongly depends on neuronal depth. Moving from superficial layer 2 down to layer 4, the percentage of visually responsive neurons nearly doubled, ultimately reaching levels similar to what is seen in other species. Over this span, the amplitude of neuronal responses also doubled. Moreover, stimulus selectivity was also modulated, not only with depth but also with response amplitude. Specifically, we found that orientation and direction selectivity were greater in stronger responding neurons, but orientation selectivity decreased with depth whereas direction selectivity increased. Importantly, these depth-dependent trends were found not just between layer 2/3 and layer 4 but at different depths within layer 2/3 itself. Thus, neuronal depth is an important factor to consider when pooling neurons for population analyses. Furthermore, the inability to drive the majority of cells in superficial layer 2/3 of mouse V1 with grating stimuli indicates that there may be fundamental differences in the micro-circuitry and role of V1 between rodents and other mammals. Studies frequently pool responses of neurons from different cortical depths in population analyses. Here, we show that population neuronal response characteristics in mouse primary visual cortex (V1) vary dramatically across depth planes separated by just 50 μm. We also demonstrate that the stimulus selectivity of neuronal responses varies with both cortical depth and the response amplitude of neurons. These findings highlight the importance of considering cell depth and response amplitude as important factors contributing to the overall characteristics of neurons in sensory cortex.With the emergence of two-photon imaging as a tool for systems neuroscience over the past 15 years, there has been an enormous increase in the use of the mouse as a model system to study cortical neuronal physiology. Mice are easily genetically modified to label specific populations of neurons with fluorescent indicators and opsins, allowing the study of cell type-specific circuitry . AdditioA further difference that has become apparent in the literature is the percentage of responsive neurons in the cortex. An early electrophysiological study reported that most neurons (87%) in mouse V1 were responsive to grating stimuli . HoweverIn addition to these differences in responsiveness and functional connectivity, the mouse neocortex is also much thinner than that of cats and primates. As a result, neurons from layers 1 through 4 can be easily accessed with conventional two-photon imaging techniques in the mouse visual cortex, whereas in cats and primates two-photon imaging is limited to the upper portion of layer 2/3. Although the ability to image more of the neocortex in the mouse can certainly be an advantage, it raises important considerations about how populations of neural activity should be analyzed and the functional differences of these populations across species. Layers of the neocortex have different anatomical and functional characteristics and play different roles in the basic circuitry of information processing . Yet, moIn the present study, we sought to determine how the response properties of neurons in mouse V1 depend on cortical depth. We show that although in superficial layer 2/3 only around half of the neurons are responding to grating stimuli, deep in layer 2/3 and in layer 4, nearly all the neurons are responsive, similar to what is seen in cats and primates. We demonstrate that the amplitude and the selectivity of neuronal responses to drifting grating stimuli depend on imaging depth – not only across laminae but even within layer 2/3 itself. Neuronal selectivity to orientation and direction across the population changes little with cortical depth, in agreement with previous studies . Howevern = 7 male, postnatal day 90–111) were initially anaesthetized with a bolus infusion of fentanyl citrate (0.04–0.05 mg kg−1), midazolam (4–5 mg kg−1), and dexmedetomidine (0.20–0.25 mg kg−1). During two-photon imaging, continuous intraperitoneal infusion with a lower concentration mixture  was administered using a catheter connected to a syringe pump. The heart and respiration rates of the animals were continually monitored throughout the surgeries and imaging. Craniotomies (2–3 mm) were opened over the primary visual cortex (V1) centered ∼2.5 mm lateral to the lamda suture and 1–1.5 mm anterior to the transverse sinus. A pipette containing a solution with Oregon Green 488 Bapta-1 AM (OGB-1 AM) and a red dye (Alexa Fluor 633 or Alexa Fluor 594) was inserted into the craniotomy and the dye was injected with pressure puffs under continuous visual guidance using two-photon microscopy . All experiments were performed at MUSC. C57Bl/6J mice (croscopy . PipetteFluorescence was monitored with a custom-built microscope (Prairie Technologies) coupled with a Mai Tai (Newport Spectra-Physics) mode-locked Ti:sapphire laser (810  or 920 nm) with DeepSee dispersion compensation. Excitation light was focused by a 40×  water immersion objective. Full frame imaging of ∼300-μm square windows was obtained at ∼0.8 Hz.−2 mean luminance, 1.5-Hz temporal frequency, and 0.033–0.063 cycles/°. Stimuli were optimized for retinotopic position and spatial frequency preference right at the layer 1/layer 2 border . Our injection site usually yielded receptive fields close to eye level (0° elevation) and roughly perpendicular to the eye (azimuth around 50–70°). At each of our depth planes, drifting gratings were presented at 16 directions of motion in 22.5° steps (except 1 of 35 runs which used 8 directions in 45° steps) for 6.5 s with 13 s of blank before each stimulus. Each condition was repeated at least 8 times except for two runs with only five repetitions due to the removal of later repetitions on account of large movements.Drifting square-wave grating stimuli were presented to the contralateral eye on a 17-inch LCD monitor. The gratings were presented at 100% contrast, 30 cd mC of the response that is attributable to out of focus contamination. Then the fluorescence time course for soma masks were corrected by subtracting this fraction of the surrounding neuropil fluorescence. So:C were between 0.35 and 0.72 (median = 0.56). Neuropil masks were created by expanding a spherical shell 15 μm beyond the soma masks. The inner 3 μm were excluded as a buffer zone around each neuron. Pixels were also excluded from the neuropil masks if they belonged to other soma masks and their 3 μm shells, blood vessels, non-neuronal cell bodies (such as astrocytes), or neuronal somas that were too out of focus to be included in the population. The radius of the neuropil mask was expanded, if necessary, until the neuropil area was greater than 10 times the soma area. The median radius of the neuropil masks was 14 μm and the range was 12–32 μm.Images were analyzed in MATLAB (MathWorks) and ImageJ . Data with significant movements  in XY or Z were excluded. Data with small drift movements were realigned by maximizing the correlation between frames. Cell masks were automatically created based on morphologic features and then subsequently refined by hand. Astrocytes were removed from the data based on morphologic criteria  and in 21-F0)/F0 where F1 was the average fluorescence across all 5 stimulus frames and F0 was the average of the last four frames of each blank interval. Neurons were defined as responsive using ANOVA across the 16 directions and the blank intervals (p < 0.01). Because different studies have used different criteria for responsiveness, we did two additional analyses using different criteria: (1) with p set to 0.001, and (2) where responsiveness was defined as average ΔF/F > 5% for at least one stimulus direction. The data showed the same depth-dependent trends in both cases  were computed as (Fth cases . The resThe Orientation Selectivity Index (OSI) was defined as:stimulus . Note thSI gOSI; . The OSIp < 0.01). If so, each neuron’s responses were then fit with a least-squares method to a Von Mises function:2 for the fit had to be at least 0.5 for inclusion in the selective population. For neurons that passed these criteria, we computed a population average response for each animal and depth plane. Each neuron’s responses were normalized to the peak response of that neuron and the preferred orientation was set to 0° before averaging.To analyze the tuning width of the neurons, we first screened the population for selective neurons. Neurons were tested to see if any of the stimulus conditions evoked a significantly different response from any other  was defined as:Stimulus-evoked “shadowing” from large surface vessel dilation can cause dimming of fluorescence from neurons during stimulus presentation windows. When neurons are not responding or responding weakly, this shadowing may lead to negative ΔF/F values from these neurons . This isR2 = 0.70, p < 10−9, linear regression, n = 35 imaged regions in seven mice; R2 > 0.79 and p < 0.04 for all animals, n = 5 regions per animal). Nearly all neurons (95%) within layer 2/3 responded to pharmacological stimuli . A pipette (schematized in pink) was filled with 50 mM glutamate and 100 μM Alexa Fluor 633 . The pipette tip size was ∼1 μm in diameter. This glutamate pipette was positioned approximately in the center of a region of cortical layer 2/3 that was bulk-loaded with OGB-1 A.M. The dashed ring shows the 100-μm diameter region around the pipette tip. Glutamate was locally injected into the cortical tissue four times, each puff of glutamate was applied for three seconds with 22 s between the puffs (each puff 10–20 psi). In this example, 100% of the neurons in the 100-μm diameter region around the pipette tip were activated by glutamate. Further away from the tip where the glutamate did not reach, most of the neurons were unresponsive. Across four regions in three animals  95% of the neurons in a 100-μm diameter window around the pipette tip were significantly responsive . Scale bar, 100 μm. B, Example responses to the glutamate injections from two cells labelled in A with yellow numbers/arrows. The blue traces are the individual repetitions and the black trace is the average. The vertical gray band indicates the glutamate injection period. Download Figure 1-2, EPS file.Control experiment; 95% of cells in superficial cortical layers of mouse V1 respond robustly to exogenous glutamate application. R2 = 0.66, p < 10−8, n = 35 regions neurons from seven animals; The depth dependence of neuronal responsivity to visual stimuli was observed not only in the proportion of responsive neurons but also in their maximum response amplitude. Responses to the preferred stimulus direction nearly doubled from 15.5 ± 2.2% (mean ΔF/F ± SD across runs) in superficial layer 2/3 to 28.9 ± 4.7% in layer 4 B,C. ThisR2 = 0.12, p = 0.04, n = 35 regions neurons from seven animals; n = 1672; middle responders 15% ≤ ΔF/F ≤ 24%, n = 1673; strong responders, ΔF/F > 24%, n = 1675), for each group, neurons were much less orientation selective deeper in the cortex  and middle responders  compared with weak responders . Additionally, independent of depth, strong responders were more orientation selective than middle responders  which, in turn, were more selective than weak responders (p < 10−9). Thus, orientation selectivity is affected by two competing trends. On the one hand, for a given level of responsiveness, orientation selectivity decreases when depth increases. On the other hand, at a given depth, orientation selectivity increases when neuronal responses increase. The two trends cancel each other out because depth covaries with neuronal responsivity . We observed the same trends as for the OSI: namely that stronger responders tended to be more orientation selective than weak responders, and that their tuning curves varied more with depth  located within layer 2/3.To accurately determine the percentage of responsive neurons in a region of cortex, one needs to monitor all the neurons in the tissue without bias. Electrophysiological recordings cannot achieve this since electrodes will only pick up neurons that fire action potentials and only in the region around the tip of the electrode. This limitation has led to the argument that most of V1 may be unresponsive to simple stimuli like oriented bars and gratings . In vivoUse of the synthetic dye OGB-AM was crucial to our study, because it labels all cells within a 300- to 400-μm diameter volume, including those that are unresponsive to visual stimuli. In contrast, genetically encoded calcium indicators, e.g., GCaMP6, can have heterogeneous expression levels in a local region of tissue , and thePrior studies comparing the amplitude of responses between layer 2/3 and layer 4 of mouse V1 have typically reported few differences. One electrophysiological study found that in awake mice, evoked firing rates were higher in layer 4 than in layer 2/3 . HoweverAlthough previous electrophysiological and imaging studies in mice have reported little variation in orientation selectivity with depth , a trendOne possible explanation for the increase in responsiveness with increasing depth in the mouse visual cortex may be the distribution of thalamic inputs. Genicular inputs are not as tightly constrained to layer 4 in mouse V1  as they selectivity of the boutons themselves could also play a role in setting the selectivity for orientation and direction across layers. One study reported that geniculate boutons in layer 4 have lower orientation selectivity but greater direction selectivity than boutons in layer 2/3 (In addition to the laminar distribution of thalamic boutons in visual cortex, the ayer 2/3 . Howeverayer 2/3 . Thus, mayer 2/3  by a relAnother possibility is that the full screen gratings we used caused greater surround suppression in the superficial depths compared with layers 3 and 4. Although past studies have reported that surround suppression is weaker in the infragranular layers than in layer 2/3 or layer 4 , none reAlthough weaker thalamic drive may explain the smaller percentage of responding neurons in superficial layer 2/3, there is still the question of what stimuli might activate the silent half of the neurons. Previous studies have found neurons in mouse V1 that are unresponsive to single gratings but do respond to two overlapping gratings  or to coThe depth dependence of stimulus selectivity we have found may have important implications for how information is encoded in mouse visual cortex. Theories of sparse coding have proposed that neurons in higher areas respond more sparsely than neurons in lower areas because they become selective for increasingly complex stimulus features . BecauseFunctional properties may vary across cells located at different depths within layer 2/3 in other sensory systems as well. Studies in the somatosensory and auditory cortices of mice have described differences in layer 2 and layer 3 in terms of their response properties as well as in their neuronal cell types and connectivity . This su


Asthma is a disease characterised by hyper responsiveness and bronchoconstriction of airways, and is a major health burden globally. A dysfunction of the oxidant-antioxidant balance, termed oxidative stress, has been implicated in the pathophysiology of asthma. The present study aims to assess the changes in oxidative stress markers, namely nitric oxide metabolites and antioxidant capacity, in children with poorly controlled and well controlled asthma, in comparison to healthy controls.x]) were determined using the Griess test, and the total antioxidant capacity (TAOC) was determined using the ABTS decolorisation method. The concentrations of these markers were compared across the three groups.The present study enrolled 72 children (ages 5-15 years) classified into three groups: (1) poorly controlled asthma (n = 20), (2) well controlled asthma (n = 24) and (3) healthy controls (n = 27). An interviewer-administered questionnaire was used to record socio-demographic data of the participants. The serum concentrations of the oxidant markers  and serum TAOC. Further analyses showed that the disparity for nitrate and NOx concentrations was greatest between poorly controlled asthma and healthy controls (p = 0.001 and p < 0.001) compared to the well-controlled asthmatics and healthy controls (p = 0.036 and p = 0.049). A significant difference in serum nitrate and NOx concentrations was not observed between the two asthma groups (p = 0.311 and 0.203). The TAOC were significantly lower in poorly controlled asthmatics as compared to well-controlled asthmatics (p = 0.003) and healthy controls (p < 0.001). However, there was no significant difference in the serum TAOC between healthy controls and well-controlled asthmatics (p = 0.496). These findings may indicate that it is perhaps the higher TAOC that contributes to the well controlled state of asthma.The three study groups were similar in terms of socio-demographic data. The differences across the three groups were statistically significant for serum concentrations of nitrate and NOThe present study indicated that an imbalance of oxidants and antioxidants in the serum may have an underlying role in asthma pathophysiology, and how these markers may be effective in asthma management. Asthma is a disease characterised by airway hyper-responsiveness, airway remodelling and reversible bronchoconstriction, affecting an estimated 235 million individuals worldwide, with a mortality of 383,000 deaths annually , 2. The The full scope of molecular mechanisms underpinning the changes seen in asthma is still unclear. Plausible contributory mechanisms include genetic predisposition –9, epige2−) and nitrate (NO3−) are metabolites of NO oxidation by the superoxide anion, and serve as proxy measures of the unstable ROS [Recently, there has been an interest in the role of oxidant-antioxidant balance (oxidative stress) in the pathophysiology of asthma. Oxidative stress arises from an imbalance between the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) accompanied by a depletion of the antioxidant defense mechanisms . RNS mayable ROS . Antioxiable ROS . Increasable ROS . Oxidatiable ROS –26. As table ROS . The perable ROS .This pilot study aims to determine the markers of oxidative stress in children with poorly controlled (symptomatic) and well controlled asthma (asymptomatic) patients in comparison to an age-sex matched group of healthy controls, in order to determine if greater oxidative stress does occur in uncontrolled asthma. This could highlight the potential use of oxidative stress measurement as an indicator of disease severity and a plausible role of antioxidants in asthma management.This comparative study was carried out from June to October 2019, amongst children aged 5–15 years presenting to the outpatient clinic of the University Paediatric Unit of the Lady Ridgeway Children’s Hospital, which is the premier, tertiary level paediatric hospital in Sri Lanka. Three groups of children were enrolled: (1) poor asthma control while on corticosteroid/combined inhalers  (n = 20)  as well as those without parental consent were excluded.There have been no previous studies that had measured oxidative stress in asthma in Sri Lanka. Hence the sample size was calculated based on an analogous study that assessed oxidative stress in fever patients in Sri Lanka using the same assays . The meaAn interviewer-administered questionnaire was used to record socio-demographic data (including monthly family income and parents level of education), and to clarify the degree of asthma control focussing on the GINA criteria. A general health check up was carried out for all children after consenting to participate. A stool smear examination, Full Blood Count and ESR was done free of charge. No parasites or parasitic stages were found on examination of saline and iodine smears. There was no significant difference in the ESR levels between the asthmatic children and healthy children.Trained experienced nurses in the clinic collected blood under aseptic conditions. Since serum nitrate is dependent on dietary factors –32, blooAll laboratory tests were carried according to previously published protocols , 35.x denoting NO2− and NO3−) in test sera. The sera were deproteinised chemically by adding 1.5 g/ml zinc sulfate and mixing for 1 min (by vortex). The mixtures were centrifuged at 10 000 g for 15 min, and the supernatant was separated and centrifuged at 10 000 g for 10 min. The serum nitrite concentration was analysed by mixing 100 μl of deproteinised serum and 100 μl of Griess reagent (prepared as a 1:1 mixture of 1% sulphanilamide in 5% phosphoric acid and 0.1% N-(1-naphytyl) ethylenediamine hydrochloride) followed by an incubation of 15 min in dark at room temperature. The serum NOx concentration was analysed by mixing 70 μl of deproteinised serum, 70 μl of 8 mg/ml vanadium (III) chloride and 70 μl of Griess reagent, followed by an incubation of 30 min at room temperature in dark.In brief, the Griess reaction was used to determine the concentrations of nitrite and total nitric oxide metabolites  at 540 nm in 96-well microplates in duplicates alongside series of sodium nitrite two-fold dilutions (100 μM–0.195 μM) and reagent blanks. The concentrations of nitrite and NOntration .+) by oxidation, 7 mM ABTS solution and 2.4 mM potassium persulphate (K2S2O8) were mixed in equal volumes and maintained in dark for 5 h. The ABTS working solution was prepared by diluting the mixture with 5 mM phosphate/0.145 M NaCl pH 7.4 phosphate buffered saline (PBS) according to the dilution factor which produces an optical density of ≈0.400 at 734 nm (determined using an ABTS:PBS dilution series). The test sera were analyzed in 96-well microplates in duplicates alongside a series of 6-Hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid (Trolox) two-fold dilutions (400 μM–5 μM), reagent blanks  and controls (a 1:19 mixture of PBS and working ABTS solution).The total antioxidant capacity (TAOC) of test sera was analyzed using the 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) decolourisation method. To facilitate the formation of radical cations  at 734 nm. The Trolox standard curve was used to determine the serum TAOC as expressed as a Trolox equivalent antioxidant capacity (TEAC).The data were transferred into a Microsoft Excel database and checked for accuracy. The analysis was done using Statistical Package for the Social Sciences/Statistical Product and Service Solutions (SPSS) version 23.0 . Descriptive statistics were summarised with measures of central tendency and measures of dispersion. Discrete variables across study groups were compared with Chi square test. The laboratory assay results were arranged into data arrays, tested for normal distribution and differences across groups were tested with the Kruskal–Wallis test, Mann–Whitney U test and Spearman’s correlation as appropriate. A p value < 0.05 was considered as statistically significant.A total of 72 children were enrolled in the study; 20 children with poorly controlled asthma, 25 children with well controlled asthma and 27 healthy children.When the baseline socio-demographic characteristics in the three groups of children were considered, there was a similar age and gender distribution across all three groups. The mean age (expressed as mean ± standard deviation in years) of children with well controlled asthma, poorly controlled asthma and controls was 8.96 ± 2.58, 8.80 ± 2.52 and 9.97 ± 2.82, respectively. There were no significant differences in the socio-demographic characteristics, namely mother’s and father’s education and monthly family income between the three groups (p > 0.05). Considering differences in co-morbidities between poorly and well controlled asthmatic children, except for higher number of allergic rhinitis in former, there were no significant difference in food or drug allergy, eczema or obesity.x concentrations were highest in the poorly controlled asthma group and lowest in the healthy controls. The mean serum nitrite, nitrate and NOx concentrations in the well-controlled asthma group was lower than in the poorly controlled asthma group, but higher than the healthy controls. In contrast, the highest mean serum TAOC was observed in the healthy controls group while the lowest mean serum TAOC was observed in the poorly controlled asthma group  and serum TAOC  compared to the well-controlled asthma and healthy controls groups (p = 0.036 and p = 0.049 respectively). There were no significant differences in these parameters between the well-controlled asthma and the poorly controlled asthma groups (p = 0.311 and 0.203 respectively). The Mann–Whitney U Test analyses showed that the serum TAOCs were significantly different across the two asthma groups (p = 0.003) and across the poorly controlled asthma group and healthy controls (p < 0.001). There was no statistically significant difference between the well-controlled asthma and healthy controls groups (p = 0.496)  concentrations at the 0.01 level  and NOx, two biomarkers for oxidative stress, were significantly elevated in patients with asthma regardless of the level of disease control compared to healthy, non-asthmatic children. Conversely, serum TAOC was highest in healthy controls, and this was statistically significantly different from that of patients with poorly controlled asthma. There was no significant difference between healthy controls and well-controlled asthmatics with regard to serum TAOC.The present study is the first in Sri Lanka to measure the biomarkers of oxidative stress in patients with asthma. The results have clearly demonstrated that the concentrations of serum nitrate and NOOxidative stress can be a manifestation of inflammation as well as an initiator of inflammation . There al-arginine-NO pathway [In this study, the approximate cost of carrying out serum NO levels was estimated at 3 CAD per sample. Thus the current study has demonstrated that measurement of serum NO may be a more cost-effective and practical approach in paediatric populations to demonstrate the occurrence of an underlying pathology in asthmatics as compared to a healthy population taking into consideration that measurement of eNO levels costs approximately 32 CAD per test , 45. The pathway . The totOn the contrary, as demonstrated in other studies , 48–50, The beneficial effects of consuming an antioxidant-rich diet have also been studied , 53. OneThis study had some limitations. Even though healthy children with no apparent acute illness were selected, there is a possibility that the children suffered from some sort of acute illness in the recent past. The assays were done on serum biomarkers which may have been influenced by pathologies other than asthma . The study assessed the plasma values of NO but as the primary pathophysiology is in the airway it would have been better to sample the airway NO levels. A study by Morris et al. , showed As hypothesised, a significant increase in serum NOx concentration was observed in children with asthma  regardless of whether the asthma is well controlled or not. Similarly, Healthy controls and those who had well-controlled asthma had significantly higher TAOC of serum compared to poorly controlled asthma patients.It can be concluded that serum TAOC assessment has potential to be developed as a cheap, easy to perform assay to objectively confirm good asthma control. However, these findings need to be verified with a larger prospective study with accurate recording of clinical exacerbations (and not just by history taking) enabling control of multiple confounding factors.


Arabidopsis thaliana ENHANCED RESPONSE TO ABA1 (ERA1) encodes the β-subunit of farnesyltransferase and regulates ABA signaling and the dehydration response. Therefore, ERA1 is an important candidate gene for enhancing drought tolerance in numerous crops. However, a rice (Oryza sativa) ERA1 homolog has not been characterized previously. Here, we show that rice osera1 mutant lines, harboring CRISPR/Cas9-induced frameshift mutations, exhibit similar leaf growth as control plants but increased primary root growth. The osera1 mutant lines also display increased sensitivity to ABA and an enhanced response to drought stress through stomatal regulation. These results illustrate that OsERA1 is a negative regulator of primary root growth under nonstressed conditions and also of responses to ABA and drought stress in rice. These findings improve our understanding of the role of ABA signaling in the drought stress response in rice and suggest a strategy to genetically improve rice.Abscisic acid (ABA) signaling components play an important role in the drought stress response in plants.  The increased occurrence of extreme weather events due to climate change severely hinders crop production. Drought is a major abiotic stress limiting rice productivity in rainfed lowland rice agro-ecosystems , 2. A nuArabidopsis thaliana ENHANCED RESPONSE TO ABA1 (ERA1), which encodes the β-subunit of the protein farnesyltransferase, regulates ABA signaling and the dehydration response  × 100. In preliminary tests, we confirmed that plants were subjected to drought stress in WS pots with a soil water content of 40%  filled with 2.2 kg of mixed soil (1.56 kg of soil dry weight per pot) made by mixing Bonsol No.2 , which is an artificial granular cultivation soil, and red clay at a volume ratio of 1:1 with a soil water content of 29.5% (weight of moisture content/soil dry weight + weight of moisture content + plant weight). Twelve, ten, and twelve pots were prepared for the t of 40% . As the t of 40%  and a pr2O m–2 s–1) was measured for the longest leaf of each plant with a LI-6400XT portable photosynthesis system . As the ambient CO2 concentration measured using the LI-6400XT was 480 ppm, the CO2 concentration of the input flow, chamber block temperature, and intensity of the LED light source were set to 480 μmol mol–1, 28.5°C, and 1,800 μmol m–2 s–1, respectively. Relative stomatal conductance rates during the period from p to q days after sowing were calculated as (CWSq/CWSp) × 100, where CWSp and CWSq are stomatal conductance in WS pots at p and q days after sowing, respectively.Stomatal conductance  at the same position in the gRNA1 target site, inducing frameshift mutations that introduce premature stop codons in the OsERA1.1 transcript  at the same position at the gRNA1 target site were selected for further study. The seedlings were treated with or without 1 μM ABA for five days to evaluate the effect of mutations at the gRNA1 target site on ABA sensitivity. The leaf and root growth of the M1G and M2T mutant lines were significantly more sensitive to ABA treatment than the WT , cuticle-dependent leaf permeability is regulated by ABA signaling pathways Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: Partly**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: No**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: The manuscript ” CRISPR/Cas9-targeted mutagenesis of OsERA1 confers enhanced responses to abscisic acid and drought stress and increased primary root growth under nonstressed conditions in rice” by Takuya Ogata et al, describe the making and characterization of rice era1 mutants.I have a few major and minor comments:Major comments:http://www.plantcell.org/content/12/8/1267), pathogen responses (http://www.plantphysiol.org/content/148/1/348.short), heat stress (https://nph.onlinelibrary.wiley.com/doi/full/10.1111/nph.14212). The ERA1 protein has been placed to regulate farnesylation of an enzyme involved in brassinosteroid synthesis [https://www.nature.com/articles/nplants2016114]. Thus the results presented in the current manuscript that some of the obtained rice mutants are lethal (the gRNA2 and gRNA3 target sites) is not surprising as ERA1 regulates protein farnesylation which is involved in many different biological pathways. I suggest that the authors expand the Introduction to more broadly illustrate that ERA1 is doing much more than only regulate ABA signaling. This can also help to explain why some of the mutants were lethal.1. The description of ERA1 protein function and era1 mutant phenotypes in Introduction is very short. The only role described role for ERA1 relates to ABA signaling. This is unfortunate as there is considerable information available for Arabidopsis era1, both mutant phenotypes and about protein function. For example, the Arabidopsis era1 mutant is defective in meristem and flower formation (https://academic.oup.com/jxb/article/70/20/5971/5536716). This would influence loss of water and drought responses. Have the authors considered testing if the rice era1 also has permeable cuticle? If rice era1 has permeable cuticle it might change the conclusions from some of the experiments. For example interpretation of stomatal conductance data might change if rice era1 mutants have permeable cuticles. The toluidine blue stain to test for cuticle permeability is a very easy experiment to do.2. The Arabidopsis era1 mutant has a permeable cuticle . This is opposite to the Arabidopsis era1 phenotype (drought tolerant). This is not surprising as the Arabidopsis era1 mutant is highly pleiotropic and involved in many signaling pathways. However, similar to Introduction I am missing in the Discussion some text related to the many functions that ERA1 and protein farnesylation has in different plant signaling pathways. See also point 2 above – if rice era1 has permeable cuticles this could also be an explanation for why it is drought sensitive.Reviewer #2: Major Concerns/Suggestions:1) The assumption of single base insertion at gRNA1 site in “M1T, M1G, M2T, and M3T” led to premature termination of stop codon, and that is the cause of the phenotype appears to wrong due to the following reasons:Singe base insertion at the site shown in “M1T, M1G, M2T, and M3T” lead to the premature stop codon after 45 amino acid. Whereas mutations in gRNA2 and gRNA3 will produce a longer proteins. The authors need to translate the mutant sequence and show the protein produced in each mutant. I have translated the “M1T, M1G, M2T, and M3T” mutant sequence and found that in reading frame 2, the mutant mRNA can produce a protein with complete homology to ERA1 except for the first 25 amino acid. Since mutants of gRNA2 and gRNA3, which produce longer protein than “M1T, M1G, M2T, and M3T” mutant, are unable to grow normally, the assumption that premature termination is cause of the phenotype in sRNA1 is wrong. A Western blotting may show what is the length of protein produced. I think that the protein produced from reading frame2 without the first 25 amino acid may be responsible for the phenotype. whether the the gain of function rather than the loss of function is responsible for the phenotype?2) The single base insertion mutants are more sensitive to ABA  and drought stress . Earlier studies have shown that reduction in expression levels of ERA1 leads to enhanced drought tolerance : 1381–1392). How this can be explained?3) Line #98: “ ----- 550 ppm CO2, and a light intensity of 150” Currently ambient CO2 concentration if only 410 ppm? Why a higher CO2 was used?4) Some problem with soil moisture content and stress imposition: Line #108-109: “red clay with a soil water content of 29.5% ” – Whether the “total soil weight” is total soil dry weight? Figure 4A. Please check the formula used for soil moisture content. The correct formula for soil moisture content % = (Soil water content/soil dry weight)*1005) Line #113: It says “Water-holding capacity of the soil was 49%” This value at what soil matric potential? Why 60% for well watered (about 11% higher than the WHC) and Drought 40% (about 9% lower than the WHC) were selected? Whether 40% SMC was stress? What was the soil type and soil matric potential? Since for seed germination only 29.5% was used (Line #108-109), 40% may not be a stress.6) Line #129-130: Why a CO2 concentration of 480 ppm was used? Currently ambient CO2 concentration if only 410 ppm?Minor comments:Line #113, 216, 237 and other places: Replace “water stress” with “water-deficit stress”Line #131-134: “days after sowing were calculated as (CWSq/CWSp) × 100, where CWSp and CWSq are stomatal conductance in WS pots at p and q days after sowing, respectively, and CWWp and CWWq are stomatal conductance in WW pots at p and q days after sowing,” - in the formula CWWp and CWWq are not mentioned.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  5 Nov 2020RESPONSE 1:We thank the Editor and Reviewers for their valuable comments and suggestions to improve our manuscript. All the changes and additions made in response to the Editor’s and Reviewers’ comments are in red font in the revised manuscript.The following minor errors have been changed: We replaced “enhance” with “improve” (Line 37); “Several” with “A number of” (Line 43); “resistance” with “tolerance” (Lines 52 and 303); and “farnesyltransferase” with “the protein farnesyltransferase” (Line 54), respectively.COMMENT 2:Reviewer #1: The manuscript ” CRISPR/Cas9-targeted mutagenesis of OsERA1 confers enhanced responses to abscisic acid and drought stress and increased primary root growth under nonstressed conditions in rice” by Takuya Ogata et al, describe the making and characterization of rice era1 mutants.I have a few major and minor comments:Major comments:http://www.plantcell.org/content/12/8/1267), pathogen responses (http://www.plantphysiol.org/content/148/1/348.short), heat stress (https://nph.onlinelibrary.wiley.com/doi/full/10.1111/nph.14212). The ERA1 protein has been placed to regulate farnesylation of an enzyme involved in brassinosteroid synthesis [https://www.nature.com/articles/nplants2016114]. Thus the results presented in the current manuscript that some of the obtained rice mutants are lethal (the gRNA2 and gRNA3 target sites) is not surprising as ERA1 regulates protein farnesylation which is involved in many different biological pathways. I suggest that the authors expand the Introduction to more broadly illustrate that ERA1 is doing much more than only regulate ABA signaling. This can also help to explain why some of the mutants were lethal.1. The description of ERA1 protein function and era1 mutant phenotypes in Introduction is very short. The only role described role for ERA1 relates to ABA signaling. This is unfortunate as there is considerable information available for Arabidopsis era1, both mutant phenotypes and about protein function. For example, the Arabidopsis era1 mutant is defective in meristem and flower formation . COMMENT 3:https://academic.oup.com/jxb/article/70/20/5971/5536716). This would influence loss of water and drought responses. Have the authors considered testing if the rice era1 also has permeable cuticle? If rice era1 has permeable cuticle it might change the conclusions from some of the experiments. For example interpretation of stomatal conductance data might change if rice era1 mutants have permeable cuticles. The toluidine blue stain to test for cuticle permeability is a very easy experiment to do.2. The Arabidopsis era1 mutant has a permeable cuticle  and corresponding text in the Results section (Lines 222–226). COMMENT 4:Minor comment:3. The rice era1 mutant was sensitive to drought . This is opposite to the Arabidopsis era1 phenotype (drought tolerant). This is not surprising as the Arabidopsis era1 mutant is highly pleiotropic and involved in many signaling pathways. However, similar to Introduction I am missing in the Discussion some text related to the many functions that ERA1 and protein farnesylation has in different plant signaling pathways. See also point 2 above – if rice era1 has permeable cuticles this could also be an explanation for why it is drought sensitive.RESPONSE 4:Thank you for your helpful comment. As we describe in RESPONSE 6, our results show that the osera1 mutant plants respond to drought stress more rapidly than do WT plants through accelerated stomatal closure , consistent with the hypersensitive ABA response seen in primary root length of osera1 mutant plants . Therefore, our results are not inconsistent with those described in the previous papers mentioned by the reviewer. Considering your comment, it occurred to us that a more accurate description of our results would be “enhanced response to drought stress”, which we used in the title, rather than “sensitive to drought stress”. We have thus altered the descriptions of our results throughout our manuscript . In addition, we have added a sentence discussing our results (Lines 328–332). COMMENT 5:Reviewer #2: Major Concerns/Suggestions:1) The assumption of single base insertion at gRNA1 site in “M1T, M1G, M2T, and M3T” led to premature termination of stop codon, and that is the cause of the phenotype appears to wrong due to the following reasons:Singe base insertion at the site shown in “M1T, M1G, M2T, and M3T” lead to the premature stop codon after 45 amino acid. Whereas mutations in gRNA2 and gRNA3 will produce a longer proteins. The authors need to translate the mutant sequence and show the protein produced in each mutant. I have translated the “M1T, M1G, M2T, and M3T” mutant sequence and found that in reading frame 2, the mutant mRNA can produce a protein with complete homology to ERA1 except for the first 25 amino acid. Since mutants of gRNA2 and gRNA3, which produce longer protein than “M1T, M1G, M2T, and M3T” mutant, are unable to grow normally, the assumption that premature termination is cause of the phenotype in sRNA1 is wrong. A Western blotting may show what is the length of protein produced. I think that the protein produced from reading frame2 without the first 25 amino acid may be responsible for the phenotype. whether the the gain of function rather than the loss of function is responsible for the phenotype?RESPONSE 5:Thank you for pointing this out. Based on your suggestion, we have translated the “M1T, M1G, M2T, and M3T” mutant sequence in silico and confirmed that the mutant mRNA can indeed produce a protein with homology to ERA1 except for the first 28 amino acids (S3 Table). This suggests that the N-terminal truncated region may influence the phenotype. In addition, as pointed out, the difference at the protein level may also affect the phenotype. However, ERA1 has not been analyzed at the protein level, nor have its antibodies been reported. Therefore, more research is needed to determine why this mutation results in the observed phenotype. In the revised version of our manuscript, we have thus added a new supplementary table (S3 Table), phrases (Lines 194–196), and sentences (Lines 332–342) to explain the current situation and have altered a sentence for clarity . In addition, we have removed “that cause premature stop codons” in the Introduction (Line 80). COMMENT 6:2) The single base insertion mutants are more sensitive to ABA  and drought stress . Earlier studies have shown that reduction in expression levels of ERA1 leads to enhanced drought tolerance : 1381–1392). How this can be explained? RESPONSE 6:Thank you for raising this good point. As we described in RESPONSE 4, our results show that the osera1 mutant plants respond to drought stress more rapidly than do WT plants through accelerated stomatal closure , consistent with the hypersensitive ABA response seen in primary root length of osera1 mutant plants . Therefore, our results are not inconsistent with those described in the previous papers mentioned by the reviewer. Upon further consideration, it occurred to us that a more accurate description of our results would be “enhanced response to drought stress”, which we used in the title, rather than “sensitive to drought stress”. Accordingly, we have altered the descriptions of our results throughout our manuscript . COMMENT 7:3) Line #98: “ ----- 550 ppm CO2, and a light intensity of 150” Currently ambient CO2 concentration if only 410 ppm? Why a higher CO2 was used?RESPONSE 7:Thank you for pointing this out. Our group  and other independent groups  have shown that CO2 concentrations inside growth chambers that comprise a closed and limited space are much lower than those of the ambient atmosphere during the growth phase of rice and soybean, and that CO2 supplementation enhances rice and soybean growth in plant growth chambers. In our case, by setting the CO2 concentration to 550 ppm, the actual CO2 concentration inside the chamber used to grow rice and soybean plants was maintained above 400 ppm. We have altered this sentence to convey this information and have added a reference in the Materials and Methods section (Lines 115–118). COMMENT 8:4) Some problem with soil moisture content and stress imposition: Line #108-109: “red clay with a soil water content of 29.5% ” – Whether the “total soil weight” is total soil dry weight? Figure 4A. Please check the formula used for soil moisture content. The correct formula for soil moisture content % = (Soil water content/soil dry weight)*100RESPONSE 8:Thank you for your comment. In this paper, “total soil weight” does not mean total soil dry weight but soil dry weight plus the weight of the moisture content. Therefore, the formula for soil water content (%) was calculated as [weight of moisture content/total soil weight (soil dry weight plus weight of moisture content) + plant weight] × 100. To use round numbers in this study, the values calculated on a total mass basis are presented as soil water contents; however, we have also added a dry weight value that readers can use to calculate the dry weight-based soil water content. We have provided clear definitions of these terms to avoid confusion . COMMENT 9:5) Line #113: It says “Water-holding capacity of the soil was 49%” This value at what soil matric potential? Why 60% for well watered (about 11% higher than the WHC) and Drought 40% (about 9% lower than the WHC) were selected? Whether 40% SMC was stress? What was the soil type and soil matric potential? Since for seed germination only 29.5% was used (Line #108-109), 40% may not be a stress.RESPONSE 9:Thank you for raising this question. We measured the soil matric potential at the water-holding capacity of the soil using a tensiometer. Our measurements confirmed that a soil water content of 30%, 40%, and 49% was equivalent to a soil matric potential of –54 kPa, –11 kPa, and 0 kPa, respectively. We used mixed soil, made by combining Bonsol No.2 , which is an artificial granular cultivation soil, and red clay at a volume ratio of 1:1. We have added sentences to provide this new information . In preliminary tests, we confirmed that plants were subjected to drought stress in WS pots with a soil water content of 40% . Based on the results of our preliminary tests  and a previous report , we determined that the soil water content was adjusted to 60% in WW pots and 40% in WS pots, respectively. In pots with a soil water content of 60%, about 1–2 cm of water accumulates on the soil surface. The weight of individual pots was recorded every few days, and the soil water content was adjusted to approximately 60% for the WW treatment and 40% for the WS treatment to compensate for water loss due to transpiration at the time of weight measurement. The soil water content was roughly maintained between 30% and 40% in WS pots from 35 days after sowing onwards, and between 50% and 60% in WW pots. Therefore, the water content in WW pots was usually maintained above the water-holding capacity during the test. As shown in Fig. 4A, since the plants are grown in sunlight, the temperature rose depending on the weather and, consequently, the soil water content occasionally dipped below the water-holding capacity. We have added these data  and the corresponding descriptions (Lines 146–148). Seeds were germinated in plastic dishes with enough water for 5 days and then the germinated seedlings were transferred to soil-filled plastic tubes as described in Lines 125–126. For clarity, we have added descriptions of this procedure in the Materials and Methods section (Line 114–115). COMMENT10:6) Line #129-130: Why a CO2 concentration of 480 ppm was used? Currently ambient CO2 concentration if only 410 ppm?RESPONSE 10:Thank you for raising these questions. For the experiments, we set a CO2 concentration of LI-6400XT at 480 ppm, since 480 ppm was the ambient CO2 concentration monitored by LI-6400XT in the greenhouse where we grew our plants. We have added text to explain this point (Lines 159–160). COMMENT 11:Minor comments:Line #113, 216, 237 and other places: Replace “water stress” with “water-deficit stress”Line #131-134: “days after sowing were calculated as (CWSq/CWSp) × 100, where CWSp and CWSq are stomatal conductance in WS pots at p and q days after sowing, respectively, and CWWp and CWWq are stomatal conductance in WW pots at p and q days after sowing,” - in the formula CWWp and CWWq are not mentioned.RESPONSE 11:Thank you for your suggestions. In response to your comment, we have replaced “water stress” with “water-deficit stress” . In addition, we have removed the unneeded phrase (Lines 162–164). 20 Nov 2020CRISPR/Cas9-targeted mutagenesis of OsERA1 confers enhanced responses to abscisic acid and drought stress and increased primary root growth under nonstressed conditions in ricePONE-D-20-25974R1Dear Dr. Fujita,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Keqiang Wu, Ph.DAcademic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.Reviewer #1: All comments have been addressedReviewer #2: All comments have been addressed**********2. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: Partly**********3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: Yes**********4. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: Yes**********5. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********6. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: In the revised manuscript, the authors have answered all of my previous comment. I have only one additional comment:1. In the revised version, lines 192 - 196 , there is a description of the effect of the Crispr mutations on the OsERA1.1 transcript and corresponding protein. This was very difficult to understand, and would benefit from careful editing. As far as I can tell from S3 Table, the crispr mutations leads to a truncated protein - if translation is initiated from the first ATG. However, if translation is started at the second ATG, the only effect is a somewhat shorter protein. If this is a correct interpretation, then I suggest to edit the text on lines 192-196 to make this easier to understand. For example, instead of trying to have all this information in one long sentence, it could be edited to several shorter sentences.Reviewer #2: Most concerns were addressed.1) The statistical significant in Fig 3 and and in few bars of Figure 4 may be checked as the error bars indicate the different may not be significant in few cases.2) Relative stomatal conductance is also not a good measure of drought stress response as even in the well watered plants, stomatal conductance will change as the VPD changes.**********what does this mean?). If published, this will include your full peer review and any attached files.7. 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Mytilus trossulus edulis) from Kvädöfjärden (collected from a depth of 5–10 m), a reference area along the Swedish coast in the Baltic Proper from 25 years during 1987–2016, and of stable isotopes in five individuals (2 cm mussels) per year during the time period 1981–2017. The data has been co-analysed with environmental (oceanographic) data in “The importance of adjusting contaminant concentrations using environmental data: a retrospective study of 25 years data in Baltic blue mussels” published in: Science of the Total Environment (https://doi.org/10.1016/j.scitotenv.2020.143913).Blue mussels are a target species in contaminant monitoring regarding Polycyclic Aromatic Hydrocarbons (PAHs) in biota, and also used as an isotope baseline for trophic position assessment in other biota. The latter is crucial for calculating biomagnification potential of environmental contaminants. This data set comprises long-term time series of PAHs  in Baltic Sea blue mussels ( Long-term time series from monitoring is often the most important source of information for understanding marine ecosystem dynamics, since long-term ecological research programs only rarely exist globally. Hence, both contract holders for monitoring programmes and researchers will greatly benefit from long, uninterrupted time series data.•Long-term data on contaminants provide baselines for anthropogenic pressure in the environment and is needed for assessing ecosystem health. Stable isotopes of carbon and nitrogen can be used to infer large scale biogeochemical changes in the environment 1−1 shell-free dry weight) measured in a pool of mussels  during 1987–2016 from station Kvädöfjärden, Baltic proper. Full name and abbreviation for each PAH is given in the specification table above and in the first row of Table 1. For acenaphthene, values are rarely found above the level of quantification (LOQ), values below this level are marked in grey and represent absolute values divided by the square root of 2.Data in supplementary n = 5 per year) nitrogen and carbon stable isotope data  as well as the carbon: nitrogen elemental ration (weight: weight) during 1981–2017. Data is organized after the unique sample ID from the Environmental Specimen Bank, year collected, the δ13C values, δ15N values, C:N ratio and a column with comments .Data in supplementary 22.1Mussel collection was conducted annually during October-November from 5–10 m depth (bottom substrate clay and gravel) at exactly the same position every year  using coastal survey nets; mussels are bycatch from sampling of fish from this site and position. For the period 1981–2017 data is missing for six years . PAHs are measured since 1987 . Mussels were immediately frozen after collection and stored at −25 °C at the Environmental Specimen Bank at the Swedish Museum of Natural History, Stockholm Sweden.n = 20–75 individuals, depending on size of sampled mussels a particular year; within a year only similar sized individuals were used) was prepared each year and homogenised using an IKA T25 digital ULTRA TURRAX homogenizer. The homogenate was analysed for 15 PAHs at the Swedish Environmental Research Institute (IVL) as described below.During sample preparation prior to analyses in the laboratory, individual specimens were thawed and carefully opened using a metallic scalpel. The soft tissue (not including the adductor muscle) was removed and placed in a glass beaker. For each specimen maximum shell length was registered. One pooled sample , homogenised in acetone and extracted in an ultrasonic bath. The extract was safeguarded and the samples were extracted once more with acetone and twice with pentane/ether. The extracts were combined and the organic compounds were extracted to an organic phase by liquid/liquid extraction with water and pentane, and further concentrated under nitrogen. The samples were hydrolysed and pre-treatment procedures, such as fractionation of the organic compounds on silica, were performed as additional "clean-up" procedures. Laboratory blanks followed the same procedures as samples in the analytical work.Determination of PAH components was carried out using a high performance liquid chromatograph . A linear gradient elution program was used, starting with acetonitrile/water 50:50 and ending with 100% acetonitrile (Rathburne HPLC-grade). A fluorescence detector (Agilent 1260) with a wavelength program optimised for each PAH was used for quantification. The peak heights were registered with a chromatographic system from Thermo (Chromeleon 7.0). The concentrations of 15 different PAH compounds were calculated by comparison to a certified standard, NIST, SRM 1647f (Priority Pollutant Polycyclic Aromatic Hydrocarbons in Acetonitrile).Mytilus edulis). Concentrations are expressed as ng g−1 shell-free dry weight; a subsample of the mussel soft tissue homogenate was dried at 105 °C at 20 h.All of the standards used (both the recovery standard and quantification standards) are certified with known purity and precision. Certified reference material (CRM) was run in parallel to the mussel samples and used to check the method performance  were placed in tin cups (1 mg dry weight) and sent for stable isotope ratio analyses (13C:12C and 15N:14N) and elemental analyses  at the UC Davis Stable Isotope Facility, US, through combustion using a PDZ Europa 20–20 isotope ratio mass spectrometer . Isotope data are expressed in the delta notations, δ15N and δ13C, which is ‰ deviations from an international standard . Reference materials measured every tenth sample are Bovine Liver, Glutamic Acid, enriched Alanine and nylon 6.Shells from thawed mussels from the Environmental Specimen Bank were dissected and subsamples of homogenized dried (60 °C for 48 h) mussel soft tissue (individual samples, Data originate from invertebrates which require no special permits for sampling in the Baltic Sea.Agnes ML Karlson: Conceptualization, Data curation, Writing- Original draft preparation; Suzanne Faxneld: Investigation, Reviewing and Editing.The authors declare that they have no known competing financial interests or personal relationships which have or could be perceived to have influenced the work reported in this article.


The groups were as follows: “Normal”: direct resin composite; “Ring”: glass fiber-reinforced strip (Dentapreg) wrapped around buccal and lingual walls followed by direct resin composite; “Inlay”: indirect CAD/CAM resin composite inlay; “Onlay”: indirect CAD/CAM resin composite onlay; “Intact”: Intact teeth (Control). Tetric EvoCeram and Adhese Universal (Ivoclar Vivadent) were used for direct restorations and Tetric CAD (Ivoclar Vivadent) adhesively luted with Adhese Universal and Variolink Esthetic LC (Ivoclar Vivadent) were used for indirect restorations. All teeth were submitted to thermo-mechanical cyclic loading. All samples were then submitted to a compressive load until fracture. Fracture load was noted and teeth were analyzed to classify the failure mode as either catastrophic (C) or non-catastrophic (NC). No statistically significant difference was found between fracture strength of the five groups when all specimens were considered (p = 0.1461). Intact group showed the lowest percentage of catastrophic failures (41.67%). Ring group presents less catastrophic failures (75%) than Normal group (83.34%), and failures of indirect restorations—Inlay and Onlay—were almost all catastrophic .The purpose of this in-vitro study was to evaluate the fracture resistance and failure mode of non-invasively reinforced endodontically treated mandibular molars. Sixty freshly extracted defect-free mandibular molars were divided into four experimental groups with extensive MOD cavities on endodontically treated teeth with different restoration types and one control group with intact teeth ( Fragility of endodontically treated teeth (ETT) is mostly due to accumulated tissue loss resulting from carious lesions, endodontic access cavities and canal preparation . The firSixty freshly extracted human mandibular molars of similar dimensions were used for this study. A digital caliper  was used to exclude teeth that do not have the following dimensions: mesio-distal width of 11 mm (± 0.5 mm), bucco-lingual width of 9 mm (± 0.5 mm) and crown length of 7 mm (± 0.3 mm). The teeth were inspected under a stereomicroscope and inclusion criteria was absence of carious lesions, visible fracture lines in the root and a complete root formation. All samples were anonymously collected in accordance with the Swiss Human Research Act, article 2.Teeth were stored in a sodium azide solution (0.2%) at 4 °C until the experiment onset. Each sample was bonded on a metallic holder —in a vertical position—with light-curing composite; then, the root base was embedded with self-curing acrylic resin  to complete the tooth stabilization.Twelve teeth were left intact for positive control group (“Intact”), and one operator performed endodontic treatment on all remaining 48 test teeth, starting with a traditional endodontic access cavity. A diamond round bur was used to access the pulp chamber, followed by a tungsten carbide tapered bur  to remove the entire roof of the pulp chamber without unnecessary mutilation of dentine. Canal preparation was performed with manual instruments until K-file 20 , then rotary instruments  to an apical diameter of 30. The canal preparation was accompanied by sodium hypochlorite 3% irrigation, and final obturation was made with the warm vertical compaction technique .n = 12) according to the restoration technique: direct resin composite restoration , glass fiber-reinforced strip wrapped around buccal and lingual walls then direct resin composite restoration (“Ring”), CAD/CAM resin composite inlay restoration (“Inlay”), cusp reduction and CAD/CAM resin composite onlay restoration (“Onlay”).Standardized extensive MOD cavities were made on test teeth using coarse diamond burs , and the margins were finished with fine diamond burs. The cavities extended to 1 mm above the cemento-enamel junction (CEJ) in the occluso-cervical direction, and to the cusp tips in the bucco-lingual direction. All axial dentin was removed in that predefined region. The 48 test teeth were randomly divided into four groups . For “Ring” group, a 0.3 mm thick custom-made S2-glass fiber-reinforced resin pre-impregnated strip  was wrapped twice around the remaining buccal and lingual walls. The band was passed through the cavity in an “X” shape and a thin layer of nano-optimized flowable composite  was used to cover the exposed fibers in the buccal and lingual, then light-cured for 20 s from buccal, lingual and occlusal. Cavities of both “Normal” and “Ring” groups were then filled with a universal nanohybrid composite  following the anatomical layering technique. For the “Onlay” group, the buccal and lingual cusps were reduced by measuring 2 mm from the lowest point of the walls, using coarse diamond coated burs  and finished with fine-grained burs of the same shape under profuse water spray cooling. The cavity surfaces of all samples of groups “Inlay” and “Onlay” were sealed with a layer of the adhesive system  then light-cured for 20 s. The pulp chamber was with a universal nanohybrid composite  and light cured for 20 s. Enamel surfaces were then exposed using fine-grained diamond burs.For direct restorations, selective etching of enamel was made with 37% phosphoric acid etchant gel  for 30 s. For the “Ring” group, additional etching of enamel 2 mm above the CEJ of the buccal and lingual walls was done on the 1 mm wide region underlying the fiber-reinforced strip. A single-component dental adhesive  was then applied to the dentin and to etched enamel and light-cured for 20 s with high power light-curing unit (LCU) of 1640 mW/cmDigital impressions were taken using an intraoral scanner , and restorations were designed using a CAD software (Cerec SW 4.5.1). Inlays and onlays were then fabricated using recently-launched A2 shade resin composite CAD/CAM material .All restorations and adhesively sealed cavities were then sandblasted, exposed enamel was etched with 37% phosphoric acid etchant gel  for 30 s and a layer of adhesive system  was then applied on all surfaces. A light-curing resin cement  was then applied into the cavity and the restorations were inserted followed by a removal of excess material, and light-curing for 270 s . Figure The margins of the restorations of all four test groups were then polished with fine diamond burs followed by polishing discs  and polishing points .All samples were submitted to a chewing simulator for thermo-mechanical cyclic loading (TMCL) which consisted of 600,000 cycles of axial 49 N loads at 17 Hz delivered by a 4 mm stainless steel sphere to the occlusal surface and 3000 thermal cycles of temperature varying between 5 and 55 °C. Metallic holders were placed on a support resting on a rubber base, which induced sliding movements during mechanical cyclic loading to some extent mimic the clinical situation. Following the TMCL, a testing machine  applied a continuous compressive load at 1 mm/min through a 3.7 mm diameter spherical stainless steel indenter pointed in the center of the occlusal surface until fracture.Fracture load was noted and teeth fragments were then inspected by three different operators under stereomicroscope  to determine fracture mode. Fractures extending apical to the CEJ were considered as catastrophic while repairable fractures above the CEJ were considered as non-catastrophic.The different incidence of Catastrophic (C) vs non Catastrophic (NC) failures among the different groups have been checked by Fisher’s exact test. Difference among the average fracture load of the five groups has been tested using a one-way analysis of variance (ANOVA) followed by a Sheffé post-hoc test for pairwise comparisons. Normality assumptions for ANOVA were tested by means of Kolmogorov–Smirnov tests. The analyses have been run both on all the specimens and on the specimens with C failures only. Finally, a Kaplan–Meier analysis was performed on specimens with C failures only to evaluate survival functions of the five groups. All analyses have been made using STATA 15.0.p = 0.009). When the means of both catastrophic and non-catastrophic failures were considered, one-way ANOVA showed no statistically significant difference at the 95% confidence level (p = 0.1461). When considering catastrophic failures, mean fracture loads were 3217 N (752), 2783 N (488), 2470 N (569), 2431 N (413) and 2185 N (391) for Intact, Inlay, Onlay, Ring and Normal respectively, and a statistically significant difference was observed between groups (p = 0.0061) (Table p = 0.017). Survival analysis and smoothed hazard estimates showed a dominance of Intact resistance over the other groups and that Normal group results to be dominated by almost all the groups.All teeth survived TMCL. Fracture mean values (SD) were 2671 N (715) for Intact, 2713 N (525) for Inlay, 2470 N (569) for Onlay, 2386 N (456) for Ring and 2211 N (359) for Normal and the numbers of catastrophic failures were 5, 11, 12, 9, 10 respectively (Table p = 0.002). Hazard estimates plots shows three distinct sets: Normal (the less resistant), Onlay–Ring–Inlay  and Intact   could be partially rejected since the reinforcing technique did have a certain effect on the fracture strength of the tooth. The ring group also showed the second highest number of NC failures after intact teeth indicating that ring reinforcement does influence the fracture mode, therefore null hypothesis (2) was rejected.Mandibular molars were chosen for this test to be in agreement with the first study that first described this technique . In thatSince no closed-loop fiber-reinforced ring exists on the market, and it could be probably challenging to manufacture, the fiber-reinforced strip was wrapped twice around the tooth to first increase the number of fibers opposing to the cuspal deflection, and then to avoid detachment of the two extremities of the strip by increasing the overlapping surface of the strip. Covering the fibers in buccal and lingual with a thin layer of flowable resin composite would be clinically necessary since exposed fibers can increase plaque accumulation and gingivitis . Pre-imp. [. [Such an addition would probably not cause discomfort for the patients. From the biological point of view, the transition between the tooth surface and the flowable resin composite was continuous, and thus no impediment of plaque control and cleaning would be caused. However, if that excess thickness turns out to be problematic, a shallow and narrow 0.3 mm groove on the buccal and lingual walls can be created to place the fibers and cover them flush with resin composite without altering the original contour of the tooth. Further investigations on the effect that this groove would have on the fracture resistance would be needed since this would become a minimally-invasive approach instead of a non-invasive one, but a certain point is that less tissues would be removed compared to current cusp reduction techniques. Cuspal deflection was shown to induce micro-cracks due to cyclic bending loads and the limited elasticity of dental hard tissues . The FEA.  and 3048. [. . The sliMode of failure could be an even more important indicator that should be considered while examining a restorative technique, especially when no large difference is present between groups in terms of fracture strength. Ring group presented a lower percentage of catastrophic failures (75%) compared to Normal group (83.34%). Almost all failures in Inlay and Onlay groups were catastrophic . None of the restorative techniques was able to restore the predominantly non-catastrophic failure pattern of intact teeth that showed 41.67% of non-repairable fractures. The smoothed hazard estimates show three clear regions in the graph. From a descriptive point a view, Onlay–Inlay–Ring present similar curves, with the Normal group on the lower boundary and Intact group on the higher side Fig. . This coWithin the limitations of this in-vitro study, it is possible to conclude that fiber-reinforcing rings present comparable fracture resistance to Inlays and Onlays, and that they increase the percentage of repairable fractures.