The integration of synaptic inputs onto dendrites provides the basis for neuronal computation. Whereas recent studies have begun to outline the spatial organization of synaptic inputs on individual neurons, the underlying principles related to the specific neural functions are not well understood. Here we perform two-photon dendritic imaging with a genetically-encoded glutamate sensor in awake monkeys, and map the excitatory synaptic inputs on dendrites of individual V1 superficial layer neurons with high spatial and temporal resolution. We find a functional integration and trade-off between orientation-selective and color-selective inputs in basal dendrites of individual V1 neurons. Synaptic inputs on dendrites are spatially clustered by stimulus feature, but functionally scattered in multidimensional feature space, providing a potential substrate of local feature integration on dendritic branches. Furthermore, apical dendrite inputs have larger receptive fields and longer response latencies than basal dendrite inputs, suggesting a dominant role for apical dendrites in integrating feedback in visual information processing. The integration of synaptic inputs onto dendrites provides the basis for neuronal computation. Here the authors perform two-photon dendritic imaging with a genetically-encoded glutamate sensor in awake monkeys, and map the excitatory synaptic inputs on dendrites of individual V1 superficial layer neurons with high spatial and temporal resolution. Whereas in vitro patch-clamp and intracellular recordings have advanced our understanding of how dendritic synaptic integration occurs in single neurons3, and in vivo studies have extended those methods to highlight how dendritic activity contributes to cortical functions such as orientation and direction selectivity5, sample size and spatial resolution have limited the utility of these methods. High-resolution two-photon calcium imaging of dendrites, in contrast, can achieve long-term functional mapping of individual dendritic inputs in the intact brain13. As the temporal sequence of synaptic inputs has been verified to be of vital importance to dendritic nonlinear integration15, high-speed dendritic imaging can provide important new insights into dendritic computational mechanisms. A recently developed genetically encoded glutamate-sensing fluorescent reporter, iGluSnFR16, which has a high signal-to-noise ratio (SNR) and fast kinetics, promises to map the spatiotemporal functional organization of dendritic excitatory inputs. In this study, we performed two-photon dendritic imaging with iGluSnFR in awake macaque monkeys, and obtained fine functional spatiotemporal maps of dendritic excitatory inputs in individual V1 neurons.Cortical neurons sample from their dendritic synaptic inputs as the basic unit of computationWe performed two-photon imaging on dendrites of sparsely labeled individual V1 neurons expressing SF-iGluSnFR.A184S Fig. , bottom.z-axis, when using the 25x Olympus objective used for these experiments. This is a typical range of measurements compared to other work in the field, and we have achieved the same diffraction-limited microscopic resolution—near the physical limit also found in other similar two-photon systems—reported in prior imaging studies of dendrites, spines, and other filaments and small cells in the neuropil. In our dendritic imaging experiments, the sampling resolution was set to about 4 pixels per μm. This setting magnified beyond the physical limitations of the two-photon imaging system. This standard approach12 provided single-ROI signals that were robust and repeatable  was measured at 0.4 μm X-Y plane resolution, and 2.0 μm z-axis fluorescence leaks across different depths, we captured a series of continuous Z-stack images having 3 μm steps along the z-axis , establishing conclusively that the fine imaging resolution along the z-axis was <3 μm. Notably, to maximize our yield of dendritic inputs in each experimental session, we targeted focal planes with the longest possible continuous dendritic trees  and color Figs.  and 2b–dλ = 4.9 and 3.3 μm respectively, see Methods for analysis details; Fig. λ = 4.3 and 5.6 μm). Recent two-photon dendritic imaging studies have revealed that, despite understandable excitement in the field concerning the discovery of functional input clustering18, highly scattered distributions of inputs are also common13. There is no known explanation for this difference, but combined with our results in macaques, the evidence suggests that dendrites on visual neurons within orientation maps receive functionally clustered synaptic inputs12, whereas neurons that are not embedded within orientation mapped cortex have less clustering in their synaptic inputs11.We first examined how excitatory synaptic inputs—tuned to each visual property tested—are spatially arranged on dendritic shafts. We found moderate clustering within local dendrites along single feature dimensions of receptive field (RF) and orientation  with no optical zoom (1×) that had a field of view of 850 × 850 μm , and had similar summed orientation preferences , the half-life (1/2) decay distance is <1 μm . In other words, whereas iGluSnFR can be used to detect glutamate spreading from up to 3.6 μm away, robust detection of glutamate occurred primarily within 1 μm. In our experiments, we did not detect significant spillover effects between nearby synapses as measured with differential stimulation. Further, significant spillover would result in homogenous nearby synaptic responses, whereas we found great diversity between even neighboring synapses stimulated at high-strength. Thus, our results are consistent with Kazemipour et al.’s findings, and we obtained similar sizes of inputs onto neuronal dendrites from our iGluSnFR imaging experiments .iGluSnFR has a high affinity to glutamate, which could potentially make it even sensitive to glutamate arising from spillover between nearby synapses. Note that whereas the 1/33. The depth of our collected neurons ranged from 100 to nearly 400 μm, and we recorded neurons with both spiny and smooth dendrites , we only used saturated colors. We later realized the need to eliminate the effect of luminance in our experiment, and conducted control experiments to compare the difference between saturated vs. equiluminant color-evoked dendritic responses (on Monkey 3). Color preferences of 79% dendritic inputs stayed stable when comparing saturated vs. equiluminant stimuli . This result makes intuitive sense, given that our saturated condition was not much brighter than the equiluminant condition . Thus, we did not expect a significant difference between the saturated and equiluminant condition responses.Consistent with previous results that superficial V1 neurons encode both orientation and chromatic selectivity, we found functional integration and trade-off between orientation and color inputs in V1 neuron dendrites. Critically, the higher the proportion of orientation-selective inputs received by a given neuron, the more uniform those inputs tended to be, which presumably contributed to the provenance of orientation selectivity in V1 neuronsWe found that dendritic inputs had a wide scattering of functional properties within the multidimensional feature space of the visual system. Individual V1 neurons receive thousands of synaptic inputs, which seems redundant if they all shared similar feature properties. The organization patterns revealed in our data may facilitate integration both within and across visual feature domains, and provide a dendritic computational mechanism for neuronal multidimensional feature integration.28, and brings additional insight into the function of the most iconic feature of cortical pyramidal cells. It remains a challenge to directly dissociate feedback vs. feedforward inputs, and our results provide indirect evidence for the provenance of the signals. Future studies will aim to obtain direct evidence by combining new techniques, such as optogenetics or electrophysiology, and specific behavioral tasks, such as a top–down modulated selective attention task. Our functional observations, in combination with the first successful application of a newly developed glutamate sensor in behaving non-human primates, provide a bridge to deeper understanding of the dendritic mechanisms and computational principles of visual information processing.Our finding that the latency of apical inputs is temporally lagged compared to basal inputs further indicates that apical dendrites receive feedback inputs, whereas basal dendrites receive feedforward inputs within the visual hierarchyMacaca mulatta) were purchased from Beijing Prima Biotech, Inc. and housed at Peking University Laboratory Animal Center. Three healthy adult male monkeys (aged 3–4 years and weighing 4–6 kg) were used in this study.All experimental protocols followed the Guide of Institutional Animal Care and Use Committee (IACUC) of Peking University Laboratory Animal Center, and were approved by the Peking University Animal Care and Use Committee (LSC-TangSM-5). Rhesus monkeys  with a small pore (0.3 mm) was used for targeting the injection pipette and stabilizing the cortical surface during the injection procedure. The injection pipette was made from a quartz pipette  and pulled with a 15–20 μm tip using a laser-based pipette puller . Through the pipette, 120 to 150 nl of AAV1.hSynap.SF-iGluSnFR.A184S.WPRE.SV40  were pressure-injected at a depth of ~350 μm. After AAV injections, we inserted a GORE membrane (22 mm in outer diameter) under the dura, sutured the dura, installed back the removed skull bone with titanium pieces and sutured the scalp. The animal was then sent back to its cage for recovery. Ceftriaxone sodium antibiotic  was administered for one week. We performed a second surgery 45 days later to implant the head posts and imaging window. Three head posts in total were implanted on the animal’s skull, with two of them on the forehead and one on the back of the skull. A T-shaped steel frame was fixed to these head posts for head stabilization during training and experiments. Also during this surgery, the skull was re-opened and the subjacent dura was cut off (diameter 16 mm) to exposure the brain cortex. A glass cover-slip (19 mm in diameter and 0.17 mm in thickness) with a titanium ring (12 mm in outer diameter and 10 mm in inner diameter) glued on it was inserted and gently pushed down onto the cortical surface. The titanium ring was glued to the dura and skull using dental acrylic, to establish an imaging chamber. The whole chamber, reinforced by additional thick dental acrylic, was then covered by a steel shell to protect the cover-slip when the animal was returned to the home cage. See Li et alAfter 10 days of recovery from the second surgery, we trained each monkey to sit in a primate chair with its head restrained, and to perform a fixation task, where keeping fixation on a small white spot (0.1°) within a 2° window for over 2.5 s resulted in a juice reward. Eye position was monitored with an infrared eye-tracking system  at 120 Hz. Whenever the eye fixated within a scope of 2° of the center white point, a trial started.Visual stimuli were generated using a ViSaGe system (Cambridge Research Systems) and presented on a 17-inch LCD monitor , positioned 45 cm away from the animal’s eyes. A small drifting grating  was first used to estimate the RF position of each recorded neuron. For synaptic input tuning measurements  and full saturated colors. The background luminance was set to 25 candela per m2 for all our experiments. Full saturated colors were generated by setting the values of the RGB channel to —red, —orange, —yellow, —green, —cyan, —blue, —purple, —white, and —black. We then measured the chromaticity and luminance of each saturated color using SpectroCAL MKII Spectroradiometer (Cambridge Research System). Accordingly, we set all equiluminant colors to match the blue channel, which had the weakest luminance value at 8.2 candela per m2. To calibrate the other equiluminant colors, we adjusted the RGB channel values for each color to a luminance intensity as 8.2 candela per m2, while maintaining chromatic saturation of each color channel.We tested both equiluminant colors  and a Ti: Sapphire laser . A 25× objective lens  with zoom 4× scanning model was used for high-resolution dendritic imaging, which covered a field of view of 136 × 136 μm. Fast resonant scanning mode  was used in all functional characterization experiments. An even faster scanning mode (64 by 64 pixel at 226 frames per second) while zoomed 8× achieved high temporal resolution imaging . We assumed ROIs as synaptic inputs on neuronal dendrites if they had a 50% intersection with the dendritic domain. To obtain the morphology of each dendritic branch, we first collected an averaged image throughout each experiment, and then filtered this image with a band-pass filter . With this processed image, we identified the dendritic domain with PS (Adobe Photoshop CC Ver. 19.1.6) and determined the contour of the dendrites using the software’s magic stick tool (tolerance ranging from 10 to 50 in different conditions to optimize fit). Responses of synaptic inputs were computed as the ratio of fluorescence change (ΔF/F0), where ΔF = F – F0 and F0 is the baseline fluorescence intensity during the blank screen before stimulus onset in each trial, and F is the intensity during stimulus presentation.All imaging data were analyzed with custom MATLAB code . To correct the image shifts caused by the relative displacement between the objective and the cortex during the imaging process, we first determined a template image by averaging 1000 frames in the middle of each imaging session and then realigning each frame in a session to the template image, using a normalized cross-correlation-based translation algorithmTo minimize contamination from back-propagation action potentials (BAPs) while Ca-imaging in dendrites from GCaMP6s, we implemented a subtraction procedure  of 12 orientations under the preferred spatial frequency, and a color SNR index was determined as the ratio of fluorescence variance between averaged ON and OFF frames of nine colors. The SNR value of each pixel was then set as the maximum between the corresponding orientation SNR index and color SNR index.p < 0.05 to obtain inputs with significant difference in response strength across the 12 orientations. Finally, to measure orientation tuning, tuning curves with step sizes of 15° were fit with the circular Gaussian function:39a is the amplitude, θ0 is the maximum angles and k is the width parameter. Based on the fitted curves, the orientation selectivity index (OSI)41 of individual synaptic input was calculated as:max is the maximum response strength in the optimal orientation and Rsportho represents the response value in the orthogonal orientation . Synaptic inputs with OSI exceeding 0.3 were then assigned as orientation-selective inputs.To collect orientation-selective inputs, the signal-to-noise ratio (SNR) of single synaptic inputs was defined as the ratio of fluorescence variance between the averaged ON and OFF frames of 12 orientations under the preferred spatial frequency:42 was calculated as:max and Rspmin are the maximum and minimum response strength to color patch stimuli. Also, a signal-to-noise ratio (SNR) index of single synaptic inputs was defined as the ratio of fluorescence variance between averaged ON and OFF frames of all colors. Moreover, we conducted a balanced one-way analysis of variance (ANOVA) on single-trial response values to obtain inputs with significant differences in response strength across various colors. When the CSI was larger than 0.8, SNR was higher than 1 and response variation across colors was significant , we assigned these synaptic inputs as color-selective.To determine the color selectivity of each synaptic input, a color selectivity index (CSI)Orien and max_RspColor are the maximum response intensity to orientation and color respectively. The color vs. orientation index was defined as the negative value of the orientation vs. color index.For each single ROIs, the orientation vs. color index was defined as:For each orientation-selective input, responses to three different spatial frequencies were fit using the Gaussian function. The preferred spatial frequency for each input was the one corresponding to the maximum of the fitted tuning curve.43θk are the orientations in radians and rspk are the response intensities. Each single pixel in the orientation map was then assigned to one of 360 bins according to its orientation preference, marked by different colors  low-pass Gaussian filter. The orientation preference of each pixel on the orientation map was then deduced from the vector sum of its response intensities under 12 orientations:x- and y-axis, and set as the square root of the sum of squares along these two axes. This matrix of gradient was then resized  and filtered by a Gaussian filter (100 pixels) to generate the final gradient map.We then determined the local orientation gradient with the following procedure. We binned pixels into groups of 8 and averaged the orientation preference (vector average) for the binned pixel. The gradient of each pixel was calculated along the 44, we first averaged differential images across high-SF drifting grating stimulus conditions, and obtained orientation selectivity maps “Orien_diffIm”. By averaging differential images across seven color stimulus conditions, we obtained color selectivity patterns “Color_diffIm”. Then, we subtracted Orien_diffIm from Color_diffIm and filtered the resultant differential image with a Gaussian filter (25 pixels) to generate color-dominant regions. Those regions having a value larger than 0 were identified as blobs, whereas the remaining regions were identified as interblobs.To estimate the positions of blob and interblob regions2) and black (<0.1 candela per m2) squares were presented randomly at one of 25 (5 by 5 matrix) or 49 (7 by 7 matrix) positions, covering a total area ranging from 0.8 × 0.8° to 2.6 × 2.6° depending on retinotopic eccentricity of target imaging area. Responses to each single square stimulus were recorded and used to define the ON and OFF subfields of spatial RFs. The 5 × 5 or 7 × 7 response matrix was zoomed in 60 or 40 times, respectively, and spatially filtered with a Gaussian filter (40 or 26 pixels). Next, a colormap changing from blue to green to red was used to denote ON/OFF signals  among orientation-selective inputs were used as a measure of RF similarity.Distance-dependent correlations of individual response properties were calculated along each single dendritic branch. Synaptic inputs on the same single dendritic branch were first combined into input pairs , on orientation-selective ROIs , or on neither . In ROC curves for each single neuron  curveSeveral preferred stimuli for each dendritic branch were used in experiments with high-imaging speed, up to 226 Hz. For each synaptic input, the mean fluorescence intensity through ten frames prior to the stimulus onset across trials was assigned as the fluorescence baseline. To get an averaged time curve of the overall inputs on one dendritic branch, temporal tunings of each synaptic input under its optimal stimulus with maximum response intensity were weighted summed by each ROI’s area size. Time curves of apical and basal dendrites were then normalized according to the peak response value and fit with a fourth-order Fourier function Fig. . The timp-value or R2 from linear regression analysis (Figs. Sampling neuron sizes and synaptic input numbers are like those in other studies in this field, and the precise details are described in the main text. The degree of correlation was calculated as Pearson’s linear correlation coefficient, unless otherwise specified. A ROC analysis was conducted to illustrate the spatial interlacing relationship of orientation-selective and color-selective inputs. The linear dependence between two distinct variables was characterized by the is Figs. , 8. The Further information on research design is available in the Supplementary InformationPeer Review FileReporting SummaryDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2


Background: Foot–ankle motion is affected by chronic ankle instability (CAI) in terms of altered kinematics. This study focuses on multisegmental foot–ankle motion and joint coupling in barefoot and taped CAI patients during the three subphases of stance at running. Methods: Foot segmental motion data of 12 controls and 15 CAI participants during running with a heel strike pattern were collected through gait analysis. CAI participants performed running trials in three conditions: barefoot running, and running with high-dye and low-dye taping. Dependent variables were the range of motion (RoM) occurring at the different inter-segment angles as well as the cross-correlation coefficients between predetermined segments. Results: There were no significant RoM differences for barefoot running between CAI patients and controls. In taped conditions, the first two subphases only showed RoM changes at the midfoot without apparent RoM reduction compared to the barefoot CAI condition. In the last subphase there was limited RoM reduction at the mid- and rearfoot. Cross-correlation coefficients highlighted a tendency towards weaker joint coupling in the barefoot CAI condition compared to the controls. Joint coupling within the taped CAI conditions did not show optimization compared to the barefoot CAI condition. Conclusions: RoM was not significantly changed for barefoot running between CAI patients and controls. In taped conditions, there was no distinct tendency towards lower mean RoM values due to the mechanical restraints of taping. Joint coupling in CAI patients was not optimized by taping. Lateral ankle sprains (LAS) are one of the most frequently occurring musculoskeletal injuries ,2,3, notMany publications have shown that patients with CAI have different foot and lower limb kinematics when compared to non-symptomatic subjects ,11,12,13Next to altered foot–ankle segmental motion, patients with CAI have also been associated with alterations in joint coupling, both in walking conditions and in running. Joint coupling is a measure for determining the degree of synchronization of motion between well-defined joint segments. Identifying changes in joint coupling enables gaining insight into movement disturbances and continued dysfunctionality . It is aTaping is a popular treatment in patients with CAI and helps preventing recurrent LAS ,11,17,18The overall changes in gait kinematics of patients with CAI reported in different publications largely agree but differ somehow in temporal observations. Some authors observed the kinematic changes during the complete stance phase and others only during certain periods of stance phase and/or swing phase. A possible explanation could be the difference in research setup . The shank–rearfoot coupling has been well studied in a rigid foot model but offers a rather incomplete evaluation with respect to the other joints of the foot . Given tTwenty-seven recreationally active university students (defined by at least 1.5 h of cardiovascular activity per week) were recruited in this study and are identical to the study of Deschamps et al. . This se2, RSscan International, Olen, Belgium). Data from the force plate and pressure plate were sampled at 200 Hz.Running analysis was performed at the Clinical Motion Analysis Laboratory of the University Hospitals Leuven. A 10 m walkway was used with the following measurement devices: a 3D motion analysis system, a plantar pressure platform, and a force platform. A passive optoelectronic motion analysis system  with 10 T-10 cameras was used to track the kinematic data (100 Hz). In the middle of the walkway, a custom-made force plate was imbedded , covered with a pressure plate . Running speed was assessed by monitoring the velocity of the reflective marker on the sacrum. A heel strike pattern was imposed in all conditions to minimize the influence of striking pattern. The control group only performed barefoot running trials whereas the CAI group additionally performed trials with low-dye (LD) and high-dye (HD) taping until at least three valid trials were registered for each condition. The order of these test conditions was randomly assigned. Taping with non-elastic sports tape  was applied by one investigator (BD) [Joint kinematics were estimated by first placing retroreflective markers according to the Rizzoli 3D multisegment foot model using double-sided tape . Once futor (BD) . Subjecttor (BD) , whereastor (BD) . This prThe collected spatiotemporal variables were running speed, stride time, stance time, and swing time. Statistical analysis on demographical and spatiotemporal parameters was performed using Wilcoxon Test and ANOVA. Kinematic waveforms associated to the Instituto Ortopedico Rizzoli 3D multisegment foot model were computed using the Vicon Foot model Plug-in  using Nexus 1.8 software. This five-segment model defines the 3D rotations between the shank (Sha), calcaneus , midfoot (Mid), metatarsus (Met), and hallux as rigid segments. The sagittal plane angle of the first metatarsophalangeal joint (F2Ps) was also computed. Ground reaction forces collected during the running trials provided an objective basis for defining the stance phase of each running cycle.t-test and comparison between the different CAI conditions was performed using a one-way repeated measures ANOVA. In order to retain a prescribed family-wise error rate alpha in the current statistical analysis, the error rate for each comparison was set at 0.5/3 = 0.017.Following data processing, temporal normalization of all waveforms for a complete gait cycle was performed with an in-house made Matlab software platform . Subsequently, the stance phase of the running cycle was subdivided into three specific subphases: The first phase, called the peak impact phase (PIP), encompassed the time window from initial contact (0%) to 5% of the running cycle. The second phase, called the absorption phase, included the time window from 6% of the running cycle to the peak vertical ground reaction force (GRF). The last phase went from the aforementioned peak GRF to toe-off (generation phase (GP)). Swing Phase (32–100%) was not included in our research. Range of motion, defined as the difference between the maximum and minimum value in each subphase, was first calculated for each trial and subsequently a mean value was calculated for the respective subject and this for each condition. Testing for differences between the control group and CAI_BF was performed using a two-sample The level of kinematic coupling during the stance phase of the running cycle was evaluated by calculating the cross-correlation coefficient between the following four inter-segmental angles ,25,26: (p > 0.05). There were some significant differences in spatio-temporal parameters between the control group and the CAI_BF condition. The demographic and spatiotemporal data are summarized in Demographic data showed no significant differences among both groups and conditions  on CAI. It is assumed that elastic taping has less mechanical restraint and exercises its effect more through sensorimotor control ,31,32. MNext to multisegmental RoM analysis of different CAI conditions, we also examined the degree of joint coupling. To our knowledge, this is the first study to evaluate multisegmental joint coupling in taped CAI subjects. Pohl et al. studied multisegmental ankle-foot joint coupling in normal running subjects and discovered a strong coupling between rearfoot  frontal plane (Inv/Eve) and transverse plane motion (Add/Abd) . The reaAlthough new insights are provided, especially concerning multisegmental joint coupling in taped CAI subjects, there are several limitations to this study. First, the sample size was rather small in the current study. Second, analysis focused on barefoot running, which may not reflect realistic conditions. Therefore, extrapolation of our results to shod conditions should be done cautiously. However, unfortunately multisegmental foot kinematics are difficult to examine in shod conditions. Third, participants were forced to adopt a heel strike pattern, probably interfering with the natural landing strategy of some participants. Consequentially, the full biomechanical picture can be obscured. Future challenges to further improve the external validity of the research in this field could be a larger sample size in which participants use their natural landing strategy with analysis of barefoot and shod running. Furthermore, the control subjects can also be analyzed with taping to obtain a complete picture of taping effects on multisegmental foot kinematics.The multisegmental kinematic research in this study was unable to demonstrate significant RoM differences between the CAI_BF condition and controls during the stance phase of running with a heel strike pattern. Moreover, in taped conditions there was no distinct tendency towards lower mean RoM values due to the mechanical restraints of taping compared to CAI_BF. The observed RoM differences in taped conditions were slightly more pronounced with LD taping, probably attributed to a different proprioceptive stimulation. In all CAI conditions, there was a trend towards a weaker joint coupling, despite application of non-elastic taping. Further research is needed to confirm our findings and possibly establish guidelines for adequate therapeutic interventions in patients with CAI.


The genetic alterations in cancer cells are tightly linked to signaling pathway dysregulation. Ras is a key molecule that controls several tumorigenesis-related processes, and mutations in RAS genes often lead to unbiased intensification of signaling networks that fuel cancer progression. In this article, we review recent studies that describe mutant Ras-regulated signaling routes and their cross-talk. In addition to the two main Ras-driven signaling pathways, i.e., the RAF/MEK/ERK and PI3K/AKT/mTOR pathways, we have also collected emerging data showing the importance of Ras in other signaling pathways, including the RAC/PAK, RalGDS/Ral, and PKC/PLC signaling pathways. Moreover, microRNA-regulated Ras-associated signaling pathways are also discussed to highlight the importance of Ras regulation in cancer. Finally, emerging data show that the signal alterations in specific cell types, such as cancer stem cells, could promote cancer development. Therefore, we also cover the up-to-date findings related to Ras-regulated signal transduction in cancer stem cells. Since the discovery of Ras as a key regulator of retrovirus-induced cell proliferation, a massive scientific effort has been devoted to identifying its critical roles in biology, especially in oncogenesis. Ras proteins are specialized guanine nucleotide-binding and hydrolyzing molecules that belong to the small G-protein superfamily . The humThe majority of the mutations in RAS genes that lead to oncogenic events are single-base substitutions in well-described positions in the G-domain. The most frequent oncogenic mutational hotspots are at codons 12, 13, and 61 in all four Ras isoforms. Less frequently, other codons in RAS genes are also affected by mutations, as summarized in Table Several studies have demonstrated that the Ras protein level is elevated in cancer tissues and that increased Ras expression is correlated with poor prognosis , 33. As The active state of wild-type Ras depends on its intrinsic ability to bind and hydrolyze GTP via the conserved 20-kDa G-domain. The Ras intrinsic GTPase activity is accelerated by the GEF/GAP system , which fuel the cycling between inactive GDP-Ras and active GTP-Ras. GEFs are the main activators of Ras via catalyzation of GDP release and GTP loading in the GTPase domain. Ras can bind GTP with high affinity, and GEFs can sterically displace the catalytic magnesium ion from the G-domain and restructure the nucleotide binding site, thus leading to the exchange of GDP to GTP. To downregulate Ras signaling, GAP proteins accelerate Ras-dependent GTP hydrolysis. A common feature of oncogenic Ras mutations is that they affect the nucleotide-binding site of Ras and lead to aberrant constitutive activation. The most common mutations, which are in codons 12, 13, and 61, lie in the G-loops of the protein by which Ras interacts with GDP and GTP. Although it is widely accepted that most Ras mutations stabilize the protein in its active state and prolong its downstream signaling, less is known about the mechanisms by which each unique nucleotide substitution can influence Ras signalome activation. Here we assembled the current evidence regarding how specific single-nucleotide substitutions in mutant Ras help to maintain the protein in its active state. It is already known that point mutations can directly prolong the GTP-bound state of Ras as reported in two independent studies comparing the features of different Ras mutants. Stolze et al. reported (based on the work in MCF10A cells) that the G13C, G12C, and G12V substitutions, along with two rare substitutions (Q61H and K117N), lead to higher levels of GTP binding relative to that of wild-type Ras . One mecIn the presence of growth factors (GFs), several intracellular signaling pathways are initiated in normal cells. In the first step, GF binding converts the receptor into an active state via autophosphorylation and dimerization, leading to recruitment of adaptor proteins such as Grb2 and Shc to the dimer’s cytoplasmic tail. Via Grb2, GEF proteins  can localize to the membrane and facilitate Ras activation. When wild-type Ras is in its GTP-bound state, it can assemble different signaling molecules at the membrane. The various Ras-associated effector proteins then promote canonical and non-canonical downstream Ras signaling Fig. .Fig. 3SiWhen Ras is activated by either GEFs or mutations, GTP-bound Ras undergoes conformational changes in the switch regions that allow RAF binding. RAF activation requires dimerization of GTP-bound Ras molecules. In the case of constitutively active mutant Ras and its dimers, the size of the membrane nanoclusters increases and more RAF molecules can be recruited. Therefore, under Ras mutant oncogenic conditions, Ras-dependent RAF signal transduction can be robustly amplified. RAF is a serine/threonine kinase with three paralogs, i.e., A-RAF, B-RAF, and C-RAF. Among them, A-RAF has the lowest and B-RAF has the highest kinase activity . NotablyThe ultimate outcome of the wild-type Ras-driven MAPK pathway is context dependent and can also lead to the initiation of diverse genetic programs associated with cell growth, cell migration, cell cycle, and cell survival. Moreover, Ras and its downstream MAPK effectors were also connected to the regulation of circadian rhythm . Since mTaken together, the data summarized here show that single amino acid substitutions at codons 12, 13, or 61 in RAS can stabilize the active GTP-bound state of RAS protein but that not all mutations result in elevated MAPK signaling -bisphosphate (PIP2) to phosphatidylinositol -trisphosphate (PIP3) at the cell membrane. PI3Kα can directly bind Ras via the Ras-binding domain (RBD) in its p110 catalytic subunit, and this interaction leads to PI3Kα activation. The mechanism of this activation remains unclear; however, it has been established that PI3Kα recruitment to the cell membrane by Ras has a key role since Ras can promote the formation of a preorganized PIP2-binding-favored state in the catalytic subunit . PIP3 isIt is not easy to investigate the involvement of Ras in the PI3K pathway because PI3K can also be activated by many other proteins independently from Ras, including G-protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs) . HoweverWe have already discussed how different amino acid substitutions have distinct consequences for MAPK pathway activation. The same is true for PI3K/AKT signaling, as mutations in Ras isoforms activate PI3K/AKT signaling with varying intensities. In an earlier work, Yan et al. studied the PI3K-activating potential of G12V mutant Ras isoforms, and they found that in transduced COS cells, H-Ras G12V could more robustly activate PI3K than could K-Ras G12V . While aIn addition to the two primary Ras-mediated pathways, Ras is also involved in several other signaling modalities. One of the less frequently studied pathways is RAC/PAK signaling. Activated Ras can interact with the Ras-binding domain of the TIAM1 protein (T lymphoma invasion and metastasis protein) at the plasma membrane . TIAM1 iMutant Ras can also upregulate RAC/PAK signaling. For example, several studies found elevated RAC activity in mutant HRAS-transformed fibroblasts , 67. In It is plausible that combination therapies targeting members of these pathways and PAKs could work by preventing cross-activation; however, PAKs can act independently of the two abovementioned signaling pathways, since in mutant KRAS-driven colon cancer, knockdown of either PAK1 or PAK4 inhibits cancer cell proliferation and increases apoptosis . UnfortuThere is an additional Ras effector pathway in which the initiation step of the signaling is triggered by an interaction between Ras and a guanine nucleotide exchange factor. RalGDS is a GEF for Ral and has also been identified as a Ras-binding partner. When Ras is in its active, GTP-bound state at the plasma membrane, RalGDS can bind to it to facilitate GDP to GTP exchange in Ral . This meRas is mainly known because of its role in promoting cell survival and proliferation; however, in certain cases, mutant Ras can inhibit cell growth, and it can lead to cellular senescence or apoptosis , 77. MatSome key regulators of Ras-modulated cell death signaling pathways belong to the RASSF protein family . The memRASSF1A is one of the most frequently inactivated tumor suppressors in human cancer, presumably because it connects Ras signaling with apoptotic mechanisms , 91. OneNORE1A is the closest RASSF1A homolog, and while it is also a tumor suppressor, there are functional differences between the two proteins. While the main function of RASSF1A is to mediate Ras-driven apoptosis, it seems likely that NORE1A is primarily involved in cellular senescence  . Cellula2) in the plasma membrane. This reaction produces two important intracellular second messengers, i.e., diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3), that lead to the initiation of several different downstream signaling pathways [Phospholipase C (PLC) proteins are enzymes that can hydrolyze phosphatidylinositol 4,5-bisphosphate  concept proposes that tumor growth, like the renewal of healthy tissues, is fueled by a small subset of tumor cells endowed with stem cell characteristics. Over the past decades, most solid and non-solid tumors have been found to harbor CSCs or cells with stem cell (SC) features, such as self-renewal and quiescence (with exceptions) as well as expression of an often tumor-specific subset of SC markers. It has been demonstrated that CSCs contribute to tumor initiation, progression, metastasis, and therapy resistance and that this processes could be linked to aberrant Ras activation in CSCs , 120.In colorectal cancer (CRC), a number of sequential genetic alterations drive tumorigenesis. Activating mutations in the KRAS gene alone do not usually induce transformation. Initiating genetic mutations, such as a loss-of-function mutation in the adenomatous polyposis coli (APC) gene, followed by accumulation of activating mutations in the KRAS gene are needed to drive tumorigenesis during the early and intermediate phases of CRC. The resulting aberrant activation of Wnt/β-catenin and Ras/ERK signaling are critical factors in the transformation and disease progression . Loss ofThe therapeutic response to gemcitabine (GEM), a standard chemotherapeutic agent used in the treatment of pancreatic adenocarcinomas, has proven to be unsatisfactory. Chemoresistance to GEM is associated with poor prognosis, and the reacquisition of CSC-like features is considered to be a main causative factor in the development of chemoresistance. Zhao et al. demonstrated that the mechanism behind GEM’s ineffectiveness was that after GEM induces NADPH oxidase activation via nuclear factor κB (NFκB), activated NADPH oxidase upregulates ROS production, which targets the K-Ras/MAPK pathway. In KRAS knockdown experiments, K-Ras was shown to be responsible for the GEM-mediated metabolic reprogramming and stemness of CSCs . The K-RRas proteins coordinate multiple downstream effectors, many of which are aberrantly activated during cancer development. Although the regulatory roles of Ras proteins in signaling networks are mostly linked to the RAF/MEK/ERK kinase pathway and PI3K/AKT signaling, there is a growing evidence that Ras also mediates other signaling routes under normal and oncogenic conditions Fig. .It has been recently established that one of the ultimate challenges in anti-Ras therapy resides in Ras mutation-specific differences; Therefore, studies investigating the distinct roles of each Ras mutant in signal transduction should be prioritized to discover additional promising Ras mutation-selective therapeutic candidates.


Stokes phase is the phase difference between orthogonal component states in the decomposition of any polarization state. Phase singularities in the Stokes phase distribution are Stokes singularities of an inhomogeneous polarization distribution. Under circular decomposition, Stokes phase distribution  Spin-orbit beams are beams embedded with polarization singularities6 and can be described as a superposition of orthogonal OAM states in orthogonal spin angular momentum (SAM) states. In recent years, beams with single polarization singularity at the center of the beam are called by special names such as Poincaré beams7, cylindrical vector beams8, vector vortex beams9, Majorana beams10. Here, we deal with higher order Poincaré sphere (HOPS) beams13 and hybrid order Poincaré sphere beams (HyOPS)17 that contain a polarization singularity at the center of the beam and only two dimensional field distributions are considered. In recent years, many methods have been reported to generate HyOPS19 and HOPS beams24. It is also possible to have multiple polarization singularities in a single beam like in random fields26 and lattice fields28. Stokes phase is more fundamental to deal singularities appearing in polarized light fields. In this article, we discuss the degeneracy in Stokes index, that is used to characterize the Stokes phase distribution. Although the discussion presented here is valid for Stokes singularity with any index, the degeneracies are explained by choosing a particular index.Increased interest in the use of spin-orbit beams invites simple detection methods. Phase singular beams carrying orbital angular momentum (OAM) have immense applications30. The predominant state of polarizations (SOPs) around an ellipse field and vector field singularities are ellipses and linear states respectively. C-points and L lines are two types of ellipse field singularities whereas V-point is vector field singularity. C-points are points of circular polarization in an inhomogeneously polarized ellipse field where azimuth of polarization is indeterminate and L line is a continuous line of linear polarization states in ellipse field where handedness is not defined. Since HOPS beams are constant ellipticity fields, they are useful to represent V-points. In HyOPS beams, both ellipticity and azimuth are variables and are useful to represent C-points.The endowed polarization singularities are broadly classified as ellipse and vector fields singularities. Ellipse field singularities are isolated points or lines where some property of elliptical state of polarization is indeterminate whereas vector field singularities are point singularities where the orientation of the electric field vector is not defined32. For inhomogeneously polarized beams, complex field distribution 33. The polarization singularities which are phase singularities in 36. This azimuth distribution is related to 34.Polarization of light can be described using four measurable Stokes parameters 42. But for polarization singular beams, the diffraction patterns alone will not suffice48 for their unambiguous discrimination. The signature diffraction patterns of singular beams are due to the disintegration of higher charge singularity into multiple lower ones. In scalar singularities, the phase vortex of higher charge disintegrates into multiple generic lower charge singularities, where topological charge conservation is observed. For polarization singularities, owing to multiple parameters associated with polarization of light, the disintegrated fragment singularities have multiple attributes. The V-points and higher-order C-points transform into lower-order more stable elliptic field singularities under small perturbation following index and sign conservation rules49. Reports on V-point diffraction show that there is index and helicity conservation47.Phase singular beams with unequal topological charges are orthogonal. These are beams embedded with scalar field singularities. The orbital angular momentum content in these beams can be found by the distinct diffraction patterns they producedl is the closed path of integration around the singular point. Both the indices are defined by using the azimuth 34. Stokes phase distribution Ellipse field singularities have C-point index defined as 50 governed by the mode indices of the component states is different for the respective HyOPS beams. The propagation properties of these beams are entirely different from each other and the distinction between such dark C-point beams is needed to deploy it for a specific application. Notably, partially coherent dark C-point beams carrying different OAM content are reported to show different strength of anti-depolarization effect although the SOP distribution is alike51. Another case where one can encounter alike polarization distribution for two different beams is when the relative weightage of the two OAM states comprising the HyOPS beam is different. In both the above cases, if HyOPS beams are diffracted by an appropriately chosen aperture the distinction in term of OAM content and the relative weightage of OAM states can be made. Following polarization transformation one can further accurately predict the Stokes phase and SOP distribution of the HyOPS beams as well. To the best of our knowledge, we are the first to present a novel technique based on diffraction and polarization transformation to detect and efficiently distinguish these degenerate Stokes index states. These polarization structured spin-orbit beams find potential application in optical activity measurement52, achieving isotropic and directional edge enhancement5, designing robust beams for propagation in turbulent medium53, optical trapping and micro-manipulation54, Mueller matrix polarimetry55, and polarization speckle generation56.The only method that can detect/lift the Stokes phase/Stokes index degeneracy present in such inhomogeneous beams is Stokes polarimetry. But that has also not been discussed in the context of degenerate Stokes index states anywhere in the existing literature. Moreover, there are cases wherein the beams composed of different OAM state combination (or different relative weightage of OAM states) possess alike polarization distribution where even Stokes polarimetry fails to distinguish between such beams. For example, a dark C-point of given Stokes index  diffraction of polarization singularities through equilateral triangular aperture and (ii) polarization transformation. Five representative degenerate Stokes phase states for a given Stokes index mentclass2pt{minima can be mathematically written as:The transmittance function of an equilateral triangular aperture with side length f is given by:u, v) are the coordinates at the Fourier plane, A is a constant and l and m) in orthogonally circularly polarized states, the resultant diffraction intensity of polarization singularities is found to be the sum of diffraction intensities of scalar vortices.The field just after the aperture is given by:58 and spiral phase plate (SPP) is used to generate different combinations of topological charges l and m in orthogonal spin-orbit states. A collimated beam from He Ne laser passing through the SWP produces V-point singularity of one type  depending on the input polarization state . A half-wave plate after SWP can be used to invert the sign of Stokes index59. The spiral phase plate after S wave plate converts the V-point into bright or dark C-point depending on the charge of the SPP. For example, a spiral phase plate placed at charge 1 (2) will give a combination of vortices in orthogonal spin-orbit states with charges 0 (3) and 2 (1) and hence produce bright (dark) C-point. A triangular aperture is inserted in the beam path to produce diffraction pattern and polarization transformation is carried out by inserting linear or circular polarizers in the setup. A Stokes camera is used to capture the diffraction intensity patterns and resultant intensities after polarization transformation at the output. For plotting the polarization distribution of the input beams, Stokes parameters are measured using Stokes camera . Using the experimentally measured Stokes parameters, the ellipticity and azimuth parameters are calculated at each of the pixel. From these polarization ellipse parameters, the SOP distribution is obtained. The setup used here for generating input beams, can be used to produce polarization singularities with even Stokes index values. The input beams with any arbitrary Stokes index value can be generated using modified Mach-Zehnder type interferometer57.The experimental setup used for detecting degenerate Stokes index states is shown in Fig. In Fig. mentclass2pt{minimTwo Stokes phase distributions from each sphere are considered, although an infinite number of Stokes phase distributions share the same Stokes index. Going outward, the SOP distributions and the diffraction patterns are presented. Note that in Fig. l and m as long as Experimental results for beams with index mentclass2pt{minim24 and higher index C-points60 can be experimentally generated, they are not stable and tend to disintegrate under strong perturbation. In the experimental figures we observed that the Dark C-points of same Stokes index, same Stokes phase, same handedness and identical polarization distribution can be obtained using many combinations of optical vortices such that Figure In this work, we have identified the various possible Stokes index degenerate states in the context of inhomogeneous HOPS/HyOPS beams. The other form of subset degeneracies occurring in Stokes phase, diffraction pattern, and polarization distribution associated with a fixed Stokes index are also discussed. We have shown that Stokes index state degeneracy can be lifted by a method based on combination of diffraction and polarization transformation in linear and circular basis. A triangular aperture is used to diffract these spin-orbit beams and it is realized that the overall diffracted intensity pattern can be used to decide to which sphere, (HyOPS or HOPS) the Stokes index is attributed. Further, polarization transformation that segregates the component intensities in the diffraction pattern, pin-points the location on the sphere that represents the SOP distribution. Distinction among beams endowed with V-points, bright C-points, dark C-points singularity and their handedness is demonstrated through simulation and supported by experimental results. Apart from being simple and elegant intensity based measurement, our method successfully distinguishes polarization degenerate HyOPS beams where even Stokes polarimetry fails. This study provides a systematic guide for intensity based detection techniques for the unambiguous detection of spin-orbit beams.


Preoperative characteristics of living kidney donors are commonly considered during donor selection and postoperative follow-up. However, the impact of preoperative uric acid (UA) levels is poorly documented. The aim of this study was to evaluate the association between preoperative serum UA levels and post-donation long-term events and renal function.This was a single-center retrospective analysis of 183 living kidney donors. The donors were divided into high (≥5.5 mg/dl) and low (< 5.5 mg/dl) UA groups. We analyzed the relationship between preoperative UA levels and postoperative estimated glomerular filtration rate (eGFR), as well as adverse events , over 5 years after donation.p = 0.003); this was true even after the exclusion of hyperuricemia-related events (p = 0.047). UA emerged as an independent risk factor for adverse events (p = 0.012). Donors with higher UA levels had lower eGFRs after donation, whereas body mass index, hemoglobin A1c, blood pressure, and low-density lipoprotein cholesterol did not have any impact on the eGFR.In total, 44 donors experienced 52 adverse events over 5 years. The incidence of adverse events within 5 years was significantly higher in the high UA group than in the low UA group (50% vs. 24%, The findings suggest that preoperative UA levels should be considered during donor selection and postoperative follow-up. Living donor kidney transplantation is one of the established treatment options for patients with end-stage renal diseases (ESRDs) , 2. GiveUric acid (UA) is known to induce endothelial dysfunction by reactive oxygen production,  inflammaThe impact of serum UA levels on the residual kidney of living kidney donors has been an emerging topic of interest. A large cohort study including 4650 living donors showed that donors with post-donation gout had a higher risk of acute kidney failure and progression of CKD . Other sAlthough no statement regarding hyperuricemia was included in the Amsterdam Forum on the Care of the Live Kidney Donor published in 2005,  given thn = 57, UA level ≥ 5.5 mg/dl) and the low UA group . The median value of UA in the present study was 4.9 [4.2–5.7] mg/dl. The definitive cutoff value of serum UA level as the risk factor of metabolic syndrome or CKD was still controversial  mg/dl) to 1-year postoperative (5.8 [5.0–6.8] mg/dl) in all donors. The degree of increment was similar in male  and female  donors. In addition, the degree of increment was similar in the high UA  and low UA groups . The annual value of postoperative UA levels were 5.8 [4.8–6.9] mg/dl at 2 years, 6.1 [4.9–6.9] mg/dl at 3 years, 6.1 [4.9–6.9] mg/dl at 4 years, and 6.2 [5.0–6.7] mg/dl at 5 years after donation.A significant increase in UA levels  donors experienced 52 adverse events in the 5 years after donation. Eight cases experienced more than two adverse events. The observed adverse events were as follows: two cases with myocardial infarction, one with cerebral infarction, one with the induction of hemodialysis due to de novo IgA nephropathy, and 48 with de novo prescriptions . Xanthine oxidase inhibitors were prescribed for all cases with hyperuricemia. Calcium blockers and angiotensin-converting enzyme inhibitors were prescribed for eight and six cases with hypertension, respectively. In dyslipidemia, statins were prescribed for 21 cases, whereas eicosatetraenoic acid and intestinal cholesterol transporter inhibitor were prescribed for one case each. Biguanide antihyperglycemic agent and dipeptidyl peptidase-4 inhibitors were prescribed for one and two cases of hyperglycemia, respectively. There was no mortality in this cohort.p = 0.003, Fig. p < 0.001, Fig. p = 0.047, log-rank test).Fig. p < 0.001), BMI , BP , and LDL-C  were significantly associated with a higher incidence of adverse events in the 5 years after donation (Table p = 0.012), BP , and LDL-C  levels emerged as independent risk factors for the development of adverse events. The ROC curve revealed that the cut-off value of preoperative UA levels was 5.5 mg/dl but the area under the curve was 0.659, which was not reliable. Table In the univariable analyses, preoperative UA levels .Fig. A post-hoc power calculation was conducted to detect the significance of UA levels. The incidence rate in the two groups was used for the dichotomous endpoint, and the median and interquartile range for the continuous endpoint. An alpha error rate of 5% was set. This calculation revealed sufficient power of the effects of UA on postoperative adverse events (93.2%) and the 1-year eGFR (90.7%).In the present study, we showed that higher UA levels may be associated with post-donation adverse events and negative effects on eGFR in living donors. There are only a few short-term studies available that discuss the correlation between preoperative UA levels and postoperative renal function –28. Cho Although higher UA had a negative impact on the postoperative eGFR recovery within the 4 years after donation in the present study, other factors such as BMI, BP, HbA1c, and LDL-C were found to be unrelated to the poor recovery of renal function. Nevertheless, hypertension, –43 hyperP < 0.05) [p = 0.047, log-rank test). Although the population in the present study was different from the CKD population in previous studies, we have provided new insight into the possible impact of an immediate increase in UA levels on new-onset hypertension and hyperlipidemia after donation. According to a recent report, new-onset obesity might be related to preoperative UA level [Based on the existing literature, we speculate that the sudden increase in UA after living donation might result in the poor recovery of renal function through endothelial dysfunction,  inflamma < 0.05) . In the  < 0.05) . A meta- < 0.05) . In addi < 0.05) . The preUA level . FurtherSimilarly, in the CKD population, a sudden cessation of anti-hyperuricemia drugs  is known to induce renal and endothelial dysfunction . FurtherWe set the arbitrary cutoff value for the UA level according to the distribution in the present study. This cutoff value (5.5 mg/dl) was slightly lower than the standard value for patients with asymptomatic hyperuricemia. While the Japanese Society of Gout and Nucleic Acid Metabolism has suggested that the cutoff value for serum UA be 7.0 mg/dl regardless of sex and age,  the AmerHbA1c was not associated with either adverse events or reduced eGFR. Normal HbA1c may not affect postoperative renal functions. A previous Japanese study at a single center reported that prediabetic donors without diabetic complications who showed an abnormal pattern in the 75 g oral glucose tolerance test did not develop ESRD after donation in the long-term . Another2), higher BP (> 130 mmHg), and higher LDL-C levels (> 120 mg/dl) were associated with a greater occurrence of adverse events. These thresholds can be conceived as pre-disease status. The Japan Society for the Study of Obesity has defined obesity as a BMI of > 25 kg/m2 [2 [Other clinical parameters, apart from HbA1c, were found to predict the postoperative incidence of adverse events in the present study. Higher BMI (> 22 or 25 kg/m25 kg/m2 . A Japankg/m2 [2 . The Amekg/m2 [2 . A randokg/m2 [2 . The Japkg/m2 [2 . These aMost of the donors (92.0%) were comprehensively followed up for 5 years in the present study. The Japanese Society for Clinical Renal Transplantation and the Japan Society for Transplantation reported in a follow-up survey in 2018 that 20.9% of Japanese living kidney donors’ data could be aggregated by web-based methods and 79.5% of them were properly followed up for 5 years after donation . The UniThere are some limitations to this study. First, this was a single-center retrospective study with a relatively small sample size, and there were only 44 donors with 52 adverse events. This limited number of events may not be adequate to support meaningful multivariable analyses. In the present study, most of the living donors were limited to Japanese living in a single prefecture. Analysis of multiethnic and heterogeneous populations should be considered in further studies. Second, although de novo prescriptions are regarded as hard outcomes, both the timing and indication of the prescriptions are dependent on individual doctors. Living donors generally hesitate to take a new medication because they think they do not require it. Therefore, it is likely that the incidence rate in the present study was underestimated.In conclusion, we showed that preoperative UA levels may be one of the negative factors affecting the incidence of lifestyle-related adverse events and renal function after living kidney donation. Preoperative uric acid level should be considered when selecting living kidney donors. Further studies are warranted for mitigating the risk of morbidity and mortality in living donors.


Human retroviruses and the hepatitis B and C viruses  share routes of transmission; thus, coinfections occur andcould alter subsequent disease outcomes. A preliminary study on humanT-lymphotropic virus types 1 and 2 (HTLV-1/2) in serum samples from HBV- andHCV-infected individuals in São Paulo revealed 1.3% and 5.3% rates ofcoinfection, respectively. These percentages were of concern since they weredetected in HTLV-endemic regions and in high-risk individuals in Brazil. Thepresent study was conducted to extend and confirm these data.  HTLV-1/2 and human immunodeficiency virus (HIV) infection status wereidentified in 1,984 sera for HBV and HCV viral load quantification - 1,290samples from HBV-infected individuals  and694 samples from HCV-infected individuals .HTLV-1/2 antibodies were detected by enzyme immunoassay, followed by westernblotting and line immunoassay; HIV infection was detected by enzymeimmunoassay. p<0.05).  HTLV-1/-2 infection was detected in 1.9% HBV-infected individuals (0.7%HTLV-1 and 1.2% HTLV-2) and in 4.0% (2.4% HTLV-1 and 1.6% HTLV-2)HCV-infected individuals; HIV infection was detected in 9.2% and 14.5%,respectively. Strong associations with HTLV and HIV, male sex, and older agewere found in HBV/HTLV and HCV/HTLV-coinfected individuals( HTLV-1 and HTLV-2 were confirmed to be prevalent in individuals with HBV andHCV in São Paulo; coinfected individuals deserve further clinical andlaboratory investigation. Although there aresome controversies, the majority of studies worldwide have described worse diseaseoutcomes in HIV-, HBV- and HCV-infected individuals who were coinfected with HTLV-1,compared to those who were not coinfected. For instance, in HIV/HTLV-1-coinfectedindividuals from Salvador, Bahia, Brazil, rapid progression to acquired immunedeficiency syndrome (AIDS) and death were observed,,-,Brazil has the largest absolute number of human T-lymphotropic virus types 1- and 2-(HTLV-1 and HTLV-2) infected individuals and, consequently, the largest number ofHTLV-associated diseases in Latin AmericaEpidemiologic Bulletins of HIV/AIDS and ViralHepatitis, there were 926,742 cases of AIDS (1980 to 2018), 218,257cases of hepatitis B (1998 to 2017), and 491,960 cases of hepatitis C (1999 to2017); the majority of which were reported in the Southeast region of Brazil,Due to the high mortality and morbidity of HIV, HBV and HCV infections worldwide,together with the high transmission/dissemination capacity of these viruses,infection notification is compulsory in Brazil. According to the Brazilian Ministryof Health’s (MH) ,In contrast, HTLV-1 and HTLV-2 are neglected infectious diseases in Brazil. There isno obligatory notification policy and these infections are not included on the listof neglected diseases; they are similarly mistreated in other parts of theworldConsidering the data described above and the expertise of the Instituto Adolfo Lutz(IAL) of São Paulo from the studies on HTLV-1/2, two years ago, we decided toinvestigate the prevalence of HTLV-1 and HTLV-2 in patients with hepatitis B and C.The preliminary results concerning because included both HTLV-1 and HTLV-2: anoverall prevalence of HTLV-1/2 of 1.3% in individuals with hepatitis B and 5.3% inindividuals with hepatitis C, and an association with HIV in the HBV/HTLV-coinfectedindividualsThe Instituto Adolfo Lutz (IAL), a public health laboratory in São Paulo, Brazil,and the reference laboratory for viral hepatitis, HIV and HTLV-1 and HTLV-2infections, carried out the diagnosis and surveillance of these viruses. Theyalso evaluated the epidemiological trends of the infections and coinfections.This institute has a central laboratory located in the city of São Paulo;analyses of high complexity are performed there. It also has 12 regionallaboratories spread throughout the state of São Paulo. Patients who attended IALcame from different specialized health centers; this included STI/AIDS and viralhepatitis centers. A cross-sectional/transversal anonymous study was conductedusing the data obtained from 1,984 plasma/serum samples. These were collectedfrom 1,290 patients analyzed for HBV viral load , and 694 patients analyzedfor HCV viral load . The collection occurred from June 2015 to June 2016 atthe central IAL; the samples were stored at -20 °C.The majority of samples were collected from the health centers in São Paulo cityand its vicinity (metropolitan area) and in the cities in the eastern region ofthe state (up to 200 km from São Paulo city) .The serological diagnoses of hepatitis B and C were performed at the specializedhealth centers where the patients were seen for clinical follow-up. Theseservices used different immunoassays for the diagnoses, depending onavailability . In some cases, HCV infections were confirmed using RNA quantificationat IAL, using the Abbott Real-Time HCV assay .The samples were screened for HTLV-1/-2-specific antibodies at the HTLV ResearchLaboratory of the central IAL using enzyme immunoassay . The reactions and interpretations of the results were performedaccording to the manufacturer’s instructions. The cut-off values and the grayzones were calculated; the samples considered reactive or inconclusive in thescreening tests were further confirmed by Western blotting (WB) assay . For the WB confirmatory assay, theresults were interpreted according to the stringent criteria provided by themanufacturer. Briefly, HTLV-1-positive serum samples were defined as thepresence of gag (p19 with/without p24) and two env (GD21 and rgp46-I) bands.HTLV-2-positive samples were defined as reactive to gag (p24 with/without p19)and two env (GD21 and rgp46-II) bands. Samples showing antibodies to both gag(p19 and p24) and env (GD21) bands were considered HTLV-positive but untypeable.Any other patterns of bands were denoted as indeterminate.In an attempt to confirm and/or to discriminate the samples with WB-inconclusiveresults , the sampleswere tested by line immunoassay . The strips used in LIA contain antigens for validating,confirming and discriminating the results. For validation, the line marked byeach sample was compared with the control line; a score ranging from +/- to +3was assigned. The confirmatory antigens included gag p19 I/II, gag p24 I/II, envgp46 I/II, and env gp21 I/II. No bands, or the occurrence of a single band , denoted a negative result. Thepresence of one band (env gp21 I/II) or two bands (except env gp21 I/II)indicated indeterminate results, while two bands  indicated HTLV positivity. Threediscriminatory bands  were considered asfollows: HTLV-1 positivity was indicated by the reactivity to gag p19-I and/orenv gp46-I, while HTLV-2 positivity was considered when the samples showed envgp46-II or a higher intensity of the env gp46-II band than the gag p19-I and theenv gp46-I bands. In addition, HIV infection was investigated by an EIA of high sensitivity andspecificity, capable of detecting both HIV-1- and HIV-2-specific antibodies andthe p24 antigen of HIV-1 . This assayallows the identification of early HIV infections. The reaction was carried outaccording to the manufacturer’s instructions; the results were defined aspositive or negative for HIV infection.p-value of ≤0.05 was considered significant.Logistic regression analysis was used to identify the factors associated withHTLV-1/2 infection by calculating odds ratios (ORs) and 95% confidence intervals(CIs).The laboratory results and the patients’ demographic data were recorded andanalyzed according to the Epi Info version 3.5.4 software .Differences in the number of males and females in each group were statisticallyevaluated using the chi-square test or Fisher’s exact test, as appropriate.GraphPad Prism software version 5.03  was employed for agecomparisons among groups using the Mann-Whitney U test (two groups), theKruskal-Wallis ANOVA test, and Dunn's Multiple Comparison Test (three or moregroups). A The study was approved by the Ethics Committee for Research at IAL (CTC#21I-2016)and received the MH protocol number: CAAE - 55837316.0.0000.0059. All procedureswere performed in accordance with the principles established in the Declarationof Helsinki of 1975, as revised in 1983. The study was conductedanonymously.During the HTLV-1/-2 screening of the 1,984 plasma/serum samples, 55 reactedpositively and were tested by WB; 43 had confirmed HTLV-1 or HTLV-2 positiveresults, 11 had WB-inconclusive results , and one had WB-negative result. After INNO-LIAanalysis of the 11 WB-inconclusive samples, HTLV-1 or HTLV-2 was confirmed in ninesamples, one sample was negative, and one remained indeterminate. The distributionof these samples, according to group (HBV or HCV), and the final HTLV results arepresented in Overall, 52 HTLV-positive samples were detected; 26 samples had confirmed HTLV-1infections , and 26 samples had confirmed HTLV-2infections . The HTLV-1/-2 and HIV serological resultsin each group (HBV and HCV) are presented in Three regions of the state of São Paulo that sent samples to the IAL accounted forthe positive HTLV-1/-2 results. In the HBV-infected individuals, 2.4% came from theeast region, 1.8% came from the metropolitan area of São Paulo city, and 1.8% camefrom São Paulo city. In the HCV-infected individuals, 4.7%, 3.2%, and 3.8% came fromthe regions described for HBV, respectively.p<0.0001), 1.7 in the HBV/HTLVgroup (p=0.257), 6.2 in the HCV/HIV group(p<0.0001), and 3.7 in the HCV/HTLV group(p=0.004). The number of individuals in each group and thepercentage of patients according to sex are depicted in According to sex, there were more males in both groups of this study , with a relationship between males and females close to 1.0 in theHBV-infected and HCV-infected patients . Howeverp=0.0434). The majority of coinfected individuals had ages rangingfrom 41 to 60 years old, however, most of them were 41 to 50 years (p=0.046); this was ten years above the median age ofHBV/HTLV-coinfected patients . This finding is alsoconcerning as HTLV-1 and HTLV-2 seem to influence hepatic disease outcomesdifferently. Once more, the LIA was identified as the best assay for confirmingHTLV-1/2 infections in such patients; the same finding was previously detected forpatients with HIV/AIDS in São Paulo,Interestingly, although the WB assays have improved over time, the number ofWB-inconclusive results in Brazilian blood samples currently remains. In addition,they include samples from high-risk populations , and samples of HTLV isolated from the outpatient clinics in HTLV-1endemic areas-Another important finding that emerged from the present study was the increased riskof HIV infection in HBV/HTLV- (OR: 15.48) and HCV/HTLV-coinfected patients (OR:7.73). In fact, when the blood samples from HBV- and HCV-infected individuals weretested for HIV infection, 9.2% and 14.5% were positive, respectively. This wassomewhat above the percentages , in HBV- andHCV-infected individuals reported by the southeast region to the Brazilian MH(2018),,,,In the present study, although we did not know the routes of viral transmission orthe temporal order of the infections, we could speculate that transmissions occurredmostly through parenteral routes. Prior to laboratory testing being commerciallyavailable, and HIV and HBV serology (1988) and HTLV and HCV serology (1993) becamemandatory in blood banks throughout BrazilAnother important point that emerged from the present study is that, while bothHTLV-1 and HTLV-2 were detected in HBV- and HCV-infected patients, there were moreHTLV-2 cases in the HBV-infected individuals, and more HTLV-1 cases in theHCV-infected individuals. Whether these findings were beneficial or not, regardingthe hepatitis B and hepatitis C outcomes, remains to be determined.This study had one major limitation. The results obtained could not allow us todetermine the precise prevalence of HTLV-1/2 in this state for the several reasons.First, it was a cross-sectional/transversal study that employed samples frompatients who were initiating treatment for hepatitis B and C or who were infollow-up. Second, the samples came mostly from the east region of the state of SãoPaulo; as such, they may not be generalizable to the rest of the state.In conclusion, HTLV-1 and HTLV-2 were confirmed to be prevalent in individuals withviral hepatitis B and C in São Paulo. We are now investigating the HBV and HCV viralloads and clearance data from these patients to enhance the information concerningthis matter. This will better assist physicians in performing accurate patientfollow-ups. Confirming the virological prognostic values of these infections mayhelp convince the Brazilian MH to add HTLV-1/2 serology to the battery of tests usedin the follow-up of patients with viral hepatitis B and C.