Cellulose nanocrystals (CNCs) are a biorenewable resource, which may be chemically modified to impart specific properties. Modified CNCs have found use in imaging applications, as rheology modifiers, polymer reinforcements, barrier and/or optical films, and nanocomposites. Nanoparticle dimensions of CNCs are typically 5–10 nm in width, with lengths of <100–300 nm. However, the physical properties are dependent upon the number and nature of the surface charge groups imparted during preparation. In the case of CNCs produced from sulfuric acid hydrolysis, the sulfated surface groups may be partially removed prior to further functionalization. This gives more available hydroxyls yet renders the CNCs less colloidally stable. Furthermore, conditions vary significantly and there is no consensus about the optimal conditions for partial removal of sulfate functionality or conditions developed to give specific surface charge. In the following, alkali hydrolysis of sulfate half-esters was quantified by conductometric titration of the strong acid groups, and using a design of experiments (DOE), optimal conditions were determined to produce CNCs with tailored surface charge. Thuely used ,18,19,203PO4 hydrolysis of Whatman filter paper [3PO4 hydrolyzed CNCs [2SO4 hydrolysis. Typical conditions found in the literature are 64 wt% H2SO4 at 45 °C for 30–45 min [−1)—are often not reported. For instance, Lin and Dufresne used 65 wt% H2SO4  to obtain CNCs with available surface –OSO3– of 209 mmol·kg−1 and a ζ-potential of –40.7 mV; however, the yield was not reported [2SO4, 55 °C, 60 min based on constraints of maximal CNC yield at a given acid concentration and the minimal retention of cellulosic solid residues (CSR) and other decomposition products (vide infra). Experimental results were in agreement with the kinetic model and gave yields as high as 70%. At a lower acid concentration, insufficient depolymerization occurred and at higher concentrations, excessive degradation to monosaccharaides occurred. However, minimal product characterization was done, and only CNC and CSR yields were reported [2SO4; sulfur contents were observed between 93–311 mmol·kg−1. However, at >62 wt% H2SO4, CNCs were further degraded into mono and oligosaccharides; thus, further sulfation reduced yields to <20% indicating typical conditions are not optimal [−1.Extensive work has gone into investigating the hydrolysis conditions required to optimize yield, surface charge and nanocrystallite size ,22,23. Fer paper . The reazed CNCs . There ireported . Wang etThe decomposition (hydrolysis) products of cellulose were investigated in sub-critical and super-critical water; the main degradation products produced are furfural and 5-hydroxymethyl-2-furfural (HMF) derivatives ,25. This3– groups has a marked effect on  the thermal stability of CNCs and limits their use in some applications  when the –OSO3– concentration is very high [3– functional groups can impede the incorporation of other functionalities onto the CNC surface; for instance, the ability to graft photocatalysts onto CNCs was inversely correlated with the presence of sulfur on the CNC surface [The inclusion of –OSOery high . Moreove surface . Abitbol surface . The eff3– prior to post-functionalization. There are three potential pathways for the cleavage of sulfate half-esters on the CNC surface, one of which is favored dependent upon on the acidity of the conjugate acid (or nucleophile) used [It is, therefore, much more common to decrease the amount of surface –OSOle) used ,30. To ale) used ,32,33. Lle) used . Dorris le) used .3– groups, while solvolytic desulfation gives the CNC product as a pyridinium salt [2SO4-hydrolyzed CNCs; the partially desulfated CNCs were functionalized with a fluorescent marker at ~2.5 times the concentration of sulfate CNCs [Jiang et al. observed that the acid-catalyzed desulfation of CNCs resulted in increased degradation products and the incomplete removal of surface –OSOium salt . The forate CNCs . Acid-caate CNCs . Ellebraate CNCs . Pandey ate CNCs .3– groups by Zoppe et al. allowed the post-functionalization of CNCs to give biomimetic nanostructures [3– groups) of CNCs, to control the nucleation and growth of silver nanoparticles (AgNP) [3– groups and optimal within a specific range; too much –OSO3– gave a large size distribution while too little led to larger AgNP. Further use of alkali hydrolysis of –OSOructures . In thisructures . An alkas (AgNP) . The sizAs 3– when prepared from H2SO4 hydrolysis [−1) given a known starting value of sulfate .Although numerous methods are used to prepare CNCs, successful surface modification requires CNCs of moderate thermal and colloidal stability  but with minimal surface functionalization, that is, minimal amounts of –OSOdrolysis . Thus, i−1, typically ~0.30–0.50 µS·cm−1. Ice water was generated from a house filtration system with a conductivity of 25–30 µS·cm−1.Cotton gin motes were obtained from the USDA Research Facility in Stoneville, Mississippi. Chemicals and supplies were purchased from MilliporeSigma Corporation, or VWR-USA and were used as received. All water sources used deionized water with a maximal conductivity of ≤1.0 µS·cm2) solution (1.0% acetic acid (v/v) and 0.25% NaClO2 (w/v)) was used for 2 h at 75 °C; bleaching was repeated until the fibers were fully white. In both instances, the fiber to liquor ratio was 1:40 (w/v). The recovered fibers were washed thoroughly with water until the eluent was near neutral (pH ≈ 6–7) and then oven-dried (70 °C) to a constant mass.Cellulose nanocrystals were prepared as previously described . Briefly2SO4 at a material to liquid ratio of 1:15 (w/v). The resulting suspension was quenched four-fold by dilution with ice water and then washed until pH ≥5 by successive centrifugation cycles at 16,000 g for 15 min each cycle. The crystals were then dispersed using a 750 W ultrasonic processor  with a power amplitude of 60% (20 kHz frequency) for 5 min. The obtained suspension was centrifuged at 5000 rpm for 5 min (to remove larger particulates and contaminates), filtered under vacuum with a Whatman glass microfiber filter  and dialyzed using a regenerated cellulose dialysis tubing  for several days until the solution conductivity stabilized for two successive bath changes . The suspension was subsequently stored in a sealed container and refrigerated (4–8 °C) between uses. The yield and concentration of the nanocrystals was determined gravimetrically.Hydrolysis of cellulose extracted from gin motes was conducted for 30–150 min at 45–60 °C with 55–65 wt% H2SO4-hydrolyzed CNCs. The maximum final concentration of NaOH used in this work was kept ≤8 wt% (≈2 M). For the alkali treatment, concentrated stock CNC suspensions (~3.2 wt%) were diluted with an appropriate volume of water before the addition of a variable amount of concentrated NaOH solution (7.30 M). The final concentration of obtained CNC suspensions was 0.5–2.0 wt%, and NaOH was 0.0–2.0 M. Solutions were heated to 25–75 °C for 0.0–6.0 h. At the conclusion of the reaction, suspensions were quenched by the addition of ice water to bring the volume to 49 mL and 1 mL of 4.0 M sodium chloride (NaCl) was added to aid flocculation. Samples were centrifuged , the supernatant decanted and washing repeated. After, the CNC pellets were suspended by probe sonication with 30 s pulses and exhaustively dialyzed until the solution conductivity stabilized .An alkaline (NaOH) treatment was employed to obtain the surface desulfation of H3H). CNCs were converted from their sodium (Na+) to their acid from by chromatography over a large excess of Dowex® Marathon™ C Hydrogen from ion-exchange resin (23–27 mesh). While the concentration of the stock CNC suspensions varied, the concentration must be known accurately for meaningful determinations of the surface charged groups. For the stock CNC suspension, 5 mL of ~1.0 wt% CNCs was diluted to 99 mL and to this was added 1 mL of 100 mM NaCl. For analysis of hydrolysis experiments, the obtained CNC suspensions were diluted to 0.02 wt% suspension in 100 mL of water containing 50 µM NaCl as added electrolyte. Throughout each titration, the conductivity was continuously monitored, and 100 µL aliquots of standardized 1.03 mM NaOH were added over 45–60 min. The volume-corrected conductivity was plotted using OriginPro 2018b  and the equivalence point was determined by the intersection of least-squares regressions from the positive and negative sloped regions. Data was collected in (at least) triplicate for each sample.The sulfate half-ester content was measured by conductometric titrations as described previously, with minor modifications ,51,52. TThe atomic force microscopy (AFM) measurements were performed with an Agilent 500 atomic force microscope for native CNCs only. Data was collected in contact mode using a triangular-shaped Pyrex nitride cantilever with a gold reflex coating and silicon nitride tips . For the determination of the CNC length, and diameter (height), mica discs  were pretreated with 100 µL of a poly-L-lysine solution (0.01 wt%) and rinsed thoroughly with water after two minutes and blown dry with a stream of Argon. CNC suspensions (0.01 wt%) were applied by the drop cast method and rinsed after two minutes and blown dry. AFM height measurements were determined by use of the section analysis tool provided with the AFM software  on a 4 × 4 µm image from at least 100 individual observations. Image J  was used to determine the CNC length. Results were then fit with a Gaussian function using OriginPro2018b to determine the mean length and height of the CNCs.The CNC suspensions were diluted to 0.20 wt% and filtered through a 0.45 µm polyvinylidene fluoride (PVDF) filter to remove any larger particulates and dust . The sust), temperature (T), NaOH concentration (molarity) and CNC wt% (factors) were simultaneously varied within a prescribed range and the resulting CNC absolute sulfate concentration (response) was measured. A software program by Stat-Ease, Inc., Design Expert , provided statistical analysis of the experimental response, based upon the four input process factors. A quadratic model was selected from the software program and a regression analysis performed to eliminate insignificant terms, which strengthen the model. The experimental data passed standardized diagnostic tests, including the normal plot of residuals and Box–Cox  was 134–308 mmol·kg−1 as measured by conductometric titration (2SO4-hydrolyzed CNCs of 80–350 mmol·kg−1 [2 (corresponding sulfate is 40–44 mmol·kg−1) is unusually lower than typical values (80–350 mmol·kg−1). Moreover, after acid-catalyzed desulfation, a complete absence of sulfate was indicated by a lack of negative slope in the conductometric titrations; in both instances, dialysis was conducted prior to treatment with a mixed-bed resin, while no strong acid resin was employed, undoubtedly, as a direct consequence of using an acid-catalyzed desulfation. Thus, the obtained results are erroneously lower than expected [The sulfate half-ester content . Significant differences are observed after a few µmol added H3O+  in as little as 2 h . Even mi% in as lexchange . ExhaustH3O+ See .–OH, and a statistically insignificant amount (within error) when dilute NaOH was employed in a few hours [3–) ≈ 15% when using as little as 10 mM NaOH over 30 min [3– from work-up for CNCs in sodium form (exp #3) or after elevated heating (exp #4). The results are shown in By contrast, CNCs in sodium form are stable; Beck et al. showed no desulfation over several days in the absence of excess ew hours . This isr 30 min . The sta3–, particle size by AFM, and ζ-potential, while products were analyzed for –OSO3– and ζ-potential as response variables.To remove sulfate half-esters, an alkali (NaOH) treatment is used ,31,45; tKloser and Gray showed that harsher reaction conditions  resulted in a yield of only 6% and removal of about 80% of the sulfur content [3– that minimizes the use of reagents, and ultimately simplifies or shortens work-up and energy requirements. content . By contT ≤ 75 °C, and t ≤ 6 h, to allow some reactions to (presumably) reach equilibrium prior to work-up and also to minimize product losses due to glycosidic bond cleavage at elevated temperature or longer reaction times [3– of about 2000:1 on the more dilute (0.5 wt%) samples. The maximum molar ratio of NaOH to –OSO3– was limited to about 500:1 on the more concentrated samples to fall within the experimental bounds. It was not possible to perform the experiments on a larger diluted sample to increase the available NaOH and still test the requisite CNC concentration for the DOE. The results are given in Based on these results and literature precedent, the bounds for the DOE were set at on times . AdditioFor conductometric titrations, the addition of some electrolyte improves the stability of the measurements for dilute solutions, and CNC suspensions are commonly measured in the presence of 1 mM NaCl as were the stock solutions (vide supra) . However3– and the ζ-potential. Generally, there is a grouping of high remaining –OSO3– at low temperature, and for reactions that occurred at similar temperature profiles, Δ(–OSO3–) correlates with longer reaction times and/or a higher NaOH concentration.Returning to + condensation on the negatively charged CNC surface. Thus, intermediate concentrations of NaOH are preferred (vide infra), and below this regime, insufficient NaOH concentration attenuated the observed response. Furthermore, an equation (Equation (1)) was generated which predicted the desulfation response (DS), based upon input responses and significant model terms (Equation (1), See S is a constant which is dependent upon the initial sulfate concentration, t is the time in min, T is the temperature (°C), and C is the equivalence of NaOH:A quadratic model was used to fit the data; the DOE found that time, temperature and a two-factor interaction between time and NaOH concentration were significant. Additionally, the significance of the NaOH concentration was nonlinear; this could be explained in part by a visual observation of rapid flocculation of the CNCs at very high NaOH concentration, promoted by Na3– hydrolysis. Moreover, since the hydrolysis is an equilibrium between the sulfated and desulfated CNCs, higher working concentrations of CNCs (increased wt%) elevated temperatures, and increased NaOH concentration (to a point), favor desulfation. Although not considered a significant factor, CNC wt% and thus the initial concentration of –OSO3– was used as a model term. At lower concentration of CNCs greater equivalents of NaOH are needed, while at higher concentrations, the optimal window is shifted towards lower equivalents NaOH (the blue region is shifted to the left in t = 6 h) there is a broad range of conditions to provide sufficient hydrolysis of sulfate half-esters  solution of NaOH. Much more manageable conditions can be employed: 0.8 wt%, 1.6 M NaOH, 60 °C, and 6 h. Thus, from ~0.75–2.0 wt%, the amount of NaOH can vary from 1500–500 equivalents per unit –OSO3–. Under these conditions there was little observed difference for reactions with T ≥ 60 °C, while lower temperatures attenuate the hydrolysis of the sulfate half-esters. Furthermore, there is a mild dependence on the initial concentration of CNCs, albeit more concentrated samples (greater wt%) undergo a greater absolute change in sulfate concentration upon hydrolysis. This explains the contradiction in results obtained by Beck et al., and Lokanathan et al. which showed 8% and 22% hydrolysis, respectively, with 100 mM NaOH [Generally, within a given temperature range, increasing temperature and/or time results in an increase in –OSOf-esters ; however mM NaOH ,46. In t3–) [3–  NaOH concentrations were used ,42. Suff3–) . At a fi3–) [3– , and yet3–), increased concentration of NaOH increases costs and time associated with work-up and purification, while increasing temperature results in significantly increased energy requirements. There is very little difference observed in desulfation above a threshold temperature of ~60 °C; additionally, while some amount of NaOH is necessary for hydrolysis, high concentration is not required, especially since complete desulfation is often not the goal (nor possible).However, it is apposite to note, while these conditions are optimal in terms of maximal Δ(–OSO3–), and time allowed to float freely (0–6 h), a number of possible solutions within 1–2% Δ(–OSO3–) were obtained. Under these restrictions, optimal conditions are 60–120 equivalents NaOH and 3–6 h to obtain 58–62% Δ(–OSO3–). Conditions of 0.13 M NaOH, 0.78 wt% CNC suspension (–OSO3– = 211 mmol·kg−1) were selected, in this instance, corresponding to 80 equivalents of NaOH per unit –OSO3–, and a total solution volume of 50 mL was used. Gratifyingly, results from this test condition were Δ(–OSO3–) = 57%, which corresponds to –OSO3– of 211 and 91.6 mmol·kg−1 initial and final values, respectively. This is in very close agreement to the expected results from the DOE.If parameters are selected at a fixed temperature (60 °C) and CNC concentration (2.0 wt%), and the DOE solved to minimize NaOH concentration, maximize Δ, CNC wt% (~0.5 to ≥2.0 wt%) and time (>0 to ≤6 h).3– removal.Above 0.1–0.2 M NaOH there is only a minor observed difference in overall efficacy of –OSOBased upon DOE analysis, reaction time, temperature, and NaOH concentration are significant factors for effective sulfate half-ester removal. There is a two-factor interaction between reaction time and NaOH concentration. The significance of the NaOH concentration is non-linear.3–) of ~60%.Optimal conditions may vary depending on the initial and targeted sulfate concentration: 60–120 equivalents NaOH and a reaction time of 3–6 h gives Δ.The traditional conditions  typically remove about one-third to one-half of the available –OSOMore desirable conditions still yield colloidally stable CNCs at significantly lower use of NaOH, thus improving work-up.


Chronic rhinitis (CR) is divided into two main categories: allergic rhinitis (AR) and nonallergic rhinitis (NAR). These conditions are more recognizable to an experienced clinician, as they can be more clearly demarcated diagnostically. However, an additional 30% to 50% of patients with CR might have an overlap of NAR and AR, referred to as mixed rhinitis (MR). Progress in elucidating the pathophysiologic mechanisms behind MR and NAR has been made in the past several years, and there are now several guidelines published to assist the clinician in accurately diagnosing AR, NAR, and MR. Clinical history and subjective symptoms can provide clues for differentiating AR from MR and NAR, but allergy testing is recommended to confirm these conditions. Progress in accurately diagnosing patients with CR will be made as studies incorporate subjective , medication responsiveness, and quality-of-life tools) and objective  methods characterizing rhinitis subtypes. Chronic rhinitis (CR) is an often-trivialized condition that is frequently poorly characterized, resulting in an incorrect diagnosis and subsequent treatment recommendations. As a result of its improper classification, a spectrum of comorbidities including sinusitis, otitis, conjunctivitis, eustachian tube disorders, chronic cough, obstructive sleep apnea, headaches, and fatigue can occur, leading to a poor quality of life, increased healthcare utilization, and an unnecessary economic burden to society . SymptomAllergic rhinitis is a ubiquitous condition estimated to affect approximately 20% of the U.S. population and can be triggered by seasonal allergens, perennial allergens, or both . SeasonaWhereas confirmation of AR requires positive skin-prick or serologic sIgE testing that correlates with symptoms after exposure to the sensitizing aeroallergen, a diagnosis of NAR is made after negative skin-prick or serum sIgE testing to aeroallergens. Patients with NAR often exhibit similar symptoms to AR patients but complain less of sneezing and itching and more of worsening nasal congestion, rhinorrhea, sinus headaches, and post-nasal drip ,7. TriggThere is another subset of patients who present with CR symptoms that worsen in response to both allergic and non-allergic triggers referred to as MR. This third category of CR is fairly common, with prevalence rates ranging anywhere from 35–50% . A retroDistinguishing between the various rhinitis phenotypes can be confusing as the clinical presentation for each can be similar. Understanding the subtle differences between these conditions allows for a more accurate diagnostic approach, leading to better symptom control and improvement of quality of life. Bernstein et al. reported that when an objective irritant index questionnaire is used to diagnose chronic rhinitis patients, almost 25% of patients previously assigned a physician diagnosis of AR were reclassified as MR because of their high total irritant burden . ReclassA comprehensive past medical history and focused physical exam serves as the cornerstone for a complete workup that can provide clues for what rhinitis phenotype the patient may have. The focus of the exam should be on the nasal mucosa but also include the oropharynx, eyes, ears, neck, lungs, and skin. Classic physical findings suggestive of seasonal AR include “allergic shiners” ; “allergic salute” or horizontal nasal crease across the lower half of the bridge of the nose caused by repeated upward rubbing of the tip of the nose by the palm of the hand; mouth breathing; conjunctivitis; and edematous, pale, boggy nasal mucosa. In addition, AR is often associated with other comorbid allergic conditions such as asthma, atopic dermatitis, and food allergy. Physical exam findings for perennial AR and NAR can be more subtle with normal or erythematous nasal mucosa, often with evidence of prominent post-nasal drip manifesting as “cobblestoning”, which refers to streaks of lymphoid tissue on the posterior pharynx. Fiber-optic nasal endoscopy provides the best visualization of intranasal structures, but this more invasive exam is not always available or necessary. Many patients with chronic rhinitis, especially MR and NAR, have a decreased sense of smell and sometimes distorted taste. A persistent absence of smell after proper treatment could suggest nasal polyps, which are often visualized by nasal exam if they are advanced but can easily be missed if they are not anterior and prolapsed. Finally, patients with MR and NAR also experience ear popping and pressure due to eustachian tube dysfunction, manifesting as retraction of the tympanic membranes that are caused by increased negative pressure in the middle ear. There seems to be distinguishing characteristics that can clue the observer on which rhinitis phenotype the patient might be suffering from. A number of studies have attempted to classify clinically reproducible features of each rhinitis phenotype to aid in differentiating between AR and NAR. Di Lorenzo et al. evaluated 1511 adult patients with chronic rhinitis using various diagnostic parameters and determined that AR patients have more severe symptoms with increased sneezing, nasal pruritus, and recurrent conjunctivitis . CompareEstablishing the diagnosis of AR requires symptoms upon exposure to sensitizing aeroallergen, whereas NAR seems to be a diagnosis of exclusion based on the absence of allergic triggers and negative confirmatory skin or serologic testing to aeroallergens. With limited diagnostic testing specific to NAR, focusing on pertinent odorant and irritant triggers has been considered an important diagnostic criterion for establishing the correct diagnosis. Some have even postulated that patients with NAR have abnormalities in their olfactory transduction pathway, leading to pathologic changes in the olfactory receptors or mucosa, making them more susceptible to these triggers ,22. RezvAn environmental exposure chamber (EEC) models has been used to simulate weather conditions in a controlled setting to objectively diagnose NAR subjects and ultimately to investigate novel NAR therapies . Others In an attempt to better evaluate clinical control of CR, the Rhinitis Control Assessment Test was developed, which is outlined in Patients with a physician diagnosis of AR or MR and a low IIQ score were reclassified as having low-burden AR.Patients with a physician diagnosis of AR or MR and a high IIQ score were reclassified as having high-burden AR.Patients with a physician diagnosis of NAR and a low IIQ score were reclassified as having low-burden NAR.Patients with a physician diagnosis of NAR and a high IIQ score were reclassified as having high- burden NAR.Although the clinical history and physical exam can be helpful aspects of the diagnostic workup, several studies suggest that that clinical symptoms and triggers alone might not be as reliable for differentiating between rhinitis subtypes. Incorporating patient-centric questionnaires into the patient evaluation is one way clinicians have attempted to more reliably characterize CR subtypes. Brandt and Bernstein distributed a questionnaire listing symptoms and common allergic and nonallergic triggers to 100 randomly selected patients with CR symptoms that ultimately was highly predictive of NAR on the basis of responses concerning triggers, but less helpful in the diagnosis of AR . PatientResults revealed that 52% of all patients with a physician diagnosis of AR and MR fit into the high-burden AR group. Of note, 47% of those with a physician diagnosis of AR were later reclassified as having high-burden AR. These findings suggest that patients with AR experience a high burden of non-allergic triggers that are not being recognized by the patient or treating clinician. The high irritant burden rhinitis group seemed to have more severe and frequent rhinitis symptoms and a significantly greater prevalence of physician diagnosed asthma, suggesting that the IIQ successfully reclassified rhinitis patients into distinct subgroups with unique clinical differences that may represent more severe phenotypes than previously recognized by current diagnostic modalities ,24. ThisAs demonstrated in previous studies highlighted above, depending exclusively on the medical history to determine the appropriate rhinitis subtype can be confusing. Patients often refer to their diagnosis as “hay fever” or AR on the basis of pre-conceived notions of CR and triggers emphasized in the news and by advertisements. Because of the ambiguity of clinical histories, objective assessment by allergen skin prick testing (SPT) or specific in vitro IgE testing is essential. Skin testing involves introducing measured amounts of seasonal and perennial aeroallergens, indigenous to the geographic location the patient resides, in conjunction with a positive histamine and negative saline control onto the skin followed by a prick with a special bifurcated needle. If the SPT is negative but the clinical history is concerning for aeroallergen sensitivity, intracutaneous testing (ICT) can also be performed. However, several studies have found that ICT has a high sensitivity but is less specific than SPT, and as a result might lead to false positive testing if previous percutaneous testing was unremarkable ,30. The Serum-specific IgE antibody testing to aeroallergens is often ordered when the patient is dermatographic, has a severe skin disease , or is on medications that interfere with the histamine response  necessary for skin test interpretation. It is important to remember that a positive skin or serum sIgE test only implies sensitization and should always be correlated with the patient’s reaction history upon exposure to that allergen before the diagnosis of “allergy” can be made. Although the testing modalities currently available are typically accurate, discrepancies can occur, as in the case of patients with localized AR (LAR) or “entopic” rhinitis. This subset of patients has negative skin and/or serologic testing to aeroallergens but has all the classic symptoms of AR upon exposure to a specific allergic trigger that can be reproduced with nasal provocation to the specific allergen. Furthermore, they respond to traditional treatments used for AR such as nasal corticosteroids and second generation antihistamines. Rondón et al. estimates that local AR affects 25.7% of the rhinitis population and more than 47% of patients previously diagnosed with NAR, but these numbers have not been consistently confirmed by other investigators ,31. GiveA wide number of methods for classifying and diagnosing rhinitis subtypes have been proposed, including nasal cytology, peak nasal inspiratory flow rates, anterior rhinomanometry, acoustic rhinometry, irritant index scales, specific biomarkers, nasal provocation testing with environmental exposure chambers, and nasolaryngoscopy . All of Imaging is not typically part of the initial diagnostic workup of CR; however, in refractory or more severe cases, computerized tomography (CT) scan of the sinuses can help further elucidate sinus anatomy and/or structural changes, including obstruction or narrowing of the osteomeatal complexes and septal deviation, as well as identify the presence of nasal polyps. Sinus plain films are not recommended as they are less sensitive than sinus CT scans and are not reliable enough to assist with clinical decision making. It is important to recognize that CR can be associated with various comorbidities, which can significantly impact management and further impair quality of life. Commonly associated comorbidities include acute and chronic sinusitis, tension and migraine headaches, asthma, chronic cough, conjunctivitis, Eustachian tube dysfunction, otitis media with or without effusion, nasal polyps, hearing impairment, changes in smell and taste, nasal dyspnea, obstructive sleep apnea, and other sleep disturbances/related complications resulting in fatigue . ComplicProgress in elucidating the pathophysiologic mechanisms of MR and NAR has been made in the past several years, and there are now several guidelines published to assist clinicians in accurately diagnosing rhinitis subtypes ,39. Clin


The truel is a three person competition that generalises the classic duel. In this game three players try to eliminate each other in a series of one-to-one duels until there is only one survivor. The players’ marksmanship, shooting order and strategies for choosing a target play a significant role in individual’s survival probability. Strategies such as shooting into the air (abstention), shooting at the strongest opponent, and shooting at the weakest opponent have been analysed in the previous literature. In this paper, for the first time, we consider suicidal and random strategies that can be chosen by the weaker player. We show that although there is no possible highest probability region for weakest player adopting suicidal strategy, the player may increase the survival probability via switching between suicidal and abstention strategies randomly. In addition, we demonstrate that there is a narrow survival area for the weakest player when the player aims randomly at two other opponents, and eventually the area fades away if the player fires randomly at himself or the other two opponents. However, in certain cases the fittest does not necessarily survive, the extinction of the dinosaurs being a classic example.“Survival of the fittest” originated from Darwinian evolutionary theory as a way of describing the mechanism of natural selection3, then the truel became used in the social sciences as a model of strategic interactions that can lead to seemingly paradoxical results6.A duel consists of two gunmen, where the probability of winning relies on marksmanship and speed in unbiased conditions. A truel is a three-person expansion of a duel, in which each of the truelists can attempt to survive by shooting at opponents in order to eliminate them. Initially, the concept of a truel was introduced as a mathematical puzzleThe outcome of the game is strictly dependant on the rules of the truel. In many cases truels can generate counterintuitive results and the player with the highest marksmanship does not necessarily have the greatest chance of survival. In some cases, probability-based calculations may lead to the surprising situation of “survival of the weakest”.8. Another possibility, as a generalisation of the classic duel, is a simultaneous truel when all three players fire their bullets at the same time9. Moreover, winning strategies are investigated for a random truel where each player is chosen at random among the survivors in every round10.A wide variety of possible conditions and settings for a truel is explored in the literature, such as biasing the game by allowing the weakest player to shoot first and the best player to shoot last7. The possibility of the situation in which the worst and better players are both willing to reverse the order of their moves has been investigated11. Previous studies investigated the concept of a truel in the case where probabilities are governed by the rules of quantum mechanics13. Other different strategies such as cooperative vs non-cooperative truels14 and shooting in the air by all players15 have also been considered.The strategies that can be potentially chosen by players have been extensively investigated in previous studies. The Nash equilibrium suggests that the best action players can optimise their chances of survival via firing the bullets at the strongest remaining opponent. Surprisingly, it is shown that under certain conditions the chances of survival are improved if the weakest player fires a bullet into the air rather than targeting the strongest player16. Some notions of equilibrium in the truels have been proposed to model political conflicts15. The truel has also been reinterpreted as a model for opinion spreading during debate17. In terms of commercial competition, it is argued that if firms or business units value their future payoffs high enough, each concentrates more on fighting the strongest opponent. Consequently, weaker companies grow stronger and the strongest grow weaker with all the parties eventually converging and remaining in the game18. In addition, implications for the maintenance of variation in natural populations are investigated based on N-player versions of the duel under constant selection19. The truel has also been considered to elucidate questions about war strategies, politics, marriage and reproduction20.A model of behaviour in an intense conflict situation is modelled using the theory of truels in psychological studies21. On the other hand, to the degree that the future seems to stretch out indefinitely, people are more likely take responsible strategies toward each other regarding the fact that tomorrow they may pay the price of any untoward behaviour. In the area of legal studies, the concept of truel is used as a model for exploring the issue of equality22, where people respond in strategic ways to legislation to maximise their interests. Therefore, the fittest party attempts to aggravate the legislature’s mistaken belief that it needs help. The party will be in the forefront of legislative activity, hiring lobbyists and buying advertisements to communicate the impression that it is the victim of disadvantageous treatment, when in reality it has the greatest chance for survival.Moreover, the literature draws some interesting inferences regarding the behaviour underlying truels. Considering the eschatological views of the players in bounded and unbounded truels, it is suggested that in a finite competition players who take a more short-term or bounded view may act less responsibly, even immorally23. It was concluded that an initial decrease in support for a candidate later results in an upsurge in the polls for the candidate. Moreover, some studies have employed the rock-scissors-paper model to systems with three species that interact with each other and ultimately create a competitive loop25. The paradoxical effect in this model is that the least competitive species may potentially become the largest population in the ecosystem. Moreover, Parrondo’s games illustrate a paradoxical situation where losing gambling games played in a random or periodic order can result in a winning outcome26.Previous studies have also applied the concept to a voting model showing a paradoxical result27. In this case low density populations thrive while high density leads to ecological suicide. In addition, the number of the cells in a multicellular organism is tightly regulated by rate of cell death. In case cells are no longer required, they commit suicide using an intracellular death mechanism that is called programmed cell death or apoptosis28. Also suicidal reproduction or semelparity has evolved in a few mammals. Mating in the species can continue for many hours with multiple partners and after that the males all die. In fact, the males compete not by fighting, but by mating themselves to death because their sperm is in competition with the sperm of many other males. The immense pressure to produce better sperm leads to immune system failure and eventually death29.A number of suicidal strategies can be seen in a variety of species in biology. Ecological suicide is a common phenomenon in microbes, for instance, bacteria can modify the environmental pH to such a degree that it leads to a rapid extinction of the whole populationIn this paper, we propose some different tactics such as a suicidal strategy in the sequential truel. A sequential truel is one where three players take turns to fire one shot until the last man stands. Using the suicidal strategy the first player attempts a shot at himself whenever both the other players are alive. We also consider the case of a random strategy for the first player while other players use the strategy of targeting the strongest opponent. Note that the truel is sequential. Thus, the players take turns to shoot one bullet at a time until the last man stands.a, b, and c respectively, independent of their target and with Players A, B, and C have a probability of hitting their chosen target of Each player’s marksmanship does not depend on other players.Each player fires sequentially in alphabetic order, and the winner is the one left standing.When all three players are alive, players B and C target the strongest opponent, which means B fires at C and C fires at B.In this paper we shall make the following assumptions:a, player B with marksmanship b, and player C with marksmanship c fire the bullets at their chosen targets, where In a sequential truel, three players shoot it out and the winner is the one left standing. Respectively, player A with marksmanship 7. In particular, at any moment the sequential truel can change to a sequential duel. In some cases being the first shooter in a sequential duel is more advantageous than marksmanship, because in sequential duel the opponent’s marksmanship does not come into play until he takes his turn. In Fig. a, b), setting a and b with step size of 0.001 when c is forced to possess the value of 1.The strategy of firing at the opponent with highest marksmanship as a target appears to be rational. In this strategy, when all three players are alive, player A fires at C, player B fires at C, and player C fires at B. Although, the naive expectation is that the strongest opponent strategy is the optimum strategy for player A, previous studies show that player A can increase his chance of survival by shooting into the air in first round or whenever both the other players are aliveComparing Fig. a, whenever all other opponents are alive. The probabilities of A, B, and C surviving against their opponent using this strategy may be calculated. Players B and C still shoot at the best opponent . Therefore, player A will not be shot at until B or C are dead, and A will be left standing in a duel with either B or C as a first shooter. All possible survival scenarios are shown in Fig. We can add an interesting twist to the truel problem by allowing player A to shoot himself, with probability A1, player A survives his own shot, and player B kills player C in a sequential duel. Then in the next state, SA2, via a sequential duel, A kills B.In first state, SA3, player A survives his own shot, and this time player C kills player B in a sequential duel. In next the state, SA4, in a sequential duel A kills C.Similarly, in first state, SA1 is given by Eq. The probability of SCorrespondingly, the probability that C kills B conditioned on A being alive is given byPA, may obtained byTherefore, the probability of player A surviving with suicidal strategy, In order to obtain the probability of player B surviving when A adopts a suicidal strategy, it is necessary to calculate the probability of the winning scenarios shown in Fig. B1, player A survives his own shot, and player B kills player C in a sequential duel. This state is exactly same as SA1. But in the next state, SA2, in a sequential duel, B kills A.In the first state, SA3, player A is killed by his own shot, and player B kills player C in a sequential duel.In the first state, SThe probability B kills C under the condition A is killed by either of their bullets is obtained byB, the probability of player B surviving when A adopts a suicidal strategy is obtained byTherefore, PFinally, the possible scenarios for player C surviving when A chooses a suicidal strategy shown in Fig. C1, player A survives his own shot, and player C kills player B in a sequential duel. This state is exactly same as SA3. Then, in the next state, SC2, in a sequential duel, C kills A.In the first state, SC3, player A is killed by own shot, and player C kills player B in a sequential duel.In the first state, SThe scenarios for player A surviving with suicidal strategy illustrated in Fig. The probability that C kills B under the condition A is killed by either of their shots is given byC, the probability of player C surviving when A chooses a suicidal strategy is given byThus, Pa, b), setting a and b  decide the target randomly between B and C, and (ii) decide the target randomly among A, B, and C thus allowing the possibility of suicide.Players may decide to fire the bullets randomly for different reasons such as lack of information about another opponent’s marksmanship. The distribution of winners with a random strategy in the sequential truel is investigated in previous studiesa, b), setting Figure Now we may add a twist to the random strategy and combine it with the suicidal strategy as given by (ii) above. The parameter space for this strategy that A randomly fires at himself or at the other two opponents is shown in Fig. It is known that under different circumstances, the fittest player in a truel does not necessarily survive. Considering different strategies in a sequential truel, it is surprising that the player with the best marksmanship does not always have the highest chance of survival.It is already known that if the weakest player in a sequential truel begins with an abstention (e.g. by shooting his bullet in the air), he greatly increases his chance of survival. Therefore, it may seem that if he were to fire at himself (with a finite accuracy) he may still be able to survive but with a reduced parameter space—however, our results show the parameter space totally vanishes in this case. For the case when the weakest player adopts a random strategy the parameter space shrinks.As a first step this work has considered the case where the probability of a bullet hitting its target is the same for suicide or aiming at an opponent. It would therefore be of interest for future studies to decouple this and assign different probabilities to suicidal and adversarial strategies.Finally, we note that the literature has found the study of the truel of interest across the physical, biological, economic, and social sciences as a toy model that describes the situation where three factors conflict with each other. There are a number of important three-way conflicts  that may potentially benefit from future study in this area.https://github.com/Dorraki/truel.We have made the Matlab code for the truels openly available on Github at The corresponding author is responsible for submitting a competing financial interests statement on behalf of all authors of the paper.


Adventitious root (AR) formation is a critical developmental process in cutting propagation for the horticultural industry. While auxin has been shown to regulate this process, the exact mechanism and details preceding AR formation remain unclear. Even though AR and lateral root (LR) formation share common developmental processes, there are exist some differences that need to be closely examined at the cytological level. Tomato stem cuttings, which readily form adventitious roots, represent the perfect system to study the influence of auxin on AR formation and to compare AR and LR organogenesis.Here we show the progression by which AR form from founder cells in the basal pericycle cell layers in tomato stem cuttings. The first disordered clumps of cells assumed a dome shape that later differentiated into functional AR cell layers. Further growth resulted in emergence of mature AR through the epidermis following programmed cell death of epidermal cells. Auxin and ethylene levels increased in the basal stem cutting within 1 h. Tomato lines expressing the auxin response element DR5pro:YFP showed an increase in auxin distribution during the AR initiation phase, and was mainly concentrated in the meristematic cells of the developing AR. Treatment of stem cuttings with auxin, increased the number of AR primordia and the length of AR, while stem cuttings treated with the pre-emergent herbicide/auxin transport inhibitor N-1-naphthylphthalamic acid (NPA) occasionally developed thick, agravitropic AR. Hormone profile analyses showed that auxin positively regulated AR formation, whereas perturbations to zeatin, salicylic acid, and abscisic acid homeostasis suggested minor roles during tomato stem rooting. The gene expression of specific auxin transporters increased during specific developmental phases of AR formation.These data show that AR formation in tomato stems is a complex process. Upon perception of a wounding stimulus, expression of auxin transporter genes and accumulation of auxin at founder cell initiation sites in pericycle cell layers and later in the meristematic cells of the AR primordia were observed. A clear understanding and documentation of these events in tomato is critical to resolve AR formation in recalcitrant species like hardwoods and improve stem cutting propagation efficiency and effectiveness.The online version of this article (10.1186/s12870-019-2002-9) contains supplementary material, which is available to authorized users. The root has multiple functions during plant growth and development including water and nutrient absorption. Depending on when and from which tissue they originate, roots can be defined as embryonic or post-embryonic . AdventiArabidopsis thaliana have not been well-described. Tomato stem cuttings readily form adventitious roots, which makes them an ideal system to study AR formation in detail.Adventitious root formation is generally divided into three developmental phases: induction, initiation and extension . During PIN gene family has eight members in Arabidopsis and every member seems responsible for different functions in auxin efflux  accumulation in a dose- and time-dependent manner  + or [M − H] −) are listed in Additional file n = 3). Each biological replicate was composed of three pooled stem sections. Fisher’s LSD statistical analysis was performed .For auxin extraction and quantitation in tomato shoots, the method described in Zhang et al. , was usehttp://www.appliedbiosystems.com/) using 5 μL of 10-fold diluted cDNA, 1× SYBR green master mix , and 1 mol/L each of target gene-specific primers . qRT-PCR was performed using an ABI PRISM 7900HT instrument  gradient parameters in mobile phases. (DOCX 13 kb)Additional file 2:Table S2. Conditions used for protonated or deprotonated plant hormones of Zeatin, ABA and SA ([M + H]+ or [M − H]−). (DOCX 14 kb)Additional file 3:Table S3. Accession numbers of the genes used in this study. (DOCX 13 kb)Additional file 4:Table S4. Primers for qRT-PCR. (DOCX 14 kb)Additional file 5:Figure S1. The detailed elaboration of DR5pro:YFP localization in initiation phase of developing AR. (DOCX 301 kb)


AR point mutations) in circulating tumor cells (CTCs). We devised a single-tube assay to detect AR-V7 splice variants and AR point mutations in CTCs using immunomagnetic cell isolation, followed by quantitative real-time PCR and DNA pyrosequencing. We prospectively investigated 47 patients with PSA progression awaiting therapy switch. Comparison of response to newly administered therapy and CTC-AR-status allowed effect size estimation. Nineteen (51%) of 37 patients with detectable CTCs carried AR-modifications. Seventeen patients carried the AR-V7 splice variant, one harbored a p.T878A point mutation and one harbored both AR-V7 and a p.H875Y mutation. We estimated a positive predictive value for response and non-response to therapy by AR status in CTCs of ~94%. Based on a conservative calculation, we estimated the effect size for molecularly-informed therapy switches for prospective clinical trial planning to ~27%. In summary, the ability to determine key resistance-mediating AR modifications in CTCs has the potential to considerably improve prostate cancer treatment.Molecular modifications of the androgen receptor (AR) can cause resistance to androgen deprivation therapy (ADT) in prostate cancer patients. Since lack of representative tumor samples hinders therapy adjustments according to emerging AR-modifications, we evaluated simultaneous detection of the two most common AR modifications (AR-V7 splice variant and  Therapy of advanced prostate cancer is based on interference with androgen receptor (AR) signaling and androgen deprivation therapy (ADT) has been firmly established as the principal therapeutic approach . UnfortuAR gene carry clinical importance due to their specific functional differences  that renders cancer cells resistant to otherwise effective therapy  . For patferences  4-7]. I-7. IAR g that renAR gene in CTCs, while there is an implicit understanding that treatment decisions based on both AR-splice variants and point mutations would significantly improve therapy response rates. This point formed a rationale for our study, and we propose that treatment decisions based on CTC assessment should consider both AR-modifications (Circulating tumor cells (CTC) hold the promise to provide easy access for cancer characterization. The number of CTCs has been demonstrated as an indicator of disease aggressiveness and tumor burden and Miyamoto et al., for example, determined AR signaling status in patients under ADT as a possible indicator for therapy success  9]. CTC. CTC9]. ications . AR hotspot mutations - in CTCs from a single blood sample. Beside demonstration of general feasibility, we assess predictive properties with respect to subsequent therapies, which collectively underscore the potential of liquid biopsies in the treatment of advanced prostate cancer.Here, we evaluated whether it is possible to simultaneously detect the two most common AR modifications - the AR-V7 splice variant and AR point mutations.A prerequisite for a clinically valid assay in this setting is the ability to reliably detect and characterize prostate cancers cells in peripheral blood. First, we demonstrated absence of qPCR products for prostate-specific mRNAs  in blood from a healthy male and female donor. In spiking experiments, using various dilutions of prostate cancer cells in blood from the same donors, we successfully detected prostate-specific mRNAs, the AR-V7 splice variant, as well as the c.2632A > G; p.T878A mutation in LNCaP-spiked blood (see Supplementary Methods). These findings provide proof-of-principle that our method allows simultaneous detection of AR-V7 and p = 0.03) with one notable exception. Briefly, in this patient with undifferentiated metastasized prostate carcinoma, we detected CTCs although the PSA level was 0.1ng/ml, underscoring the assumption that PSA level alone does not always accurately reflect tumor burden [p-range = 0.69-0.72). Clinico-pathological data of the 47 patients that fulfilled enrollment criteria are summarized in r burden . Hallmarp = 0.046), but not with other parameters classically associated with aggressive clinical course , previous treatment with abiraterone (p = 0.007), enzalutamide (p = 0.02), or docetaxel (p = 0.02), as well as with the number of prior therapies (p = 0.004). Specifically, examining the percentage of patients harboring the AR-V7 in relation to prior therapy combinations showed the following results: 28.6% primary ADT alone, 66.7% primary ADT + docetaxel, 80% primary ADT + next-generation ADT , and 80% primary ADT + next-generation ADT + docetaxel. The significantly higher rate of AR-V7 in patients with multiple prior therapies (p = 0.005), and our finding that no therapy-naïve patient was AR-V7-positive argues for acquisition of the splice variant during therapy.AR-V7 qPCR revealed splice variants in 18 of 37 CTC-positive patients (49%). Presence of AR-V7 correlated significantly with metastatic disease  [gure 1A) . The p.Tn = 10/14) whereas only one AR-V7 positive patient  responded to the following therapy. While these differences reached statistical significance (p < 0.001), the response characteristics of subgroups by treatment type were not as clear. For example, two of three AR-V7-negative patients who received docetaxel responded, while the AR-V7 positive patient had no benefit from docetaxel treatment. With respect to follow-up in the p.T878A-positive patient, we observed a response to subsequent treatment with enzalutamide (PSA decline from 32 to 16ng/ml). In contrast, the p.H875Y/AR-V7-positive patient, showed no biochemical response to subsequent enzalutamide treatment.During follow-up after therapy switch, we observed a biochemical response in 71% of AR-V7 negative patients (n = 29), we dichotomized therapy outcome into either response or non-response. We defined response as reduction of serum PSA to < 50% maintained for ≥4 weeks at any time after the initiation of therapy. When we assumed that assessment of the AR modification status predicts response as well as non-response, the ‘positive’ predictive value would add up to ~94%. To determine predictive properties of our assay in patients with outcome information (n = 11/29). When splitting up patients into subgroups with molecularly matched (n = 14) and –unmatched therapy switches (n = 15), we observed 71.4% and 6.6% response rates, respectively . Despite the small number of patients, we noted a measurable benefit of a molecularly unmatched therapy  [Correlating AR modification status with treatment response, we made three key findings. First, we noted that parameters of tumor aggressiveness at the time of diagnosis  did not correlate with the presence of AR-V7. On the other hand, presence of AR-V7 was associated with the number of prior therapies. Together we interpret these associations as evidence for development of AR-V7 under therapy. This interpretation is supported by recently published -V567es) . Further-V567es) . However-V567es) .AR mutations. Specifically, both patients with AR point mutations were pre-treated with abiraterone. This observation supports recent reports about the emergence of these mutations under abiraterone therapy [in vitro [The second key finding results from analysis of the two patients with  therapy , where p therapy -22. Notain vitro , we intein vitro . The third key finding is related to the observation that two of the three AR-V7-negative patients who received docetaxel responded to therapy. While the number of examined patients precluded formal statistical testing of response rates of AR-V7-positive vs. AR-V7-negative patients to docetaxel, one docetaxel-treated patient harbored the AR-V7 splice variant and showed no therapy response. In light of recent findings indicating that AR-V7 expression may also constitute a mechanism of resistance to taxanes , a more The small number of patients included in this first report has to be acknowledged as a clear limitation to the study; moreover, the predictive value of the assay is limited to patients with detectable CTCs. Finally, focusing on AR hotspot regions might lead to a miss of other mutations with possible clinical implications. From our point of view, since CTCs were detectable in 79% of patients, a pragmatic and cost-effective approach focusing on well-documented AR-modifications holds great clinical promise, all the more since an overall benefit of 27% could already be demonstrated in this small sample size.In summary, our results demonstrate the ability to reliably characterize the two main AR-modifications in CTCs of patients with advanced prostate cancer in a single-tube assay. This finding lays the foundations for a liquid biopsy-based, personalized treatment decision that can prevent patients from receiving inefficient systemic therapy.We designed a prospective observational study  and obtaWe prospectively enrolled patients with advanced prostate cancer who underwent treatment at the university medical center Ulm. To compose the study cohort, we applied the following inclusion criteria: histologically confirmed prostatic adenocarcinoma, progressive disease, and signed consent form. We defined progressive disease as PSA progression and/or radiographic progression (according to RECIST 1.1) irrespective of the stage of disease. PSA-Progression was defined as ≥50% decline in PSA from baseline maintained for ≥4 weeks at any time after the initiation of therapy. We enrolled patients irrespective of type, line, or sequence of prior therapies; however, therapy switch to any of the approved anticancer drugs had to occur after blood draw. Inclusion in other prospective clinical trials was allowed. For each enrolled patient we obtained epidemiological, clinical, and tumor-specific data from the medical record. Study start was February 2014 and we followed patients until October 2014.AR point mutations in pyrosequencing experiments, we used undiluted or admixed cDNA from prostate cancer cell lines harboring either wildtype sequence (VCaP) or different AR point mutations . To determine limits of detection for CTC isolation from peripheral blood, we performed spiking experiments with defined numbers of cultured prostate cancer cells (LNCaP). To determine detection limits  of We collected 7.5 ml of peripheral venous blood in EDTA collection tubes and stored samples immediately at 4°C. CTC isolation procedure started within 1-24 hours after blood draw. For CTC isolation, we employed the AdnaTest ProstateCancerSelect Kit . Initial tests revealed that washing steps with PBS+0.05% bovine serum albumine reduced the loss of magnetic beads to plastic surfaces. Otherwise we followed the instructions provided by the manufacturer. Isolation of RNA from CTCs employed the AdnaGen ProstateCancerDetect Kit . AR Exon 8 expression level served as internal reference). To achieve mRNA specificity , we designed qPCR primers that span exon-exon boundaries. We assessed mRNA levels of AR-V7, KLK3 (PSA), ALAS1, G6PD, and AR exons 5 and 8 with the QuantiFast Multiplex RT-PCR+R Kit  on a ViiA™ 7 Real-Time PCR system . To rule out contamination, we analyzed patient samples along with their corresponding reverse transcription and qPCR non-templates. To determine the relative AR-V7 expression levels, we applied the ΔCt method (To allow simultaneous mutation analysis, we designed the qPCR products of AR exons 5 and 8 to specifically cover known AR hotspot point mutations . SpecifiPrimer sequences and further experimental details are given in the Supplementary Material section.p < 0.05 as statistically significant. We assessed correlations between clinico-pathological parameters  and 3 us


Androgen-deprivation therapy was shown to improve treatment outcome of external beam radiation therapy (EBRT) for locally advanced prostate cancer (PCa). DNA damage response (DDR) was suggested to play a role in the underlying mechanism, but conflicting results were reported. This study aims to reveal the role of the androgen receptor (AR) in EBRT-induced DDR and to investigate whether next-generation AR inhibitor apalutamide can radiosensitize PCa. PCa cell lines and tissue slices were treated with anti-androgen alone or combined with EBRT. The effect of treatments on cell growth, tissue viability, DDR, and cell cycle were investigated. RAD51 and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) levels were determined by Western blotting. Homologous recombination (HR) capacity was measured with the directed repeats-green fluorescent protein (DR-GFP) assay. We report the radiosensitizing effect of anti-androgens, which showed synergism in combination with EBRT in AR-expressing tumor slices and cell lines. Moreover, a compromised DDR was observed in AR-expressing cells upon AR suppression. We found that AR inhibition downregulated DNA-PKcs expression, resulting in reduced non-homologous end-joining repair. DDR through HR was a secondary effect due to cell-cycle change. These data provide a mechanistic explanation for the combination regimen and support the clinical use of apalutamide together with EBRT for localized PCa patients. Prostate cancer (PCa) is the most common malignancy and the second most common cause of cancer-related death among men . ExternaIonizing radiation (IR) induces DNA double-strand breaks (DSBs), which can be repaired via two main pathways: Non-homologous end-joining (NHEJ) and homologous recombination (HR). NHEJ involves recruitment of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to DNA ends by the Ku70/80 heterodimer, followed by coupling of the DNA ends. HR is mediated by the RAD51 protein in combination with many other factors such as breast cancer 1 and 2 (BRCA1 or 2) . AR acti®) is a next-generation AR inhibitor which was approved in 2018 for treatment of non-metastatic castration-resistant prostate cancer (CRPC). Apalutamide blocks AR activation by competing with androgen binding and preventing AR translocation to the nucleus. It has a 5–10-fold higher binding affinity than the first-generation anti-androgen bicalutamide, which is widely used for PCa patient treatment [Apalutamide  tissue slice models. Furthermore, we identified NHEJ downregulation as the underlying mechanism of apalutamide-enhanced radiosensitivity in AR-expressing PCa models.The impact of AR suppression treatments on PCa cell response to IR exposure was studied by applying ADT, enzalutamide, or apalutamide 24 h prior to IR. The IR dose per cell lines was chosen such that cell proliferation was not fully inhibited. Since the intrinisic radiosensitivity varied between the tested cell lines, a direct comparison of the observed radiosensitizing effects between these cell lines was difficult. AR suppression alone reduced the total cell number by 50% in LNCaP and 40% in PC346C cells seven days post-IR, and IR treatment alone reduced the total cell number by 20% in LNCaP and 35% in PC346C cells A. The coSubsequently, we investigated the effects of apalutamide on IR sensitivity in androgen-dependent, AR-positive PC295 ex vivo tumor slices . Tumor sNext, we investigated whether AR suppression treatment-enhanced IR sensitivity could result from a DSB repair defect by measuring DSB repair kinetics of PCa cells. AR suppression treatment alone did not increase DSB foci numbers compared to untreated controls . IR expoIn addition to indirect measurement of the level of DSBs using phospho-H2A histone family member X (γH2AX) and p53-binding protein 1 (53BP1) foci quantifications, we also measured the number of DNA breaks by pulsed-field gel electrophoresis (PFGE). AR suppression as single treatment did not cause increased levels of broken DNA, while IR exposure did. No difference was observed between control and AR suppression treatment directly after IR exposure, indicating that the same level of DNA breaks was induced C,D. WhenSubsequently, we investigated whether AR suppression directly affected HR. Since previous studies reported reduced RAD51 expression after enzalutamide treatment , we measAfter excluding the HR pathway as the direct cause of AR suppression induced radiosensitization, we investigated the involvement of the NHEJ pathway. Previously, Goodwin et al. showed that the AR pathway regulates the NHEJ factor DNA-PKcs . TherefoHere, we demonstrate that different forms of AR suppression treatment can act as a radiosensitizer in AR-expressing androgen-dependent and CRPC preclinical PCa models. The mechanism of action causing this radiosensitization involves regulation of the NHEJ DNA repair pathway.We and others found that AR suppression and IR treatment have an additive effect on cell proliferation in AR-expressing PCa ,13. BasePrevious preclinical studies investigated the molecular mechanism of radiosensitization by ADT and discovered an interplay between AR signaling and the DNA repair machinery ,10,11,16After excluding AR-regulated HR repair, we further investigated the NHEJ repair pathway. A previous study discovered that AR directly regulates the level and function of the NHEJ factor DNA-PKcs . Our datPrevious preclinical studies reported the synthetic lethality between AR inhibitors and PARP inhibitors ,11. UnfoEmerging preclinial and clinical studies show that AR variants (AR-Vs) can play an important role in the development of resistance to AR suppression treatment ,23. AR-VWe did not find significant differences in response between ADT, enzalutamide, and apalutamide treatment, probably because ADT in an in vitro setting is a simplified and optimal scenario, while effects in patients are less optimal due to adrenal androgen production being unaffected under ADT. Apalutamide recently received Food and Drug Administration (FDA) approval for the treatment of non-metastatic CRPC. Several clinical trials are underway to test the efficacy of apalutamide with EBRT . The preclinical data presented here fully support the ongoing clinical trials. Furthermore, our study showed benefit of the combination in AR-expressing CRPC cells, a situation also found in patients with advanced metastatic disease that are progressive on ADT. Therefore, we propose that an alternative treatment approach should be considered for these patients, consisting of apalutamide in combination with prostate-specific membrane antigen (PSMA)-targeted radioligand therapy [Reagents were purchased from Sigma-Aldrich , unless otherwise specified. The following antibodies were used: AR , RAD51 , DNA-PKDU145 and LNCaP cells were purchased from American Type Culture Collection  and cultured as described ,29. PC34Cells and tissue slices were irradiated with different X-ray doses using Xstrahl Cabinet Irradiator RS320 .®-Multi Detection System .Cell growth was measured using the sulforhodamine beta (SRB) assay. Briefly, all cells were plated in 96-well plates coated with FNC coating mix  in 100 μL of steroid-stripped medium ). The next day, 100 µL of culture medium was added either alone (ADT condition) or supplemented with R1881  or R1881 (0.1 nM) plus apalutamide/enzalutamide  24 h before irradiation (IR). Cells were allowed to grow until different time points, and fixed and stained with SRB as previously described . Absorba2, 300 mM sucrose) at room temperature (RT) for 1 min before fixation. After permeabilization and blocking, cells were incubated with anti-RAD51 antibody and subsequently incubated with secondary antibody followed by Click-iT Alexa Fluor 594 cocktail buffer for 30 min.The γ-H2AX and 53BP1 staining was performed as previously described . BrieflyCell-cycle distribution was measured by flowcytometry . BrieflyAll animal experiments were approved by the Animal Welfare Committee of the Erasmus MC, and all experiments were conducted in accordance with accepted guidelines . PC295 PDXs were previously established in our laboratory . PDX tumEdU at a concentration of 3 μg/mL was added to the tissue slice culture medium 2 h before fixation. Simultaneous TUNEL and EdU staining was performed as described previously . BrieflyHistological tumor architecture was examined by H&E staining as previously described . BrieflySections were deparaffinized in xylene followed by rehydration in graded alcohols. Antigen retrieval was performed with target retrieval buffer . Sections were treated with 3% hydrogen peroxide solution in methanol at RT for 20 min, followed by incubation in 5% BSA in PBS for 30 min at RT. Sections were incubated with anti-AR antibodies overnight, and horseradish peroxidase (HRP)-conjugated anti-rabbit immunoglobulin G (IgG) secondary antibody  for 1 h at RT. AR-positive cells were visualized using a diaminobenzidine (DAB) staining kit  and hematoxylin counter-staining.https://imagej.nih.gov/ij/index.html) with background subtracted and normalized to unsaturated signals of intact DNA. For each treatment, these values were normalized against their respective untreated controls to obtain the fold-change in DSBs.Pulsed-field gel electrophoresis (PFGE) was performed as previously described . BrieflyThe DR-GFP assay was performed to measure HR as previous described . BrieflyCells were lysed and lysates were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) , transferred to polyvinylidene difluoride membranes, and analyzed using antibodies described above. Quantification was conducted using ImageJ software.PC346C-Flu1 cells were transfected with either control or DNA-PKcs siRNA  using Lipofectamine RNAi MAX . Transfected cells were cultured for 48 h and then treated as specified.For 53BP1, γH2AX, and RAD51 foci quantification, at least three random fields of view of Z-stack images were captured with a LSM700 confocal microscope  and quantified as previously described . AR and t-test was used to analyze the differences between two groups. Statistical analysis and generation of graphs was performed using GraphPad Prism 6.0 .Experiments were performed in duplicate (foci kinetics) or triplicate . One-way ANOVA was used to compare more than two groups, and an unpaired Student’s In summary, we demonstrate for the first time that the next-generation AR inhibitor apalutamide acts as a radiosensitizer in AR-expressing androgen-dependent PCa and CRPC models. This radiosensitization is caused by inhibition of DNA repair by NHEJ, not HR. Our results suggest that apalutamide can not only be used in combination with EBRT for the treatment of androgen-dependent localized PCa, but also for AR-expressing CRPC patients.


The major challenge in castration-resistant prostate cancer (CRPC) remains the ability to predict the clinical responses to improve patient selection for appropriate treatments. The finding that androgen deprivation therapy (ADT) induces alterations in the androgen receptor (AR) transcriptional program by AR coregulators activity in a context-dependent manner, offers the opportunity for identifying signatures discriminating different clinical states of prostate cancer (PCa) progression. Gel electrophoretic analyses combined with western blot showed that, in androgen-dependent PCa and CRPC in vitro models, the subcellular distribution of spliced and serine-phosphorylated heterogeneous nuclear ribonucleoprotein K (hnRNP K) isoforms can be associated with different AR activities. Using mass spectrometry and bioinformatic analyses, we showed that the protein sets of androgen-dependent (LNCaP) and ADT-resistant cell lines (PDB and MDB) co-immunoprecipitated with hnRNP K varied depending on the cell type, unravelling a dynamic relationship between hnRNP K and AR during PCa progression to CRPC. By comparing the interactome of LNCaP, PDB, and MDB cell lines, we identified 51 proteins differentially interacting with hnRNP K, among which KLK3, SORD, SPON2, IMPDH2, ACTN4, ATP1B1, HSPB1, and KHDRBS1 were associated with AR and differentially expressed in normal and tumor human prostate tissues. This hnRNP K–AR-related signature, associated with androgen sensitivity and PCa progression, may help clinicians to better manage patients with CRPC. In the last years, experimental evidence has supported the role of the androgen receptor (AR) in castration-resistant prostate cancer (CRPC) development. Almost all patients with metastatic prostate cancer (PCa) initially treated with androgen deprivation therapy (ADT) progress to CRPC, in many cases following the reactivation of the AR pathway. Several mechanisms, which are not necessarily mutually exclusive, have been proposed to explain CRPC development . They inMulti-omics studies showed that CRPC is a heterogeneous group of diseases characterized by different genotypes and phenotypes ,5,6. EmeA new scenario to develop alternative targeted therapies was recently proposed by Liu et al. , reportiThe heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a multifunctional protein playing a pivotal role in regulating numerous cellular functions, such as transcription, signal transduction, alternative splicing, and chromatin remodeling . This fuKelly et al.  underlinGiven the above findings, in this study, we investigated the role of hnRNP K in androgen-resistance using in vitro models of androgen-dependent or castration-resistant PCa. We hypothesized that hnRNP K, working like an AR transcriptional collaborator, could participate in regulating the different AR transcriptional programs during PCa development and progression. Consequently, a hnRNP K signature associated to AR activity could be useful to identify clinically distinct PCa subgroups.We previously reported that in the androgen-dependent cell line LNCaP, changes in the AR binding property of hnRNP K was associated with cell growth and AR activity ,20. HereHnRNP K silencing decreased both AR and PSA expression in LNCaP cells a,b, whilBIC removal and restored availability of androgen in the culture media determined comparable features in prostate-specific antigen (PSA) expression in both our ADT-resistant cell lines, with a maximum effect after 3–4 weeks d,e. HoweOverall, these results suggest that hnRNP K in prostate cancer cell lines is likely to act as an AR transcriptional collaborator that regulates AR activity through different molecular mechanisms depending on cell differentiation, supporting the hypothesis of the involvement of hnRNP K in ADT resistance.The mechanistic role of phosphorylation in regulating hnRNP K transcriptional activity ,22 as weUnexpectedly, using quantitative western blotting (WB) analysis a, we detBy evaluating the total hnRNP K phosphorylation status by means of the monodimensional phosphate affinity gel electrophoresis (1D Phos-tag), it was possible to identify a non-phosphorylated isoform (phK0) and two species characterized by intermediate (phK1) and maximal (phK2) phosphorylation b. As showww.phosphosite.org) are closely related, we evaluated the role of the subcellular distribution of hnRNP K phosphorylated isoforms in PCa evolution. Using Phos-tag bidimensional gel electrophoresis (2D Phos-tag) and WB  and WB , we analThe level of nuclear hnRNP K phosphorylated isoform 1 was higher than that of isoform 2 in resistant cell lines compared to androgen-dependent LNCaP, while, in the cytoplasm, alternatively spliced isoforms phosphorylation was regulated in an opposite manner. Interestingly, pS353, localized in the nuclear shuttling (KNS) domains regulating hnRNP K intracellular localization, was detected exclusively in isoform 2 .Quantitative analysis of nuclear hnRNP K phosphorylated forms in all cell lines revealed that: (i) pS353 decreased in resistant cell lines compared to LNCaP; (ii) an over 36% pS284 increase was observed in cell lines with active AR axis, while pS284 decreased to about 23% in MDB and PC3 cells; (iii) both high percentages of non-phosphorylated forms and low pS116 percentages were detected in androgen-independent cell lines MDB (AR-positive), 22Rv1 (ARv7), and PC3 (AR-negative), independently of the AR status.The cytoplasmic hnRNP K isoforms distribution in the different cell lines showed that pS116 could discriminate between LNCaP and CRPC lines, regardless of AR functionality. Cytoplasmic pS353 showed an opposite trend in PDB and 22Rv1 cell lines with aberrant AR activation, in comparison to LNCaP. The pS284 isoforms were overrepresented in androgen-independent MDB and 22Rv1 cell lines.These findings suggest that differential phosphorylation at specific serine residues and compartmentalization of hnRNP K splicing isoforms 1 and 2 correlate with different resistant phenotypes, providing further evidence for hnRNP K involvement in CRPC evolution.HnRNP K may regulate several cellular functions, such as transcription and signal transduction, by PTMs that modify its binding partners ,27. UsinAs shown in p < 0.05) with hnRNP K (DIhKP), as reported in By comparing hnRNP K co-IP abundance values obtained from LNCaP, PDB, and MDB, we identified three sets of differentially interacting proteins. The data were linearly modeled using Limma R package, and from the three contrasts generated  we identified 51 proteins differentially interacting  determines AR transcriptional program modifications . By perfAs LNCaP, PDB and MDB cell lines can mimic distinct stages of PCa progression , we can Overall, these findings indicate that, in cell lines with different androgen-resistant phenotypes, decreased hnRNP K expression and modified subcellular distribution of its isoforms were associated with dynamic remodeling of the hnRNP K interacting protein network depending on cellular differentiation, thus providing a rationale for DIhKPs analysis in human prostate cancer tissues.From the above results, we hypothesized that some DIhKPs could differentiate distinct stages of PCa progression to CRPC, thus conferring clinical relevance to the findings obtained in our CRPC cell lines PDB and MDB. In this section, we describe the analytical strategies used to identify, among the 51 DIhKPs, a protein set that might putatively discriminate among clinical stages of PCa progression.http://www. proteinatlas.org/), we verified that only three proteins , four AR-controlled proteins , and three TFs associated with AR activity  . In bothUnderstanding the context and the functional role of key proteins in ADT resistance could allow the identification of a molecular signature, rather than single proteins, that could overcome the main limit of prostate cancer treatments: tumor heterogeneity. Previously, we demonstrated that, in our CRPC model, the AR transcriptional program was modified by ADT . In thisThe eight proteins highlighted in this study  represent a signature associated with AR modulation during the transition from androgen-dependent to castration-resistant prostate cancers. These proteins might discriminate specific stages of PCa and thus predict patients’ prognosis and response to treatments. Further investigations on PCa tissues or liquid biopsies from CRPC patients are needed to validate the clinical usefulness of the hnRNP K–AR-related signature and to evaluate whether some of the proteins included in our signature might become potential targets for new drugs development.The human prostate cancer cell lines LNCaP, 22Rv1, and PC3 were purchased from the American Type Culture Collection  and cultured in RPMI  medium containing 10% fetal bovine serum, 2 mM glutamine, 1% penicillin, and 1% streptomycin. The LNCaP and 22Rv1 medium was also supplemented with 10 mM HEPES, 10 mM sodium pyruvate, and 0.25% glucose. The passage numbers at which LNCaP cells were used ranged from 22 to 30. The BIC-resistant cell lines PDB and MDB, obtained in our laboratory , were cu2S2O5, 1 mM phenylmethylsulfonyl fluoride, 0.5 mM benzamidine, 20 mg/mL leupeptin, 10 mg/mL pepstatin A, 25 mg/mL aprotinin), 1 mM Na3VO4, and 1 mM dithiothreitol. The fractionation procedures were performed at 4 °C. The cells were mechanically harvested with a sterile cell scraper and washed three times in 30 mL of PBS. Total protein extracts, obtained using RIPA buffer, and cytoplasmic and nuclear extracts were prepared as reported [All buffers were supplemented with a protease inhibitors cocktail  and separated at 5 mA/gel for 16 h at a constant temperature of 12 °C. 2D Phos-tag was performed as described [Eight micrograms of proteins extracted from different cell fractions were loaded onto an 8–14% linear gradient (1D) or 7.5% (1D Phos-tag) polyacrylamide gels, containing 25 mM polyacrylamide -bound Mnescribed . After eescribed , the cont-test within OriginPro 7.5 software , and the differences were considered statistically significant when p < 0.05.Statistical significance was evaluated by two-tailed Student’s ON-TARGET plus SMART pool for human hnRNP K  was used to knockdown the expression of hnRNP K; siCONTROL non-targeting (NT) siRNA pool  was used as a negative control. The cells were grown in their proper medium and transfected with Lipofectamine 2000  following the manufacturer’s instructions. Protein expression was analyzed by WB 72 h after transfection.To measure AR function, the cells were transfected with Lipofectamine 2000 utilizing the Cignal androgen receptor dual-luciferase reporter kit , following the manufacturer’s instructions. Luciferase activity was assayed in triplicate 48 h after transfection using the dual-luciferase reporter assay kit . To control for general effects on transcription, Renilla luciferase was co-transfected in all reporter assays, and luciferase values represent the ratio luciferase/Renilla.Co-immunoprecipitation, employed to identify proteins directly or indirectly interacting with hnRNP K, was carried out using the Pierce Direct IP kit according to the manufacturer’s instructions . Briefly, total cellular extracts, obtained with IP Lysis/Wash buffer containing a protease inhibitor cocktail, were pre-cleared with agarose resin and passed through an AminoLink plus resin with covalently immobilized anti-hnRNP K antibodies . The eluted proteins were subjected to MS for identification of the hnRNP K interactome. Control resin and quenched antibody-coupling resin without anti-hnRNP K antibodies were utilized to identify non-specific interactions. Common proteins between these groups and the co-IPs were excluded from bioinformatic analyses. For MS analysis, three independent experiments with LNCaP, PDB, and MDB were performed.The hnRNP K co-IPs were analyzed by WB for hnRNP K and AR expression evaluation in LNCaP, PDB, and MDB cell lines .3 solution at 37 °C overnight. Peptides were collected by centrifugation of the filter units for 10 min, and the filter devices were rinsed with two 40 µL washes of 50 mM NaHCO3 and 50 µL 0.5 M NaCl to eliminate the hydrophobic interactions. Each sample digest was desalted on StageTips and analyzed by liquid chromatography–tandem mass spectrometry (LC–MS/MS).The samples were processed by the FASP Protein Digestion Kit . Briefly, the co-IP samples were mixed with 0.3 mL of 8 M urea in 0.1 M Tris/HCl pH 8.5 (UA solution), loaded into the filtration devices, and alkylated in 0.1 mL of 50 mM iodoacetamide in UA solution for 1 h in darkness at room temperature. The samples were digested using, sequentially, 1 µg of LysC and 3 µg of Trypsin in 50 mM NaHCOR = 60,000. The 10 most intense peptide ions with charge states ≥2 were sequentially isolated and fragmented by collision-induced dissociation in the LTQ mass spectrometer. The raw mass spectrometric data were analyzed using the MaxQuant pipeline. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies, and proteome-wide protein quantification [All MS experiments were performed on a nanoscale high-performance liquid chromatography system connected to a hybrid linear trap quadrupole (LTQ) Orbitrap mass spectrometer. The MS instrument was operated in data-dependent mode to automatically switch between full-scan MS and MS/MS acquisition. Survey full-scan MS spectra were acquired in the Orbitrap analyzer with resolution fication . The stafication  and Cytofication  respectiCytoscape (v 3.6.0) was used to visualize the protein–protein interaction network obtained from the above proteomic data (co-IP) and to highlight functional annotations . StatistDifferential expression analyses were carried out both on protein detected through MS spectroscopy for their binding to hnRNP K and on gene expression data extracted from four datasets published and available on the GEO portal .p-value ≤ 0.05 and a |log2FC| ≥ 1 were considered differentially interacting (DIhKPs).In the first case, the log-transformed values of protein binding were imported and analyzed through linear model fitting and empirical Bayes approach provided by Limma R package . Three dMicroarray data from GEO datasets were imported through GEOquery R package , “GEOque2FC| ≥ 1 and the corresponding p-value ≤ 0.05 were considered statistically significant.For each dataset, pairwise comparisons between sample groups, as indicated in Enrichment analysis was performed on the 51 DIhKPs to identify the over-represented GO annotations regarding Biological Processes and Molecular Function, as well as the transcription factor binding sites through the category UCSC-TFBS provided by the functional annotation tool DAVID v 6.8 .


Phytochelatins react with heavy metal ions by glutathione S-transferase in the cytosol and afterwards they are sequestered into the vacuole for degradation. Furthermore, heavy metals induce reactive oxygen species (ROS), which directly or indirectly influence metabolic processes. Reduced glutathione (GSH) attributes as an antioxidant and participates to control ROS during stress. Maintenance of the GSH/GSSG ratio is important for cellular redox balance, which is crucial for the survival of the plants. In this context, sulfurtransferases (Str), also called rhodaneses, comprise a group of enzymes widely distributed in all phyla, paving the way for the transfer of a sulfur atom from suitable sulfur donors to nucleophilic sulfur acceptors, at least in vitro. The best characterized in vitro reaction is the transfer of a sulfane sulfur atom from thiosulfate to cyanide, leading to the formation of sulfite and thiocyanate. Plants as well as other organisms have multi-protein families (MPF) of Str. Despite the presence of Str activities in many living organisms, their physiological role has not been clarified unambiguously. In mammals, these proteins are involved in the elimination of cyanide released from cyanogenic compounds. However, their ubiquity suggests additional physiological functions. Furthermore, it is speculated that a member of the Str family acts as arsenate reductase (AR) and is involved in arsenate detoxification. In summary, the role of Str in detoxification processes is still not well understood but seems to be a major function in the organism.Plants and animals have evolved various potential mechanisms to surmount the adverse effects of heavy metal toxicity. Plants possess low molecular weight compounds containing sulfhydryl groups (-SH) that actively react with toxic metals. For instance, glutathione (γ-Glu-Cys-Gly) is a sulfur-containing tripeptide thiol and a substrate of cysteine-rich phytochelatins (γ-Glu-Cys) The mosidue 247  [2]:Escherichia coli (GlpE) that often interacts with thioredoxins  and arsenate [As (V)]. Both these forms of arsenic are toxic to organisms. However, As (III) is considered to be more toxic than the As (V) form. Both forms interrupt biological functions in a different manner. Arsenite binds to proteins with sulfhydryl groups interfering with their functions. It inhibits respiration by binding to vicinal thiols in pyruvate dehydrogenase and 2-oxo-glutarate dehydrogenase . Arsenit3 form through aquaglyceroporins. The majority of arsenic found in the soil is in the arsenate form bound to different elements with different solubility, also dependent on the pH of the soil. The As (V) taken up is then reduced to As (III) by the enzyme AR. The next process is arsenite complexation with free thiol groups in order to detoxify the compound. This is followed by the vacuolar compartmentalization and storage of the arsenite-thiolate complex, thus completing the arsenic phytoremediation process [The arsenic hyperaccumulator plants are potential candidates for arsenic phytoremediation. The process by which the plant accumulates arsenic is illustrated in  process .ArsC gene from Staphylococcus aureus utilizes thioredoxin as its reducing agent, while the AR from E. coli R773 (encoded by ArsC gene) and S. cerevisiae (encoded by Acr2p gene) utilize glutathione and glutaredoxin as reducing agents [The mechanism of enzymatic reduction by AR involves the formation of a thioester bond between the cysteine and As (V), and the arginine residue assists in the stabilization of the intermediate. The reduced form of AR is its active form, and the reducing agent involved in the generation of the active form of AR is either a thioredoxin or a glutaredoxin . For exag agents .A. thaliana protein. However, the comparison of growth of A. thaliana AtStr5-expressing E. coli cells and negative control cells cultured on media containing varying levels of arsenate (125 to 1000 µM) showed that AtStr5 positive E. coli transformants were resistant to arsenate . These observations further pointed towards the role of AtStr5 as an AR, maybe in an interaction process with thioredoxins.So far the AR activity could not be confirmed for the recombinant AtStr5 AtStr5 mutant plants unexposed to arsenate. Furthermore, it was confirmed that the AR activity of AtStr5 represents 36% of the total activity and is inducible by arsenate in A. thaliana roots [AtStr5 mRNA was silenced using an RNAi construct. This increased the shoot arsenic accumulation 10–16 fold more than the roots of wild-type plants grown under identical conditions [AtStr5 accumulated less arsenic in shoots than wild-type plants over a range of arsenate concentrations [In another study, the AR activity was observed in root extracts of T-DNA knockout na roots . In a sinditions . In conttrations . DespiteOryza sativa Cdc25 (OsCdc25), and 42% identity and 61% similarity with Pteris vittata AR. Although AtStr5 shares the same active site region as the human Cdc25 isoforms , it was seen that they have less overall identity. This may be considered as another indication for the possibility that AtStr5 may not act as a phosphatase but may have an important role in arsenate reduction. AtStr6 is similar to the human Cdc25 isoform A (huCdc25A) and since huCdc25A has no AR activity, the same can be expected of AtStr6. AtStr7 and AtStr8 form an entirely different clade in the phylogenetic tree, suggesting that they are not functionally related to AtStr5. Therefore, the other Str from group III, except AtStr5, may not show AR activity.A distance phylogenetic tree  of rhodanese/Cdc25 superfamily members demonstrates the relationships among putative AR proteins . AtStr5 33−) in the stem and leaves. The γ-ECS is involved in the first step of the phytochelatin synthesis pathway. γ-Glutamylcysteine complexes are formed with As (III) via its thiol groups, consequently detoxifying and preparing them for being stored away in vacuole, supporting the idea that the transgenic plants were able to accumulate 2–3 times more arsenic than wild-type plants [Phytoremediation is virtually considered as a potential solution to mitigate arsenic pollution. Certain plants known as hyperaccumulators or metallophytes have the ability to reduce heavy metal contamination by accumulating higher than normal levels of toxic heavy metals in their above-ground parts . The ovee plants . However2O2) by stimulating NADPH oxidase. In this context, β-CAS pathway has been profoundly accepted for maintaining cyanide homeostasis in higher plants. On the other hand, Str in animals has been well described as means of detoxifying cyanide. Nevertheless, Str activities are present in many living organisms, but their physiological role hare still ambiguously. Their ubiquity suggests additional physiological functions. Furthermore, it was suggested that one member of Str mimic as AR which is involved in arsenic phytoremediation and might be a promising candidate for successful removal of arsenic from soil. Henceforth, development of abiotic stress-tolerant crop via over-expression of ROS-scavenging enzymes and enzymes containing reactive sulfur groups, such as Str, may be useful.Various heavy metal ions trigger the overproduction of ROS or free radicals in plants which are toxic and highly sensitive to proteins, lipids, carbohydrates and DNA, and ultimately results in oxidative stress. Against this backdrop, cells are evolved with sophisticated antioxidant defense mechanisms to detoxify the deleterious consequences of ROS. These antioxidant defenses could be non-enzymatic  or enzymatic . There is profound evidence that cyanide leads to the yield of ROS and to escalate hydrogen peroxide (H