Eimeria oocyst counts in feces, blood metabolites, and the meat quality of growing rabbits. A total of 160 hybrid rabbits  of 30 days of age and 643 ± 8.0 g body weight (BW) were assigned to four treatments with 10 replicates each (four rabbits/replicate). The treatments were as follows: (1) CON: rabbits fed basal diet and not supplemented with BP or PRO; (2) BP500: CON + BP (500 mg/kg BW); (3) PRO50: CON + PRO (50 µL/kg BW); and (4) BP + PRO: CON + BP (500 mg/kg BW) + PRO (50 µL/kg BW). Higher daily weight gain (p = 0.04) and lower feed conversion rate (p = 0.03) were observed in rabbits supplemented with PRO50. In addition, supplementation with PRO50 and BP + PRO reduced the amount of Eimeria oocysts per gram of feces (p < 0.05). Most hematological and serum biochemical parameters were similar in rabbits of all treatments. Protein content, collagen, and meat color were similar between treatments. In conclusion, propolis supplementation (50 µL/kg BW) can prevent coccidiosis and act as a natural growth promoter in rabbits without affecting animal health and meat quality.The objective of this study was to evaluate the effect of supplementation with bee pollen (BP) and propolis (PRO) on productive performance,  Apis mellifera) products, such as bee pollen (BP) and propolis (PRO), could be used for this purpose because they contain various nutritional compounds and some bioactive metabolites with pharmaceutical properties [It has been discussed that, due to its short production cycle, rabbit production could be used as an alternative to solve the growing meat shortage in developing countries . Howeveroperties ,6.BP is a mixture of flower pollen grains collected by bees with nectar and secretions from the hypopharyngeal glands of bees ,7. AccorEscherichia coli and Salmonella spp. in the cecum of growing rabbits [Pasteurella multocida [On the other hand, PRO is a complex mixture of resinous, gummy, and balsamic substances collected by bees from plant buds, flowers, and exudates ,13,14,15 rabbits . Likewisultocida . The effultocida  or by miultocida . Howeverultocida . FurtherEimeria oocyst count per gram of feces, blood biometry, blood biochemistry, and meat quality in growing rabbits.Due to the positive effects of BP and PRO, this work hypothesizes that supplementation with BP and PRO will benefit the productive performance of rabbits without affecting health and meat quality. Furthermore, the combination of BP and PRO could act synergistically due to their chemical composition and mechanisms of action. Therefore, the objective of this study was to evaluate the effects of supplementation with bee pollen, propolis, and the combination of both products on productive performance, The experiment was conducted during April and May 2022 at the “Conejos” Centro de Investigación Científica del Estado de México A.C. (COCICEMAC). This site is located in San Miguel Coatlinchán, State of Mexico, Mexico (latitude 19°26′56″ N and longitude 98°52′20″ W). San Miguel Coatlinchán is 2240 m above sea level, the mean annual temperature is 15 °C, and the mean annual precipitation is 645 mm [BP was collected in hives located in the municipality of Contla de Juan Cuamatzi, State of Tlaxcala, Mexico, located at 2320 masl (latitude 19°20’00″ N and longitude 98°10’00″ W). The BP collection was carried out from September to November 2021 using intermediate pollen traps placed in the hives’ inner part .The PRO was collected in the village Charco de Pantoja, in the municipality of Valle de Santiago, Guanajuato State, Mexico, located at 1709 masl (latitude 20°23’34″ N and longitude 101°11’29″ W). PRO collection was carried out from October 2020 to February 2021 using the plastic net mesh technique .BP and PRO samples were analyzed in the laboratory to determine the content of dry matter (method 967.03), crude protein (method 981.10), ether extract (method 920.29), and ash (method 942.05), following the procedures previously described by AOAC . The nutTM, Waltham, MA, USA). Similarly, the total flavonoid content of the PRO extract was obtained following the colorimetric method using aluminum chloride, as previously described by Chang et al. [TM, Waltham, MA, USA). The total content of phenolic compounds of the PRO was 23.67 mg gallic acid equivalent per g dry weight of the extract. Likewise, the total flavonoid content of the PRO was 9.21 mg quercetin equivalent per g dry weight of the extract.The total phenolic compound content of the PRO extract was determined using the colorimetric method described by Folin–Ciocalteu . For thig et al. . In thisAll rabbits used in the present study were clinically healthy at the start of the experiment. One hundred sixty freshly weaned California × New Zealand hybrid rabbits , 30 days of age and with an average body weight (BW) of 643 ± 8.0 g, were used. The rabbits were weighed to form four homogeneous groups and were entirely randomized for one of four treatments. Each treatment had 10 replicates (cages), and in each replicate there were four rabbits with similar initial weights and of both sexes . The treatments used were as follows: (1) rabbits fed a basal diet and without BP or PRO in the drinking water (CON); (2) BP500: CON + BP (500 mg/kg BW); (3) PRO50: CON + PRO (50 µL/kg BW); and (4) BP + PRO: CON + BP (500 mg/kg BW) + PRO (50 µL/kg BW). The BP dose (500 mg/kg BW) was chosen based on a previous report in which supplementation with this dose improved the digestibility of ingested nutrients in growing rabbits . The PROad libitum access to drinking water without treatment. The animals of the CON treatment had the same conditions as the experimental groups but without applying BP or PRO to the drinking water. Instead, drinking water was provided through nipple drinkers. The BP500, PRO50, or BP + PRO treatments were adjusted each week based on the body weights obtained. Oral administration of the treatments was performed in the morning (09:00 h).Treatments were administered five times per week (Monday through Friday) throughout the experimental period. Based on a previous experiment carried out by our work team (unpublished data), we verified that the doses of BP and PRO were well dissolved in the drinking water and that the rabbits consumed this water adequately. Therefore, the BP500, PRO50, or BP + PRO treatments were first applied to 200 mL of drinking water in each of the cages using automatic drinkers, as other authors previously used ,31,32,33Environmental, hygienic, and handling conditions were the same for all rabbits. Also, the health status of the rabbits was monitored throughout the experimental phase. The rabbits were housed in a module with natural ventilation system and thermal insulation on the walls but not on the ceiling. The rabbits were kept in galvanized metal cages 78 cm long, 56 cm wide, and 30 cm high (four rabbits/cage) arranged in a flat-deck system at 45 cm above the floor level. Each cage had a galvanized English-type hopper feeder  with a capacity of 1.5 kg and a nipple-type automatic drinker. During the entire experimental period (April and May), the average daylight was 13 h.®, Texcoco, Mexico), which came from the same production lot. According to the labeled feed package, antibiotics are not included, but 1 g of Diclazuril per ton as coccidiostat is added to prevent Eimeria spp. Each week, feed samples were taken and analyzed to determine the chemical composition of the feed following the procedures described by AOAC [ad libitum during the 42 days of the experimental phase. All rabbits were fed a commercial pelleted basal diet  for growing rabbits  located inside the rabbit facility. With the temperature and relative humidity data, the temperature and humidity index (THI) was estimated using the following equation [Ambient temperature and relative humidity were recorded daily using a Traceableequation : THI = T® brand digital scale , with an accuracy of ± 0.5 g. Subsequently, the rabbits were weighed weekly on days 7, 14, 21, 28, 35, and 42 of the experimental periods. The feed intake of the rabbits was recorded each week and divided by the number of days of the period to estimate daily feed intake . Likewise, average daily gain  was calculated using the weekly BW data. In addition, the rabbits’ feed conversion ratio (FCR) was calculated using the equation: FCR = DFI/ADG.The body weight (BW) of the rabbits was recorded before morning feeding at the beginning (day 0) of the experimental phase, using a TORREYEimeria spp. in rabbits, freshly voided feces samples were collected from eight replicates (cages) of each treatment each week . The feces were collected at the same time (9:00 h) on each sampling and were used to perform oocyst counts using the McMaster counting technique [To evaluate the parasite load of echnique : OPG = oechnique .® K2 EDTA anticoagulant  were used to collect the first sample, which was stored at 4 °C, for subsequent determination of the complete blood count and differential leukocyte count using an EasyVet® hematology analyzer , as previously reported by several authors [®  anticoagulant-free tubes were used to collect the second blood sample. These blood samples were centrifuged at 3500 rpm for 20 min to obtain blood serum using a refrigerated centrifuge . The blood serum was stored in Eppendorf tubes and frozen at −20 °C. Finally, for the analysis of the blood serum samples, an EasyVet® autoanalyzer  and Spinreact kits  were used to determine the contents of glucose (kit 41011), cholesterol (kit 41021), albumin (kit 1001020), globulin (kit 1001032), total protein (kit 1001291), urea (kit 41041), uric acid (kit 41001), bilirubin (kit 1001046), creatinine (kit 1001113), liver enzymes , lactate dehydrogenase (kit MX41274), and aspartate aminotransferase (kit MX41264)), calcium (kit 1001060), and phosphorus (kit 1001155), as described by other authors [To determine hematological and biochemical parameters, at the end of the experimental phase (day 42), blood samples were randomly taken from the ear vein of 10 rabbits  from each treatment. Two 2.5 mL blood samples were taken from each rabbit. Tubes with BD Vacutainer authors ,39,40. B authors ,41.Longissimus dorsi muscles (LDM) were removed from the hot carcass, following procedures similar to those described and recommended by Blasco and Ouhayoun [After obtaining the final body weight (day 42 of the experiment), all rabbits were kept fasting for 12 h. Subsequently, 20 rabbits  from each treatment were sacrificed following the procedures of the Mexican Official Standard (NOM-033 SAG/ZOO-2014). After completing the slaughter and bleeding of the rabbits, all internal organs, skin, feet, and heads were separated from the carcass, and the hot carcass weight (HCW) was recorded. The hot carcass yield (HCY) was estimated by the equation: HCY =  × 100. Subsequently, the leg muscles (HLM) and ® brand pH meter .Before meat quality analyses, samples were thawed at 4 °C for 24 h. Then, meat pH was determined using the procedures previously described by Orzuna-Orzuna et al. . For thi® brand thermometer , and when it reached 70 °C, the steaks were removed from the grill and allowed to cool for 1 h at room temperature (20–25 °C). The percentage of CL was estimated with the equation [Meat color parameters were measured in triplicate in LDM samples from each rabbit using the procedures described by Miltenburg . For thiequation : CL, % =HLM samples from each rabbit were separately ground and homogenized for 5 min using a Ship to Shore brand meat grinder  . SubsequAll data were analyzed by analysis of variance (ANOVA) using SAS statistical software . Before ijk represents the value observed in treatment i in week j for rabbit k; µ represents the overall mean; Ti represents the fixed effect of the i-th treatment (i = 1 (CON), 2 (BP500), 3 (PRO50), 4 (BP + PRO)); Wj represents the fixed effect of the j-th week of the experimental period ; (T × W)ij represents the fixed effect of the interaction between the j-th week and the i-th treatment; Rk represents the random effect of a cage (repetition) within treatment ; and eijk represents the random error.In this model, YThe variables of blood biometry, blood biochemistry, meat quality, and HCY were analyzed using a completely randomized design. For this, the GLM procedure of SAS  was usedij represents the observations, µ represents the overall mean, Ti represents the fixed effect of the i-th treatment, and eij is the random error. For all variables analyzed, treatment means were compared using Tukey’s test. The mortality rate was analyzed as a percentage using chi-square analysis [p ≤ 0.05. In addition, p > 0.05 and ≤ 0.10 was considered as tending to be significant.In this model, Yanalysis . A statip < 0.05) than rabbits assigned to the CON treatment. ADG was 8.8% greater (p = 0.04) in the PRO50-group rabbits than in the CON-treatment rabbits. FCR was 7.1% lower (p = 0.03) in PRO50-treatment rabbits than in CON-treatment rabbits. Compared to the CON treatment, rabbits in the PRO50 and BP + PRO treatments had 50 and 55% lower OPG (p < 0.05), respectively. On the other hand, mortality was higher (p = 0.05) in the BP500-treatment rabbits than in the rabbits assigned to the other treatments (eatments .p = 0.02) percent hematocrit was observed in BP500-supplemented rabbits than in CON rabbits (p = 0.009). Mean corpuscular volume was lower (p = 0.02) in PRO50-treatment rabbits than in CON rabbits. Lower (p = 0.001) blood concentration of monocytes was observed in BP + PRO-treatment rabbits than in rabbits assigned to the other treatments. Rabbits in the PRO50 treatment had higher (p < 0.05) blood concentrations of band neutrophils and eosinophils than in the CON, BP500, and BP + PRO treatments.Higher ( rabbits . Comparep = 0.001) serum urea concentration compared to rabbits in the CON, BP500, and BP + PRO treatments. In addition, rabbits supplemented with BP500 had a higher albumin/globulin ratio (p = 0.04) and lower serum total protein and globulin concentration (p < 0.05) than CON-treatment rabbits. Serum aspartate aminotransferase concentration tended to be lower in BP500-supplemented rabbits than in CON-treatment rabbits  HCY was observed in rabbits supplemented with BP500 and PRO50 than in rabbits assigned to the CON and BP + PRO treatments (p = 0.001) in meat from rabbits supplemented with BP + PRO than in meat from rabbits in the other treatments. PRO50 supplementation reduced (p < 0.05) the CL of meat compared to the other treatments. However, lower fat content (p = 0.05) and higher moisture content (p = 0.03) were observed in meat from rabbits supplemented with BP500 than in meat from CON rabbits  between BW and Eimeria spp. OPG count [Eubacteriaceae, Peptococcaceae, and Natranaerobiaceae) that are positively correlated with ADG. Likewise, the addition of quercetin in diets for rabbits increases up to 7.6% the serum concentration of growth hormone [Zeedan et al.  observedPG count . FurtherPG count . PreviouPG count ,18 have PG count  reported hormone . SimilarFCR was not affected by supplementation with BP500 and BP + PR, suggesting that the doses of BP and BP + PR used in the present study do not improve feed efficiency in growing rabbits. In similar studies, Attia et al. ,10 also Erysipelotrichaceae and Haloplasmataceae) that are negatively correlated with FCR. Consequently, similar PRO and flavonoid consumption effects observed in our study could partially explain the lower FCR in rabbits on PRO50 treatment. The lower FCR observed in rabbits supplemented with PRO50 suggests that PRO could improve the profitability of rabbit production systems since FCR is a crucial indicator for judging an animal production system [On the other hand, FCR was lower in rabbits supplemented with PRO50. PRO supplementation has been reported to improve rabbits’ digestibility of organic matter and ingested crude protein . North en system .Eimeria protozoa, which in rabbits causes growth retardation and high mortality [Eimeria decreased in rabbits supplemented with PRO50 and BP + PRO. This result suggests that these products could be used as natural coccidiostats for growing rabbits. This hypothesis is supported by the average OPG values observed in rabbits supplemented with PRO50 and BP + PRO, which were lower than the range (4000–5000 OPG) at which the application of prophylactic treatment against Eimeria protozoa is required [Coccidiosis is an infection caused by ortality . In the required .On the other hand, although mortality was higher in rabbits supplemented with BP500, the mortality observed in the present study was high (between 17.5 and 40%) for all treatments. In the present study, a seasonal effect and climatic conditions might have an essential role in the high mortality rates observed , particuWhen evaluating a new feed additive for domestic animals, it is essential to analyze the effects of the consumption of the additive on animal health . Accordi3/mL) [Eimeria parasites [The blood concentration of leukocytes was similar between treatments. However, the blood concentration of leukocytes in CON treatment rabbits was higher than the normal range reported for healthy rabbits . In addi3/mL) . These rarasites . Furtherarasites . On the arasites . ConsequSerum glucose concentration and lipid metabolites are indicators of rabbits’ energy status . In the Supplementation with BP500 reduced serum globulin and total protein levels. However, rabbits from all treatments had serum urea, albumin, globulin, and total protein concentrations within the reference range considered normal for healthy rabbits . This reIf analyzed at appropriate reference intervals, serum uric acid and creatinine concentrations can be used as biomarkers of renal function status in rabbits . For exaSerum levels of hepatic enzymes are often used as markers of liver disease . In addiIt has been mentioned that serum calcium and phosphorus levels can be used as good indicators of nutritional status in domestic animals because their variability is low ,73. In tIn the present study, supplementation with BP500 and PRO50 increased HCY. In a similar study, Zeedan et al.  also obsThe pH is considered one of the most critical parameters determining rabbit meat quality because it is related to its color, flavor, water-holding capacity (WHC), tenderness, and shelf life . For exaPrevious studies have reported that meat color can be modified by changes in fat content and pH ,77. In tIn the present study, PRO50 supplementation reduced the CL of meat. This result suggests that PRO supplementation can improve the WHC of rabbit meat because CL and WHC are negatively correlated . LimitedThe chemical composition of rabbit meat from all treatments had values within the normal range . This reThe results of this study indicate that propolis supplementation (50 µL/kg BW) can be used to prevent coccidiosis and as a natural growth promoter in rabbits without affecting animal health and meat quality. However, more research is needed to understand and explain the impact of bee pollen and propolis supplementation on productivity, parasite load, health status, and the meat quality of growing rabbits.


Indium selenide (InSe) is an emerging van der Waals material, which exhibits the potential to serve in excellent electronic and optoelectronic devices. One of the advantages of layered materials is their application to flexible devices. How strain alters the electronic and optical properties is, thus, an important issue. In this work, we experimentally measured the strain dependence on the angle-resolved second harmonic generation (SHG) pattern of a few layers of InSe. We used the exfoliation method to fabricate InSe flakes and measured the SHG images of the flakes with different azimuthal angles. We found the SHG intensity of InSe decreased, while the compressive strain increased. Through first–principles electronic structure calculations, we investigated the strain dependence on SHG susceptibilities and the corresponding angle-resolved SHG pattern. The experimental data could be fitted well by the calculated results using only a fitting parameter. The demonstrated method based on first–principles in this work can be used to quantitatively model the strain-induced angle-resolved SHG patterns in 2D materials. Our obtained results are very useful for the exploration of the physical properties of flexible devices based on 2D materials. Two-dimensional 2D) materials have drawn much attention since the discovery of graphene in 2004 . SubsequD materiaSHG is a second-order nonlinear process that only occurs in crystals without inversion symmetry. In a thick crystal, the conversion efficiency of SHG is maximized in the condition of phase matching. For thin crystals with a thickness below 100 nm, a few methods have been proposed to increase the efficiency of SHG recently ,18,19. Firection ,22,23. Iirection ,24,25. Sequences . SHG is ructures ,28,29,30In this work, we investigated the strain dependence on angle-resolved SHG from mechanically exfoliated −4 Pa. The ampoules were placed in a horizontal furnace at 550 °C for two days to obtain a partially mixed crystal. Later, the mixed crystals were used to grow the pure InSe single crystal with the help of a vertical Bridgman setup. The furnace during the Bridgman process was maintained at a high temperature of about 850 °C. The mixed crystal in the ampoule was hung and heat treated at 850 °C for 24 h; once the mixed crystal melted entirely in the ampoule, the ampoules were lowered through a temperature gradient of 1 °C at a rate of 0.1 mm/h to obtain a large-sized single crystal. Using this procedure, we obtained over 5 cm long and 1 cm in diameter InSe single crystals in bulk. InSe crystals were grown by the vertical Bridgman method. We purchased 99.999% pure Indium (In) and Selenide (Se) chemicals from Sigma Aldrich. The growth of the InSe single crystals was performed in quartz ampoules by mixing In and Se powders under a vacuum of 10Powder X-ray diffraction (PXRD) was employed to study the lattice phases. The result of the Rietveld refinement of X-ray diffraction was carried out by the software GSAS, while the high orientation of the powdered InSe was taken into account for the refinement. The lattice structure was determined as γ-InSe (R3m) with lattice constants a = b = 4.006 Å, and c = 24.969 Å, respectively. The crystal phase was also confirmed by oblique incident SHG measurements . AlthougThe sample structure of γ-InSe is shown in Flakes of InSe in a few layers were prepared by the mechanical exfoliation of the γ-InSe crystal with polydimethylsiloxane (PDMS). Multiple flakes were subsequently transferred to a polyethylene naphthalate (PEN) flexible substrate, with an SU-8 photoresist film on top. The thickness of the PEN substrate was about 125 We referred to the experimental configuration in the literature  to desigThe schematic of the experimental setup is shown in s, p, and d orbitals, respectively. We adopted a cutoff radius of 7 bohr for the In atom, denoted as In7.0-s3p2d2. Similarly, for the Se atom, Se7.0-s3p2d2 was adopted. A cutoff energy of 500 Ry was used for numerical integrations and for the solution of the Poisson equation. An 8 × 8 × 2 k-point sampling was used. The structures were fully relaxed without considering spin-orbit coupling at the specified strain, that is, only the components of the lattice vectors associated with the desired strain were fixed during the calculations. For the unstrained ground-state structure, no constraint was applied to the lattice vectors. Once the optimized lattice constants were obtained, the atomic positions were relaxed again with the effect of spin-orbit coupling. All of the atomic forces in the resulting lattice satisfied the criterion of being smaller than 0.0001 Ha/bohr. The electronic self-consistent field (SCF) criterion was set to 10−9 Ha, which guaranteed that the difference between the band energies in the last two consecutive SCF steps was smaller than the criterion.To theoretically obtain the geometrical structures of InSe under strain and calculate the SHG susceptibilities of the strained InSe, we performed first–principles calculations based on the density functional theory (DFT) using the OpenMX code , where gThe spectra of SHG susceptibilities were calculated via the momentum matrix elements using the basis of the adopted pseudoatomic basis functions , followiulations , the calulations . irection . The angWe further applied compressive uniaxial strain on the samples along the AC direction d=[dThe parallel SHG intensity for unstrained InSe isSince the variation in SHG susceptibilities is linear to the compressive strain, the strain effect on the SHG susceptibilities could thus be modelled as follows.The coefficients To quantitatively understand the appropriateness of the aforementioned model, we used the strain values in the experiments to calculate the angle-resolved The model should be modified to appropriately fit the experimental data. Here, we additionally introduced a correction parameter We mapped the SHG images of


Here, we characterized lysosomal Ca2+ signals in mouse renal collecting duct (CD) cells and we assessed their putative role in aquaporin 2 (AQP2)-dependent water reabsorption. Bafilomycin A1 and ML-SA1 triggered similar Ca2+ oscillations, in the absence of extracellular Ca2+, by alkalizing the acidic lysosomal pH or activating the lysosomal cation channel mucolipin 1 (TRPML1), respectively. TRPML1-dependent Ca2+ signals were blocked either pharmacologically or by lysosomes’ osmotic permeabilization, thus indicating these organelles as primary sources of Ca2+ release. Lysosome-induced Ca2+ oscillations were sustained by endoplasmic reticulum (ER) Ca2+ content, while bafilomycin A1 and ML-SA1 did not directly interfere with ER Ca2+ homeostasis per se. TRPML1 activation strongly increased AQP2 apical expression and depolymerized the actin cytoskeleton, thereby boosting water flux in response to an hypoosmotic stimulus. These effects were strictly dependent on the activation of the Ca2+/calcineurin pathway. Conversely, bafilomycin A1 led to perinuclear accumulation of AQP2 vesicles without affecting water permeability. Overall, lysosomal Ca2+ signaling events can be differently decoded to modulate Ca2+-dependent cellular functions related to the dock/fusion of AQP2-transporting vesicles in principal cells of the CD.Lysosomes are acidic Ca Lysosomes play key roles in the molecular machinery that enables normal renal physiology. One critical function of lysosomes is the catabolism of low-molecular-weight proteins that freely cross the glomerular filter into the lumen of the nephron. These small proteins are immediately endocytosed by the cells of the proximal tubules with a receptor-dependent mechanism and targeted to the lysosomes for degradation, thus avoiding their waste with urine excretion . A seconThe importance of lysosomes in renal physiology is also highlighted by the evidence that diseases involving abnormal lysosomal functionality, such as lysosomal storage disorders (LSD), often manifest severe renal phenotypes . For ins2+ (0.5 mM) into cellular organelles with the sole exception of the endoplasmic reticulum (ER) [2+ within lysosomes still puzzles scientists. On one hand, the lysosomal matrix can be refilled with Ca2+ in a pH-dependent manner owing to the presence of a putative H+− Ca2+ exchanger [2+ uptake through the extracellular space via endocytosis or store-operated Ca2+ entry seems to be the most reasonable scenario [2+ could also be redirected towards the lysosomal matrix via inositol-1,4,5-trisphosphate receptors (InsP3Rs) [2+ by gating two pore channels TPC1 and TPC2 [2+ stores via TPC1 and TPC2 has long been known to evoke ER-dependent Ca2+ release and, therefore, shape intracellular Ca2+ signals in different cell types [2+ release events into global Ca2+ elevations to finely tune distinct physiological processes, such as membrane fusion and vesicular trafficking [2+ release events triggered by local lysosomal Ca2+ mobilization via TRPML1 [2+ signaling often associates with life-threatening diseases, such as cancer [2+ signaling in full.This study was inspired by recent discoveries that led to a re-evaluation of the mere digestive functions of this organelle, which is now regarded as a sophisticated regulatory hub that integrates multiple signals to finely tune different cell functions, such as nutrient sensing, intracellular signaling, vesicle trafficking and cellular growth . The mullum (ER) . The accxchanger ,12,13. Oscenario . HoweverInsP3Rs) . As descand TPC2  and Tranand TPC2 , respectll types ,20,21,22fficking  and prolfficking . Conversa TRPML1 ,26. Of ns cancer , viral is cancer , hypertes cancer  and lysos cancer ,30,31, u2+ signaling in renal collecting duct cells by either blocking the vacuolar H-ATPase (V-ATPase) with bafilomycin A1 [2+ pool or activating TRPML1 with the synthetic agonist ML-SA1 [2+ oscillations sustained by ER Ca2+ release through Ins3PRs. In addition, we aimed at understanding whether lysosomal Ca2+ release drives AQP2-dependent water reabsorption. Of note, we showed that ML-SA1 and bafilomycin A1 differentially affected AQP2 translocation to the apical membrane and actin polymerization in the cytosol, and that only ML-SA1 induced maximal water reabsorption in collecting duct cells. In our experiments, even though ML-SA1 was strikingly able to increase water permeability to an extent similar to that induced by submaximal doses of the cAMP increasing agents forskolin and IBMX, TRPML1 activation did not affect the cAMP/PKA pathway. Conversely, TRPML1-dependent AQP2 translocation and actin depolymerization were inhibited by buffering intracellular Ca2+ changes with BAPTA or blocking the Ca2+-dependent phosphatase calcineurin (CaN) with cyclosporine A. Finally, ex vivo stimulation of freshly isolated mouse kidney slices with the same dose of ML-SA1 confirmed the existence of the same modulatory action of TRPML1 activation on AQP2 accumulation even in a more complex physiological system.Therefore, the aim of this work is to provide the first evaluation of lysosomal Camycin A1  to deplet ML-SA1 . We foun2+-sensitive fluorophore, Fura-2-AM, each M1 cell showed asynchronous Ca2+ oscillation in terms of frequency and amplitude  cells are densely packed with acidic endosomes/lysosomes, as evident by the positive loading with the weak base of the Lysosome Staining Kit (LSK) A. Lysosomplitude C.2+ coupling, as reported for inner medullary collecting duct cells [2+ oscillations ceased after the removal of extracellular Ca2+, indicating a high resting membrane permeability for Ca2+. In agreement with this observation, the prompt decrease in fluorescence observed at the isosbestic point for Fura-2 (360 nm) when extracellular Ca2+ was replaced with Mn2+ hints at the presence of a constitutive Ca2+ entry pathway . Parallel measurements of cytosolic Ca2+ concentration in cells loaded with Fura-2 showed that acute addition of 200 µM GPN induced, in about 70% of the cells, asynchronous Ca2+ transients that were qualitatively compared in each experiment with 100 μM ATP-induced Ca2+ increases . This effect has been previously associated with the fact that GPN is a weak base that can potentially alkalize the cytosol, thereby inducing ER-dependent Ca2+ changes unrelated to lysosomal membrane permeabilization and lysosomal Ca2+ release [2+ and that the Ca2+ signaling events evoked upon activation of lysosomal Ca2+ channels were specifically inhibited by GPN via lysosomal permeabilization [To dissect lysosomal Cal matrix ,21,22. Ial cells . This weal cells . In acconcreases B. Note t release . Howeverlization .2+ signaling events in M1 cells, we decided to avoid the use of GPN and, therefore, selected two alternative strategies to induce lysosomal Ca2+ release: (i) we alkalized the intravesicular pH using a specific blocker of the V-ATPase, bafilomycin A1 [2+-permeable TRPML1 channel, which is localized in the late endosomal and lysosomal compartment [p = 0.0001), but it did not change the cytosolic pH . Conversely, perfusion with ML-SA1 in free Ca2+ induced Ca2+ oscillation in a reduced number of cells . The apparent discrepancy between these findings could be due to the requirement of spontaneous Ca2+ oscillations for extracellular Ca2+  and ML-SA1  without affecting the response induced by ATP (data not shown). In addition, pretreatment with ML-SI1, a specific antagonist of TRPML1 that did not affect Ca2+ homeostasis per se [2+ oscillations (p = 0.0062). This finding clearly confirms the specificity of ML-SA1 as a synthetic agonist for TRPML1. To further investigate if Cas per se , signifi2+-signaling events mediated by the activation of TRPML1 are reported in collecting duct cells. Taken together, these findings show for the first time that lysosomal Ca2+ release may be sustained by either lysosomal Ca2+ uptake from the ER [2+ mobilization through the “Ca2+-induced Ca2+ release” (CICR) mechanism, which results in the appearance of regenerative Ca2+ waves [2+ oscillation after agonist removal suggests that similar mechanisms might take place in the collecting duct [2+ sequestration into ER lumen and depletes the ER Ca2+ content. Pretreatment with 40 μM CPA for 20 min in the absence of extracellular Ca2+ is sufficient to empty the InsP3-sensitive ER Ca2+ store since subsequent addition of ATP did not increase the intracellular Ca2+ level in M1 cells  and ML-SA1 (p = 0.0064), clearly indicating the presence of a functional coupling between lysosomes and ER, as previously reported [2+ release by either CPA or 2-APB strongly suggests that tonic ER Ca2+ release through InsP3Rs is required to refill lysosomes with Ca2+ [On the other hand, we can safely exclude a direct effect of bafilomycin A1 and ML-SA1 on the ER Careported ,45. The ith Ca2+ ,45.2+ increase and cAMP production can promote the fusion of intracellular vesicles harboring AQP2 with the plasma membrane [2+ allows the accumulation of AQP2 on the apical plasma membrane, thereby increasing membrane water permeability. Therefore, we evaluated if the activation of local lysosomal Ca2+ release events could modulate AQP2 accumulation at the plasma membrane of M1 cells stably expressing AQP2 (known as MCD4 cells). The panel in It is known that vasopressin-induced intracellular Camembrane ,47,48. I2+ signaling events, redistributed AQP2 in a perinuclear region without affecting phalloidin staining compared to control conditions (bafilomycin A1). The same perinuclear distribution of AQP2 under bafilomycin A1 stimulation was reported by the group of Dennis Brown in LL-CPK1 cells [What we found even more interesting is that bafilomycin A1, which exerted similar CaK1 cells .AQP2 translocation at the plasma membrane should be matched by a change in the rate of M1 cell swelling measured as calcein quenching in response to hypotonicity. As shown by the representative traces A and by 2+ from lysosomes is not the only determinant involved in AQP2 translocation/actin depolymerization/water reabsorption since ML-SA1 and bafilomycin A1 exert opposite effects, and (2) ML-SA1 share with a submaximal dose of forskolin a strong ability in activating both AQP2 membrane translocation and the increase in water permeability. The idea that the local TRPML1 activation  could significantly affect a tissue-defining cellular function of the collecting duct, such as AQP2-mediated water reabsorption, is completely unexplored. Therefore, at first, we tested whether the activation of TRPML1 could be associated with increases in the cAMP level and/or PKA activity and whether the antidiuretic effect of TRPML1 relied on PKA. Therefore, we first transfected M1 cells with Epac-S-H187, a loss of FRET cAMP probe that reports the increase in the intracellular cAMP as a reduction in the FRET emission ratio [All together, these pieces of evidence underline that (1) the discharge of Caon ratio . As showon ratio . Again, 2+ release. It is well known that lysosomal calcium efflux through the lysosomal Ca2+ channel TRPML1 can activate the Transcription Factor EB (TFEB) through CaN-dependent dephosphorylation [2+/calmodulin-dependent serine/threonine protein phosphatase that regulates cytoskeleton organization in different cell types, such as neurons, myocytes, podocytes and renal tubular cells. Of note, the control of cytoskeleton organization can be exerted by CaN in two ways: a direct control of filament organization through dephosphorylation of cytoskeletal organizing proteins  and a long-lasting transcriptional effect mediated by the nuclear factor of activated T-cells (NFAT).We next wondered whether the increased amount of AQP2 localized at the apical membrane could be the direct result of the depolymerization of the actin cytoskeleton that we observed upon TRPML1-induced lysosomal Carylation . CaN is 2+-dependent. Pretreatment with the Ca2+ chelator BAPTA-AM, that did not change AQP2 intracellular distribution and the polymerization state of the actin cytoskeleton per se (2+ release induced by ML-SA1 in the presence of extracellular Ca2+ (data not shown). In addition, cyclosporine A (CsA), which is a selective CaN inhibitor [2+/CaN pathway. In addition, to further address whether CaN also mediates the TRPML1-induced nuclear translocation of NFAT, M1 cells were transfected with NFAT-GFP. Then, its subcellular localization, expressed as the ratio between the number of cells expressing the probe in the nucleus . As shown in 2+-releasing activity of the endo/lysosomal non-selective cation channel TRPML1 on the AQP2-mediated water handling of the collecting duct (CD) in the absence of vasopressin stimulation.This study highlights the relevance of the Ca2+ mobilization. Consequently, these vesicles dock and fuse with the apical membrane of principal cells, increasing AQP2 accumulation and water permeability so that the CD can significantly reabsorb water and concentrate urine. The transcription factors, cAMP-responsive binding protein (CREB) and CaN-dependent NFAT, are also associated with AQP2 homeostasis during antidiuresis [AQP2 redistribution back and forth the apical membrane of CD cells relies on the activation of a multifactorial signaling network. Once activated by AVP, V2R increases cytosolic cAMP levels which in turn activate PKA and stimulate PKA tethering to AQP2-containing vesicles, thereby leading to AQP2 phosphorylation. In addition, vasopressin triggers intracellular Cadiuresis .2+ signaling events elicited specifically by TRPML1 activation also exert significant effects on in vitro and ex vivo AQP2 accumulation at the apical membrane of principal cells lining the CD P2) or synthetic ligands (like ML-SA1) [2+ signaling events locally regulate various lysosomal functions, including lysosomal exocytosis, membrane trafficking and lysosomal biogenesis [2+, we found that intracellular Ca2+ level either oscillates or undergoes a persistent increase until agonist removal  . TRPML1-ogenesis ,59. Unli removal . If the  release ,24, a ra pattern . Whether2+ compartment in renal cells is likely to be refilled by the ER, as also reported in other cell types [2+ release events elicited by both bafilomycin A1 and ML-SA1  and a long-lasting transcriptional effect mediated by NFAT [c14 murine collecting duct principal cells, where AQP2 expression is regulated by the Ca2+/CaN-NFAT pathway in response to intracellular Ca2+ signals evoked by hypertonicity and other extracellular stimuli [Lysosomal Ca by NFAT . For ins stimuli .2+ signaling events are significantly influenced by intracellular pH changes [2+ release. The weak base GPN was firstly used as a lysosomotropic agent to evaluate lysosomal Ca2+ storage and signaling events in renal cells [2+ events has been limited in this study because it alkalizes the intracellular pH potentially leading to the onset of intracellular Ca2+ responses completely unrelated to lysosomes, as already demonstrated in primary cultured human fibroblasts [2+ transients likely associated with lysosomal matrix alkalization , (cat no. D9754), ammonium chloride (cat no. 213330), adenosine 5′-triphosphate , cyclopiazonic acid (cat no. C1530), cyclosporin A (cat no. C3662), 1-deamino-8-arginine vasopressin , ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid , GW405833 , H-89 (cat no. B1427), 3-isobutyl-1-methylxanthine , ML-SA1 (cat no. SML0627) and Nigericin (cat. # N7143) were from Merck KGaA . Bafilomycin A1 (cat no. S1413) was from Selleckchem . BAPTA AM (cat no. B1205), BCECF AM (cat no. B1170), Fura-2 AM (cat no. F1221), Lipofectamine 2000 (cat no. 18324012), Phalloidin-488 (cat no. A12379) and Calcein-AM (cat no. C1430) were from Thermo Fisher Scientific . Forskolin (cat no. FOR010b) was from Fermentek . Glycyl-N-2-naphthalenyl-L-phenylalaninamide  was from Cayman , and the Lysosome Staining Kit was from Abnova . The rabbit affinity-purified polyclonal antibody against human AQP2 was previously described [Immortalized mouse cortical collecting duct M1 cells  and MCD42+ evaluations were performed with the ratiometric dye Fura-2 AM and intracellular pH changes were recorded using the ratiometric dye BCECF AM. Cells were loaded with either 4 µM Fura-2 or 2.5 µM BCECF for 15–30 min at 37 °C and then rinsed with saline solution to wash out dye retained extracellularly. Coverslips with dye-loaded cells were analyzed with a set-up described in detail in previous papers of our group [2+-free experiments were carried out in a Ringer’s solution without Ca2+ with the addition of 50 µM EGTA. Fura-2 and BCECF ratios were acquired using a ratio imaging set-up running Metafluor software . Each coverslip, mounted in an open-top perfusion chamber, was placed on the heated stage of a Nikon TE200 inverted microscope . For Fura-2 experiments, cells were excited alternately at 340 nm and 380 nm through a 40×  oil immersion objective. For BCECF experiments, cells were excited alternately at 440 nm and 490 nm. The excitation wavelengths were generated using a monochromator system in the path of a 75-W xenon light source. Pairs of fluorescence images for both dyes (emission collected at 520 nm) were captured by a cooled CCD camera CoolSNAP HQ  every 5 s and converted to a ratio image by the Metafluor software. We used a specific positive internal control at the end of each run. For Fura-2 experiments, cells were stimulated with a submaximal dose of ATP (100 µM), and ammonium chloride was used to induce maximal pH changes in BCECF-loaded M1 cells. To analyze Ca2+ membrane resting permeability, an Mn2+ quench experiment was performed [2+ entered cells through Ca2+ pathways and quenched Fura-2 fluorescence. In the presence of Mn2+, the quenching of Fura-2 fluorescence excited at the Ca2+-insensitive wavelength of 356 nm can be considered as evidence of Ca2+ influx. Finally, the same set-up was used to acquire images of the acidic endo-lysosomal system of M1 cells. The Lysosome Staining Kit (LSK) is a permeable hydrophobic compound that visualize the acidic compartment of live cells in orange fluorescence (Ex/Em = 575/600 nm). LSK enhances its fluorescence upon entering the lysosome, while a reduction in the lysosomal pH gradient causes a drop in the fluorescence signal. Therefore, transmitted light images of cells were used to draw the region of interest corresponding to positive staining to gain information regarding LSK fluorescence as an index of lysosomal pH acidity. The probe was loaded in M1 cells following the manufacturer’s instructions. For calculation of the slope upon treatments with lysosomal agents, the data recorded were fitted to a line using non-linear regression analysis. Slopes are reported in the result section as mean ± SEM.Cells were seeded on glass coverslips (Ø 15 mm). Cytosolic Caur group ,66,67,68erformed . Mn2+ enm) and the number of responsive/total cells (n). For each experiment, the fluorescence signal acquired was normalized to the basal fluorescence recorded in the absence of the stimulus. Significant differences were calculated by Student’s t-test for paired or unpaired data.Regardless of the probe used in our experiments, we decided to show traces of 5 out of the total recorded cells for each protocol. Figure legends report the number of times each protocol has been repeated  and AKAR4 for PKA activity  were gifts from Kees Jalink and Jin Zhang, respectively. Cells were seeded on Ø 15 mm glass coverslips and transiently transfected with Epac H187 or AKAR4 sensors using Lipofectamine® 2000. Cells were mounted in a perfusion chamber and imaged using 40XPlan Fluor (NA 1.30) oil immersion objective lens. Ringer’s solution was used to perfuse cells during the experiment, containing 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 10 mM HEPES, 5 mM glucose and 1.0 mM CaCl2 at pH 7.4. Real-time FRET imaging experiments were performed at room temperature using a fluorescence ratio imaging system built around a Nikon Eclipse TE2000-S inverted fluorescence microscope  equipped with a cooled CCD camera CoolSNAP HQ . MetaFluor software was used to acquire the FRET emission ratios  every 5 s. The trace reported in each figure depicts the mean emission ratio ± SEM of cells in the same coverslip. The figure legend reports the number of times each protocol has been repeated (m) and the number of responsive/total cells (n).The FRET-based sensors Epac H187 for intracellular cAMP  containing each treatment (as described above). Fluorescence signal changes in calcein-loaded glial cells after hypotonic gradient were recorded on a Flex Station3 plate reader  equipped with an integrated automatic liquid handling module, as previously described [A total of 40,000 MCD4 cells were seeded in each well of a 96-well black, clear bottom Corning microplates  and grown to 90% confluence. Cells were treated overnight with indomethacin 50 µM. The day after, cells were loaded with 10 μM calcein AM  dissolved in complete medium with indomethacin for a total time of 45 min at 37 °C. After 15 min, during calcein incubation, cells were left untreated (CTR) or stimulated for the remaining 30 min as follows: 5 µM FK+ 500 µM IBMX; 100 µM ML-SA1; 25 µM ML-SI, 100 nM bafilomycin A1. When we used both the agonist and the antagonist in combination, ML-SI1 was added to the incubation mixture 20 min before ML-SA1. Cells were then rinsed in isosmotic PBS  were used for kidney slice experiments as previously reported [v/v) and killed by cervical dislocation. Kidneys were collected and thin transversal slices (250 μm) were obtained using a Mcllwain Tissue Chopper . Slices were left at 37 °C in Dulbecco’s modified eagle/F12 medium (CTR) or stimulated for 30 min with dDAVP (100 µM) or ML-SI1 (25 µM) or ML-SA1 (100 µM), the latter given alone or after 30 min of preincubation with ML-SI1. Slices were fixed in 4% paraformaldehyde, and then treated an analyzed as previously described [Sex-, age- and weight-matched C57BL/6J male mice  for 30 sec at 4 °C and insoluble material was pelleted at 13,000× g for 30 min at 4 °C. Then, 20 μg of supernatant was separated by standard SDS-PAGE using Mini-PROTEAN® TGX Stain-Free™ Precast Gels Bio-Rad. The immunoreactive bands were detected with a ChemiDoc™ System .M1 cells were seeded on a 6-well culture plate. Cells were washed with ice-cold PBS and scraped with RIPA buffer  containing protease and phosphatase inhibitors . Lysates were then sonicated at 60 amplitude with the microprobe sonicator Vibra-cellt-test for unpaired data (Fura-2 and LSK experiments) or paired data (BCECF experiments). We conducted ordinary one-way ANOVA followed by the Dunnett’s multiple comparison test to compare stimulated vs. control condition depending on the dataset .GraphPad Prism 8 was used for statistical analysis. Whenever needed, statistical analysis was performed using Student’s 2+ signaling events mediated by TRPML1 as a new actor in the regulation of AQP2-mediated water homeostasis.Collectively, our results point to local lysosomal Ca


Clinical examination showed that one of the characteristics of vvIBDV-infected chickens was severe metabolic disorders, and the chemical examination showed that their serum Ca2+ level decreased significantly. However, there are limited studies on how vvIBDV infection modulates the cellular Ca2+ level and the effect of Ca2+ level changes on vvIBDV replication. In our study, we found Ca2+ levels in the endoplasmic reticulum (ER) of vvIBDV-infected B cells were higher than that of mock-infected cells, and the expression level of stromal interaction molecule 1 (STIM1), an ER Ca2+ sensor, was significantly upregulated due to vvIBDV infection. The knock-down expression of STIM1 led to decreased Ca2+ level in the ER and suppressed vvIBDV replication, while the over-expressed STIM1 led to ER Ca2+ upregulation and promoted vvIBDV replication. We also showed that the inhibition of Ca2+-release-activated-Ca2+ (CRAC) channels could reduce vvIBDV infection by blocking Ca2+ from entering the ER. This study suggests a new mechanism that STIM1 promotes the replication of vvIBDV by mobilizing Ca2+ in the ER.Infectious bursal disease virus (IBDV) is one of the main threats to the poultry industry worldwide. Very virulent IBDV (vvIBDV) is a fatal virus strain that causes heavy mortality in young chicken flocks. Ca Chickens and eggs are important sources of human food and are extensively consumed worldwide ,2. Howev2+ content of the serum i, was measured by using the fluorescent Ca2+ indicator fura-2/AM  i was calculated by the following equations:380T and F380E are the emission fluorescence values at 380 nm excitation in the presence of Triton X-100 and EGTA, respectively. The equilibrium dissociation constant (Kd) for the Ca2+-fura-2 complex was 278 nM. Increases in fura-2 fluorescence indicated increased intracellular free calcium levels, because cellular cytosolic esterases cleave the membrane permeant ester group off the fura-2/AM derivative and leave the membrane-impermeant fura-2 trapped in the cytosol. The binding of Ca2+ shifts the absorbance spectra to shorter wavelengths (from 380 to 340 nm).Intracellular free Ca2+-ATPase pump)  was used to release Ca2+ from ER stores. A Ca2+-ATPase pump in ER membrane transports Ca2+ from the cytoplasm back into ER. When the ATPase pump in the ER is inhibited by TG, Ca2+ that leaks from the ER is not re-sequestered by the pumps, and Ca2+ accumulates in the cytosol. The TG-induced Ca2+ increase in cytosol is thought to represent Ca2+ released from the ER stores i was measured as described above.According to , thapsigR stores ,27. Brie2+ influx was used to measure plasmalemma permeability in vvIBDV infected DT40 cells, and 2-APB treated DT40 cells. Ba2+ is a Ca2+ surrogate that is transported across the cytoplasmic membrane through Ca2+ channels but is not a substrate for the carriers and pumps that normally transport Ca2+ across cell membranes [2+ will bind to fura-2 and change fluorescence in a fashion analogous to Ca2+ [2+-free HBSS immediately before measurements were made. Then, 10 mM Ba2+  was added to the cells 2 min later. The fluorescence was measured at excitation wavelengths of 350 and 390 nm. An increased Ba2+ influx was indicated by an increase in the 350/390nm fluorescence ratio.According to , Ba2+ inembranes . However to Ca2+ . Briefly6 cells were resuspended with 1640 complete medium. Inoculated cell suspension (100 μL/well) in a 96-well plate (repeated each sample 3 times), and added 10 μL CCK-8  solution to each well of the plate. Next, incubated the plate for 2 h at 37 °C away from light. Finally, measured the absorbance at 450 nm using a microplate reader .Synta66  was freshly prepared from stock solutions in DMSO. First, DT40 cells were pre-treated with Synta66 for a period of time at specific concentrations, then 1.5 × 102 to let viruses enter the cells; then, the virus inoculum was removed. Synta66 was diluted in DMSO and added to cells at indicated concentrations. An equivalent percentage of DMSO was used as vehicle control. Cells were then incubated at a 41 °C, 5% CO2 incubator for 48 h.DT40 cells were infected with Gx strain of vvIBDV at an MOI of 5 for 4 h at 41 °C, 5% COThe specific-pathogen-free (SPF) chickens were purchased from the State Resource Center of Laboratory Animal for Poultry . This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Ministry of Science and Technology of China. The use of SPF chickens and animal experiments were approved by the Animal Ethics Committee of Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences and performed in accordance with the animal ethics guidelines and approved protocols (SYXK (Hei) 2017-009).n = 19) and the vvIBDV challenged group (n = 19). The mock group was received 200 μL PBS via the ocular and intranasal routes as the negative control; the challenged group was infected with 103 ELD50 (200 μL) of Gx strain of vvIBDV via the same route. From 1 to 3 d p.i., three chickens randomly selected each day from all groups were euthanized for necropsy and examination of pathological changes. The remaining chickens in each group were used to calculate the survival rate. Some parts of bursae at 3 d p.i. were fixed immediately in 10% neutral buffered formalin and were stained with haematoxylin and eosin for further histopathological, and some parts at 2 d p.i. were used for electron microscope examination. Serum was collected to test Ca2+ concentration, which was performed in Third Affiliated Hospital, Heilongjiang University of Chinese Medicine.3-week-old SPF chickens were randomly divided into two groups, the mock group .Statistical analysis was performed with the unpaired 2+ in the serum and found the Ca2+ concentration of infected chicken decreased by 45.72% at 3 d p.i. . Our results showed that Ca2+ levels in the cytoplasm remained stable, while Ca2+ levels in the ER increased in a dose-dependent manner in vvIBDV-infected cells  to detect cell membrane permeability. As a positive control for measuring changes in the membrane permeability, 0.02% Triton X-100 was used. Our results showed that, in mock and infected groups, the addition of Ba2+ to cells incubated in Ca2+-free HBSS buffer led to an insignificant increase in the fluorescence ratio, and their Ba2+ influx was much lower than the positive control. These results suggest that the cell membrane permeability does not differ between vvIBDV-infected cells and mock-infected cells (p > 0.05), and changes in cell membrane permeability are not responsible for increased ER Ca2+ levels observed in vvIBDV-infected cells.To rule out the cell membrane permeability change as the cause of intracellular Ca2+, we performed iTRAQ labelling of differentially expressed proteins in vvIBDV-infected and mock-infected B cells. Our results showed that viral infection led to overexpression of many host proteins, among which we found STIM1 to be of particular interest as STIM1 modulates ER Ca2+ levels directly , pCAGGS-HA-GxVP3 (756-1012 aa), pCAGGS-HA-GxVP4 (513-755 aa), and pCAGGS-HA-GxVP5, followed by co-immunoprecipitation (CO-IP) assays with anti-Flag antibody, and WB with anti-Flag, anti-MYC, or anti-HA monoclonal antibodies. Results showed that when lysates of cells expressing both HA-GxVP2 and Flag-STIM1 were immunoprecipitated with anti-Flag antibody, pCAGGS-HA-GxVP2 was detected in the precipitate. Similarly, in cells expressing both HA-GxVP4 and Flag-STIM1, the HA-GxVP4 was detected in the precipitate. These results indicated that both VP2 and VP4 interacted with STIM1 A,D. OtheTo determine the subcellular localization of VP2/VP4 and STIM1, HEK293T cells were transfected to express HA-GxVP2/HA-GxVP4 and Flag-STIM1. After 24 h, cells were fixed and incubated with mouse anti-HA antibody, rabbit anti-Flag antibody, and DAPI, and analysed by confocal microscopy. We observed complete co-localization of VP2 with STIM1 C, whereaTo study whether vvIBDV infection activates STIM1 and CRAC channels, we performed Airyscan confocal microscopy assays to observe whether STIM1 shifts due to vvIBDV infection. Briefly, DT40 cells were infected by vvIBDV (MOI = 10) for 24 h. Following this, the fixed cells were incubated with mouse anti-VP2 antibody, rabbit anti-STIM1 antibody, and DAPI and analysed using Airyscan confocal microscopy. The results showed that in mock-infected cells, STIM1 was evenly distributed in the cytoplasm. However, vvIBDV-infection led to redistribution and oligomerization of STIM1 in the cell membrane of infected cells . These r2+ levels and the replication of vvIBDV [2+ levels were checked in DT40 cells after infected with vvIBDV (MOI = 5), followed by Synta66 treatment for 24 h. The results showed that Synta66 treatment significantly decreased ER Ca2+ levels in a dose-dependent manner , indicating that change in cell membrane permeability was not responsible for the decrease in Ca2+ levels observed in Synta66 treated cells.To further confirm that CRAC channels were activated in vvIBDV infection, we used Synta66  to evaluate the inhibition of CRAC channels on the Caf vvIBDV . First, t manner B. To rulshown in C, the ad50 assays. The results showed that Synta66 treatment  of infected cells resulted in 15-fold, 28-fold, and 31-fold decreases in extracellular viral yields at 48 h p.i.  resulted in a respective 3-fold, 11-fold, and 21-fold decrease in the relative VP5 RNA transcript D. At the8 h p.i. G,H. In c2+ concentration in the blood serum is tightly regulated, and any abnormalities in serum Ca2+ level are associated with many dangerous diseases [2+ levels in chickens’ serum. This result was consistent with previous research that Cosgrove found the most prominent finding in the chemical constituents of IBDV-infected chickens was the serum’s low Ca2+ content [2+ homeostasis would severely affect the normal physiological function of the host [2+ is related to some major diseases in humans, such as cardiac disease, schizophrenia, bipolar disorder, and Alzheimer’s disease [2+ levels were also found to be significantly correlated with dengue severity [2+ levels in vvIBDV-infected chickens.The Cadiseases . Our res content . Such a the host ,33,34. T disease . Serum Cseverity . Hence, 2+ levels and found that the Ca2+ levels of the ER in vvIBDV-infected cells were significantly higher than in mock-infected cells. In the normal resting state of cells, the Ca2+ concentration inside and outside the cell maintains a dynamic balance [2+ homeostasis of the host to fulfil their life cycle, such as coxsackievirus, human cytomegalovirus, and rotavirus [2+ in the IBDV life cycle [2+ homeostasis of the host, which might perform essential roles in the viral life cycle and pathogenesis.Cells live in extracellular fluid, of which serum is a part . Thus, w balance ,14,16. Hotavirus ,39,40. Pfe cycle . Taken t2+ levels. For example, constitutive STIM1 activation observed in rotavirus-infected cells reflects an effect of its non-structural protein 4 on ER Ca2+ permeability [2+ signalling, triggered by viral activation of STIM1 and Orai1, is among the key host mechanisms that orchestrate the later stages of filovirus and arenavirus assembly and budding [2+ levels were caused by vvIBDV infection. Our results showed that vvIBDV infection induced upregulation of endogenous STIM1 protein levels. STIM1 overexpression has been reported to play important roles in the progression of many severe diseases, including several malignancies, such as pancreatic, breast, and cervical cancer [2+ regulation and vvIBDV replication, we examined the effects of overexpression and knockdown of STIM1. Our results showed that overexpression of STIM1 increased Ca2+ levels in the ER, which improved vvIBDV replication, while knockdown of STIM1 decreased Ca2+ levels in the ER, which suppressed vvIBDV replication. In conclusion, we demonstrate that vvIBDV infection upregulates the expression of STIM1, and the elevated expression of STIM1 leads to increased Ca2+ levels in the ER, which facilitates vvIBDV replication. It suggests that increased Ca2+ levels in the ER are not related to Ca2+ store depletion but are related to the upregulated expression of STIM1 caused by vvIBDV infection. Studies have shown that except the decreased Ca2+ levels in ER can activate STIM1, STIM1 can also be activated by other factors, such as oxidant stress and temperature, which does not depend on Ca2+ store depletion [The results of iTRAQ showed that the expression level of STIM1 significantly increased after vvIBDV infection. To date, several studies have suggested that many viral infections result in STIM1-mediated changes in Caeability . Ziying  budding . Therefol cancer ,44,45. Tepletion ,47. Our 2+ levels in ER is unknown. Studies have shown that increased expression levels of STIM1 correlated with a gain in function of CRAC channel activity [2+ influx was observed [2+ levels in a STIM1-dependent manner and identify the viral proteins that interact with STIM1. CO-IP results indicated that VP2 and VP4 interacted with STIM1. It has been extensively studied that, in resting cells, STIM1 and Orai1 distribute independently in the ER and PM, respectively. However, activated STIM1 proteins oligomerize and accumulate at ER-PM junctions where it binds, traps, and opens CRAC channels for extracellular Ca2+ to enter the ER stores [2+ storage is emptied, subsequent steps will take place [2+ entry [2+ entering the ER, resulting in the decrease in ER Ca2+ levels that impaired vvIBDV replication. This result can strongly demonstrate the previous conclusion that vvIBDV infection activated CRAC channels and allowed extracellular Ca2+ to enter the ER through CRAC channels. Furthermore, since CRAC channels are ubiquitously expressed, and like ion channels in general, they represent potentially pharmacologically accessible (cell surface) therapeutic targets with application in severe diseases with Ca2+ imbalance [Our results demonstrate that STIM1 plays a crucial role in vvIBDV–host cell interaction, but how this interaction influences Caactivity , while iobserved . Thus, wR stores ,29. The R stores ,51. STIMke place . In our 2+ entry . Our resmbalance ,51.2+ signals triggered by the activation of STIM1 orchestrate the steps of vvIBDV replication. Viral proteins VP2 and VP4 interact with STIM1 and activates STIM1, leading to the opening of CRAC channels, which facilitates the entry of the extracellular Ca2+ into the ER. Indeed, this process may contribute to the decrease in serum Ca2+ level severe disturbance of Ca2+ homeostasis, which is worthy of further study in the future. Indeed, the pathogenesis of vvIBDV is quite complicated, and even metabolic disorder is the result of a combination of factors, such as difficulty feeding in late-infected chickens. Our study may provide new insights in the study of vvIBDV pathogenesis.In conclusion, our study provides the first direct evidence that host Ca


While the underlying genetic alterations and biology of acute myeloid leukemia (AML), an aggressive hematologic malignancy characterized by clonal expansion of undifferentiated myeloid cells, have been gradually unraveled in the last decades, translation into clinical treatment approaches has only just begun. High relapse rates remain a major challenge in AML therapy and are to a large extent attributed to the persistence of treatment-resistant leukemic stem cells (LSCs). The Hedgehog (HH) signaling pathway is crucial for the development and progression of multiple cancer stem cell driven tumors, including AML, and has therefore gained interest as a therapeutic target. In this review, we give an overview of the major components of the HH signaling pathway, dissect HH functions in normal and malignant hematopoiesis, and specifically elaborate on the role of HH signaling in AML pathogenesis and resistance. Furthermore, we summarize preclinical and clinical HH inhibitor studies, leading to the approval of the HH pathway inhibitor glasdegib, in combination with low-dose cytarabine, for AML treatment. Within the supporting bone marrow microenvironment HSCs are maintained in a delicate balance between self-renewal and differentiation to ensure life-long steady-state hematopoiesis and HPC and effector cell replenishment upon, e.g., blood loss or infections . Upon trc blasts . These sc blasts . Due to c blasts . In thisThe HH signaling pathway, initially discovered as a regulator of segment polarity in the fruit fly, is a highly conserved signaling cascade that regulates several aspects of embryonic development while being largely silenced in adult tissues except for i.e., stem and progenitor cell maintenance, metabolic control, inflammatory processes and tissue repair after injury . AberranR), which translocates to the nucleus and inhibits HH-induced target gene expression , Indian hedgehog (IHH) or Desert hedgehog (DHH), to the twelve-transmembrane domain receptor Patched (PTCH) . In an upression   forms. Eventually, GLIA shuttles to the nucleus where it acts as a transcription factor and induces the expression of HH target genes including regulators of cell differentiation, survival and proliferation as well as components of the HH/GLI pathway itself, such as GLI1 and PTCH1  mice.Hematopoietic stem and progenitor cells (HSPCs) isolated from human newborn cord blood, frequently termed primitive HSPCs, express PTCH1, SMO, SHH, IHH, GLI1, GLI2 and GLI3 mRNA . While fPtch1 deletion (Ptch1+/−), which results in increased HH pathway activity, revealed an expansion of HPCs in the bone marrow under steady-state conditions compared to wild-type littermates. When treated with 5-fluorouracil, which depletes HPCs and effector cells, thereby leading to a strong activation and proliferation of HSCs  is a heterogeneous clonal hematologic disorder characterized by multiple cytogenetic and molecular abnormalities leading to the production and accumulation of undifferentiated myeloid progenitors, so-called leukemic blasts, which displace the normal hematopoietic system in the bone marrow . The estferative . This phThe early clinical manifestation of AML is directly attributable to the loss of functional hematopoietic effector cells with patients exhibiting fatigue, pallor, anemia, susceptibility to infections, easy bruising, or hemorrhage. Secondary organ infiltration of leukemic cells provokes the development of additional symptoms, such as splenomegaly, hepatomegaly, or lymphadenopathy . AML is The standard induction therapy for young and/or fit AML patients has remained largely unchanged for the last decades and consists of an intense chemotherapy with 7 days of cytarabine and 3 days of an anthracycline, such as daunorubicin or idarubicin (7 + 3 regimen), resulting in about 70% of patients <60 years and less than 50% of patients >60 years achieving complete remission (CR) . Once CR+ cells and/or bone marrow tissue. Consistent with AML heterogeneity, it was shown that the core components that mediate the HH signal response, PTCH1, SMO, GLI1, GLI2, and GLI3, are differentially expressed among AML patient samples , these cells were resensitized to irradiation by downregulation of the PI3K/AKT/NFκB and DNA repair pathways . This isfunction  but alsofunction . HMA decferation . Furtherferation   was the first HH pathway inhibitor approved by the FDA for the treatment of locally advanced or metastatic BCC in 2012 . A singltro data , no datain vitro assays demonstrated a synergistic effect between sonidegib and azacytidine in AML, combination treatment resulted in promising progression-free and overall patient survival rates but surprisingly did not enhance complete remission rates in AML patients in a phase I/Ib study (NCT02129101)  is the second SMO inhibitor approved for treatment of advanced BCC and is currently being investigated for treatment of other cancers . Althoug2129101) . In a phAfter clinical trials for the use of previously approved SMO inhibitors in AML therapy failed, glasdegib  in combination with low-dose cytarabine (LDAC) was the first SMO inhibitor to receive FDA approval for the treatment of AML at the end of 2018.As determined during safety and pharmacokinetic profiling of orally applied glasdegib, the maximum tolerated dose was 400 mg per day with a mean plasma half-life of 24 h. In light of the commonly observed AEs, such as taste distortion, decreased appetite and hair loss, the recommended dose was lowered to below 200 mg per day (NCT00953758) . CombinaThe combination of glasdegib with already established inhibitors targeting IDH1/2, BCL-2 or FLT3 in AML are obviously of high interest and have been incorporated into a clinical trial draft (NCT04655391). Unfortunately, this study was stopped prior to patient enrollment due to limited drug availability. An additional AML study investigating the combinational effect of glasdegib with, e.g., venetoclax on CR rate and mortality is currently recruiting patients (NCT03226418).Adapting AML treatment to a patient’s individual needs requires a growing arsenal of small molecule inhibitors, HMAs, and chemotherapeutic agents to simultaneously target multiple oncogenic pathways and tumor-promoting processes. As reviewed here, successful preclinical and clinical AML studies inhibiting HH signaling have recently led to the FDA approval of the SMO inhibitor glasdegib in combination with LDAC for the treatment of newly diagnosed and unfit AML patients. Ongoing clinical trials are further evaluating the benefit of SMO inhibitor combination therapies with HMAs, IDH inhibitors, tyrosine kinase inhibitors and/or pyrimidine analogues. These studies will reveal if and how AML patient subgroups benefit from HH inhibiting treatment approaches and position glasdegib and other HH inhibitors in the landscape of AML therapy. Furthermore, combination therapies targeting canonical and non-canonical HH signaling simultaneously are envisioned and might offer new therapeutic approaches preventing the development of drug resistance. Although the clinical efficacy of glasdegib is undeniable, the exact tumor targeting mechanism of HH signal inhibition in AML is still not clear. Whether the anti-leukemic effects are exclusively attributable to abrogating AML (stem) cell intrinsic HH signaling or to blocking HH signaling within the tumor supporting bone marrow microenvironment remains to be clarified and calls for a more detailed analysis of HH signaling components and regulators in AML patient-derived blasts, LSCs and bone marrow stromal and endothelial cells. This will also be crucial for the identification of biomarkers predictive for HH inhibitor response of AML patients.


A complex toolkit of Ca2+ pumps and exchangers maintains the fluctuation of cytosolic Ca2+ concentration within the appropriate threshold. Two ubiquitous (isoforms 1 and 4) and two neuronally enriched (isoforms 2 and 3) of the plasma membrane Ca2+ATPase (PMCA pump) selectively regulate cytosolic Ca2+ transients by shaping the sub-plasma membrane (PM) microdomains. In humans, genetic mutations in ATP2B1, ATP2B2 and ATP2B3 gene have been linked with hearing loss, cerebellar ataxia and global neurodevelopmental delay: all of them were found to impair pump activity. Here we report three additional mutations in ATP2B3 gene corresponding to E1081Q, R1133Q and R696H amino acids substitution, respectively. Among them, the novel missense mutation (E1081Q) immediately upstream the C-terminal calmodulin-binding domain (CaM-BD) of the PMCA3 protein was present in two patients originating from two distinct families. Our biochemical and molecular studies on PMCA3 E1081Q mutant have revealed a splicing variant-dependent effect of the mutation in shaping the sub-PM [Ca2+]. The E1081Q substitution in the full-length b variant abolished the capacity of the pump to reduce [Ca2+] in the sub-PM microdomain (in line with the previously described ataxia-related PMCA mutations negatively affecting Ca2+ pumping activity), while, surprisingly, its introduction in the truncated a variant selectively increased Ca2+ extrusion activity in the sub-PM Ca2+ microdomains. These results highlight the importance to set a precise threshold of [Ca2+] by fine-tuning the sub-PM microdomains and the different contribution of the PMCA splice variants in this regulation.Calcium concentration must be finely tuned in all eukaryotic cells to ensure the correct performance of its signalling function. Neuronal activity is exquisitely dependent on the control of Ca The finding that the ER Ca2+ content was not changed in cells overexpressing the wt pump compared to those overexpressing the mutant PMCA3a suggests that the mutation did not modify the ability of the pump to influence the ER Ca2+ filling or its basal level.The CaPA) Fig. , and Ca2ion Fig. . As show content , 29, 31,2+ handling, we applied a different protocol. We pre-depleted the intracellular Ca2+ deposits by treating cells for 5 minutes with thapsigargin and histamine in a medium containing 1 mM EGTA. Then, upon the addition of a solution containing 2 mM CaCl2 which induces fast Ca2+ entry from the extracellular ambient, we exclusively monitored the effect of PMCA on Ca2+ influx. As shown and quantified in Fig. 2+ and in the absence of SERCA pumps inhibitors . This was not surprising as under these conditions the massive opening of Ca2+ influx channels could easily overwhelm the PMCA activity. When the Ca2+ peaks declined, a plateau was reached , to further explore the role of the E1081Q mutation on the pump activity, the same mutation was introduced in the full-length (b) variant of the PMCA3 and its effects on Ca2+ extrusion activity of the pump further evaluated.To gain further insights into the role of the E1081Q mutation on the pump activity and having excluded a role of the ER Caors Fig.  and was hed Fig.  at about2+ pumping ability of the PMCA3a variant to oppose the histamine-induced Ca2+ transients compared to its wt counterpart. Therefore, we decided to check whether the E1081Q mutation could impact on the full-length b variant of the PMCA3 differently. To this aim we have introduced the point mutation in the rat full-length b variant of the PMCA3 and, in analogy to what we did for the human C-terminally truncated a variant, we checked its effect on the expression, subcellular localization and Ca2+ handling.The experiments above have indicated that the introduction of a E1081Q substitution activates the Caa and b variants of the PMCA3 pump were compared by Western blot analysis and immunocytochemistry, respectively. As shown in Fig. a variant, however, to our surprise, the expression level of the full-length b variant was strongly affected by the E1081Q mutation , however, cells overexpressing the mutated E1081Q b variant substantially reduced their ability to extrude Ca2+, as the average Ca2+ peak value was equal to that measured in control cells . At first glance, on the contrary of what found with the human truncated PMCA3a variant, these results might suggest that the mutation completely abolished the pump activity of the full-length b variant by affecting its ability to extrude Ca2+ in the extracellular medium. However, it must be noticed that the decay of the traces in Fig. b wt and the mutant and faster than in the control cells, indicating that also the E1081Q PMCA3b mutant retains some Ca2+ extrusion activity. It must be also considered that the low expression of the mutated protein might explain this phenotype. On the other hand, when cytosolic Ca2+ transients were monitored in cells stimulated with histamine in the absence of extracellular Ca2+ , the overexpression of either the wt or the mutated pump reduced the amplitude of the peak to the same extent in respect to control cells, as shown in Fig. 2+ fluxes deriving from the intracellular stores .The expression levels and the subcellular localization of the wt and E1081Q mutant n Figure . Insteadn Figure . We thenb expression on the free [Ca2+] in the ER lumen. As shown in Fig. 2+]ER reached in control cells was significantly higher than in those observed in cell overexpressing the PMCA3b pump ; however, it was not different in cells overexpressing the wt or the mutant PMCA3b pump; suggesting, again, that the mutation does not impinge on the ability of the pump to influence the ER Ca2+ filling or its basal level.We also assessed the effect of mutant PMCA32+ transients were also monitored in cells treated with histamine in KRB supplemented with 100 μM EGTA and 10 μM thapsigargin followed by Ca2+ re-addition. As shown in Fig. 2+ influx nor the cytosolic plateau levels were affected by the overexpression of the wt and the mutated pump .The Caump Fig.   was identical for yeast cells expressing the wt or mutant PMCA3 protein, in Ca2+-depleted medium the E1081Q substitution did not affected cell viability, as indicated by the growth rate of cells expressing the mutant PMCA3 with respect to yeast expressing the wt pump. The expression levels of the mutated pump detected by Western blot analysis, in agreement with the results showed above in mammalian cells, were also affected compared to the amounts of wild-type isoform  was monitored. Ca2+ influx through plasma membrane channels was induced by perfusing KRB supplemented with 1 mM CaCl2. Figure 2+]pm between control HeLa cells and wt PMCA3a expressing cells could not be appreciated. On the other side, a strong and significant difference in the formation of the Ca2+ microdomain in the sub-PM region could be observed upon overexpression of the E1081Q PMCA3a pump mutant , suggesting the possibility that the mutation positively influenced the activity of the truncated a but not the full-length b PMCA3 splicing variant by selectively affecting the ability to locally control [Ca2+]pm. Interestingly, a different scenario emerged when the full-length b variant of the PMCA3 pump was considered. As shown in Fig. b led to a statistically significant decrease in the formation of the Ca2+ microdomain under the PM compared to control cells, indicating a variant-dependent role of this PMCA isoform in shaping the sub-PM [Ca2+]. Interestingly, the E1081Q mutation completely abolished the observed phenotype of its wt counterpart . These results could also be interpreted as a lack of difference in the sub-PM effect due the low expression level of the PMCA3b E1081Q mutant. However, the ability of this mutant to behave like the wt pump in counteracting the Ca2+ release from the intracellular stores or in regulating the total ER Ca2+ content independent on its expression level  binding –35. As sTo explore the effect of the E1081Q mutation on PMCA3 structure and function, homology modeling by using the SERCA pump as a template could not be used since, to date, it has proven impossible to model the C-terminal domain of the PMCA due to its flexible nature and to the disordered region at the C-terminal tail. We took advantage of the AlphaFold database , where tCerebellar ataxias are rare neurological disorders characterized by the lack of voluntary coordination of muscle and gait abnormality . Alterat2+ homeostasis. Their main role is the regulation of Ca2+ signalling in selective sub-plasma membrane microdomains: the important point is that these microdomains host a number of important enzymes, which activity is directly or indirectly regulated by Ca2+ 40. The results described in this contribution underline once more the concept that the function of the PMCA pump, at least in specialized cells like neurons, is next to irrelevant to the global regulation of cytosolic Ca2+. It has been shown, for instance, that the ablation of PMCA4 isoform from cardiac cells has no effect on the Ca2+-dependent contractility properties of the cardiomyocytes  in the sub-PM microdomains by the full-length b and the truncated a variant, respectively. Mechanistically this could be due to their different regulation of the activity by specific partners or by impaired allosteric regulation through differences in Ca2+ binding. Although we were able to replicate the Ca2+ binding to the A18 peptide by using the stains all, other A18-Ca2+ binding experiments failed to reveal any binding. Thus, whether this peptide binds Ca2+ or not was unclear, as was the impact of the E1081Q mutation on the Ca2+ binding feature of that region of the CTD of PMCA3. Possibly, however, the Ca2+ affinity of the A18 peptide could have been too low to reveal Ca2+ binding under the experimental conditions and/or, the length of peptide could have been insufficient to construct a Ca2+-binding module. It is felt that future work on these aspects of the defects of the PMCA3 pump that would include the development of currently unavailable experimental protocols, could possibly shed light on the link between PMCA pump defects and the ataxic phenotype.As mentioned in the Introduction, the PMCA pumps only offer a minor contribution to the global cytosolic Caodomains . The samfunction , 43. Oneolerated , 44. We ATP2B3 was performed as in . Briefly, Stains-all was dissolved in ethylene glycol at a concentration of 500 μM, while the spectra acquisition solution was 2 mM MOPS pH 7.2, 30% ethylene glycol (spectra buffer). The peptides, in the presence or absence of CaClp value ≤0.05 was considered statistically significant.All the data are representative of at least three independent experiments unless otherwise indicated. Values are expressed as mean ± SEM. Statistical significance was determined using the multiparametric one-way ANOVA test. A Figure S1Figure S2aj-checklist


Colpodella species, Alphamonas species, and Voromonas pontica are predators that prey on ciliate, bodonid, and algal prey using the process of myzocytosis. During myzocytosis, the pseudoconoid is used to attach to the prey leading to aspiration of cytoplasmic contents of the prey into a posterior food vacuole formed in the predator, aided by secretions from the apical complex organelles. The conoid and associated proteins are conserved among the apicomplexa. However, the organization and function of the pseudoconoid during myzocytosis are not well understood. In this study, we investigated the morphology and ultrastructure of Colpodella sp. (ATCC 50594) during the stages of myzocytosis and cyst formation in the life cycle using light microscopy and transmission electron microscopy (TEM) in order to identify the organization of the tubular tether involved in nutrient aspiration by Colpodella sp. Tubular tethers of varying lengths were identified by light microscopy. We report that initial contact by Colpodella sp. trophozoites with Parabodo caudatus prey is by an area posterior to the apical tip of the rostrum that engulfs the membrane of the prey pulling it into the cytoplasm of the predator. The tubular tether that forms contains membranes of both predator and prey and is facilitated by microtubule organization and the cytoskeleton at the point of contact. Cytochalasin D treatment of diprotist cultures resulted in morphological distortions of trophozoites and the tubular tether suggesting a role of actin in the formation of the tubular tether. This mechanism of predation may provide insight into the mode of invasion observed in pathogenic apicomplexan zoites during host cell entry.Free-living relatives of the Apicomplexa such as  Colpodella species, Chromera velia, Vitrella brassicaformis, and Voromonas pontica have also been described and share the features found in the apical complex [C. velia and V. brassicaformis and the process of microgametogenesis are considered ancestral forms of schizogony and endodyogeny and microgamete development in the Apicomplexa [Perkinsus and Psammosa species possess an apical complex with a pseudoconoid, rhoptries, and micronemes [Chromera velia, in addition to being photosynthetic, the apical complex is thought to be instrumental in establishing endosymbiosis [Cryptosporidium species to intracellular parasitism found in pathogenic Apicomplexa such as in Plasmodium species, Toxoplasma gondii, and other coccidia [Colpodella species, the trophozoite with the food vacuole and nucleus can be recognized. The Colpodella sp. (ATCC 50594) trophozoite, containing aspirated contents from the prey and the cell nucleus, differentiates into a cyst stage that divides to produce asynchronous symmetric or asymmetric trophozoite progeny [Colpodella species include Colpodella sp. (ATCC 50594), C. vorax, C. tetrahymenae, C. pugnax, and C. turpis [Colpodella pseudoedax divides by longitudinal fission and only produces resting cysts [Colpodella species have been implicated in two cases of human infection, characterized by neurological symptoms and babesiosis-like symptoms [Colpodella species has not been investigated. The life cycle stages of Colpodella sp. within the human host cells have not been described and the stages responsible for pathogenesis are unknown [Colpodella gonderi was identified in urine samples in a case of urinary tract infection [Colpodella species identified in animal hosts and in ticks have also been described, but characteristics of the identified organisms are unknown [Colpodella species [Colpodella vorax, Colpodella gonderi, Colpodella tetrahymenae, and Colpodella sp. (ATCC 50594) trophozoites, cysts, and cells attached in myzocytosis [Colpodella sp. (ATCC 50594) and investigated the role of actin in the formation of the tubular tether. We used staining and light microscopy to demonstrate tubular tethers with varying lengths and to identify the effects of Cytochalasin D on the tubular tethers. We performed ultrastructural studies to further our understanding of the organization of the point of attachment and the formation of the tubular tether. We show that the tubular tether consists of the membranes and cytoskeleton of both predator and prey. Following attachment of the predator to prey, the prey’s membrane is not pierced but is engulfed into the cytoplasm of the predator before the prey’s membrane is destroyed and cytoplasmic contents are aspirated into the posterior food vacuole of the predator.Apicomplexans comprise important pathogens causing human and animal infections such as malaria, toxoplasmosis, and babesiosis. The mechanism of invasion among pathogenic apicomplexans involves direct contact of the invasive stages (zoites) with the host cell using the apical end ,2,3. Thi complex ,5,6,7,8.complexa ,9. Amongcronemes ,11,12. Acronemes . The apicronemes ,5,6,7,8.ymbiosis ,14. The  progeny . Resting. turpis . Colpodesymptoms ,17. The  unknown ,17. Colpnfection  although unknown ,20,21,22ocytosis ,24,25,26Colpodella sp. (ATCC 50594) [Colpodella sp. (ATCC 50594) could be identified. In reports of human infections caused by Colpodella species, infective stages were not described [Colpodella gonderi was not reported [Colpodella species [Colpodella sp. (ATCC 50594). Formalin-fixed cells of Colpodella sp. (ATCC 50594) stained with Giemsa, Kinyoun’s carbol fuchsin, and Sam-Yellowe’s trichrome stains identified the tubular tether with varying lengths as shown previously [Colpodella sp. (ATCC 50594) (yellow arrow) in myzocytosis with the prey P. caudatus (red arrow). Different lengths of the tubular tether are shown (black arrows). The flagella of the predator (white arrows) and prey (green arrows) were identified. Single Copodella sp. (ATCC 50594) attachments to prey (Copodella sp. (ATCC 50594) trophozoites are shown  ,24. In pescribed ,17 and treported . The sta species ,7,8,23.  to prey A,C–F andre shown B. Two maThe cysts are non-motile. Colpodella sp. (ATCC 50594) are shown in Colpodella sp. (ATCC 50594) with four trophozoites and a clear zone free of bacteria is shown (Colpodella sp. (ATCC 50594). Trophozoites (yellow arrow) are shown in myzocytosis with P. caudatus (red arrow).Single attacks B,C,H,I ais shown K. The adP. caudatus is shown in Colpodella sp. (ATCC 50594) trophozoite, darkly stained, is seen in P. caudatus (red arrow) can be distinguished from Colpodella sp. (ATCC 50594) cysts. During myzocytosis, the predator aspirates the cytoplasmic contents out of its prey and into a posterior food vacuole. Multiple predators can attach to a single prey in the most active phase of the in vitro life cycle as shown with Sam-Yellowe’s trichrome staining in The use of staining for light microscopy is not routinely performed for the identification of the free-living apicomplexan relatives. In previous studies , and in Colpodella sp. (ATCC 50594) in vitro was shown to be between 20 and 28 h [P. caudatus in the in vitro life cycle resulting in few trophozoites, multiple predators can be identified attached to single P. caudatus trophozoites in close proximity on the surface of the prey as shown in our previous studies [Colpodella sp. (ATCC 50594) are shown in Colpodella sp. (50594) are shown in Colpodella sp. (ATCC 50594) trophozoite [Colpodella sp. (ATCC 50594) in Colpodella sp. (ATCC 50594) cysts and not around P. caudatus cysts [Colpodella species cysts have not been reported previously. A mature cyst with multiple juvenile trophozoites is shown (black arrow).In previous studies, the most active phase of the life cycle of and 28 h . With th studies  and in tphozoite . The antphozoite . Bacterius cysts ,24 The dColpodella sp. (ATCC 50594) cysts, both immature and mature cysts, and not around P. caudatus cysts [Further investigations will demonstrate if the clear zones are the result of antimicrobial activity or artifacts of fixation. The clear zones are identified only around us cysts ,24.Colpodella species and their prey have been described using transmission electron microscopy [Colpodella sp. (ATCC 50594) in vitro [Colpodella sp. (ATCC 50594) trophozoites in the apical end of the trophozoite for attachment to prey. The ultrastructure of the attachment site and tubular tether was investigated. Colpodella sp. (ATCC 50594) (yellow arrow) is not at the apical tip (orange arrow) but in an area posterior to the apical tip of the rostrum  trophozoites and the membrane of P. caudatus (boxed area). Bacteria (B) and mitochondria (m) present in the cytoplasm of P. caudatus were identified. We showed previously [Colpodella sp. (ATCC 50594) does not lead to penetration of the plasma membrane of P. caudatus. Attachment to the prey’s plasma membrane is followed by engulfment of the prey’s membrane into the cytoplasm of the predator, to facilitate dissolution of the membrane to allow the flow of cytoplasmic contents into the predator’s cytoplasm. Colpodella species showed the steps after the initial contact between predator and prey [Colpodella vorax. A clear opening was reported between the attached trophozoites of Colpodella gonderi and the prey Colpoda fastigata [Colpodella vorax, C. gonderi, and C. tetrahymenae [Colpodella sp. (ATCC 50594) is the plasma membrane of Parabodo caudatus  surrounding and engulfing the plasma membrane and cytoplasm of P. caudatus. Pellicular microtubular organization at the point of contact supports the tether as the prey’s cytoplasm is pulled into the cytoplasm of Colpodella sp. (ATCC 50594) (black arrows). The thickening of the pellicle in the predator is shown by the three arrows on either side of the tether  does not occur at the apical tip of the rostrum with a piercing of the plasma membrane of P. caudatus. We show in the current study that the process of myzocytosis in Colpodella sp. (ATCC 50594) is sequential and may involve signal transduction and the release of enzymes and secreted molecules from the rhoptries and micronemes to digest the plasma membrane of the prey, including macromolecules and organelles from the prey’s cytoplasm, similar to events in the pathogenic apicomplexan [Colpodella sp. (ATCC 50594) trophozoite shown in The plasma membrane of the prey is destroyed by enzymes and secreted contents from the apical complex organelles allowing for cytoplasmic contents from the prey to flow into the cytoplasm of the predator B. The rhATCC 5059 does notColpodella sp. (ATCC 50594) (Precyst stages of C 50594) B,C to shC. gonderi trophozoites feeding on prey [Colpodella sp. (ATCC 50594) with asymmetric and asynchronous division as reported previously [The food vacuole in the cell enlarges during myzocytosis and differentiates into a cyst  after fe on prey . Cell dieviously .Colpodella sp. (ATCC 50594) and P. caudatus trophozoites, over the body of both cells.In order to determine the role of the cytoskeleton in the formation of the tubular tether during myzocytosis, cells from a diprotist culture were labelled with Alexa Fluor 488 phalloidin, which targets F-actin. A Volocity video prepared from a 3D reconstruction of Z-stacks from Volocity videos show that the distribution of actin is not limited to the point of attachment but rather present in the body of the trophozoites in both predator and prey. In order to determine the role of actin in the formation of the tether in the present study, diprotist culture was treated with cytochalasin D. Treatment of cultures with cytochalasin D resulted in morphological distortions of cells during myzocytosis  as well P. caudatus trophozoite shown in panel J appear stretched apart. It is currently unclear why all cells were not equally affected by cytochalasin D treatment. These types of studies have not been performed with other Colpodella species or other free-living relatives of the Apicomplexa. This is the first study to investigate the role of actin during myzocytosis in Colpodella species. Our hypothesis was that motility would be affected and that attachments would not occur following treatment with cytochalasin D. However, the results obtained showed that attachments did occur with distortions to the tubular tether following cytochalasin D treatment, suggesting actin involvement in the attachment process. Differences in life cycle stages and the duration of the attachment between predator and prey may account for the differences observed. Additional investigations are needed to understand the role of the cytoskeleton in tether formation. Diprotist cultures are asynchronous at different life cycle stages and may account for the differences observed. The actinmyosin motor is essential for motility and host cell invasion among zoites of pathogenic Apicomplexa such as in Toxoplasma gondii and Plasmodium species [Plasmodium falciparum cultures are treated with cytochalasin B [Toxoplasma gondii cultures are treated with cytochalasin D [Gregarina garnhami [G. garnhami with cytochalasin D resulted in the inhibition of motility [Cells in myzocytosis that appeared “normal” were also observed H,I. The  species . Actin i species ,3. The palasin B  and whengarnhami . The tremotility . The useParabodo caudatus trophozoites (Colpodella sp. (ATCC 50594) trophozoites (Colpodella sp. (ATCC 50594), Colpodella sp. (ATCC 50594) are sequential and resemble the steps described for trophozoite stages in Archigregarines where the mucron attaches to the host cell and aspirates the host cell cytoplasm using myzocytosis [Colpodella sp. (ATCC 50594) trophozoites and P. caudatus is followed by the engulfment of the plasma membrane and cytoskeleton of P. caudatus forming the tubular tether between predator and prey. Destruction of the prey’s membrane by enzymes from the predator leads to the formation of an open channel through which the prey’s cytoplasmic contents are aspirated into the predator. Myzocytosis in Colpodella sp. (ATCC 50594) may serve as the immediate precursor to myzocytosis observed among the gregarines and Cryptosporidium species [Colpodella sp. (ATCC 50594) trophozoites and P. caudatus. Future ultrastructural studies will clarify the cytoskeletal contributions to the different lengths of the tubular tethers observed. The identification of molecules participating in myzocytosis will provide important insights to aid in the determination of the origins of the invasion and nutrition acquisition mechanisms among zoites of pathogenic apicomplexans.hozoites A, Colpodhozoites B, and otocytosis , as well species ,30. AddiColpodella sp. (ATCC 50594) was maintained in the Hay medium as a diprotist culture containing Parabodo caudatus in the supplied culture. The Hay medium was bacterized with Enterobacter aerogenes in tissue culture flasks as described [Parabodo caudatus in the diprotist culture served as prey for Colpodella sp. (ATCC 50594). Cultures were examined using an inverted microscope to observe different stages of trophozoites and cysts as described [escribed . Ten milescribed .Colpodella sp. (ATCC 50594) in diprotist cultures was fixed using 5% formalin as described [escribed . Brieflyescribed . The fixescribed . FollowiColpodella sp. (ATCC 50594) from a diprotist culture was permeabilized for 5 min in 10 mL of 0.1% Triton-X100 in Dulbecco’s PBS (dPBS). Slides were washed twice with dPBS and blocked in 3% bovine serum albumin for 30 min. Following 2 washes in dPBS, the ActinGreen 488 Ready Probes reagent  was applied to slides and incubated for 30 min in a covered humid chamber protected from light following manufacturer’s instructions. Slides were washed in dPBS followed by the addition of DAPI  mounting solution to the smear and the application of a cover slip. The edges of the coverslip were sealed with clear nail polish. For the application of antibodies to actin488-labeled smears for immunofluorescence assay (IFA) colocalization studies, diluted primary antibodies were applied to the slides after ActinGreen 488 staining and dPBS washes. The Anti-RhopH3 [For direct staining of F-actin with a fluorescent label, formalin-fixed i-RhopH3  primary Cells in diprotist Hay medium culture were treated with cytochalasin D (cytoD) (Sigma Aldrich). Cells cultured in 24- or 6-well microtiter plates or T25 tissue flasks were treated with the drug reconstituted in dimethyl sulfoxide (DMSO). Cells were incubated with Cyto D at 1 µM, 2.5 µM, 5 µM, and 10 µM in duplicate in microtiter plates. Three independent experiments were performed and then repeated at concentrations of 10 µM. CytoD was added to cells subcultured from resting cyst stages, scraped with cell scrapers, and inoculated into a bacterized Hay medium (treatment before excystation), or cytoD was added into actively growing cultures with young trophozoites undergoing myzocytosis (treatment after excystation). Cultures incubated with DMSO at the same volumes and untreated cells served as controls. Cultures were monitored by observing cells under an inverted microscope. At 48 h, cells were fixed in 5% formalin, collected, and centrifuged. Supernatants were discarded, the pellets were washed in 1x dPBS, and smears were prepared on glass slides for the Giemsa stain. DMSO-treated cells and untreated control cells were observed in culture during the 48 h incubation period, then formalin-fixed and collected for centrifugation, smear preparation, and staining with the Giemsa stain.For light microscopy, formalin-fixed cells were stained with Giemsa, Kinyoun’s carbol fucshin, and Sam-Yellowe’s trichrome stains . All staColpodella sp. (ATCC 50594) in the diprotist culture was harvested and fixed as described previously [eviously . After e