Euglena suspension was then allowed to flow through the central channel. The two side channels were filled with either, 0.1% peptone as a negative control, or a Listeria suspension respectively. The membrane design prevented direct interaction but allowed Euglena cells to detect Listeria cells as secretions diffused through the nitrocellulose membrane. A significant number of Euglena cells migrated toward the chambers near the bacterial cells, indicating a positive chemotactic response of Euglena toward chemical cues released from Listeria cells. Filtrates collected from Listeria suspension with a series of molecular weight cutoffs  were examined in Euglena chemotaxis tests. Euglena cells were attracted to all filtrates collected from the membrane filtration with different molecular weight cutoffs, suggesting small molecules from Listeria might be the chemical cues to attract protozoa. Headspace volatile organic compounds (VOC) released from Listeria were collected, spiked to 0.1% peptone and tested as the chemotactic effectors. It was discovered that the Euglena cells responded quickly to Listeria VOCs including decanal, 3,5- dimethylbenzaldehyde, ethyl acetate, indicating bacterial VOCs were used by Euglena to track the location of bacteria.In nature, protozoa play a major role in controlling bacterial populations. This paper proposes a microfluidic device for the study of protozoa behaviors change due to their chemotactic response in the presence of bacterial cells. A three-channel microfluidic device was designed using a nitrocellulose membrane into which channels were cut using a laser cutter. The membrane was sandwiched between two glass slides; a  Protozoa are unicellular eukaryotes that are nearly ubiquitous in various natural and man-made ecosystems including terrestrial, aquatic, abiotic and biotic habitats. They plPseudomonas being favored over other genera such as Streptomyces and Bacillus. Jezbera et al. observed the preferable predation by the flagellates Bodo saltans and Goniomonas sp. on Aeromonas hydrophila over Pseudomonas fluorescens. One recent study suggested that protozoa preferentially graze non-viable and particle-associated forms over viable and free cells[Tetrahymena sp. tended to preferentially remove the more dominant bacterial species from the community while Poterioochromonas sp. and Acanthamoeba sp. grazed non-specifically[There is a good deal of evidence suggesting that planktonic protozoa are capable of selective feeding although their feeding preferences are not well understood. A relearee cells. Anothercifically. It appecifically.Flectobacillus sp. produces long filamentous cells, which are harder to ingest, in the presence of the bacterivorous flagellate Ochromonas sp.[Acanthamoeba spp. and some ciliates possess the ability to package bacterial pathogens including Legionella pneumophila, Salmonella enterica, Listeria monocytogenes, and Escherichia coli O157:H7, into multilamellar bodies (MLBs)[L. pneumophila packaged in MLBs by the ciliate Tetrahymena was more infectious and displayed greater resistance to gentamicin and longer survival in a nutrient-poor environment, compared to the planktonic state[Campylobacter in intensively reared poultry[In turn, bacteria have adapted multiple strategies to counteract predation by protozoa through both intracellular and extracellular defense strategies. Intracemonas sp.. Some pres (MLBs). Bacterinic state. It has d poultry.There appear to be molecular interactions between protozoa and bacteria which may employ dissolved cues for chemical-mediated prey selection. Chemicals involved in chemosensory attraction could include proteins, amino acids, and other dissolved inorganic or organic nutrients, 20. OthCo-culture based methods, incubating a mixture of protozoa and bacteria species in a saline buffer and counting the cell numbers over the incubation period, are typically used to study interactions of protozoa and bacteria, 24–27. Euglena gracilis and Listeria were selected as the model protozoa and bacterial organisms respectively; Euglena gracilis is a unicellular flagellated photosynthetic alga; the body is approximately 10 μm wide and 50–100 μm long. E. gracilis is a freshwater flagellate found in many aquatic habitats especially shallow eutrophic ponds[Euglena can grow photoautotrophically, photo-heterotropically or heterotropically. It has emerged as an excellent source of dietary protein, pro(vitamins), lipids, and the β-1,3-glucan paramylon[Listeria spp., including two pathogenic species Listeria monocytogenes and Listeria ivanovii, can thrive in various environments and are often isolated from water, soil and detritus[In the current study, we studied the interaction between protozoa and bacteria in a microfluidic device made of a porous nitrocellulose membrane. Microfluidic devices have been developed to generate and maintain a stable chemical gradient for bacterial chemotaxis studies–33. Euglhic ponds. Euglenaparamylon. Meanwhi detritus. Given t detritus.2 laser cutter (Hermes LS500XL) was used to generate a pattern of channels into a nitrocellulose membrane with a thickness of 120 μm (EMD Millipore). A fabricated membrane was soaked in the sterilized and 0.22 μm filtered 0.1% peptone buffer. The wetted membrane was then sandwiched between two pre-cleaned glass microscope slides that were secured with clips. The top slide contained six holes connected to reservoirs made with glass tubes. These holes aligned with the input and output of the three channels in the membrane respectively. The side channels were filled with a control buffer (0.1% peptone) and a chemoeffector solution respectively by adding 100 μl of each solution into the reservoirs connected with side channels. The liquid flows in the side channels were achieved through a capillary force and reached a static equilibrium before the chemotaxis experiment started. A syringe pump was used to control the flow of liquid in the center channel. The device was then fixed on the stage of an inverted confocal microscope (Nikon Eclipse TI) where the movement of protozoa could be observed and recorded.Details of the design and fabrication of the microfluidic device have been described previously. BrieflyEuglena was obtained from Carolina Biological Supply Company  and maintained in a pre-made Euglena media distributed by Carolina Biological Supply Company. The pre-existing bacterial cells inside the Euglena suspension were removed by passing the protozoa suspension through a syringe filter with 5 μm pore size and exchanging Euglena growth medium with sterilized 0.1% peptone. The cleaned Euglena sample was kept in the dark at 21°C for 2 hours before use. A 4-chip hemocytometer from Bulldog Bio  was used to quantify Euglena cells. A concentration of Euglena suspension of ~ 106 cell/ml was employed in the chemotaxis experiment.Polystyrene beads with a diameter of 6.1 μm were obtained from ThermoSci. . They were diluted in 0.1% peptone and 100 μL of 10 ppm of polystyrene beads were tested in the microfluidic device as a negative control to examine the effect of liquid flow on the distribution of beads inside the microfluidic channel.Listeria monocytogenes (ATCC 13932) was obtained from Microbiologics . All growth media and buffers were autoclaved and filtered through 0.22 μm syringe filters. Cultures of each isolate were incubated for 20 hours at 37°C in Trypticase Soy Broth . Overnight cell cultures were centrifuged three times and pellets were suspended in 0.1% peptone to produce a concentration of 109 cfu/ml. Optical densities (600 nm) of bacterial cultures were adjusted to produce a suspension containing 109 cfu/ml, and suspensions were serially diluted in 0.1% peptone to produce a suspension of the desired concentration. Suspensions of dead bacteria were prepared by autoclaving suspensions of live bacteria at 121°C for 15 minutes.Bacterial samples from overnight cultures underwent buffer exchange with a 0.22 μm syringe filter from growth media to 0.1% peptone. Bacteria remained in the 0.1% peptone buffer for approximately 30 minutes to allow any chemicals released from cells to diffuse into the media. The sample was then loaded into an EMD centrifugal filter with molecular weight discriminations of 3kD, 10kD, or 100kD. Filtrates with a molecular weight below these thresholds were collected and used as the experimental attractant in the microfluidic device.Listeria/peptone via a PTFE tube. The Listeria vial was gently heated to around 35°C, and both vials were held for roughly 2 hours to allow for a transfer of headspace VOCs from the Listeria vial to the second vial. Afterward, the second vial was detached from the Listeria vial and gently inverted.Bacterial samples from overnight cultures underwent buffer exchange with a 0.22 μm syringe filter from growth media to 0.1% peptone in an airtight glass vial. A second airtight glass vial containing 10 ml of filtered 0.1% peptone solution was then connected to the vial containing the Four representative bacterial VOCs including 2-ethylhexyl acetate, ethyl acetate, 3,5-dimethylbenzaldehyde, and decanal were selected for further chemotaxis validations. They were purchased  and used directly. The VOC spiked samples were prepared in 0.1% peptone water individually at their saturation concentrations and 10-fold (100-fold dilution for ethyl acetate) dilution of their saturated solution in 0.1% peptone was used for euglena chemotaxis experiment.Euglena suspension was added to the center channel and the flow rate was controlled by a syringe pump, as shown in Our microfluidic design contains a center channel and two side channels, as shown in Euglena suspension was loaded into the center channel at a flow rate controlled by a syringe pump. The flow rate of the Euglena suspension was set as 3 μL/h. Both of the side channels were filled with 0.1% peptone. After allowing the system to equilibrate with Euglena in the microfluidic channel for 20 minutes, the cell counts process was started and designated at time 0, which was right before the introduction of chemoeffector in the side channel. At 20 minutes, 100 μL of the bacterial cell suspension or filtrate solution collected from bacterial samples were added into the bottom channel of the device. Videos were then taken at 10-minute intervals until 60 minutes to determine if a change in Euglena cell distribution had taken place. These experiments were performed in a dark room to prevent Euglena from responding to ambient light instead of the chemoeffector being tested.Channels in assembled microfluidic devices were washed and filled with 0.1% peptone using a vacuum pump. A volume of 100 to 200 μL Euglena cell counts. The viewing area of the chamber was measured and kept standard for all measurements. Euglena cells were counted using Nikon Elements software. After counting Euglena at the top and bottom of each channel, the percentage of a cell population in the top and bottom of each channel was compared at each time point. The total number of Euglena (the sum of cell numbers in the top and bottom chambers) at each time point was used to calculate the percentage of cells at the top and bottom chambers. If Euglena had a preference for bacteria or other attractant placed in a channel then the percentage of Euglena near that channel should increase over time to above 50% and stay above 50%. Each test was repeated at least three times and the mean value with standard deviation are reported.Videos were taken of the three perpendicular chambers of the device as identified in f Rxi-17sil column  was connected to a 2 m × 0.25 mm i.d. × 0.25 μm DB5 column  in series and separated by the cryogenic modulator so that the polar primary column separated VOCs according to their polarity and the non-polar secondary column in the secondary oven separated VOCs according to their boiling point. VOCs eluting from the secondary column were detected with TOF-MS. Helium was used as the carrier gas at 2.0 mL/min controlled via an automated pressure ramp. An initial temperature of 45°C held for 1 min, followed by a gradient at 5°C/min to 120°C, then 10°C/min to 240°C was used in all analyses. The second column was operated at 5°C higher than the primary column. The transfer line was at 240°C. The thermal modulator offset was +15°C relative to the primary oven temperature. The modulation period was 5 seconds. The MS range of mass-to-charge ratio (m/z) was 35–450 and 200 mass spectra were acquired per second. The ion source chamber was held at 200°C. The detector voltage was 15750 V with an electron energy of 70 eV.A 10 mL aliquot of bacterial VOC sample was transferred to a 20 mL capped glass vial. The vial was heated at 50°C and stirred at 200 rpm for 20 min to equilibrate liquid and vapor, then the organic vapors in headspace were adsorbed on a SPME fiber coated with 50/30 mm DVB/CAR/PDMS  for 20 min at 50°C. The sample was desorbed at 250°C for 2 min in the GC injector in splitless mode. The VOC analysis was performed using a Pegasus 4D GC×GC-TOFMS instrument , including an Agilent 6890 GC , time of flight mass spectrometer (LECO) and Gerstel MPS2 autosampler . A 30 m × 0.25 mm id. × 0.25 μm d® software, v4.50 (LECO). The baseline was obtained between the middle and the top of the noise. Peaks with a minimum signal to noise ratio of 100 were identified. Subpeaks were combined if shifts on their second dimension retention time were less than 0.1 s and their mass spectral match was greater than 600/1000. Identified peaks were assigned putative identifications based on mass spectral matching score greater than 700/1000 compared to the National Institute of Standards and Technology (NIST) 2011 mass spectral library. All post GC-MS data analysis including clustering analysis and feature identification were performed with Python programs using routines from the sklearn and scipy libraries[Alignment on chromatographic data was conducted using the Statistical compare feature of the ChromaTOFlibraries.Using rapid prototype techniques including a laser cutter based method to write patterns directly on the nitrocellulose membrane provides a simple and maskless fabrication process to produce microfluidic devices. The process is simple and the microfluidic design can be easily modified and adapted in the process of laser writing. We have developed a design containing chambers perpendicular to the center channel so the movement of studying microorganisms inside the chambers will not be influenced by the variations in the fluid flow. Our previous study indicated that a pseudo-linear chemical gradient inside the chamber can be established within 10 minutes and maintained for at least 60 minutes via a chemical diffusion through the porous nitrocellulose membrane. Using pEuglena inside the microfluidic devices was studied first with both side channels filled with sterilized 0.1% peptone. The flow rate of the Euglena suspension was set as 3 μL/h. Faster flow rates at 15 and 30 μL/h were also examined, however, Euglena cells were pulled through the device too quickly to allow for a chemotactic shift. In addition, the high shear rate caused by a faster flow produced heterogeneous cell distributions in the form of regions of accumulation and regions of depletion[Euglena cell is about 6 minutes. Although there are no chemoeffectors inside the side channels, more Euglena cells reached the topsides of the chambers after they were introduced inside the microfluidic channel for 20 minutes. Chamber 1 (the chamber nearest sample introduction) contained more Euglena cells than the chambers closer to the exit of the central channel as shown in The behavior of depletion. Based oEuglena cells occurred at a rate of 13.7 μm/sec at 6°C and increased to a maximum of 80.1 μm/sec at 30°C[Euglena was about 50 μm/sec at room temperature. Therefore, it took less than 132 sec for Euglena cells to cross the end of the top chamber to the end of the bottom chamber based on our current design. It was observed that the number of Euglena cells inside chambers gradually increased over time and slightly more Euglena cells were found in the top chambers, as shown in Euglena is observed in the first channel versus the other two channels.Forward swimming of c at 30°C. The swiEuglena cells shifted to the chambers on the top even when both side channels were filled with 0.1% peptone. This observation could be attributed to the uneven light distribution on the microscope stage as the Euglena is photosensitive [Euglena cells (about 65%) remained in the top chambers during the entire experimental duration, indicating a slightly biased Euglena accumulation in the top chambers, as shown in Slightly more ensitive , 47. AltListeria cell suspension in 0.1% peptone was added into the side channel close to the bottom chambers and the top channel was filled with 0.1% peptone as the control. The shift of Euglena cells inside the microfluidic chambers over time can be found in Euglena in the viewing section of each chamber were in the top of the chamber, as expected from the previous experiment. The percentages of Euglena cells in the top chambers were quickly reduced after Listeria was introduced into the system. It’s interesting to observe that the reductions in percentages of Euglena cells in the top of chamber 2 and 3 were similar while less reduction was observed in chamber 1. The rates of reduction in 1st, 2nd, and 3rd chambers, by linear fitting the data in Euglena inside the chambers suggests that chemicals released from Listeria attracted the protozoa. The pore size of the nitrocellulose membrane was about 0.22 μm, which is small enough to prevent Listeria cells from entering chambers housing Euglena, while allowing molecules released by Listeria to reach the chambers. The absolute number of Euglena cells inside the first chambers are much higher compared to those in the second and third chambers. The higher cell density might result in an increased cell dispersion inside the chambers due to the abiotic stress associated with higher cell densities [Euglena. The chemotactic response of Euglena to other bacterial cells including E. coli, P. aeruginosa, and S. aureus were also examined inside the microfluidic device. Similar population shifts of Euglena cells were observed indicating the response of Euglena towards Listeria might not be selective.ensities , which mEuglena, we used filtrates collected from filters with different molecular weight cutoffs including 3k, 10k and 100k. The filtrate was then introduced into the side channel instead of the whole cell suspensions. We observed similar Euglena distribution inside the microfluidic devices even with the filtrates collected using the 3k MW cutoff membrane, indicating that chemicals with molecular weight less than 3k could be attractants for Euglena. To further identify the chemical cues responsible for the population shift of Euglena cells, headspace VOCs released from Listeria cells were collected in 0.1% peptone water and used as chemoeffectors in the side channel. The percentages of cells in the top chambers were reduced from 65% to ~ 25% after Listeria VOC was introduced in the side channel next to the bottom chambers for 60 min, suggesting Listeria VOCs might be the chemical cues for Euglena. It is well known that bacteria release molecules of low molecular weight (<300 Da) and high vapor pressure (0.01 kPa at 20°C) that can readily evaporate and diffuse through heterogeneous mixtures of solids, liquids and gasses[To narrow down the chemical cues responsible for attracting nd gasses. Volatilnd gasses–53.Listeria VOCs present in 0.1% peptone buffer. Both headspace samples from Listeria VOC spiked 0.1% peptone and 0.1% peptone buffer were examined and compared. Over two hundred VOC compounds were identified in both control (0.1% peptone only) and samples (VOCs from Listeria diffused into 0.1% peptone). Typical 2-dimensional GC plots with retention times from two columns and corresponding peak intensities can be found in Headspace SPME-GCxGC-TOFMS was used to identify Listeria GC-MS datasets comparing to the datasets to the background[Euglena cells were observed towards all four tested VOCs. The rates of changes [Panagrellus redivivus and the pinewood nematode Bursaphelenchus xylophilus[Decanal has been reported as a bacteria VOC associated with baumannii, Lysobaczymogenes, lactic  cibaria), 25 stra cibaria), bacteri cibaria). In factylophilus. Decanalylophilus, which mParamecium and for its natural predator, Didinium. Naegleria fowleri amoebae demonstrated a chemotactic and chemokinetic response toward live cells and extracts of Escherichia coli and other bacterial species when experiments were performed using a blind-well chemotaxis chamber. It was also reported that both free-swimming filter feeder Tetrahymena sp. and the surface-associated predator Chilodonella sp., used dissolved chemical cues from bacterial cell extracts for preferential feeding decisions although the nature of the dissolved cues which caused the feeding preference is uncertain[Trypanosoma brucei towards E. coli indicated that the attractant is diffusible through the culture medium, and produced by actively growing bacteria. However, efforts to isolate the attractant was unsuccessful[In earlier work on the attraction of protists towards bacteria, a heat-stable chemoattractant was isolated from bacterial cultures. This component had a molecular weight in the range of 500–1000 daltons, was produced by both Gram-positive and Gram-negative bacteria, and served equally well as an attractant for both the bacterial feeding uncertain. A recenuccessful. Our stuListeria monocytogenes. Chemicals released by bacteria diffuse through the nitrocellulose membrane and act as attractants to the protozoa. The design of this device allows for quantifiable comparisons between the numbers of Euglena that move toward either side of the device. The two channels above and below the center channel allow for two different bacteria to be compared or one bacteria against a control. An observable shift of protozoa in the direction of a species bacteria over a control indicates that protozoa are attracted to that bacteria. Comparing the attraction of protozoa towards two different species of bacteria may indicate if the protozoa have a preference towards ingesting certain species of bacteria over others.We have designed and demonstrated the effectiveness of a microfluidic device for the quantitative evaluation of protozoa response to Listeria VOCs dissolved in a buffer are the chemical cues for Euglena. Several VOCs associated with bacterial samples were identified including 2-ethylhexyl acetate, ethyl acetate, 3,5-dimethylbenzaldehyde, and decanal. Their chemoattractant activities toward Euglena were confirmed. Among the four VOCs studies, decanal showed the most significant influence to attract Euglena. In the future, we can assess an organism’s tendency to make these VOCs, and use that as a measure of how useful protozoa predation could be in a control effort. We may be able to search for the genes that control VOC release and search for those in potential prey using a targeted molecular method.We have discovered that


Here, we describe the synthesis of cell-permeant caged PP-InsPs as tools to rapidly modulate intracellular levels of defined isomers of PP-InsPs in a genetically non-perturbed cellular environment. We show that caged prometabolites readily enter live cells where they are enzymatically converted into still inactive, metabolically stable, photocaged PP-InsPs. Upon light-triggered release of 5-PP-InsP5, the major cellular inositol pyrophosphate, oscillations of intracellular Ca2+ levels in MIN6 cells were transiently reduced to spontaneously recover again. In contrast, uncaging of 1-PP-InsP5, a minor cellular isomer, was without effect. These results provide evidence that PP-InsPs play an active role in regulating [Ca2+]i oscillations, a key element in triggering exocytosis and secretion in β-cells.Among many cellular functions, inositol pyrophosphates (PP-InsPs) are metabolic messengers involved in the regulation of glucose uptake, insulin sensitivity, and weight gain. However, their mechanisms of action are still poorly understood. So far, the influence of PP-InsPs on cellular metabolism has been studied by overexpression or knockout/inhibition of relevant metabolizing kinases . These approaches are,  Many imhate InsP 1.5 2 is+ (KATP) channels, which results in decreased K+-influx, membrane depolarization and subsequent voltage-gated Ca2+-influx. Even though an involvement of PP-InsPs in the regulation of oscillations of cytosolic Ca2+ levels ([Ca2+]i oscillations) seems reasonable due to their impact on insulin secretion in β-cells, their immediate effect on [Ca2+]i oscillations has not been studied before.Insulin release from pancreatic β-cells in response to changes in blood-glucose levels is the hallmark of β-cell physiology. Glucose stimulated insulin secretion involves the glucose metabolism-induced rise of the intracellular ATP/ADP ratio. Increased ATP-levels act by inhibiting ATP-sensitive KExtracellular application of non-caged metabolites comes with the drawback of unpredictable concentration gradients across the plasma membrane of live cells, especially when studying charged metabolites. Since chemical or electrophysiological permeabilization of cells causes significant perturbation, even more so when the biological effect is driven by changes in the plasma membrane potential, less invasive technologies are in high demand. The spontaneous increase of second messenger levels using photolysis is such a technology.5 using acetoxybenzylesters (AB) has been reported.5 using this prometabolite technology has never been achieved before but would additionally facilitate temporal and spatial control over the release of the active messengers and thus avoid concentration gradients. To date, this has not been achieved for any member of the inositol pyrophosphate family. Controlled intracellular release of non-symmetric PP-InsPs, such as 1-PP-InsP5, also remains an unsolved problem that it now addressed in this publication. In contrast, the delivery of a symmetric caged 5-PP-InsP5 using non-covalent association with a guanidinium-rich molecular transporter was recently reported and used to demonstrate the effect of 5-PP-InsP5 on the subcellular localization of the PH domain of Akt in living HeLa cells.27Recently, the cellular delivery of 5-PP-InsP5, 3-PP-InsP5, and 5-PP-InsP5, without the need for cell permeabilization or use of additives. The simple incubation of cells with photocaged prometabolites is sufficient. Moreover, we provide evidence that an increase of intracellular 5-PP-InsP5 concentrations profoundly impacts β-cell activity ([Ca2+]i oscillations), whereas 1-PP-InsP5 is inactive.Here, we demonstrate the efficient loading of pancreatic mouse insulinoma 6 (MIN6) cells10-DEACM 5-PP-InsP5 8 started from pentanol 5.iii) chemistry, followed by oxidation to give hexakisphosphate 6 in 52% yield.β-CE) groups were replaced by TMS esters, which were methanolized, followed by generation of a P(iii)–P(v) anhydride and oxidation to the protected diphosphate 7 (72% yield) in a one-flask procedure.iii)–P(v) anhydride was generated with a fluorenylmethyl (Fm) and DEACM modified P-amidite.10-DEACM 5-PP-InsP5 8 in 62% yield. For the prometabolite approach, it is vital that the β-phosphate only bears one protecting group (in this case: DEACM) to avoid unwanted side-product formation in the enzymatic cleavage.5 9,5 2 (The preparation of acetoxybenzyl (AB) prometabolites of [7-(diethylamino)coumarin-4-yl]methyl (DEACM) caged PP-InsPs is shown in .5 9,5 2 .5 prometabolite 13 required desymmetrization of myo-inositol, which was achieved by asymmetric phosphorylation. This strategy enables the installation of a protected phosphate in the 1- and 3-positions of myo-inositol, such as in phosphate ester 10.βCE* used in this strategy resembles the achiral βCE protecting group used in approach A  without AB groups as a reference, the AB groups were removed chemically with 33% piperidine in DMF in 68% yield. Furthermore, DEACM was removed by irradiation in a photoreactor releasing 1-PP-InsP5 3 in high purity after ion exchange and precipitation in 80% yield.The synthesis of 1-PP-InsPproach A . This sture form  as well 5 9, 14, ent-14 from AB prometabolites. Incubation of prometabolites in cell extract, followed by TiO2 enrichment and resolution of the digest on polyacrylamide gels (PAGE)5 9 was identified by comigration with a synthetic standard as the product of enzymatic hydrolysis, involving complete and selective removal of 10 AB groups in less than 10 min of incubation. Only minor amounts of InsP6 1 due to hydrolysis of the P-anhydride were identified.5 2 from the caged compound 9 in MIN6 extract in less than five minutes. A comparable outcome was observed when repeating the experiments with the 1-PP-InsP5 prometabolite 13 and its enantiomer ent-13  were lysed after prolonged exposure  or ca. 10 min (population II) after UV-irradiation ation II .10-DEACM 3-PP-InsP5 loaded MIN6 cells transiently reduced their [Ca2+]i oscillations, ca. 15 min after UV-irradiation 5 but also on the two enantiomers 1- and 3-PP-InsP5 by applying asymmetric phosphorylation. The photocaged prometabolites enter live cells and can efficiently release the caged precursor molecule involving clean enzymatic removal of ten protecting groups. The procedure is simple, as it only requires incubation of cells with the prometabolite to achieve efficient loading. Upon photolysis, the effects of a controlled intracellular increase of different inositol pyrophosphate species were read out at the single cell level and averaged on ensembles of cells. The controlled increase of the intracellular 5-PP-InsP5 concentration was found to translate into a transient decrease of [Ca2+]i oscillations within MIN6 cells. In contrast, 1-PP-InsP5, the other relevant cellular inositol pyrophosphate with seven phosphate groups, did not show any of these effects but, interestingly, its unnatural enantiomer did. The underlying mechanisms still have to be identified. This study shows for the first time the modulation of β-cell activity upon temporally defined photo-release of inositol pyrophosphate species. In addition to the described effects on cellular [Ca2+]i oscillations, we propose that the presented tools and strategies will be of high value to also study other effects that are associated with the PP-InsPs.In summary, this study provides advanced synthetic procedures for the preparation of complex inositol pyrophosphate prometabolites and of PP-InsPs that are additionally equipped with DEACM photocages. Such photocaged prometabolites of inositol pyrophosphates have not been reported before. The modifications are not only introduced on symmetric 5-PP-InsPThere are no conflicts to declare.Supplementary informationClick here for additional data file.


However, p21 function in fluoride toxicity is not well characterized. This study seeks to gain a better understanding of how p53 down-stream mediators, p21 and MDM2, respond to fluoride toxicity. LS8 cells were treated with NaF with/without MG-132 (proteasome inhibitor) or Nutlin-3a (MDM2 antagonist). NaF treatment for 2–6 h increased phospho-p21, which can inhibit apoptosis. However, phospho-p21 and p21 were decreased by NaF at 24 h, even though p21 mRNA was significantly increased at this time point. MG-132 reversed the fluoride-mediated p21 decrease, indicating that fluoride facilitates p21 proteasomal degradation. MG-132 suppressed fluoride-induced caspase-3 cleavage, suggesting that the proteasome plays a pro-apoptotic role in fluoride toxicity. NaF increased phospho-MDM2 in vitro and in mouse ameloblasts in vivo. Nutlin-3a suppressed NaF-mediated MDM2-p21 binding to reverse p21 degradation which increased phospho-p21. This suppressed apoptosis after 24 h NaF treatment. These results suggest that MDM2-mediated p21 proteasomal degradation with subsequent phospho-p21 attenuation contributes to fluoride-induced apoptosis. Inhibition of MDM2-mediated p21 degradation may be a potential therapeutic target to mitigate fluoride toxicity.Fluoride overexposure is an environmental health hazard and can cause enamel and skeletal fluorosis. Previously we demonstrated that fluoride increased acetylated-p53 and its downstream target  Fluoride is ubiquitous in the environment and is present in bones, teeth and calcified tissues. Fluoride is known as an effective caries prophylactic . The U.SDental fluorosis is a developmental disorder caused by fluoride overexposure during enamel formation. The cells of the enamel organ responsible for enamel formation are ameloblasts. Enamel development (amelogenesis) occurs in stages, pre-secretary, secretary, transition and maturation . ExposurPreviously we reported that high dose fluoride causes cell stress, endoplasmic reticulum (ER) stress ,13 and op53 increases transcription of the cyclin-dependent kinase inhibitor 1A/p21 (p21). p21 functions in cell cycle arrest, transcriptional regulation, and anti-apoptosis. These functions are largely dependent on p21 post-translational modification, p21 protein interactions, p21 subcellular localization, and on cell type and specific cellular stresses . InhibitMdm2 expression and MDM2 can inhibit p53 through a negative feedback mechanism , rabbit anti-cleaved caspase 3, rabbit anti-γH2AX, rabbit anti-phospho-MDM2 [Ser166], rabbit anti-ubiquitin, rabbit anti-β actin and mouse anti-β actin , rabbit anti-p21, rabbit anti-MDM2  and rabbit anti-phospho-p21 [Thr145] (Thermo Scientific). The membranes were then washed with Tris-Buffered Saline (TBS)-Tween (TBST) and incubated with the horseradish peroxidase (HRP)-conjugated secondary antibodies; goat anti-mouse IgG  or goat anti-rabbit IgG (Biorad) at RT for 1 h. After washing with TBST, enhanced chemiluminescence was performed with SuperSignal West Pico (Thermo Scientific) and the signal was detected by myECL imager (Thermo Scientific). Bands were quantified by MyImage analysis software (version 1.1), Thermo Scientific). At least three biological replicates for each experiment were performed and representative images are shown. Protein expression was normalized by use of the loading control protein (β-actin). Relative protein expression and statistical significance were analyzed by one-way analysis of variance (ANOVA) with Fisher’s least significant difference (g for 10 min at 4 °C. Protein concentration was determined by BCA protein assay kit (Thermo Scientific). 1 mg total protein was used for co-IP with antibodies; mouse anti-p53  and mouse anti-p21 . Mouse IgG was used as the negative control . The immunocomplexes were analyzed by Western blotting with antibodies; rabbit anti-MDM2, rabbit anti-p21 (Abcam), rabbit anti-p53, and rabbit anti-ubiquitin . 30 μg protein was used for input. At least three biological replicates for each experiment were performed and representative images are shown.The co-IP assay was performed using Pierce™ Co-immunoprecipitation Kit (Thermo Scientific), according to the manufacturer’s instructions. The cells were washed twice with ice-cold phosphate-buffered saline (PBS) and lysed with ice-cold IP Lysis/Wash Buffer and incubated on ice for 5 min. The lysate was centrifuged at 13,000× Immunocytochemistry was performed to detect p-p21 in vitro. LS8 cells were cultured on micro cover glasses  in 24-well plates and treated with NaF for 6 h or 24 h. After that, cells were washed with PBS and fixed with 4% PFA at RT for 10 min. Then, cells were incubated with 0.1% Triton X-100 at RT for 10 min followed by blocking with 1% BSA at RT for 1 h. Next, cells were incubated with rabbit anti-p-p21 [Thr145] antibody (Thermo Scientific) and mouse anti-β actin antibody  overnight at 4 °C. After washing with PBS, cells were incubated with secondary antibodies; AlexaFluor 488-conjugated goat anti-rabbit IgG  and AlexaFluor 594-conjugated goat anti-mouse IgG (Thermo Scientific) at RT for 1 h. Then, cells were incubated with DAPI (Thermo Scientific) at RT for 5 min. Cells were analyzed using fluorescence microscopy . At least three biological replicates for each experiment were performed and representative images are shown.Mouse incisors were extracted after fluoride treatment for 6 weeks and fixed in paraformaldehyde, demineralized with EDTA for 2 weeks, and embedded in paraffin. Sections were incubated with primary antibodies: rabbit anti-p-MDM2 [Ser185] (Thermo Scientific) followed by incubation with a peroxidase-conjugated secondary antibody, Vectastain ABC Regent , and DAB kit (Vector Labs). Then, sections were counterstained with 0.1% Fast Green in PBS and examined by light microscopy.LSD) post-hoc test using SPSS statistics 20 software (version 20). Significance was assessed at p < 0.05.The qPCR results and Western blot results were analyzed by one-way ANOVA with Fisher’s least significant difference (p21 mRNA levels at 24 h compared to control (0 mM) (** p < 0.01) (p21 mRNA levels were significantly increased at 24 h (Fluoride treatment increased acetylated-p53 at Lys379 (Ac-p53) in LS8 cells  and in r < 0.01) a. At ear < 0.01) b,c, whic < 0.01) c, even t at 24 h a. These Next, we investigated the role of the proteasome in fluoride-mediated p21 protein levels. NaF (5 mM) treatment for 6 h increased Ubiquitinated-p21 (Ub-p21) in LS8 cells a, indicap21 mRNA compared to control (p21 mRNA levels (p21 transcription. Previously we demonstrated that fluoride induced caspase-3 cleavage and DNA fragmentation and in LS8 cells  protein levels in vitro (MDM2 (ubiquitin ligase E3) is one of the transcriptional targets of p53 . Fluoridin vitro  and alsoin vitro . p-MDM2 in vitro , suggestp21 mRNA expression (p21 mRNA levels (p21 transcription independent manner. Nutlin-3a can induce or inhibit apoptosis [Bcl2/Bax mRNA ratio during fluoride treatment (Bcl2/Bax ration that was reduced after fluoride treatment, indicating that the proteasome plays a role in fluoride-mediated apoptosis. However, both MG-132 and Nutlin-3a inhibited caspase-3 cleavage, indicating some overlap of function between the two inhibitors.Nutlin-3a is an MDM2 antagonist that binds in the p53-binding pocket of MDM2 to inhibit MDM2-mediated p53 proteasomal degradation . Intrigupression , Nutlin-A levels c. These poptosis ,46, depereatment , indicatThis is the first report implicating MDM2-p53 and MDM2-p21 signaling pathways in fluoride toxicity. MDM2-mediated p21 proteasomal degradation plays a critical role as a pro-apoptotic factor during fluoride toxicity in ameloblast-derived LS8 cells. The MDM2-p53 and MDM2-p21 pathways may be potential therapeutic targets for fluoride-mediated health problems.


One hundred twelve participants underwent binocular vision assessment by evaluating horizontal heterophoria, horizontal and vertical fusional vergence ranges and vergence facility. The subjects were classified into two groups: binocular anomalies and normal binocular function. Neck complaints were measured with the Neck Disability Index, visual analogue scale, cervical range of motion, deep-flexor muscle activation score (AS) and performance index (PI). Our results showed that participants with low AS had significantly altered values of lateral phoria (near) (mean = -6.99 SD ± 6.96 PD) and PFV (near) blur (mean = 9.49 SD ± 5.45 PD) against those who presented normal AS  mean = -3.64 SD ± 6.37 PD; PFV (near) blur mean = 12.84 SD ± 6.20 PD). In addition, participants with NFV (near) recovery outside the norm had a significantly lower right side-bending (mean = 35.63 SD ± 8.35 PD) than those within the standard (mean = 39.64 SD ± 9 PD). The subjects with binocular vision impairment showed a diminished response to the deep cervical musculature, with low AS and PI, as well as a tendency to suffer from cervicalgia of more than three months’ evolution and a lower range of motion.The aim of this study is to establish a relationship between non The use of new technologies requires prolonged visual demand in a restricted visual space. This situation implies a continuous over-exertion of accommodation and vergence, which alter the efficiency of the visual system, and a diverse symptomatology appears that includes asthenopia and performance problems ,2. AccomDifferent authors have reported the joint prevalence of visual and cervical symptoms. Zetterberg et al. ,8 establThere is a relationship between visual dysfunction and neck muscle alterations–13. The The visual parameters that are normally measured to determine the binocular vision status are the horizontal heterophoria value, the amplitude of both the positive (PFV) (convergence) and negative (NFV) (divergence) fusional vergences, range of vertical vergences (VV), vergence facility testing (VF) and near point of convergence (NPC) ,18. OtheThe aim of our study is to determine if there is a relationship between suffering nonstrabismic binocular dysfunction and suffering neck pain. We also propose a complete and exhaustive assessment of the state of binocular function.A descriptive, cross-sectional, correlational study, conducted from March 1, 2017 until December 31, 2017 at the Faculty of Pharmacy, at the Optics and Optometry Titling facilities, Faculty of Pharmacy, University of Seville, was performed.The research followed the tenets of the Declaration of Helsinki; informed consent was obtained from the subjects after explaining the nature and possible consequences of the study; and the Institutional Review Board of the University Hospital Virgen Macarena of the University of Seville approved the research.The selected population was made up of students, professors and administrative and service personnel of the University of Seville. The proposal for participation in the present study was sent via email to the entire university community of the Faculty of Pharmacy at the University of Seville. Those interested totaled 143 subjects. All subjects were informed about the study verbally and in writing. Once informed in depth, 6 people refused to participate, and 3 did not sign the informed consent, leaving a total of 134 participants who gave their consent to participate in this research.All subjects had at least 20/20 best-corrected visual acuity and an absence of ocular motility defects, strabismus, nystagmus, corneal ectasias, suppression, diplopia, amblyopia, and any ocular or systemic disease that could affect the results. Subjects who had undergone some type of ocular surgery or had a history of head trauma, cervical fracture or surgery in this area; persons with intellectual disabilities or any problems that prevented them from completing the Neck Disability Index (NDI); or who suffered any type of degenerative disease or neurological alteration were excluded. Twenty-two out of a total of 134 potentially eligible subjects were excluded . The sample consisted of 112 subjects with a mean age of 39.8 years [standard deviation (SD) ± 14.97 probability distribution (PD)], aged from 18.0 to 65.0 years, and comprised 61 (54.5%) women and 51(45.5%) men.The measurements used in our study by physiotherapists were:The cervical joint range measured with the cervical range of motion (CROM) instrument , in degrThe condition of the deep flexor musculature [activation score (AS) and performance index (PI)], using the craniocervical flexion test (CCFT), was assessed with the ChattanoogaTM stabilizer pressure biofeedback device  .The cervical disability assessed with the NDI questionnaire (range 0–50) .Neck pain intensity was evaluated with the visual analogue scale (VAS), range 0–10.Cervicalgia evolution ,25 of thThe variables with which the binocular function was measured were:The magnitude of the horizontal heterophoria , was measured at distance and near with an occluder, a prism bar, and an accommodative target.The amplitude of both the positive (convergence) and negative fusional vergences (divergence), were measured using the rotary prisms of the phoropter (ESSILOR MPH100E S / N 000104 phoropter).Vertical fusional vergences  were measured using the rotary prisms of the phoropter, (ESSILOR MPH100E S / N 000104 phoropter).Vergence facility (VF)  was quantified with a prismatic combination 3 Δ base-in (BI) /12 Δ base-out (BO).A new variable was defined from the values of the previous variables: Sheard’s criterion , which dA trained physiotherapist evaluator, with seven years of experience in these types of measurements, assessed in this order: 1) the patient’s pain using the VAS and cervical disability using the NDI questionnaire before beginning the other evaluations; 2) cervical range of motion while sitting: the participant was asked to actively perform flexion, extension, right side-bending (RSB), left side-bending (LSB), right rotation (RR) and left rotation (LR) movements three times each to find the mean of the measurements; and 3) the activity of the deep flexor musculature with the CCFT (AS and PI). The CCFT was performed with the participant in a supine position with the neck in a neutral position (without a pillow). The device was positioned under the neck and against the occiput. It was inflated, once placed, to the 20 mmHg level. The patient moved the head as if they were saying "Yes". A trained examiner observed and corrected any substitution of movements. Everyone was instructed to perform craniocervical flexion of the neck at five pressure levels , and hold the position firmly. If they achieved this, they had to relax the muscles, and then repeated the movement for each position (obtaining the "activation score" (AS), depending on the pressure, with a range of 1 to 5). When the AS was established, the therapist asked them to maintain the pressure, with minimal superficial muscle activity, performing 10 sustained 10-second repetitions. The number of repetitions was called "performance." A performance index (PI) was calculated by multiplying the AS by the performance. Neck pain case studies using this test showed that the scores were less than 4 in AS and less than 10 in PI in patients with cervical disorders. These subjects presented neuromotor control with a deteriorated activation of the deep cervical flexor muscles. This deterioration seems generic to cervical pain disorders [All data were collected on a record sheet by another physiotherapist. At the time of data collection, the assessors did not know the level of discomfort of the participants. This was established after the data processing. The physiotherapists were blinded regarding the optometric evaluation and vice versa.Once physiotherapy assessment was finished, and after a 60-minute break, patients were moved to an adjoining room where a licensed optometrist performed an optometric examination.Horizontal heterophoria was quantified with alternate cover test (ACT), which occluded each eye alternatively for 5 seconds. At any moment, both eyes remained uncovered, so binocular vision was not allowed to adopt a rest position of the visual axes. At the same time, the prism bar was placed. The prismatic power was increased until the movement of the eyes was neutralized. This constituted the phoria value. Prisms BO neutralizes esophoria and prisms BI neutralizes exophoria . The altThe range of horizontal and vertical vergences was measured using Risley rotating prisms of the phoropter and taking three measurements spaced 15 seconds apart. Prisms were introduced at a rate of 1 Δ per second. The patient had to indicate when saw blurred text (blur point) or doubled (break point) in horizontal vergences. Double text indication (break point) was only in vertical vergences. The prismatic power was decreased until the patient merged the image again (recovery point) .VF was measured by changing between BI and BO prisms (3 Δ BI / 12 Δ BO), requiring the subjects to converge and diverge. The fixation point was a near Snellen chart located 40 cm from the subject. We presented a VA equivalent of 0.8. The measurement involved introducing the BI first. The patient clarified the image. Next, we changed to BO. The process alternated for 1 minute. The number of complete cycles (one BI and one BO prism) was the value of the VF .The data were analyzed with the SPSS 24 package for Windows . The normality of our variables was verified with the Shapiro-Wilk test. A descriptive data analysis was developed, showing the count and proportion of each category in the qualitative variables and the mean and SD or in its defect the median and the interquartile range (IQR), and the range (minimum-maximum) in the quantitative ones. Then, the relationships between the variables considered were studied by calculating the Pearson coefficient (r). Then, the values of disability, pain, mobility, AS and PI were compared in the groups in which we differentiated the subjects according to the normative values of variables that described the state of binocular vision. After this, the values of binocular vision were compared in the groups in which we differentiated the subjects according to the normative values of NDI, AS, PI and suffer cervicalgia of 3 or more months’ evolution. Finally, we analyzed if there was a relationship when comparing the categorized values (if they were inside or outside the norm) of the variables that defined the state of the vergence function relative to the normative levels of the variables that described the neck area. In all these analyses, Student t-test or Welch’s t-test was used, as required, and for the variables that were not normally distributed, the Mann-Whitney U-test was used. As a complement to the above analyses, the effect size was calculated to determine the value of the standardized difference of means (d of Cohen) when the t-test was performed, while we followed the criteria of Grissom –33 when Significant relationships were found between: 1) Neck Disability Index (NDI) and VV (near) recovery ; 2) activation score (AS) and PFV (near) blur ; 3) performance index (PI) and lateral phoria (distance) , NFV (distance) recovery , PFV (near) blur , PFV (near) recovery ; 4) flexion and PFV (near) blur ; 5) extension and lateral phoria (near) , PFV (distance) recovery , and vergence facility ; 6) right side-bending (RSB) and NFV (distance) break ; 7) left side-bending (LSB) and lateral phoria (near) ; 8) right rotation (RR) and lateral phoria (distance) , lateral phoria (near)  and PFV (near) blur ; 9) left rotation (LR) and lateral phoria (near) , PFV (near) break , PFV (near) recovery  and VV (distance) recovery . Correlations at the descriptive level were nonsignificant between NDI and PFV (distance) recovery , between PFV (near) break and AS and PI , and between LR and VV (near) recovery . In all cases the correlations found were small.Regarding the comparison of the values of the variables related to pain, and deep cervical musculature activity, in the subjects who presented a state of normal or outside-of-normal binocular vision, there were no significant differences in any case, except in the NFV (distance) break. Participants with NFV (distance) break values within the norm, which were 92, had an AS  and a PI  significantly higher  than the remaining 18 subjects who had higher-than-normal NFV (distance) break . These results are shown graphically in Regarding the levels of the variables related to neck mobility in individuals with normal values, or outside the norm, In the subjects who presented a state of normal or outside-of-normal AS, there were significant differences in the value of the variables that described the state of the binocular vision. These results are shown in Finally, at the descriptive level, we found differences that were not statistically significant when we compared the subjects with PI greater than or equal to 10 (n = 39 average = -0.03 SD ± 2.08 PD) with those who had PI with less than 10 points (n = 72 average = -0.79 SD ± 2.22 PD) in the distance of lateral phoria  = 0.35).When grouping the subjects according to the normative values of the variables that defined the state of binocular vision, we found that, in those who were not within the norm, there was a higher percentage of participants with AS below the norm. In contrast, in subjects without binocular vision impairment, there was a higher percentage with normal AS. Finally, a greater percentage of the subjects with alteration of the Vergence facility (near) suffered neck pain  than in the participants with Vergence facility (near) within the norm. These analyses are shown in In our investigation, a complete evaluation of binocular vision status was proposed, through tests that presented the highest repeatability, to determine the possible existence of nonstrabismic binocular dysfunctions and to analyze whether there was a relationship between the visual system and neck complaints.The analysis of our variables indicated that subjects with binocular vision impairment showed a decreased response of the deep cervical musculature, with low AS and PI levels. They also showed a tendency to suffer cervicalgia of more than three months of evolution. These results are consistent with previous studies demonstrating the relationship between the visual system and the musculoskeletal system of the neck –13.Several symptoms and signs can be used to diagnose binocular anomalies. However, there is a lack of consensus in scientific literature about which diagnostic criteria should be used to define each dysfunction ,34,35. ITaking into account all the variables that defined binocular vision, essentially two groups were distinguished in our sample: those who presented nonstrabismic binocular dysfunctions , insufficiency of convergence, vertical dysfunction and unstable binocular vision ,35) and We identified subjects who presented values of NFV (distance) break above the norm, a condition that causes a tendency toward greater amplitudes of divergence and is associated with greater exodeviation in far than in near, signs that characterize an excess of divergence ,37. ThisOn the other hand , we idenWhen we classified subjects according to the state of the cervical region, we detected that participants with AS below normal present diminished values of blur, break and recovery PFV (near), a situation that leads to a tendency to decrease the range fusional convergence amplitude, signs that can be associated with the presence of insufficiency of convergence  38,41–4–441–43. When analyzing the existing relationships, considering the normative values of both the variables that defined the binocular vision status and the variables referring to the state of the cervical region, we found that in subjects with a smaller amplitude of PFV near, a situation that is identified with insufficiency of convergence ,41–43, aOur literature review showed two hypotheses explaining the relationship between binocular dysfunction and neck dysfunction.Three reflexes influence head, eye and postural stability , which dA subject with altered binocular vision has reported various symptoms  and a moGiven the relationship between both systems, the visual and cervical, future research might propose an intervention consisting of a visual therapy program in subjects with nonstrabismic binocular dysfunction and neck pain, because visual therapy is a useful treatment option in subjects with binocular anomalies , and it After an evaluation of the complete and exhaustive binocular function, we conclude that there is a relationship between nonstrabismic binocular dysfunction and neck pain. The binocular anomalies detected in the sample under study were excess divergence, insufficiency of convergence, vertical dysfunction and fusional vergence dysfunction, and unstable binocular functions were correlated with low activation score and performance index of the deep neck musculature, less mobility of the neck, greater functional disability, and greater cervical pain.S1 File(XLSX)Click here for additional data file.


Although QRS duration (QRSd) is an important determinant of cardiac resynchronization therapy (CRT) response, non-responder rates remain high. QRS fragmentation can also reflect electrical dyssynchrony. We hypothesized that quantification of abnormal QRS peaks (QRSp) would predict CRT response.Forty-seven CRT patients (left ventricular ejection fraction = 23±7%) were prospectively studied. Digital 12-lead ECGs were recorded during native rhythm at baseline and 6 months post-CRT. For each precordial lead, QRSp was defined as the total number of peaks detected on the unfiltered QRS minus those detected on a smoothed moving average template QRS. CRT response was defined as >5% increase in left ventricular ejection fraction post-CRT.Sixty-percent of patients responded to CRT. Baseline QRSd was similar in CRT responders and non-responders, and did not change post-CRT regardless of response. Baseline QRSp was greater in responders than non-responders  and decreased in responders  but increased in non-responders  post-CRT. In multivariable analysis, QRSp was the only independent predictor of CRT response . ROC analysis revealed QRSp (area under curve = 0.80) to better discriminate response than QRSd (area under curve = 0.67). Compared to QRSd ≥150ms, QRSp ≥7 identified response with similar sensitivity but greater specificity . Amongst patients with QRSd <150ms, more patients with QRSp ≥7 responded than those with QRSp <7 .Our novel automated QRSp metric independently predicts CRT response and decreases in responders. Electrical dyssynchrony assessed by QRSp may improve CRT selection and track structural remodeling, especially in those with QRSd <150ms. Cardiac resynchronization therapy (CRT) restores electromechanical left ventricular (LV) synchrony and has been shown to reverse structural remodeling and improve clinical outcomes in heart failure patients with New York Heart Association (NYHA) class II-III function, LV ejection fraction (LVEF) <35%, and QRS duration (QRSd) >120ms . NonetheThe presence of QRS fragmentation has been shown to predict mortality and sudden cardiac death in patients with coronary artery disease and cardiomyopathy . QRS frade novo or upgrade from single or dual chamber ICD), according to current heart failure management guidelines were prospectively enrolled [Forty-seven consecutive patients with ischemic or non-ischemic dilated cardiomyopathy undergoing CRT-defibrillator implantation  lead placed in the RV apex and an LV lead placed in a postero-lateral or lateral tributary of the coronary sinus. Septal and apical LV stimulation were avoided where feasible. All but three patients with persistent atrial fibrillation (AF) underwent transvenous implantation of an atrial lead. Permanent CRT programming of AV delay, LV to RV delay and the LV stimulation vector was optimized for each patient as per the discretion of the treating electrophysiologist based on a combination of factors that included intrinsic PR interval, paced QRS morphology and QRSd after CRT. The study was approved by the local Research Ethics Boards and informed written consent was obtained from all subjects.A baseline ECG in native QRS rhythm was collected within the first 3 months of CRT for all patients and a follow-up study of native QRS rhythm was performed after 6 months of CRT in 38 patients. At each study, the skin was carefully prepared before recording high-resolution 12-lead ECGs for 3 minutes using a digital 12-lead Holter monitor  at 1024Hz sampling rate . To minimize ECG noise, patients were required to lie still in the supine position with their hands at their sides for the duration of the recording.Intrinsic QRSd was measured as the difference between the earliest QRS onset and the latest QRS offset on the baseline digital 12-lead ECG. The presence of intrinsic fQRS was assessed according to published criteria from a paper print out of the digital ECG using standard 12-lead ECG technical specifications  . IntrinsPrior to QRSp detection, each lead was pre-processed to reduce noise and exclude irregular beats  as described in the QRSp for each precordial lead (V1p -V6p) was computed as the greatest QRSp value found in at least 5 of the 90 individual 10-beat windows, an empirically derived threshold used to reduce the chances of a spurious result. QRSp Max and QRSp Mean were calculated for each patient as the maximum and mean of their six precordial lead QRSp values, respectively. Noise was assessed for each lead by computing the average ST segment root mean square noise value (RMS-ST) of all 100 beats .Response to CRT was evaluated functionally based on echocardiographic-derived LVEF in all patients. Two-dimensional echocardiography was performed according to standard clinical procedures within 3 months prior to CRT and 6–18 months post-CRT. LVEF was calculated from the apical 2- and 4-chamber views using the biplane Simpson’s method. Final interpretation was conducted by an echocardiographer blinded to the patient’s clinical and ECG characteristics. CRT response was defined as an absolute increase in LVEF of >5% between pre- and post-CRT assessments .th-75th percentiles) where appropriate. Student's t test or the Mann-Whitney U-test was used for unpaired comparison between CRT responders and non-responders. Categorical variables are presented as frequency or percentage and were compared by χ2 or Fisher's exact test. Amongst responder and non-responder groups, the paired t-test was used to compare continuous variables and McNemar’s test was used to compare categorical variables between baseline and follow-up. One-way analysis of variance was used to compare variables between the patients divided into 4 groups based on combined optimal QRSp and QRSd cut-points. Correlations were assessed using Pearson’s correlation coefficient.Continuous variables are presented as mean ± standard deviation or median and interquartile range .Univariable and multivariable logistic regression analysis was used to assess the predictive value of clinical variables and QRSp Max for CRT response. Amongst the potential QRSp metrics, QRSp Max was chosen Receiver operating characteristic (ROC) curves were constructed for QRSp and QRSd as predictors of CRT response. For QRSp, the optimal cut-point was assessed using Youden’s index and the point closest to , while for QRSd, the conventionally accepted cut-point of 150ms was used . The areAll statistical analyses were performed using MATLAB  or SPSS . A two-sided P<0.05 was considered statistically significant, except when multiple comparisons were made amongst the family of QRSp variables , in which cases a Bonferroni-corrected significance level of P<0.00625 was used to control for potential experiment-wise error.Patient baseline characteristics are presented in The baseline ECG study was conducted at a median 1.8 (1.3–2.4) months post-CRT and occurred at similar times in responders and non-responders (1.8 (1.3–2.4) vs. 1.8 (1.3–2.3) months, p = 1.00). As presented in Importantly, RMS-ST noise did not relate to QRSp or CRT response. There was no correlation between the QRSp and average RMS-ST noise value of each precordial lead , suggesting that noise did not significantly contribute to the QRSp signal. There was also no difference in the precordial lead RMS-ST noise values between responders and non-responders .Univariable and multivariable logistic regressions models for the prediction of CRT response are presented in Since intrinsic QRSd is an established marker of CRT response, ROC analysis was used to evaluate the performance characteristics of baseline QRSp Max compared to baseline QRSd . AlthougAs illustrated in Amongst the 38 patients with repeat ECG studies, the follow-up ECG was performed at a median 8.3 (7.4–11.9) months post-CRT with a trend toward being earlier in responders than non-responders (7.9 (6.7–10.6) vs. 11.6 (7.8–14.6), p = 0.06). The main findings of our study are as follows: (i) QRSp independently predicts functional CRT response in cardiomyopathy patients, unlike QRSd, LBBB or fQRS, (ii) QRSp ≥7 identifies CRT responders with greater accuracy than QRSd ≥150ms or fQRS, and (iii) QRSp decreases in CRT responders while increasing in CRT non-responders, whereas QRSd and fQRS do not change irrespective of CRT response.Electrical conduction delay in the LV causes dyssynchronous activation, which may respond to CRT. Currently, electrical dyssynchrony assessment for CRT eligibility is based solely on the QRSd ≥120ms and LBBB morphology . MoreoveOur study for the first time shows that QRSp independently predicts functional CRT response in cardiomyopathy patients after adjusting for QRSd, while LBBB and fQRS were not predictive. For each QRS peak detected, the odds of CRT response increase by 1.6-fold. QRSp ≥7 identified CRT responders with similar sensitivity as QRSd, but with significantly greater specificity (74 vs. 32%) and positive predictive value (82 vs. 66%). Importantly, QRSp discriminated CRT responders from non-responders among patients with QRSd >150ms as well as those with QRSd <150ms. Patients with QRSd ≥150ms who also had QRSp of ≥7 had a >2-fold higher probability of attaining functional CRT response (83% vs. 40%) and a larger LVEF improvement compared to those with QRSp <7. In addition, although a minority of our study patients had QRSd <150ms, QRSp <7 in this group predicted 100% non-response to CRT.Electrical resynchronization of the ventricles with CRT is mediated by fusion of RV and LV propagating wavefronts, which reduces global activation time and mitigates conduction block . This maFragmentation of the QRS complex on the standard 12-lead ECG reflects conduction delay and abnormal wavefront propagation caused by fibrotic infiltration and/or ventricular ion-current remodeling . A linkaAmongst the CRT population, paced fQRS has also been associated with future ventricular tachyarrhythmia events and sudden cardiac death in patients with non-ischemic cardiomyopathy . ParadoxIn our study, QRSp independently predicted functional CRT response and a higher QRSp was associated with greater LVEF recovery in both patients with QRSd <150ms as well as those with QRSd ≥150ms. On the other hand, we did not find fQRS to be associated with functional CRT response. This discrepancy from the above-mentioned fQRS-based studies suggests that the quantitative evaluation of QRS fragmentation with QRSp provides a more specific measure of electrical dyssynchrony. QRSp is a quantitative metric measured from high-resolution ECG recordings (≥1000Hz). The abnormal QRS peaks counted are typically not visually apparent  and do not include larger “normal” peaks that define the broad shape of the QRS complex . In contOur sample size is limited and effectively that of a pilot study; however, significant differences in QRSp between responders and non-responders to CRT were still demonstrable. As the proportion of patients with QRSd <150ms was low, applicability of the QRSp metric for determining CRT-eligibility in these patients should be confirmed in a larger prospective study. Second, we could not characterize whether late reduction in QRSp was principally due to reverse cardiac remodeling through CRT or co-interventions such as medical therapy for heart failure, but medical therapy had been optimized prior to CRT implantation and was systematically adjusted post-implant in all patients as needed per heart failure practice guidelines . Third, Automated quantification of abnormal QRS peaks from high-resolution ECGs is an independent predictor of CRT response in patients with cardiomyopathy. QRSp ≥7 identifies CRT responders with similar sensitivity as QRSd, but with significantly greater specificity. For each additional QRSp detected, the odds of CRT response increase by 1.6-fold. In addition, long-term change in this novel marker of electrical dyssynchrony tracks mechanical recovery in CRT responders. Larger CRT cohort studies are warranted to confirm these findings and the utility of QRSp in improving CRT selection and ventricular arrhythmia prognostication, particularly in those with QRSd <150ms and RBBB or intraventricular conduction delay.S1 Methods(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


The main purpose of the paper was to evaluate selected psychomotor abilities of handball players depending on the competition class (league), position on the court, training seniority and the dominant hand. The study covered a group of 40 handball players (age: 24.02 ± 3.99), while 50 non-training men (age: 22.90 ± 1.13) formed the control group. Studies were performed using Test2Drive computer tests. The following four tests were used for measuring psychomotor fitness: simple reaction time test, choice reaction time test, hand-eye coordination test and spatial anticipation test. An analysis revealed that handball players had better reaction times and movement times than the control group. The league, position on the court, training seniority and the dominant upper limb were analysed for their impact on the reaction time and movement time in handball players. An analysis of psychomotor abilities of handball players with regard to the league revealed that in the majority of tests the Superliga players had a shorter reaction time than players in lower leagues. Handball is an Olympic discipline game where successful performance depends on a number of basic abilities in particular strength, power, speed and endurance. Creativity in combination with speed and strength as well as coordination makes this sport very attractive but tough to play . StatistEvaluation of motor skills and anthropometric characteristics of handball players was a subject matter of a number of studies. Jensen et al. 1997)  showed a  showed Besides evaluation of competitor’s basic somatic parameters ,10,11, mThe previous research shows that reaction time is influenced by the following factors such as: body height , traininThe main purpose of the paper was to evaluate selected psychomotor abilities of professional handball players using computer systems Test2Drive. The data of handball players were evaluated in relation to the control group consisting of non-training men. The impact of the competition class (league), position on the court, training seniority and the dominant hand on the studied psychomotor abilities were analyzed.The study population consisted of 40 professional handball players , while 50 non-training men (age: 22.90 ± 1.13) formed the control group. The league in which the players played , position on the court , seniority  and the dominant upper limb  were taken into account in the analysis of the players’ psychomotor abilities. Characteristic of the handball group with analyzed sub-groups was presented in The study was carried out in 2017. The scope and project research were assessed by the Bioethics Committee of the University of Rzeszow, which declared no contraindications or ethical violations (Resolution No. 02/05/2016 of 2 February 2016).Psychometric computer tests in the Test2Drive system  were the study method . The TesSIRT—evaluation of reaction speed and its stability. The stimuli signalling field changed its colour in the right moments of time. The reaction to the stimuli involved moving the finger from the START field to the reaction time field marked in blue.CHORT—evaluation of the speed and adequacy of reaction in a complex situation. Horizontal (stimuli) benchmarks and vertical stimuli which require a reaction, and a slant benchmark  that does not require reaction were displayed in the top signalling row. The response to the stimuli involved moving one’s finger from the START field to one of the two reaction fields . During the neutral stimulus, the finger was kept on the START field.HECOR—evaluation of eye–hand coordination. The test required careful observation of the board and a quick reaction to the displayed red signalling field. The test participant was supposed to move his finger from the START field to the blue reaction field and return the finger to the START field.SPANT—evaluation of eye-hand coordination using complex spatial information. On the top, left and right of the test board there were signalling fields two of which (on in the row and one in a column) turned red simultaneously. In response to the stimulus the test participant was supposed to indicate with his finger the field on the crossing of the lit row and column, and put the finger back to the START field.Every test was performed in a standing position facilitating access to the screen area. The parameters of the tests are presented in Z—standardized value for the U-value, r—correlation coefficient where r assumes the value ranging from −1.00 to 1.00, N—the total number of observations on which Z is based. The analysis was performed with the GNU R software [Basic statistical measures i.e., arithmetic mean, standard deviation and coefficient of variability were used in the study. In order to identify the significance of differences between the groups the Mann-Whitney-Wilcoxon test was used. The effect size was calculated using formula :(1)r=ZNsoftware .The results obtained for handball players were compared with the results for the control group . Analysip < 0.05) for the studied parameters. HECOR parameters were analysed on the following stage (p < 0.05). Handball players obtained better results than the non-training group. Lower diversification inside the groups was observed among handball players for the variables. The research also showed that the RT and MT measured during SPANT among the players was significantly lower than in the control group. Moreover, it can be observed that handball players gave more correct responses in the SPANT (93.1%) than the control group (88.1%). The obtained results revealed statistical significance.ng stage . Statistp < 0.05) were observed among the 1st league and Superliga players for the MT obtained in the CHORT test. The tests also revealed that Superliga handball players had the shortest RT in HECOR (384.3 ms). Analysing the second HECOR variable it can be observed that Superliga handball players had the worst MT (251.4 ms) as compared to other players. The best MT was recorded for the 1st league players (228.0 ms). SPANT was the last analysed test. The tests revealed that Superliga players had the best RT (562.2 ms) and the longest MT (292.6 ms). The 1st league players demonstrated the highest MT level in SPANT (253.3 ms).On the following stage of the study, the RT and MT were analysed depending to the player’s position on the court . The anaA further analysis presents the RT and MT variables depending on the player’s seniority . As regap < 0.05) were demonstrated between reaction times (RT) in the hand-eye coordination test (HECOR). In most of the tests, left-handed players achieved better mean values characterising the reaction time, apart from two complex tests, i.e., MT CHORT and MT SPANT, in which the right-handed players reached slightly shorter reaction time.Studies of psychomotor abilities of handball players included the aspect of the players’ dominant hand . Based oEvaluation of the coordination level of psychomotor abilities in handball players was the subject matter of the study. The evaluation was made in the aspect of differences between training and non-training participants, as well as within the group of handball players with regard to the league , position on the court , sports seniority  and the dominant hand .The literature highlights a positive impact of physical exercises on the level of psychomotor abilities ,17,27,28The results of studies carried out with regard to the game league show that Superliga players have the highest level of psychomotor abilities. They achieved the shortest reaction times in all tests (except SIRT). The results in this group were 638.2 ms for CHORT, 384.3 ms for HECOR and 562.2 ms for SPANT test. It was also observed that Superliga players had the poorest MT in each of the analysed tests. The 1st league players reached the best MT (except SIRT). Williams and Walmslay came to similar conclusions . AccordiThe literature contains many papers investigating the relationship between position on the court and movement abilities ,32,33,34Age is a factor which greatly affects reaction time. According to the studies by Luchies et al. , reactioConsidering the issues related to the level of psychomotor abilities one shall take lateralisation into account. Evaluation of functional asymmetry of the right and left side of the human body was studied e.g., by Bryden , who exaThe presented paper has potential limitations. Study limitations are related to the number of handball players. In the group of 40 players there is only three of pivot players and four left handed players. The conclusions being drawn about those subgroups may be potentially not precise.The evaluation of psychomotor abilities using i.e., Test2Drive can help handball coaches in appropriate selection of individual abilities of players. The increasing tempo of handball game is related to the constant improvement of the technical skills of handball players, so the time of contact with the ball is shorter. Professional handball training using psychomotor abilities should be recommended for coaches to improve the tempo of game, anticipation and decisiveness in the one-on-one game play. The present findings shows that the key point is using during training exercises connected with psychomotor abilities a special reaction time. Studies also showed that reaction and movement time is related to the level of handball game. This fact shows that it is crucial to develop and improve the psychomotor abilities in handball training process.p < 0.05).Handball players demonstrate a higher level of the analysed psychomotor abilities as compared to the control group (non-training people). The differences in time indicators among groups in all tests are statistically significant (An analysis of psychomotor abilities of handball players with regard to the league revealed that in the majority of tests the Superliga players had a shorter reaction time than players in lower leagues.Both, position on the court and training seniority have got an impact on indicators of psychomotor abilities.The factor which differentiates the level of the studied skills is the dominant hand: left-handed players were characterised by shorter reaction times.The data obtained may be helpful in the development and optimization of training systems depending on the position on the court.The following conclusions were formulated based on the analysis of the data:


Alcohol consumption during pregnancy can lead to damaging effects on an infant’s health, including fetal alcohol spectrum disorders. Project Changing High-risk alcOhol use and Increasing Contraception Effectiveness Study (CHOICES), a program developed to reduce alcohol-exposed pregnancies through decreased alcohol consumption and increased birth control use, has been implemented with success in a variety of populations. The CHOICES program was structured to align with the transtheoretical model (Stages of Change), a popular public health model. Although studies have described the Stages of Change in the context of a variety of health behaviors, none have addressed the qualitatively distinct characteristics of each stage in the context of American Indian (AI) women’s alcohol and birth control use. A framework analysis of 203 participants’ written responses during their experience in the Oglala Sioux Tribe (OST) CHOICES Program was conducted. As a conceptual framework, the transtheoretical model of behavior change was applied to the participants’ experiences, with two staff reading the open-ended responses and coding based on the stage of change. Participants’ responses suggest qualitatively distinct stages as well as a progression through the stages for both behaviors during the course of the program. Many participants mentioned their children, education, and work as inspiration to decrease their unhealthy behaviors. Common barriers to behavior change were found across both behaviors. The open-ended responses uncover common themes in the experiences of the participants. These results can help inform future programs which hope to address the needs of AI communities. Fetal alcohol spectrum disorder (FASD) is the continuum of detrimental outcomes in individuals prenatally exposed to alcohol. This includes a diagnosis of fetal alcohol syndrome (FAS), which is evidenced by facial abnormalities, growth retardation, delayed brain grown, and neurobehavioral impairments . As FASDFASD occurs in all socioeconomic and racial/ethnic groups. Recent research in an Upper Midwest study reported FAS in 5.9 to 10.2 per 1,000 children with no differences by race . AlthougChanging High-risk alcOhol use and Increasing Contraception Effectiveness Study (CHOICES), an evidence-based intervention that has demonstrated success in reducing risk for AEP , the maintenance stage has been reached. Finally, in the termination stage, the individual has no temptation to return to their behavior and is confident that regardless of their circumstances, they will not relapse. MI, as used in the CHOICES Program, is particularly compatible with TTM as it allows for individualized plans that are tailored to a particular stage , commonly known as the Stages of Change, which is particularly compatible with MI . TTM proar stage .The purpose of this article is to describe movement through these Stages of Change for AI women enrolled in the Oglala Sioux Tribe (OST) CHOICES Program, a tribally run intervention where the original CHOICES program was modified to be more culturally relevant for implementation with AI women . QuantitInstitutional review boards at the tribal level and at the principal investigator’s institution reviewed the program and found it did not constitute research but was instead a program evaluation that fell outside the prevue of the review boards. The OST CHOICES Program enrolled participants at three sites: two locations were on the reservation and a third in an urban setting approximately 2 hr from the reservation. Eligibility for the program was based on race (AI), age (18 years and older), and risky behavior status: Participants had to be drinking at binging levels—four or more drinks per occasion or eight or more drinks per week . EligibiChange Plan—were then utilized to help participants identify personal change goals for both alcohol and birth control.OST CHOICES sessions were held across multiple locations and included two to four sessions approximately 1 to 2 weeks apart . During Change Plan regarding their alcohol and contraceptive use.During Session 2 or 4, the interventionist asked open-ended questions about any changes in alcohol and birth control use since the first session, and participants completed temptation and confidence scales regarding their temptation to use alcohol or not use birth control in specific situations, and alternatively, their confidence to not drink and/or to use birth control in those same situations. The temptation and confidence measures are particularly sensitive to the changes that are involved in progress in the Stages of Change and are good predictors of relapse . After bAll the qualitative data responses were transcribed and analyzed via deductive content analysis . A totalInter-rater reliability was calculated using Cohen’s kappa. The two coders separately coded the responses using the final draft of the codebook. The Cohen’s kappa value was 0.58, “moderate” (0.41–0.60) when interpreted using the standards set by Within the sample, relatively few women were in the precontemplation stage. Generally, women in the precontemplation stage did not see their drinking as problematic, or simply did not want to reduce their drinking. Many felt that their drinking levels could become problematic in the future, but that their current levels of drinking were acceptable. As one participant expressed, “It makes me think of how I can get into trouble when I do drink, but I don’t drink that much.” Alcohol consumption in certain scenarios was clearly understood to be connected to negative effects, with one woman stating, “I feel that my level of drinking is ok (but) if I did increase my drinking, it would increase the chance of getting in trouble.” Another woman did consider changing her drinking in the future, as “I don’t really think of nothing at the moment, but down the line eventually I do wanna stop drinking.”Women in the precontemplation stage for birth control felt they did not need birth control, or did not express an interest to learn more about birth control. Within the sample, even fewer women were in the precontemplation stage for birth control use than they were for alcohol consumption. Although some women were worried about the side effects of birth control, many in the precontemplation stage simply stated that they did not want to use birth control and did not provide any reason.Many participants were seen in the contemplation phase, especially when it came to alcohol reduction. Logically this makes sense, considering that they were enrolled in a program dedicated specifically to addressing substance use. Women in the contemplation stage expressed a desire to decrease their drinking, and were aware that their drinking caused negative outcomes. Women were very cognizant of the effects drinking had on their family, and often named their family as one of the main reasons they wanted to decrease their drinking. One participant put it simply, stating, “I want to control my drinking, not my drinking controlling me.” Although women definitely desired to decrease their drinking, an overwhelming number of them lamented the fact that the activity of drinking is one that is socially embedded within many of their support systems, and was also a form of stress relief. Participants often reported that “stressful situations” and “peers I associate with, negative thoughts” were obstacles in their way of decreasing their drinking.In the context of birth control use, the contemplation stage was manifested in women who expressed a desire to learn more about birth control, expressed the importance of birth control, stated that they did not want more children, or expressed a desire to use birth control more effectively. One participant was open in writing, “I am a risky drinker, and I don’t want any more children.” A large number of participants were similar; they felt that for a variety of reasons that it would be best to wait to have children. Many participants moved from contemplation to preparation from the first visit to the second. This is likely due to the emphasis on educating participants about contraception and on encouraging participants to make an appointment to see a health care provider.In the preparation stage, women had decreased their drinking since their first CHOICES session, or had taken a step to decrease their drinking in the near future. Many of the women at this stage mentioned motivations to change such as their family, school, and jobs. For example, one woman expressed her reasons for decreasing her alcohol consumption were “because of my health, my kids, my freedom.” One challenge was difficulty of stopping drinking completely. Decreasing alcohol slowly over time was found to be a common solution, with one participant explaining, “Because it’s too hard to quit drinking all at once, so I’ll just try to slow down,” and another writing, “Take small steps at a time.” Women also mentioned that they felt the need to learn new ways to confront the conflicts that frequently provoked them to drink; one woman expressed that she wanted to learn “how to deal with conflict and temptation and people who are part of the problem.” Family and friends seemed to play an important part in this process, sometimes as the instigators: “My brothers come over and want to drink at my house every day. Told my brothers to stay away if they’re drinking.”Women in the preparation stage had scheduled an appointment to meet with a health care provider, had independently learned more about birth control, or were using a birth control method, but inconsistently. Many women in this stage had appointments scheduled to begin birth control but expressed concern that they would be unable to attend their appointment due to other responsibilities or lack of transportation. Some women wanted to use condoms regularly as a form of birth control but felt worried that their partners would object to this. One woman stated she was “giving into the pressure of my boyfriend to not use condoms.” Another woman also mentioned that they would attempt to overcome this challenge by “talking with him about the importance of using condoms.”In the context of alcohol consumption, the action stage was manifested in women who had changed their lifestyle to decrease their drinking, including attending Alcoholic Anonymous (AA) meetings or decreasing their drinking to below binge drinking levels. Barriers to continued sobriety included stress and temptation to drink socially. One woman expressed, “I am now tempted to drink when something bad happens or something good, but (I) cut down.” Many women were very focused on achieving goals to keep them on their chosen paths, for example, “I can keep track of my months of being sober.” Thinking about the consequences of their actions was also mentioned as a strategy to stay away from alcohol. One participant said that she would “think about it first and think about my baby first before drinking.”For birth control use, women in the action stage had changed their lifestyle to avoid unplanned pregnancy by using a form of contraception. Many of the women in this stage expressed that they were not yet ready for children: “Not being ready for a baby is okay and don’t rush myself!” Many women also communicated an understanding that drinking while pregnant could cause damage to an unborn baby: “I’m currently on birth control and when I do decide to have my own child, he or she won’t be exposed to alcohol.” For some women, having medical appointments for a Depo shot and remembering to take the pill every day were concerns. One woman planned on “keepin’ sticky note on my mirror reminding me to take my pill.”Quantitative data from OST CHOICES, as reported elsewhere, showed a significant decrease in AEP risk from baseline at both 3- and 6-month follow-ups, indicating the significant impact of the OST CHOICES intervention . HoweverThe qualitative analysis indicates some of the reasons behind the success of the OST CHOICES intervention: that participants moved through the Stages of Change while participating in the intervention. At the first session, the majority of participants were in the contemplation stage for changing alcohol behavior, with few participants in the preparation stage and even fewer in the action stage. Most participants were likely in the contemplation stage specifically because of their inclusion into the OST CHOICES Program. At the second session, the distribution had changed, with less than half the previous number of participants in the contemplation stage, only a few participants in the preparation stage, and an increased number both in the preparation stage and in the action stage. As highlighted in the “Results” section, many participants had moved from thinking about change to actively making lifestyle changes to reduce risky drinking.Similar results were found for contraception, although like the quantitative data results, contraception behaviors changed in a more striking way. At the first session, the majority of participants were in the contemplation stage for birth control, likely because of inclusion in the OST CHOICES Program, with few participants in the other stages. At the second session, the distribution had changed with many in the action stage . We speculate this is because of the nature of the behavior change—making a birth control appointment and getting a prescription for birth control may be much more feasible to accomplish than decreasing alcohol consumption, especially when drinking is typically a social activity that many in a social network also participate in. For example, one woman explained changing her contraception was the most logical way for her to prevent FASD considering her personal situation: “I drink and I don’t want to have a FAS baby or bring someone else into this world because I can’t take care of it. So I got an IUD.”This study is unique in several aspects. Few previous studies have utilized TTM within qualitative studies, and those that do, do not focus on the use of alcohol or contraception and do not involve AI populations. The one qualitative study that incorporated TTM with AI women worked to develop a culturally specific tool for mammogram screenings . TTM hasFinally, our study is unique within the CHOICES literature, which has not yet had any results reported via qualitative means. As is noted in the introduction, the CHOICES intervention is based within the TTM. The CHOICES intervention employs several TTM methods, such as decisional balance and temptation/confidence exercises, which help to identify self-efficacy toward behavior change. However, previous CHOICES research has focused on the behavioral impact of the intervention, with no reported results on the process of change via the TTM lens nor qualitative data collected throughout the intervention .As noted from other OST CHOICES manuscripts, there were some limitations to our study. Our participants were typically self-referred, so the motivation to enter into the OST CHOICES Program may indicate that some women were already initiating behavioral change. In addition, all participants were AI women, and this sample was mainly women from one reservation community and one nontribal site, therefore results cannot be generalized.Overall, the results showed that women progressed through the course of the program. Specific activities such as decisional balance exercises, readiness rulers, and goal setting help to encourage women’s movement through the stages, and our qualitative data highlight some excellent evidence as to the “how” and “why” a participant moved through the stages. Several questions remain unanswered and can be addressed in future research. Although the program had a significant impact on contraception as shown in both the qualitative and quantitative results, more direct emphasis must somehow be placed on reducing risky drinking. The data, both qualitative and quantitative, show that CHOICES can work with AI/Alaska Native (AN) women, but additional efforts are needed for reducing alcohol use. Introducing specific interventions into the CHOICES intervention to address alcohol will take us to the next level in reducing risk for alcohol-exposed pregnancies with high-risk women.