Reversible Step Research Articles

The Influence of Thermal History on the Multistage Transformation of NiTi Shape-Memory Alloys

The multistage martensitic phase transformation of a polycrystalline NiTi shape-memory alloy (50.3 at. %Ni–49.7 at. % Ti) has been studied by means of calorimetric measurements. After a conventional thermal treatment followed by successive thermal cycles, the initial two-step forward transformation splits into four-overlapping stages. However, the reverse martensitic transformation maintains the initial two-step sequence, usually assigned to the B19′→R→B2 transformation. The correlation between the forward and reverse steps has been established by means of selected thermal cycles together with an estimation of their enthalpy and thermal hysteresis. These results have also provided information about the storage of the elastic strain energy and the frictional works associated with the variants’ nucleation. Moreover, the study around the forward transformation temperature range by means of uncompleted thermal cycles undoubtedly shows the presence of temperature memory effects in both stages.

Mono- and di-(2,3,5,6-tetrafluoro-4-N,N-dimethylaminophenyl) meso-tetraarylporphyrins: Synthesis, spectral, structural and electrochemical studies

A new series of mono-/di-aminated meso-tetraarylporphyrins has been synthesized and characterized by conventional spectroscopic methods. Crystal structure analysis shows that interactions involving halogens are the major contributors, and the relative contributions are 56% and 53% respectively for MB2c and MB3c. All the compounds were electrochemically analyzed under different reaction conditions and showed a positive shift in the reduction and oxidation potentials. The HOMO–LUMO energy gap was altered with respect to the nature of the supporting electrolyte and reference electrodes used. The linear behavior of Randles-Sevcik plots indicate that the redox processes are diffusion controlled; the first reduction/oxidation is a reversible one-electron step whereas the second reduction/oxidation is a quasi-reversible one-electron process. Results also reveal that the mono-aminated porphyrins are more electron deficient than non-aminated and di-aminated porphyrins.

FREQUENCY OF HEPATITIS C VIRUS GENOTYPES /SUBTYPES ASSOCIATION WITH RESPONSE TO THERAPY IN A SAMPLE OF HCV INFECTED IRAQI PATIENTS

Background: Hepatitis C virus (HCV) is an important human pathogen affecting 120-170 million individuals in the world. Identification of the causative virus genotype is of a significance to both clinical practices and predict the likelihood to therapy response. Objective: To determine the distribution of HCV genotypes/subtypes and its association with response to therapy among newly diagnosed HCV patients. Methods: Fifty patients with confirmed anti-HCV antibodies were included in this study for HCV genotyping in association with response to therapy. Blood samples from patients were subjected to RNA extraction and reverse transcription step; viral load of HCV was measured by polymerase chain reaction (PCR) at time zero, 3 months and 6 months of dual therapy. Response to therapy was measured as a decrease in viral load (2 log or more) and was described as: good (median log is zero after 6 months of therapy), moderate (median log declines more than 2 log but not zero after 6 months of therapy), poor (median log does not decline or decline less than 2 log after 6 months of therapy). Results: Two genotypes of HCV were detected, genotype 4 was the predominant (27/50, 54%) followed by genotype 1 (23/50, 46%). For HCV subtypes, subtype 1a was of highest percentage (28%) followed by 4e (24%), 1b (18%), 4a (14%), 4b (12%), and 4e (4%).The results revealed a significant association between HCV subtypes, but not genotypes, with response to therapy. HCV subtype 1a followed by 4a showed the highest rate of response 85.7% and 71.4%, respectively, while interestingly HCV subtype 4d showed no response and 1b showed poor response 11.11%. Conclusion: HCV subtypes of great importance in predicting success to HCV therapy and it is believed this would affect the newly emerging directly acting drugs as well. Keywords: HCV, genotypes, response to therapy Citation: Abdulhassan LJ, Abdulamir AS, Alkhalidi NM, Alwaysi SA. Frequency of hepatitis C virus genotypes /subtypes association with response to therapy in a sample of HCV infected Iraqi patients. Iraq. Iraqi JMS. 2018; Vol. 16(1): 30-40. doi: 10.22578/IJMS.16.1.6

Effect of different reverse transcription aproaches in Pru p 3 transcripts semiquantitative amplification

Reverse transcriptase transcribes the cDNA based on its previous extraction and standardization. Reverse transcription step is considered to be critical in the workflow of quantification of transcribed genes. The aim of the study was to extract total RNA by different methods and to analyse the results of the subsequent reverse transcription reaction when different commercial RT kits were used to process RNA extracted from pulp of matured peach fruit. Mature peach pulp was used in the study. The fruit of variety Vistarich was collected in summer 2017 in the orchard of Dvory nad Žitavou. Two RNA extraction methods, TRIzol® Reagent and GeneJET Plant RNA Purification Kit, were tested in to determine the suitable method for peach fruit RNA extraction. Three different cDNA reagent sets were used to transcribe 115 ng/500 ng total RNA or 11 ng/115 ng, respectively. Both variants of the primers, random hexamers as well as oligo (dT) 18, were used to anneal the target mRNA of Pru p 3 allergen following the manufacturer instructions. No specific effect was obtained in the case of peach fruit when using ethanol-extracted tissue treatment and the effect of the used extraction method was more significant. The A260/230 ratios were similar for three from four tested methods. In the case of these three methods, the A260/A230 ratios for all the extracted samples were higher than 1.9 which indicates high purity without contamination by polyphenols and polysaccharides. The specificity of obtained amplicons was proved by restriction cleavage using Tse I restriction endonuclease. This provided the cleavage of the 179 bp long product in all amplicons. Working with mature fruit meet a specific situation in the field of RNA extraction and subsequently all the downstream applications. That is, why choosing the most fitting methods and kits is a crucial step. Here, the method for the semi-quantitative analysis of the Pru p 3 allergen expressions was set up in the way that will be directly applicable for Pru p 3 expression analyses.

Differences in the energetics of collagen denaturation in connective tissue from two muscles

The thermal denaturation of collagen the perimysium of intramuscular connective tissue isolated from bovine Semitendinosus (ST) and Perctoralis profundus (PP) muscles was investigated using a range of heating rates in differential scanning calorimetry (DSC) and analyzed by application of the Kissinger–Akahira–Sunose (KAS) and Lumry-Eyring models. Thermograms showed a broadening of endotherms and a shift towards higher temperatures as the thermal scanning rate increased. These features are consistent with the two-step process of a reversible transition between native and unfolded collagen molecules followed by an irreversible transition between unfolded and denatured states. There were small differences between muscles in the onset temperatures of the thermal transitions at heating rates of 0.5 K min−1, while both the KAS and Lumry-Eyring models yielded similar values for the effective activation energy of the whole two-step process, the Lumry-Eyring model allowed a greater insight into differences in the reversible and irreversible steps between the perimysium both muscles. Reversible unwinding of the triple-helical collagen molecules in the perimysium from ST muscle required more energy than in PP muscle. It is speculated that the presence of large amounts of elastin in the perimysium of ST muscles may influence this due to a protein crowding mechanism, or by affecting the covalent cross-linking of the collagen.

Mechanistic Insights into the Activation of Soluble Guanylate Cyclase by Carbon Monoxide: A Multistep Mechanism Proposed for the BAY 41-2272 Induced Formation of 5-Coordinate CO-Heme.

Soluble guanylate cyclase (sGC) is a heme-containing enzyme that catalyzes cGMP production upon sensing NO. While the CO adduct, sGC-CO, is much less active, the allosteric regulator BAY 41-2272 stimulates the cGMP productivity to the same extent as that of sGC-NO. The stimulatory effect has been thought to be likely associated with Fe-His bond cleavage leading to 5-coordinate CO-heme, but the detailed mechanism remains unresolved. In this study, we examined the mechanism under the condition including BAY 41-2272, 2'-deoxy-3'-GMP and foscarnet. The addition of these effectors caused the original 6-coordinate CO-heme to convert to an end product that was an equimolar mixture of a 5- and a new 6-coordinate CO-heme, as assessed by IR spectral measurements. The two types of CO-hemes in the end product were further confirmed by CO dissociation kinetics. Stopped-flow measurements under the condition indicated that the ferrous sGC bound CO as two reversible steps, where the primary step was assigned to the full conversion of the ferrous enzyme to the 6-coordinate CO-heme, and subsequently followed by the slower second step leading a partial conversion of the 6-coordinate CO-heme to the 5-coordinate CO-heme. The observed rates for both steps linearly depended on CO concentrations. The unexpected CO dependence of the rates in the second step supports a multistep mechanism, in which the 5-coordinate CO-heme is led by CO release from a putative bis-carbonyl intermediate that is likely provided by the binding of a second CO to the 6-coordinate CO-heme. This mechanism provides a new aspect on the activation of sGC by CO.

Activity and selectivity of Ni nanoclusters in the selective hydrogenation of acetylene: A computational investigation

In this work, the Ni n ( n = 2–10) nanoclusters were investigated to design new catalysts for the selective hydrogenation of acetylene. Our results show that among the Ni n nanoclusters, the Ni 6 nanocluster can be used as a catalyst in the reactions of hydrogenation. In the presence of the Ni 6 nanocluster, the E a of the forward step in the reaction of conversion of vinyl to ethylene was 21.21 kJ/mol lower than that of the reverse step in the reaction of conversion of acetylene to vinyl. Also, the E a of the forward step in the reaction of conversion of ethyl to ethane was 96.59 kJ/mol higher than that of the reverse step in the reaction of conversion of ethylene to ethyl. According to the obtained results, the Ni 6 nanocluster can selectively act in the hydrogenation of a mixture of acetylene and ethylene.

Experimental and numerical studies of the stress state at the reverse step joint in heavy timber trusses

Traditional notched joints can be used in timber structures to connect two structural parts under compression loads that concur at a somehow diagonal angle. They can be used to connect the top-chord and the tie-beam of timber trusses, which have been widely in use as roofs in buildings, agricultural and industrial service constructions. Computer-aided manufacturing has again encouraged its use, resulting in an increased demand to improve the knowledge of these assemblies. In this paper, full-scale experimental tests with structural loads and cross-sections were carried out for front notched joint in reverse configuration, also named heel joint, which is commonly used to avoid the tie-beam from standing out of the meeting with the rafter. A monotonic load was applied along the rafter axis by a hydraulic actuator. The skew angle between the pieces was 30°, which is one of the most commonly used in covers solved with heavy timber trusses. Due the large cross-sections used in these trusses, the material used in the tested specimens was glue laminated timber made from Norway spruce. The ultimate force, failure form, and experimental load–strain diagrams were obtained by extensometry. Deformation and displacement values were recorded to validate the models generated by finite elements method. The behaviour observed in the experiments was captured well by the models and allowed for the study of the stress state, the distribution of loads between contact surfaces, the shear stress distribution, and the presence of tensile stress perpendicular to the grain, especially dangerous in this joints.

Kirkendall void formation in reverse step graded Si1−xGex/Ge/Si(001) virtual substrates

Formation of Kirkendall voids is demonstrated in the Ge underlayer of reverse step graded Si1−xGex/Ge buffer layers grown on Si(001) using reduced pressure chemical vapour deposition (RP-CVD). This phenomenon is seen when the constant composition Si1−xGex layer is grown at high temperatures and for x ≤ 0.7. The density and size of the spherical voids can be tuned by changing Ge content in the Si1−xGex and other growth parameters.

Highly parallel direct RNA sequencing on an array of nanopores.

Sequencing the RNA in a biological sample can unlock a wealth of information, including the identity of bacteria and viruses, the nuances of alternative splicing or the transcriptional state of organisms. However, current methods have limitations due to short read lengths and reverse transcription or amplification biases. Here we demonstrate nanopore direct RNA-seq, a highly parallel, real-time, single-molecule method that circumvents reverse transcription or amplification steps. This method yields full-length, strand-specific RNA sequences and enables the direct detection of nucleotide analogs in RNA.

On the asymmetric autocatalysis of aldol reactions: The case of 4-nitrobenzaldehyde and acetone. A critical appraisal with a focus on theory.

Under neutral conditions, spontaneous mirror symmetry breaking has been occasionally reported for aldol reactions starting from achiral reagents and conditions. Chiral induction might be interpreted in terms of autocatalysis exerted by chiral mono-aldol or bis-aldol products as source of initial enantiomeric excesses, which may account for such experimental observations. We describe here a thorough Density Functional Theory (DFT) study on this complex and otherwise difficult problem, which provides some insights into this phenomenon. The picture adds further rationale to an in-depth analysis by Moyano et al, who showed the isolation and characterization of bis-aldol adducts and their participation in a complex network of reversible steps. However, the lack of enantiodiscrimination (ees vanish rapidly in solution) suggests, according to the present results, a weak association in complexes formed by the catalysts and substrates. The latter would also be consistent with almost flat transition states having similar heights for competitive catalyst-bound transition structures (actually, we were unable to locate them at the level explored). Overall, neither autocatalysis as once conjectured nor mutual inhibition of enantiomers appears to be operating mechanisms. Asymmetric amplification in early stages harnessing unavoidable enantiomeric imbalances in reaction mixtures of chiral products represents a plausible interpretation.

A New Reversal Steps Stapedotomy.

To evaluate whether removal of the stapes head and not the entire stapes superstructure prevents footplate complications. Prospective study. One hundred and forty-five patients with otosclerosis were included. Patients were divided into 2 groups as follows: Individuals in group 1 (n = 75) underwent removal of the entire stapes superstructure; individuals in group 2 (n = 70) underwent removal of the stapes head only. These 2 groups were further subdivided into 2 subgroups based on the type of otosclerosis. Group 1a included 55 cases of marginal otosclerosis, while group 1b included 15 diffusive and 5 biscuit otosclerosis cases. Group 2a included 51 marginal otosclerosis cases, while group 2b included 14 diffuse and 5 biscuit otosclerosis cases. Estimations were made regarding statistical correlation between type of reversal steps stapedotomy, otosclerosis type, incidence of footplate complications, and hearing results. There were 9 footplate complications in group 1b and no complications in group 2b. This difference was significant. Air-bone gap closure to within 10 dB was achieved in 65% of cases in group 1b and 94.7% in group 2b. This difference was significant. The new stapedotomy prevented footplate complications and improved hearing results in diffuse and biscuit otosclerosis.

Conditions that Influence Drivers’ Behaviors at Roundabouts: Increasing Yielding for Pedestrians who are Visually Impaired

Introduction This study examined the effects on drivers’ yields of seven conditions (pedestrian behaviors) at a roundabout, measuring yielding behaviors. Methods At a roundabout, drivers’ yielding responses were recorded (none, soft, hard) when a pedestrian presented one of seven conditions: pedestrian without long cane, long cane display, hand up, reversible step (a single step forward into the street), head turn gaze, hand up plus gaze, and a reversible step plus gaze. Trials were completed at the crosswalks at the entrance and exit lanes. Results At the entry and exit lanes, the hand up and reversible step conditions received significantly higher yield percentages than all other conditions. At the exit lanes, the reversible step condition received a significantly higher yield percentage than the hand up condition. There was no significant difference among the three head turn gaze conditions: hand up, hand up plus gaze, and reversible step plus gaze. The hand up and reversible step strategies averaged 83% yielding overall, compared with 20% yielding for the other strategies. Discussion This study demonstrates that forced detectable yields may be generated when pedestrians have not entered the travel lanes of the street at a crosswalk or have only entered the very edge of the street. Gazing behaviors do not substantially influence drivers’ yielding rates. Implications for practitioners Using a hand up or reversible step can allow pedestrians who are visually impaired or blind to create circumstances to help determine if and when to initiate a crossing at a roundabout.

Immuno-Guided Laser-Capture Microdissection of Glial Cells for mRNA Analysis.

Laser-capture microdissection (LCM) allows for retrieval of specific cell populations in situ. By combining immunofluorescent labeling with LCM, mRNAs can be probed by qRT-PCR for determining in situ gene expression during health and disease. This approach permits obtaining and analyzing histologically enriched cell populations in a tissue that can be hardly obtained from other methods such as white matter astrocytes from rodents or any individual cell population from archival human or rodent brain tissues. Herein, we present our methodology of laser-captured mouse spinal cord white matter astrocytes, which can be adapted for any cell type in CNS tissue and low RNAse containing tissues. The methods presented with an emphasis on tips and advices include the cryostat section preparation from snap-frozen tissue, an adapted immunofluorescent labeling, a brief overview of LCM using a UV-based technology with polyethylene membrane glass slides, procedures for direct use of RNA from lysis buffer vs. column-based purified RNA, RNA quality/quantity assessment, the reverse transcription and preamplification steps used before real-time qPCR analysis.

Investigation of reaction mechanisms in the chemical vapor deposition of al from DMEAA

We propose a novel reaction scheme for the chemical vapor deposition (CVD) of Al films on substrates from dimethylethylamine alane (DMEAA), supported by the prediction of the Al deposition rate as a function of process temperature. The scheme is based on gas phase oligomerizations of alane which form a substantial amount of intermediates. Combined with reversible surface dehydrogenation steps, the global deposition reaction is composed of a set of 12 chemical reactions. This new scheme entails four intermediates and includes side reactions that play an important role in the formation of Al thin films. The chemistry mechanism is incorporated in a 2D Computational Fluid Dynamics (CFD) model of the CVD reactor setup used for the experimental investigation. The simulation predictions of the Al deposition rate are in good agreement with corresponding experimental measurements. The success of this novel reaction pathway lies in its ability to capture the abrupt decrease of the deposition rate at temperatures above 200 °C, which is attributed to the gas phase consumption of alane along with its increased desorption rate from the film surface.

Sample Preparation for Endopeptidomic Analysis in Human Cerebrospinal Fluid.

This protocol describes a method developed to identify endogenous peptides in human cerebrospinal fluid (CSF). For this purpose, a previously developed method based on molecular weight cut-off (MWCO) filtration and mass spectrometric analysis was combined with an offline high-pH reverse phase HPLC pre-fractionation step. Secretion into CSF is the main pathway for removal of molecules shed by cells of the central nervous system. Thus, many processes in the central nervous system are reflected in the CSF, rendering it a valuable diagnostic fluid. CSF has a complex composition, containing proteins that span a concentration range of 8 - 9 orders of magnitude. Besides proteins, previous studies have also demonstrated the presence of a large number of endogenous peptides. While less extensively studied than proteins, these may also hold potential interest as biomarkers. Endogenous peptides were separated from the CSF protein content through MWCO filtration. By removing a majority of the protein content from the sample, it is possible to increase the sample volume studied and thereby also the total amount of the endogenous peptides. The complexity of the filtrated peptide mixture was addressed by including a reverse phase (RP) HPLC pre-fractionation step at alkaline pH prior to LC-MS analysis. The fractionation was combined with a simple concatenation scheme where 60 fractions were pooled into 12, analysis time consumption could thereby be reduced while still largely avoiding co-elution. Automated peptide identification was performed by using three different peptide/protein identification software programs and subsequently combining the results. The different programs were complementary rather than comparable with less than 15% of the identifications overlapped between the three.

Magnetization Reversal of Nd‐Fe‐B Thin Film under a Nanosecond Pulse Magnetic Field

SUMMARYThe magnetization reversal process of Nd‐Fe‐B magnets has been long discussed. Since the elemental steps of the magnetization reversal process such as a nucleation of reversed embryo and a wall depinning are the events as small as nanometer scale and as fast as nano/sub‐nanosecond time scale, the study on the magnetization reversal with these space and time regions is essentially important. In this work, a generation of large amplitude nanosecond pulse field and a fabrication of Nd‐Fe‐B nanodot were examined, and then the magnetization reversal process of the Nd‐Fe‐B nanodot under the nanosecond pulse field was investigated. Combination of a Blumlein transmission line and a micro‐coil enables to generate a pulse field of 0.87 T in amplitude and 6 ns in width. It was clearly demonstrated that the reversed domain evolution in the dot under the pulse field was quite different from that under the static field, probably owing to the dispersion of energy barrier of each elemental magnetization reversal step.

Susceptibility of Human Endogenous Retrovirus Type K to Reverse Transcriptase Inhibitors.

Human endogenous retroviruses (HERVs) make up 8% of the human genome. The HERV type K (HERV-K) HML-2 (HK2) family contains proviruses that are the most recent entrants into the human germ line and are transcriptionally active. In HIV-1 infection and cancer, HK2 genes produce retroviral particles that appear to be infectious, yet the replication capacity of these viruses and potential pathogenicity has been difficult to ascertain. In this report, we screened the efficacy of commercially available reverse transcriptase inhibitors (RTIs) at inhibiting the enzymatic activity of HK2 RT and HK2 genomic replication. Interestingly, only one provirus, K103, was found to encode a functional RT among those examined. Several nucleoside analogue RTIs (NRTIs) blocked K103 RT activity and consistently inhibited the replication of HK2 genomes. The NRTIs zidovudine (AZT), stavudine (d4T), didanosine (ddI), and lamivudine (3TC), and the nucleotide RTI inhibitor tenofovir (TDF), show efficacy in blocking K103 RT. HIV-1-specific nonnucleoside RTIs (NNRTIs), protease inhibitors (PIs), and integrase inhibitors (IIs) did not affect HK2, except for the NNRTI etravirine (ETV). The inhibition of HK2 infectivity by NRTIs appears to take place at either the reverse transcription step of the viral genome prior to HK2 viral particle formation and/or in the infected cells. Inhibition of HK2 by these drugs will be useful in suppressing HK2 infectivity if these viruses prove to be pathogenic in cancer, neurological disorders, or other diseases associated with HK2. The present studies also elucidate a key aspect of the life cycle of HK2, specifically addressing how they do, and/or did, replicate.IMPORTANCE Endogenous retroviruses are relics of ancestral virus infections in the human genome. The most recent of these infections was caused by HK2. While HK2 often remains silent in the genome, this group of viruses is activated in HIV-1-infected and cancer cells. Recent evidence suggests that these viruses are infectious, and the potential exists for HK2 to contribute to disease. We show that HK2, and specifically the enzyme that mediates virus replication, can be inhibited by a panel of drugs that are commercially available. We show that several drugs block HK2 with different efficacies. The inhibition of HK2 replication by antiretroviral drugs appears to occur in the virus itself as well as after infection of cells. Therefore, these drugs might prove to be an effective treatment by suppressing HK2 infectivity in diseases where these viruses have been implicated, such as cancer and neurological syndromes.

Wanted dead or alive? Using metabarcoding of environmental DNA and RNA to distinguish living assemblages for biosecurity applications.

High-throughput sequencing metabarcoding studies in marine biosecurity have largely focused on targeting environmental DNA (eDNA). DNA can persist extracellularly in the environment, making discrimination of living organisms difficult. In this study, bilge water samples (i.e., water accumulating on-board a vessel during transit) were collected from 15 small recreational and commercial vessels. eDNA and eRNA molecules were co-extracted and the V4 region of the 18S ribosomal RNA gene targeted for metabarcoding. In total, 62.7% of the Operational Taxonomic Units (OTUs) were identified at least once in the corresponding eDNA and eRNA reads, with 19.5% unique to eDNA and 17.7% to eRNA. There were substantial differences in diversity between molecular compartments; 57% of sequences from eDNA-only OTUs belonged to fungi, likely originating from legacy DNA. In contrast, there was a higher percentage of metazoan (50.2%) and ciliate (31.7%) sequences in the eRNA-only OTUs. Our data suggest that the presence of eRNA-only OTUs could be due to increased cellular activities of some rare taxa that were not identified in the eDNA datasets, unusually high numbers of rRNA transcripts in ciliates, and/or artefacts produced during the reverse transcriptase, PCR and sequencing steps. The proportions of eDNA/eRNA shared and unshared OTUs were highly heterogeneous within individual bilge water samples. Multiple factors including boat type and the activities performed on-board, such as washing of scientific equipment, may play a major role in contributing to this variability. For some marine biosecurity applications analysis, eDNA-only data may be sufficient, however there are an increasing number of instances where distinguishing the living portion of a community is essential. For these circumstances, we suggest only including OTUs that are present in both eDNA and eRNA data. OTUs found only in the eRNA data need to be interpreted with caution until further research provides conclusive evidence for their origin.

Dynamics of P-type ATPase transport revealed by single-molecule FRET.

Phosphorylation-type (P-type) ATPases are ubiquitous primary transporters that pump cations across cell membranes through the formation and breakdown of a phosphoenzyme intermediate. Structural investigations suggest that the transport mechanism is defined by conformational changes in the cytoplasmic domains of the protein that are allosterically coupled to transmembrane helices so as to expose ion binding sites to alternate sides of the membrane. Here, we have used single-molecule fluorescence resonance energy transfer to directly observe conformational changes associated with the functional transitions in the Listeria monocytogenes Ca2+-ATPase (LMCA1), an orthologue of eukaryotic Ca2+-ATPases. We identify key intermediates with no known crystal structures and show that Ca2+ efflux by LMCA1 is rate-limited by phosphoenzyme formation. The transport process involves reversible steps and an irreversible step that follows release of ADP and extracellular release of Ca2+.