This service exclusively searches for literature that cites resources. Please be aware that the total number of searchable documents is limited to those containing RRIDs and does not include all open-access literature.
The polymerase chain reaction (PCR) is essential in nucleic acid amplification tests and is widely used in many applications such as infectious disease detection, tumor screening, and food safety testing; however, most PCR devices have inefficient heating and cooling ramp rates for the solution, which significantly limit their application in special scenarios such as hospital emergencies, airports, and customs. Here, we propose a temperature control strategy to significantly increase the ramp rates for the solution temperature by switching microfluidic chips between multiple temperature zones and excessively increasing the temperature difference between temperature zones and the solution; accordingly, we have designed an ultrafast thermocycler. The results showed that the ramp rates of the solution temperature are a linear function of temperature differences within a range, and a larger temperature difference would result in faster ramp rates. The maximum heating and cooling ramp rates of the 25 μL solution reached 24.12 °C/s and 25.28 °C/s, respectively, and the average ramp rate was 13.33 °C/s, 6-8 times higher than that of conventional commercial PCR devices. The thermocycler achieved 9 min (1 min pre-denaturation + 45 PCR cycles) ultrafast nucleic acid amplification, shortening the time by 92% compared to the conventional 120 min nucleic acid amplification, and has the potential to be used for rapid nucleic acid detection.
Protein denaturation plays a crucial role in cellular processes. In this study, denaturation of HIV-1 Protease (PR) was investigated by all-atom MD simulations in explicit solvent. The PR dimer and monomer were simulated separately in 9 M acetic acid (9 M AcOH) solution and water to study the denaturation process of PR in acetic acid environment. Direct visualization of the denaturation dynamics that is readily available from such simulations has been presented. Our simulations in 9 M AcOH reveal that the PR denaturation begins by separation of dimer into intact monomers and it is only after this separation that the monomer units start denaturing. The denaturation of the monomers is flagged off by the loss of crucial interactions between the α-helix at C-terminal and surrounding β-strands. This causes the structure to transit from the equilibrium dynamics to random non-equilibrating dynamics. Residence time calculations indicate that denaturation occurs via direct interaction of the acetic acid molecules with certain regions of the protein in 9 M AcOH. All these observations have helped to decipher a picture of the early events in acetic acid denaturation of PR and have illustrated that the α-helix and the β-sheet at the C-terminus of a native and functional PR dimer should maintain both the stability and the function of the enzyme and thus present newer targets for blocking PR function.
We used stopped-flow to monitor hypochromicity for 43 oligonucleotide duplexes to study nucleic acid kinetics and extract transition-state parameters for association and dissociation. Reactions were performed in 1.0 M NaCl (for literature comparisons) and 2.2 mM MgCl2 (PCR conditions). Dissociation kinetics depended on sequence, increased exponentially with temperature, and transition-state parameters inversely correlated to thermodynamic parameters (r = -0.99). Association had no consistent enthalpic component, varied little with temperature or sequence, and poorly correlated to thermodynamic parameters (r = 0.28). Average association rates decreased 78% in MgCl2 compared to NaCl while dissociation was relatively insensitive to ionic conditions. A nearest-neighbour kinetic model for dissociation predicted rate constants within 3-fold of literature values (n = 11). However, a nearest-neighbour model for association appeared overparameterized and inadequate for predictions. Kinetic predictions were used to simulate published high-speed (<1 min) melting analysis and extreme (<2 min) PCR experiments. Melting simulations predicted apparent melting temperatures increase on average 2.4°C when temperature ramp rates increased from 0.1 to 32°C/s, compared to 2.8°C reported in the literature. PCR simulations revealed that denaturation kinetics are dependent on the thermocycling profile. Simulations overestimated annealing efficiencies at shorter annealing times and suggested that polymerase interactions contribute to primer-template complex stability at extension temperatures.
Preventing protein aggregation is a major goal of biotechnology. Since protein aggregates are mainly comprised of unfolded proteins, protecting against denaturation is likely to assist solubility in an aqueous medium. Contrary to this concept, we found denatured total cellular protein mixture from mammalian cell kept high solubility in pure water when the mixture was nucleic acids free. The lysates were prepared from total cellular protein pellet extracted by using guanidinium thiocyanate-phenol-chloroform mixture of TRIzol, denatured and reduced total protein mixtures remained soluble after extensive dialysis against pure water. The total cell protein lysates contained fully disordered proteins that readily formed large aggregates upon contact with nucleic acids or salts. These findings suggested that the highly flexible mixtures of disordered proteins, which have fully ionized side chains, are protected against aggregation. Interestingly, this unusual solubility is characteristic of protein mixtures from higher eukaryotes, whereas most prokaryotic protein mixtures were aggregated under identical conditions. This unusual solubility of unfolded protein mixtures could have implications for the study of intrinsically disordered proteins in a variety of cells.
The PCR technique and its variations have been increasingly used in the clinical laboratory and recent advances in this field generated new higher resolution techniques based on nucleic acid denaturation dynamics. The principle of these new molecular tools is based on the comparison of melting profiles, after denaturation of a DNA double strand. Until now, the secondary structure of single-stranded nucleic acids has not been exploited to develop identification systems based on PCR. To test the potential of single-strand RNA denaturation as a new alternative to detect specific nucleic acid variations, sequences from viruses of the Totiviridae family were compared using a new in silico melting curve approach. This family comprises double-stranded RNA virus, with a genome constituted by two ORFs, ORF1 and ORF2, which encodes the capsid/RNA binding proteins and an RNA-dependent RNA polymerase (RdRp), respectively.
The outbreak of COVID-19 makes epidemic prevention and control become a growing global concern. Nucleic acid amplification testing (NAAT) can realize early and rapid detection of targets, thus it is considered as an ideal approach for detecting pathogens of severe acute infectious diseases. Rapid acquisition of high-quality target nucleic acid is the prerequisite to ensure the efficiency and accuracy of NAAT. Herein, we proposed a simple system in which magnetic nanoparticles (MNPs) based nucleic acid extraction was carried out in a plastic Pasteur pipette. Different from traditional approaches, this proposed system could be finished in 15 min without the supports of any electrical instruments. Furthermore, this system was superior to traditional MNPs based extraction methods in the aspects of rapid extraction and enhancing the sensitivity of a NAAT method, accelerated denaturation bubbles mediated strand exchange amplification (ASEA), to the pathogens from various artificial samples. Finally, this Pasteur pipette system was utilized for pathogen detection in actual samples of throat swabs, cervical swabs and gastric mucosa, the diagnosis results of which were identical with that provided by hospital. This rapid, easy-performing and efficiency extraction method ensures the applications of the NAAT in pathogen detection in regions with restricted resources.
The SARS-CoV-2 pandemic has brought to light the need for expedient diagnostic testing. Cost and availability of large-scale testing capacity has led to a lag in turnaround time and hindered contact tracing efforts, resulting in a further spread of SARS-CoV-2. To increase the speed and frequency of testing, we developed a cost-effective single-tube approach for collection, denaturation, and analysis of clinical samples. The approach utilizes 1 µL microbiological inoculation loops to collect saliva, sodium dodecyl sulfate (SDS) to inactivate and release viral genomic RNA, and a diagnostic reaction mix containing polysorbate 80 (Tween 80). In the same tube, the SDS-denatured clinical samples are introduced to the mixtures containing all components for nucleic acids detection and Tween 80 micelles to absorb the SDS and allow enzymatic reactions to proceed, obviating the need for further handling of the samples. The samples can be collected by the tested individuals, further decreasing the need for trained personnel to administer the test. We validated this single-tube sample-to-assay method with reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP) and discovered little-to-no difference between Tween- and SDS-containing reaction mixtures, compared to control reactions. This approach reduces the logistical burden of traditional large-scale testing and provides a method of deployable point-of-care diagnostics to increase testing frequency.
Mycoplasma pneumoniae is a strong infectious pathogen that may cause severe respiratory infections. Since this pathogen may possess a latent period after infection, which sometimes leads to misdiagnosis by traditional diagnosis methods, the establishment of a rapid and sensitive diagnostic method is crucial for transmission prevention and timely treatment. Herein, a novel detection method was established for M. pneumoniae detection. The method, which improves upon a denaturation bubble-mediated strand exchange amplification (SEA) that we developed in 2016, is called accelerated SEA (ASEA). The established ASEA achieved detection of 1% M. pneumoniae genomic DNA in a DNA mixture from multiple pathogens, and the limit of detection (LOD) of ASEA was as low as 1.0 × 10-17 M (approximately 6.0 × 103 copies/mL). Considering that the threshold of an asymptomatic carriage is normally recommended as 1.0 × 104 copies/mL, this method was able to satisfy the requirement for practical diagnosis of M. pneumoniae. Moreover, the detection process was finished within 20.4 min, significantly shorter than real-time PCR and SEA. Furthermore, ASEA exhibited excellent performance in clinical specimen analysis, with sensitivity and specificity of 96.2% and 100%, respectively, compared with the "gold standard" real-time PCR. More importantly, similar to real-time PCR, ASEA requires only one pair of primers and ordinary commercial polymerase, and can be carried out using a conventional fluorescence real-time PCR instrument, which makes this method low-cost and easy to accomplish. Therefore, ASEA has the potential for wide use in the rapid detection of M. pneumoniae or other pathogens in large numbers of specimens. Graphical abstract.
The effects of urea and guanidine hydrochloride (GdnHCl) on the activity, conformation and unfolding process of protein tyrosine phosphatase (PTPase), a thermostable low molecular weight protein from Thermus thermophilus HB27, have been studied. Enzymatic activity assays showed both urea and GdnHCl resulted in the inactivation of PTPase in a concentration and time-dependent manner. Inactivation kinetics analysis suggested that the inactivation of PTPase induced by urea and GdnHCl were both monophasic and reversible processes, and the effects of urea and GdnHCl on PTPase were similar to that of mixed-type reversible inhibitors. Far-ultraviolet (UV) circular dichroism (CD), Tryptophan and 1-anilinonaphthalene -8-sulfonic acid (ANS) fluorescence spectral analyses indicated the existence of a partially active and an inactive molten globule-like intermediate during the unfolding processes induced by urea and GdnHCl, respectively. Based on the sequence alignment and the homolog Tt1001 protein structure, we discussed the possible conformational transitions of PTPase induced by urea and GdnHCl and compared the conformations of these unfolding intermediates with the transient states in bovine PTPase and its complex structures in detail. Our results may be able to provide some valuable clues to reveal the relationship between the structure and enzymatic activity, and the unfolding pathway and mechanism of PTPase.
The γS1- and γS2-crystallins, structural eye lens proteins from the Antarctic toothfish (Dissostichus mawsoni), are homologues of the human lens protein γS-crystallin. Although γS1 has the higher thermal stability of the two, it is more susceptible to chemical denaturation by urea. The lower thermodynamic stability of both toothfish crystallins relative to human γS-crystallin is consistent with the current picture of how proteins from organisms endemic to perennially cold environments have achieved low-temperature functionality via greater structural flexibility. In some respects, the sequences of γS1- and γS2-crystallin are typical of psychrophilic proteins; however, their amino acid compositions also reflect their selection for a high refractive index increment. Like their counterparts in the human lens and those of mesophilic fish, both toothfish crystallins are relatively enriched in aromatic residues and methionine and exiguous in aliphatic residues. The sometimes contradictory requirements of selection for cold tolerance and high refractive index make the toothfish crystallins an excellent model system for further investigation of the biophysical properties of structural proteins.
The reversibility of thermal denaturation and catalytic efficiency of Bacillus licheniformis α-amylase were improved through site-directed mutagenesis. By using multiple sequence alignment and PoPMuSiC algorithm, Ser187 and Asn188, which located within a long loop in Domain B of Bacillus licheniformis α-amylase, were selected for mutation. In addition, Ala269, which is adjacent to Ser187 and Asn188, was also investigated. Seven mutants carrying the mutations S187D, N188T, N188S, A269K, A269K/S187D, S187D/N188T, and A269K/S187D/N188T were generated and characterized. The most thermostable mutant, A269K/S187D/N188T, exhibited a 9-fold improvement in half-life at 95°C and pH 5.5, compared with that of the wild-type enzyme. Mutant A269K/S187D/N188T also exhibited improved catalytic efficiency. The catalytic efficiency of mutant A269K/S187D/N188T reached 5.87×103±0.17 g·L-1·s-1 at pH 5.5, which is 1.84-fold larger than the corresponding value determined for the wild-type enzyme. Furthermore, the structure analysis showed that immobilization of the loop containing Ser187 and Asn188 plays a significant role in developing the properties of Bacillus licheniformis α-amylase.
TDP-43 is an important pathological protein that aggregates in the diseased neuronal cells and is linked to various neurodegenerative disorders. In normal cells, TDP-43 is primarily an RNA-binding protein; however, how the dimeric TDP-43 binds RNA via its two RNA recognition motifs, RRM1 and RRM2, is not clear. Here we report the crystal structure of human TDP-43 RRM1 in complex with a single-stranded DNA showing that RRM1 binds the nucleic acid extensively not only by the conserved β-sheet residues but also by the loop residues. Mutational and biochemical assays further reveal that both RRMs in TDP-43 dimers participate in binding of UG-rich RNA or TG-rich DNA with RRM1 playing a dominant role and RRM2 playing a supporting role. Moreover, RRM1 of the amyotrophic lateral sclerosis-linked mutant D169G binds DNA as efficiently as the wild type; nevertheless, it is more resistant to thermal denaturation, suggesting that the resistance to degradation is likely linked to TDP-43 proteinopathies. Taken together all the data, we suggest a model showing that the two RRMs in each protomer of TDP-43 homodimer work together in RNA binding and thus the dimeric TDP-43 recognizes long clusters of UG-rich RNA to achieve high affinity and specificity.
Polycomb proteins play key roles in mediating epigenetic modifications that occur during cell differentiation. The Polycomb repressive complex 2 (PRC2) mediates the tri-methylation of histone H3 lysine 27 (H3K27me3). In this study, we identify a distinguishing feature of two classes of PRC2 target genes, represented by the Nr2F1 (Coup-TF1) and the Hoxa5 gene, respectively. Both genes are transcriptionally activated by all-trans retinoic acid (RA) and display increased levels of the permissive H3K9/K14ac and tri-methylated histone H3 lysine 4 epigenetic marks in response to RA. However, while in response to RA the PRC2 and H3K27me3 marks are greatly decreased at the Hoxa5 promoter, these marks are initially increased at the Nr2F1 promoter. Functional depletion of the essential PRC2 protein Suz12 by short hairpin RNA (shRNA) technology enhanced the RA-associated transcription of Nr2F1, Nr2F2, Meis1, Sox9 and BMP2, but had no effect on the Hoxa5, Hoxa1, Cyp26a1, Cyp26b1 and RARβ2 transcript levels in wild-type embryonic stem cells. We propose that PRC2 recruitment attenuates the RA-associated transcriptional activation of a subset of genes. Such a mechanism would permit the fine-tuning of transcriptional networks during differentiation.
The interaction between human serum albumin (HSA) and arachidonic acid (AA) as an unsaturated fatty acid were investigated in the present study using methods including UV-VIS spectrophotometry, fluorescence and circular dichroism (CD) spectroscopy, lifetime measurements, fluorescence anisotropy measurements and visual molecular dynamics (MD). The thermodynamic parameters were assessed from HSA thermal and chemical denaturation in the presence and absence of AA. From the thermal denaturation, the Tm and ΔG˚(298K) magnitudes obtained were 327.7 K and 88 kJ/mol, respectively, for HSA alone, and 323.4 K and 85 kJ/mol, respectively, following treatment with a 10 µM AA concentration. The same manner of reduction in Gibbs free energy as a criterion of protein stability was achieved during chemical denaturation by urea in the presence of AA. The present study investigates HSA binding nature through MD approaches, and the results indicated that the binding affinity of AA to the subdomain IIA of HSA is greater compared with that of subdomain IIIA. Although the HSA regular secondary structure evaluation by CD exhibited a minor change following incubation with AA, its tertiary structure revealed an observable fluctuation. Thus, it appears that the interaction between AA and HSA requires minor instability and partial structural changes.
Wheat α-amylase, a multi-domain protein with immense industrial applications, belongs to α+β class of proteins with native molecular mass of 32 kDa. In the present study, the pathways leading to denaturation and the relevant unfolded states of this multi-domain, robust enzyme from wheat were discerned under the influence of temperature, pH and chemical denaturants. The structural and functional aspects along with thermodynamic parameters for α-amylase unfolding were probed and analyzed using fluorescence, circular dichroism and enzyme assay methods. The enzyme exhibited remarkable stability up to 70°C with tendency to aggregate at higher temperature. Acid induced unfolding was also incomplete with respect to the structural content of the enzyme. Strong ANS binding at pH 2.0 suggested the existence of a partially unfolded intermediate state. The enzyme was structurally and functionally stable in the pH range 4.0-9.0 with 88% recovery of hydrolytic activity. Careful examination of biophysical properties of intermediate states populated in urea and GdHCl induced denaturation suggests that α-amylase unfolding undergoes irreversible and non-coincidental cooperative transitions, as opposed to previous reports of two-state unfolding. Our investigation highlights several structural features of the enzyme in relation to its catalytic activity. Since, α-amylase has been comprehensively exploited for use in a range of starch-based industries, in addition to its physiological significance in plants and animals, knowledge regarding its stability and folding aspects will promote its biotechnological applications.
Docosahexaenoic acid (DHA) plays important physiological roles in vertebrates. Studies in rats and rainbow trout confirmed that DHA biosynthesis proceeds through the so-called "Sprecher pathway", a biosynthetic process requiring a Δ6 desaturation of 24:5n-3 to 24:6n-3. Alternatively, some teleosts possess fatty acyl desaturases 2 (Fads2) that enable them to biosynthesis DHA through a more direct route termed the "Δ4 pathway". In order to elucidate the prevalence of both pathways among teleosts, we investigated the Δ6 ability towards C24 substrates of Fads2 from fish with different evolutionary and ecological backgrounds. Subsequently, we retrieved public databases to identify Fads2 containing the YXXN domain responsible for the Δ4 desaturase function, and consequently enabling these species to operate the Δ4 pathway. We demonstrated that, with the exception of Δ4 desaturases, fish Fads2 have the ability to operate as Δ6 desaturases towards C24 PUFA enabling them to synthesise DHA through the Sprecher pathway. Nevertheless, the Δ4 pathway represents an alternative route in some teleosts and we identified the presence of putative Δ4 Fads2 in a further 11 species and confirmed the function as Δ4 desaturases of Fads2 from medaka and Nile tilapia. Our results demonstrated that two alternative pathways for DHA biosynthesis exist in teleosts.
2-Hydroxyisobutyric acid (2-HIBA) is a biomarker of adiposity and associated metabolic diseases such as diabetes mellitus. It is also formed in the bacterial degradation pathway of the fuel oxygenate methyl tert-butyl ether (MTBE), requiring thioesterification with CoA prior to isomerization to 3-hydroxybutyryl-CoA by B12-dependent acyl-CoA mutases. Here, we identify the adenylating enzymes superfamily member 2-HIBA-CoA ligase (HCL) in the MTBE-degrading bacterium Aquincola tertiaricarbonis L108 by knockout experiments. To characterize this central enzyme of 2-HIBA metabolism, ligase activity kinetics of purified HCL and its X-ray crystal structures were studied. We analyzed the enzyme in three states, which differ in the orientation of the two enzyme domains. A 154° rotation of the C-terminal domain accompanies the switch from the adenylate- into the thioester-forming state. Furthermore, a third conformation was obtained, which differs by 50° and 130° from the adenylation and thioesterification states, respectively. Phylogenetic and structural analysis reveals that HCL defines a new subgroup within phenylacetate-CoA ligases (PCLs) thus far described to exclusively accept aromatic acyl substrates. In contrast, kinetic characterization clearly demonstrated that HCL catalyzes CoA activation of several aliphatic short-chain carboxylic acids, preferentially 2-HIBA. Compared to the classical PCL representatives PaaK1 and PaaK2 of Burkholderia cenocepacia J2315, the acyl binding pocket of HCL is significantly smaller and more polar, due to unique active-site residues Y164 and S239 forming H-bonds with the OH-group of the acyl substrate moiety. Furthermore, HCL and PaaK topologies illustrate the evolutionary steps leading from a homodimeric to the fused monomeric core fold found in other ligases.
During 2009 to 2012, Thailand had encountered 4 distinctive waves of the 2009 pandemic influenza A(H1N1) (H1N1pdm) outbreaks. Considering the RNA nature of the influenza viral genome, a mutation in hemagglutinin (HA) gene which led to change in antigenicity of the strains circulating during those epidemic periods is anticipated. It is also uncertain whether the A/California/07/2009 (H1N1) (CA/07) vaccine strain still confers protective immunity against those evolved viruses, the causative agents of the later epidemic waves.
Caffeic acid O-methyltransferase (COMT) is an important protein that participates in lignin synthesis and is associated with the ratio of G-/S-type lignin in plants. COMTs are associated with the wood properties of forest trees; however, little known about the COMT family in Catalpa bungei, a valuable timber tree species in China . We performed a comprehensive analysis of COMT genes in the C. bungei genome by describing the gene structure and phylogenetic relationships of each family member using bioinformatics-based methods. A total of 23 putative COMT genes were identified using the conserved domain sequences and amino acid sequences of COMTs from Arabidopsis thaliana and Populus trichocarpa as probes. Phylogenetic analysis showed that 23 CbuCOMTs can be divided into three groups based on their structural characteristics; five conserved domains were found in the COMT family. Promoter analysis indicated that the CbuCOMT promoters included various cis-acting elements related to growth and development. Real-time quantitative polymerase chain reaction (PCR) analysis showed differential expression among CbuCOMTs. CbuCOMT2, 7, 8, 9, 10, 12, 13, 14, 21, and 23 were mainly expressed in xylem. Only CbuCOMT23 was significantly downregulated in tension wood and upregulated in opposite wood compared to normal wood. Our study provides new information about the CbuCOMT gene family and will facilitate functional characterisation in further research.
We previously reported that undernutrition in late fetal life reduced whole-body insulin sensitivity in adult sheep, irrespective of dietary exposure in early postnatal life. Skeletal muscle may play an important role in control of insulin action. We therefore studied a range of putative key muscle determinants of insulin signalling in two types of skeletal muscles (longissimus dorsi (LD) and biceps femoris (BF)) and in the cardiac muscle (ventriculus sinister cordis (VSC)) of sheep from the same experiment. Twin-bearing ewes were fed either 100% (NORM) or 50% (LOW) of their energy and protein requirements during the last trimester of gestation. From day-3 postpartum to 6-months of age (around puberty), twin offspring received a high-carbohydrate-high-fat (HCHF) or a moderate-conventional (CONV) diet, whereafter all males were slaughtered. Females were subsequently raised on a moderate diet and slaughtered at 2-years of age (young adults). The only long-term consequences of fetal undernutrition observed in adult offspring were lower expressions of the insulin responsive glucose transporter 4 (GLUT4) protein and peroxisome proliferator-activated receptor gamma, coactivator 1α (PGC1α) mRNA in BF, but increased PGC1α expression in VSC. Interestingly, the HCHF diet in early postnatal life was associated with somewhat paradoxically increased expressions in LD of a range of genes (but not proteins) related to glucose uptake, insulin signalling and fatty acid oxidation. Except for fatty acid oxidation genes, these changes persisted into adulthood. No persistent expression changes were observed in BF and VSC. The HCHF diet increased phospholipid ratios of n-6/n-3 polyunsaturated fatty acids in all muscles, even in adults fed identical diets for 1½ years. In conclusion, early postnatal, but not late gestation, nutrition had long-term consequences for a number of determinants of insulin action and metabolism in LD. Tissues other than muscle may account for reduced whole body insulin sensitivity in adult LOW sheep.
Welcome to the FDI Lab - SciCrunch.org Resources search. From here you can search through a compilation of resources used by FDI Lab - SciCrunch.org and see how data is organized within our community.
You are currently on the Community Resources tab looking through categories and sources that FDI Lab - SciCrunch.org has compiled. You can navigate through those categories from here or change to a different tab to execute your search through. Each tab gives a different perspective on data.
If you have an account on FDI Lab - SciCrunch.org then you can log in from here to get additional features in FDI Lab - SciCrunch.org such as Collections, Saved Searches, and managing Resources.
Here is the search term that is being executed, you can type in anything you want to search for. Some tips to help searching:
You can save any searches you perform for quick access to later from here.
We recognized your search term and included synonyms and inferred terms along side your term to help get the data you are looking for.
If you are logged into FDI Lab - SciCrunch.org you can add data records to your collections to create custom spreadsheets across multiple sources of data.
Here are the facets that you can filter your papers by.
From here we'll present any options for the literature, such as exporting your current results.
If you have any further questions please check out our FAQs Page to ask questions and see our tutorials. Click this button to view this tutorial again.
Year:
Count: