Thursday, August 27, 2020
Execution Practice in a World Music Ensemble Presentation One of the targets of the Indiana University International Vocal Ensemble (IVE) is to sing music of non-western societies in the local language, and to the degree conceivable, sing with trustworthiness of vocal and melodic style. An ensuing goal is to copy music precisely when given an aural model. In view of these goals I am keen on semantic discernment and PC investigation signs of vocal tone and the degree, assuming any, to which vocal tone can be imitated among societies, and how this data can be added to an interpretation to help the artist. For this specific task I concentrated on the creation of Ghanaian vocal tone by three local Ghanaians, people in IVE, and non-IVE individuals from a Ghanaian youngsters' tune. This paper will concentrate on the information of one of the Ghanaian female witnesses (Gf1) and one American female IVE part (Af1). There is no convincing hypothesis of tone recognition. Some portion of the trouble is that tone is hard to see as a detached wonder since it depends on recognition. Tone is associated with the source instead of individual and quantifiable qualities, for example, recurrence or plentifulness. Concerning vocal tone the vocal tract changes the wind stream range into conspicuous acoustical examples which we know as vowels. Vowels are a melodic component of singing beside their data conveying capacity. (Benade, 1990) Three zones of vowel creation that are regularly concentrated in PC investigation while exploring tone are the consonant spectra, formants, and assault and rot homeless people. Another way to deal with researching tone alongside PC examination is the examination of... ...oice contrasts in choral singing and solo singing, and in Western prepared singing an alternate tone is wanted for choral singing contrasted and solo singing. Another distinction is that ...choral vocalists endeavor to tune their voice tone so as to work with the tone of the remainder of the ensemble, while an independent artist would attempt to build up their own individual tone. (Sundberg, 1987) How individuals' impression of tone make an interpretation of from the person to a gathering sound? What modifications do one's ears make? These issues are open for additional examination on the tone of singing. The melody Kofi has since been performed at various shows, and numerous individuals in the ensemble will keep murmuring the tune when it flies into their head for some explanation. Next semester there will be new pieces and new tones to start to display, with new examination fit to be explored. Execution Practice in a World Music Ensemble Essay - Timbre Music E Execution Practice in a World Music Ensemble Presentation One of the goals of the Indiana University International Vocal Ensemble (IVE) is to sing music of non-western societies in the local language, and to the degree conceivable, sing with uprightness of vocal and melodic style. An ensuing goal is to emulate music precisely when given an aural model. In light of these destinations I am keen on semantic recognition and PC examination signals of vocal tone and the degree, assuming any, to which vocal tone can be imitated among societies, and how this data can be added to an interpretation to help the artist. For this specific undertaking I concentrated on the creation of Ghanaian vocal tone by three local Ghanaians, people in IVE, and non-IVE individuals from a Ghanaian kids' tune. This paper will concentrate on the information of one of the Ghanaian female sources (Gf1) and one American female IVE part (Af1). There is no decisive hypothesis of tone observation. Some portion of the trouble is that tone is hard to see as a disconnected marvel since it depends on discernment. Tone is associated with the source instead of individual and quantifiable qualities, for example, recurrence or abundancy. With respect to vocal tone the vocal tract changes the wind current range into conspicuous acoustical examples which we know as vowels. Vowels are a melodic component of singing beside their data conveying capacity. (Benade, 1990) Three regions of vowel creation that are regularly concentrated in PC investigation while exploring tone are the consonant spectra, formants, and assault and rot homeless people. Another way to deal with exploring tone alongside PC examination is the examination of... ...oice contrasts in choral singing and solo singing, and in Western prepared singing an alternate tone is wanted for choral singing contrasted and solo singing. Another distinction is that ...choral artists endeavor to tune their voice tone so as to work with the tone of the remainder of the ensemble, while an independent artist would attempt to build up their own individual tone. (Sundberg, 1987) How individuals' view of tone make an interpretation of from the person to a gathering sound? What alterations do one's ears make? These issues are open for additional examination on the tone of singing. The tune Kofi has since been performed at various shows, and numerous individuals in the ensemble will keep murmuring the melody when it flies into their head for some explanation. Next semester there will be new pieces and new tones to start to show, with new exploration fit to be researched.
Saturday, August 22, 2020
Goaste Security Objectives Macintosh which has been a current PC configuration organization since 1976 discharged its most up to date creation ipad on June 4, 2010. Mac additionally joined forces with ATJust a couple of days preceding the offer of the first ipad there was plugged news that the cutting edge gadget included a default inside its security that could interface data to potential PC programmers. The disclosure of this defect was revealed by Goaste Security firm. The firm hacked into AT&T?s site and had the option to interface the id?s of new ipad proprietors to their email addresses. Goaste security authorities had the option to seize upwards of 100,000 email addresses if not more. These email addresses comprised of legislators, government authorities, notable corporate officials and numerous other perceived and compelling individuals. Goaste Security at that point discharged the data of their discoveries to Gawker Media LLC, and afterward told AT&T of the mistake that was revealed due to the ir investigational hacking. There could have been various motivations to why Goaste Security chose to make this data open. One protest may have been for the sole reason for introduction. Since Goaste is a genuinely new security firm and there had been no acknowledgment of the firm before the ipad episode the firm could have been pulling an exposure stunt for its own promotion. This is by all accounts the case since the firm didn't make the best possible strides of contact, which would have been to initially make AT Besides Goaste Security Firm could have been attempting to build up a relationship with AT&T to influence them to come on board as one of their first customers. The firm could have been doing this by flaunting their abilities to perceive the potential perils for security programmers to use against AT&T. PC Hacking PC hacking isn't a dishonest technique for security firms to utilize anyway they ought to get assent before having the option to rehearse their hacking abilities. Particularly since the case is that their claim to fame is PC hacking and they are utilizing this extraordinary enthusiasm as a calling. Before PC hacking firms utilize these kinds of proficiencies both the organizations and the organizations wherein the firm is obliging, probably settled a composed archive ideally an agreement understanding that uncovers data with respect to what sort of administrations the firm will give and the means that an important to fill the obligations of the agreement. Besides by no means should the security firm have the option to discharge data to any outsiders about errors or whatever other gained realities that the firm has divulged because of its examination. By no means should the security firm have the option to discharge data to any outsiders about disparities or whatever other procured realities that the firm has found because of its examination. By no means should the security firm have the option to plug data about the proposed organization. It ought to be surrendered over to the predetermined advertising division to settle on the choice to advise the general population about any data that is identified with the matter of any organization and its investors. For future thoughts, there ought to be progressively severe standards and guidelines that become possibly the most important factor when observing programmers. Greater security programming ought to be created and tried to the highest caliber to forestall future programmers. Security firms ought to be the pioneers that actualize these safe apparatuses. These organizations ought to likewise keep awake to date with the a wide range of databases so they may create programming in like manner. Rubberneck Media Rubberneck Media is an online organization that is comprised of a systems administration web journals website. Onlooker was established in 2002 and its corporate base camp are situated in New York, New York. Goaste Security firm was the organization that applied data from AT&T?s sites that had the option to see private information that could have oppressed AT&T clients to focuses for unapproved programmers. After Goaste Security firm picked up this data they at that point uncovered these discoveries to Gawker Media. The firm at that point took this report and made it open information. Under no course of occasions should Gawker
Master of the Flies versus A Separate Peace Essay In the World Book Dictionary, envy is characterized as being in a desirous condition or feeling. Many can identify with this inclination, since they have by and by experienced envy previously. Despite the fact that these individuals may share a comparable inclination, the way every individual follows up on his/her inclination is unique. Some simply disregard their desirous inclination, trusting that it would rapidly leave so they can go on with their every day lives. Others become so overpowered that they really may follow up on their feelings, expecting that their activity would cause them to feel better. Typically, the opposite happens. The individual doesn't rest easy thinking about himself; rather, harsh sentiments, lost regard, or even lost fellowship are basic results. In the books Lord of the Flies by William Golding and A Separate Peace by John Knowles, the characters Jack and Gene both experience enviously towards someone else, and their activities, inspirations, and sentiments all circumnavigate around envy. In Lord of the Flies, when Jack understands that Ralph will get boss, he absolutely changes and turns into the adversary. Above all else, Jack carries on his desire by endeavoring to hurt Ralph. In spite of the fact that Jacks plan to execute Ralph is ineffective, Ralph is still near death. In the novel, the peruser realizes that Jack is plotting to murder Ralph on the grounds that Samneric advises Ralph, Theyre going to chase you tomorrow[Jack] honed a stick at the two finishes. When Samneric reports that Jack honed a stick at the two finishes, they suggest that Jacks faction intends to behead Ralph and stick the prized ownership on a finish of the stick as it did to the sow. Jack wants to utilize the head as a contribution for the Beastie, an anecdotal beast that the young men accept frequents the island. As expressed previously, Jack chooses to make a faction from his inspirations that emerge in light of his envy. Also, since Jack realizes that Ralph is continually going to be chosen boss regardless of how often the gathering of young men vote, Jack begins his own group and chooses himself as boss. At the point when he does this, he transparently tells his associates, Anyone who needs to chase when I do can come as well. By expressing this, he is just convincing individuals to help his accomplishment. As Jack designs the demise of Ralph, he doesn't feel any feeling of regret or blame for plotting to execute his old companion. Jack is recently energized that there will be a major chase for Ralph. Quality additionally changes radically in A Separate Peace after he understands that he will never be as athletic as Finny. Finny has consistently had the option to achieve objectives and accomplishments that nobody else can reach. Moreover, Gene gets desirous. In light of his avarice, Gene hurts Finny by [taking] a stage toward [Finny], and afterward [Genes] knees bowed and [Gene] bumped the appendage. Quality does this so rapidly that he doesn't understand the results in yanking the tree appendage. He doesn't have the foggiest idea about that his activity would influence the remainder of Finnys life. In contrast to Jack however, Gene doesn't look for the help of his companions. All Gene needs is for Finny to comprehend that Gene didn't hurt Finny deliberately, and he is significantly heartbroken. Be that as it may, when Gene endeavors to disclose this to Finny, Finny just gets over it and attempts to persuade Gene in any case. He says, I dont know, I should have quite recently lost my equalization. It more likely than not been thatI simply fell. Another distinction is that after Gene shakes the appendage and makes Finny fall and break his leg, he feels remorseful about his demonstration of envy. Quality really goes up to Finny various occasions to clarify that Gene was really the person who made Finny break his leg, yet Finny doesn't tune in. Finny just obstinately adheres to his motivation to [being] ungainly and not watching where [he] was venturing. At last, Gene is drenched with distress and blame. His jealousy for Finny not just reverse discharges; it makes perpetual disgrace and regret. In the books Lord of the Flies by William Golding and A Separate Peace by John Knowles, the principle characters activities, inspirations, and sentiments are for the most part results of their desire towards another. Jack and Gene share the similitude of endeavoring to hurt another because of their jealous inspiration. However, they are distinctive as observed through their definitive emotions. Both Jack and Gene seek to get equivalent to their opponents, regardless of whether they should hurt their companions to achieve self-satisfaction. At long last, Gene experiences blame while Jack can't be increasingly satisfied when his recently settled group obeys him to murder Ralph. Richard Griper says all that needs to be said: Jealousy is nothing except if you follow up on it.
Friday, August 21, 2020
Perspectives to Language Essay Language unmistakably assumes a significant job in all parts of society. The most clear is its social job of permitting individuals to identify with one another in all features of their lives: to share data, feelings and lifestyles. We use language as a methods for exploring our day by day lives and it assumes a fundamental job in the vast majority of our communications. Maybe consequently, French is viewed as an exquisite and sentimental language, while German is viewed as throaty. Furthermore, since the time humankind advanced into various language networks, it is ordinary for individuals to embrace different mentalities towards the language(s) spoken by others, just as towards the tongues of the language they talk. These perspectives are spurred by various elements, remembering pride for or disgrace with respect to oneÃ¢â¬â¢s own language, certainty or shame about how one sounds, patriotism and a feeling of individual nobility, oneÃ¢â¬â¢s status and qualities just as the notoriety a few dialects are given in universal connections. A notable mentality is the craving for remote discourse designs; another is the dismissal of specific tongues. Individuals structure impressions of your character, enthusiastic state, geographic starting point, training, encounters, age or financial status from the language you use and the manner in which you use it. We regularly witness the delight of a crowd of people when somebody talks in the creole, for not exclusively does the arrangement of sound bring out giggling, yet the suspicion that the speaker is an uneducated serf is then made. Scorn and disdain for the vernacular, creoles and lingos are normal reactions from certain citizenry, even inside the Caribbean culture, where tongues are rich, solid and the primary language. Lingos create under different conditions just as topographical areas and are assortments of dialects. A creole could be a vernacular inside a language. Due to our history, individuals of the locale will in general spot a high premium on the standard dialects (the language of intensity and monetary may). ManyÃ¢ people accept that upward versatility is to a great extent reliant on oneÃ¢â¬â¢s capacity to fit in with the transcendent financial class, and language is the fundamental signifier of this fit. Numerous Caribbean journalists have depicted situations of individuals who went abroad, were commonly expected to come back with another order of the objective language and frequently showed their recently discovered Ã¢â¬ËstatusÃ¢â¬â¢ by underlining their remote highlight of Ã¢â¬ËtwangÃ¢â¬ â¢. While some may be dazzled by the Ã¢â¬ËtwangÃ¢â¬â¢, others view such claims with criticism. Perspectives to language may fluctuate starting with one area of the general public then onto the next and a few people exhibit hesitant conduct when communicating in the standard language. This is to a great extent an aftereffect of the way that in many social orders one is frequently decided based on the assortment of language that one talks. This is considerably progressively predominant in social orders with a pilgrim inheritance, similar to the Caribbean, where certain vernaculars are related with the establishment of servitude or triumph. Progressively, instructors are turning out to be mindful that a personÃ¢â¬â¢s local language is a necessary piece of who that individual is and underestimating the language can have serious harming consequences for that personÃ¢â¬â¢s mind. Numerous etymologists reliably put forth a defense for showing local dialects nearby the objective dialects so youngsters can plainly separate among the codes ( a term utilized interchangeably with language or tongue yet for the most part alludes to a semantic arrangement of correspondence. A code can likewise be non-etymological, for example, a clothing standard or set of accepted rules) and subsequently be more averse to blend the two. This methodology has been received in Haiti, where schools show both Standard French and French Creole (Haitian) and youngsters are required to be conversant in both. Extra noticeable quality has been given to Caribbean Creoles with the distribution of Creole word references and with the interpretation of the New Testament from the Christian Bible into French Creole in St. Lucia. A comparable task is in progress in Jamaica. While perspectives to neighborhood vernaculars have been gradually changing, numerous individuals despite everything partner the utilization of Creole with negative pictures and accept that its utilization ought to be consigned to explicit conditions and events. Nonetheless, the way that non-standard language assortments are the most broadly spoken in the Caribbean settles on them the selection of people attempting to get data to enormous areas of the general public. For instance, numerous publicists utilize the Creole language to guarantee that their message offers to a great many people. Simultaneously, due to the eminence appended to the standard language, it will in general be the language of decision on formal events, similar to community gatherings. A language assortment is normally picked on account of its apparent social capacities. You may have seen that, the more formal the event, the more probable the utilization of the standard language, while for ordinary communication, well known music or enthusiastic interests, individuals will in general float towards the non-standard assortments. You would have seen that, even in a proper circumstance, non-standard vernacular may be utilized for stories, to infuse humor or in a citation. In the Caribbean, individuals changing starting with one code of language then onto the next, frequently without intuition. In any case, there are times when the utilization of standard langue would appear to be absolutely strange and would even meddle with semantics. For instance, people stories, society tunes and adages appear to lose a specific substance when converted into standard. The job of language as a vehicle for sharing society is unquestionable. Caribbean journalists, vocalists and oral artists have had a significant impact in encouraging acknowledgment of the Creole dialects of the area, by joining them into their work and presenting them to the world. In any case, negative mentalities to these dialects continue in the brains of many. Perspectives to Language. (2018, Oct 28).
Argumentative Essay Topics For High School StudentsArgumentative essay topics for high school students should be of a specific topic for discussion. The assignment should not be taken as an assignment in and of itself. It should be used to help the student to have more of a solid foundation on which to build a case in their own opinions.This type of writing will require the student to take a critical essay topic for discussion and evaluate it. It is important that the topic is very narrow, but it must be strong enough to produce something good to present to the teacher. This type of assignment will be challenging, but will be very rewarding.A persuasive essay topic for high school students should include a discussion on a particular problem or situation in a particular situation. The subject should be something where there is a conflict. There are a number of ways to tackle this, including reading a news article, watching a television commercial, or getting advice from a professional who has knowledge on the topic.While in a classroom situation the student should be able to discuss the subject matter in detail. Argumentative essay topics for high school students should also focus on the person or group that is experiencing a conflict. For example, a father who had recently lost his job will want to find ways to help his son be successful, while a business owner who had been out of work would need to convince the CEO of the company to hire him.The topics will be as broad or as specific as the student wants. They should focus on the person, place, or group being confronted with the problem. This will help them to create a more descriptive essay that is accurate to the situation.An essay that is informative will focus on specific facts about the people or situation that is being analyzed. In the former case, the writer may explain how the losing business owner had been fired, but they should do so in a manner that is brief and to the point. An informative essay wi ll focus on the facts and not attempt to look for conflict.Argumentative essay topics for high school students will all take the same format, however, the goal is to use the information presented as a way to support one's own argument. The facts are presented in a short and concise manner to provide a clear picture of the situation or group being analyzed. The writing should be clear and concise, and the ideas must be properly introduced.Writing an argumentative essay for high school students should be something that is fun. An essay that is well written will produce a strong argument that will not only be of value to the reader, but also to the author. Students should try to write each essay in a way that will help them to reach a high school graduation with a great academic and writing grade.
Sunday, June 28, 2020
The genome editing using engineered nuclease has strategically transformed the idea of gene therapy for monogenic diseases including in hematopoietic stem and progenitor cells (HSPCs) (Osborn et al., 2016; Yu et al., 2016). The genome editing technology enables to create a site specific double-strand break (DSB) by the engineered nucleases that programmable triggering the cellÃ¢â¬â¢s endogenous repair machinery to edit the genome in a site-specific manner via the non-homology end joining repair (NHEJ) and the homology directed repair (HDR) mechanisms(Branzei and Foiani, 2008). The approach allows the precise alteration of the disease-causing alleles at the specific locus making it a permanent event that maintains the phenotypical gene expression under the control of endogenous regulatory elements.. Over the past decade, three major classes of engineered nucleases have been used for genome editing, including zinc-finger nucleases (ZFNs) (Kim et al., 1996; Urnov et al., 2010), transcription activator-like effector nucleases (TALENs) (Li et al., 2011; Miller et al., 2011) and CRISPRÃ¢â¬âCas9 (clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) protein 9) (Hsu et al., 2014; Sander and Joung, 2014; Tsai and Joung, 2016; Wiedenheft et al., 2012). ZFNs and TALENs are fusions between arrays of ZF or TALE DNA-binding domains and the dimerization-dependent FokI nuclease domain. The both of ZFN and TELENs nucleases exclusively rely on proteinÃ¢â¬âDNA interactions to mediate site-specific recognition of genomic DNA sequences which requires complex protein engineering for each new targets (Kim and Kim, 2014). By contrast, CRISPRÃ¢â¬âCas9 nuclease is a RNA-guided endonuclease. Through the guidance of a 23 nucleotides linked to CRISPR-domain RNA (gRNA), CRISPR-Cas9 finds the complementary protospacer DNA target in a genome where it cuts the double stranded DNA precisely 3 base pairs upstream of a PAM (Protospacer Adjacent Motif). The broken DNA ends generated by those nucleases are repaired either by NHEJ resulting in small insertion/deletions (indels) to disrupt target allele, or by HDR to precisely replace desired nucleotides required with delivery homologous DNA template. Compared to ZFNs and TALEN, the CRISPR/Cas9 system has rapidly become the most promising genome editing tool with demonstrated advantages including simplicity, easy programming, low cost and potential multiplexed editing (Bannikov and Lavrov, 2017; Brunetti et al., 2018; Salsman and Dellaire, 2017; Tsai and Joung, 2016) (Minkenberg et al., 2017). Despite of the genome editing holds tremendous promise for the developing novel gene therapy, the technique has been shown to be more refractory in HSPCs than any other cell types due to their quiescent stat us associated with low activity of the HDR machinery, and prone to DSB induced toxicity. However since first publication of using the ZFNs editing on human CD34+ cell (Genovese et al., 2014), the substantial developments have been made in last few years to circumvent the problems. Optimization of gene editing efficiency in HSPCs In vitro expansion of HSPCs Since all nucleases targeted gene editing occurs through cell cycle progress, the increased stimulation HSPCs ex vivo can make them more permissive to editing components. However, increasing stimulation can also promote cell differentiation. To circumvent this, the compounds that agonist HSPC self-renew while maintain their primitive phenotypes have been discovered and applied in the culture (Boitano et al., 2010; Fares et al., 2014; Goessling et al., 2011). Using the compounds in HSPCs culture, researchers have achieved significantly increased percentage of edited HSPCs in vitro and also increased human cell engraftment in vivo (Charlesworth et al., 2018; Genovese et al., 2014). In a recent published study, Psatha et al. have described 5 days HSPC culture condition, in which StemRegenin 1(SR1) was used with a small molecule Ly2228820 (SL), the p38?MAPK14 inhibitor (Psatha et al., 2017). Using this culture condition, they haveÃ successfully expanded highly engraftable CD34+/CD38?/ CD90+Ã primitive HSPC cells. They then tested if using SR1 and SL condition can also expand edited HSPCs effectively. For this, they cultured edited HSPCs for additional 5 days after the editing, and found that the edited CD34+/CD38?/CD90+ primitive HSPCs can be effectively expanded in vitro without any loss of editing efficiency. Moreover, the expansion of edited cells gave rise to a more than 2-fold higher engraftment compared to their unexpanded counterparts, showing the same editing rate (Psatha et al., 2018). The study highlights a possible way to obtain sufficient engraftable HSPCs by expanding edited cells effectively ex vivo in presence of SR1 and SL.Ã However, this study was conducted using the NHEJ directed gene editing strategy for disrupting the genomic locus. It would be important to know if the presence of SR1 and SL in culture can also increase the HDR directed gene editing. And also convincing evidence on long-term in vivo engraftment from significantly expande d HSPCs is needed to ensure no oncogenic burden associated with ex vitro expansion. Delivering the editing components In clinical application setting, the approach for delivering nucleases or other components into HSPCs should be transient to avoid the cytotoxicity engendered by prolonged endonucleases activity and immune responses. Therefore, a Ã¢â¬Å"hit-and-runÃ¢â¬ approach is used, whereby the nuclease complex is transient expression. The mostly used method for delivering DNA or RNA encoding engineered nucleases is via nuclear transfection. The transfection of plasmid DNA encoding the nucleases to HSPCs has been used with success on editing targeted loci (Holt et al., 2010; Mandal et al., 2014). However the main concern from this approach is its potential random integration into the genome which could lead to cytotoxicity in HSPCs and their progenies. And DNA related cytotoxicity, such as cyclic GMP-AMP synthase induced pathway (Sun et al., 2013), could lead to high toxic to primitive HSPCs. Therefore, the transfection of mRNA encoding nucleases synthesized in vitro has become an optimal alte rnative approach (Liang et al., 2015; Wang et al., 2015). It has emerged from recent studies that the mRNA transfection approach indeed has provided an increased efficiency in genome editing in HSPCs (De Ravin et al., 2016; Kuo et al., 2018; Schiroli et al., 2017). In addition, Cas9 can be delivered as the protein or as the precomplexed ribonucleoproteins (RNPs) by mixing gRNAs with Cas9 protein (Dever et al., 2016; Liang et al., 2015). The approach serves in protecting gRNAs from degradation, and reducing cytotoxicity caused by naked RNA stimulated innate immunity. The improved editing efficiency based on such approach has been achieved in targeting HSPCs shown in recent studies (Bak et al., 2017; Kuo et al., 2018; Schiroli et al., 2017; Vakulskas et al., 2018). Apart from above components, a safe and efficient delivering DNA donor template into edited cells is crucial for achieving HDR process. Several donor template platforms have been used. Single-stranded DNA oligonucleotide (ssODN) donor has been shown as a simple and effective approach in genome editing for correction of single-nucleotide mutation in HSPCs, such as for SickleÃ cell disease (SCD) (DeWitt et al., 2016).Ã Integration defective lentiviral vector (IDLV), that allow incorporating large DNA template, has been used in the ZFN genome editing to target the IL2RG mutations and theÃ adenosineÃ deaminaseÃ (ADA) gene (Genovese et al., 2014; Joglekar et al., 2013). However, those early studies showed a very limited gene targeting efficiency in HSPCs, suggesting that IDLV could be more cytotoxicity to HSPCs. The efficiency of IDLV in targeted integration in HSPCs can be significantly improved by using cyclosporine H, which is shown in a very recent study (Petrillo et al., 2018). Certain adenoviral serotypes (Ad5) can transduce human HPSCs and deliver large transgene cassettes (Li et al., 2013) (Saydaminova et al., 2015). However the concern that residual of adenovector particles could be highly immunogenic which may prevent its potential use in clinical application therapy. Recombinant adeno-associated viral vectors (rAAVs) have been shown to naturally mediate HR in mammalian cells without stimulating DSB (Barzel et al., 2015; Mingozzi and High, 2011; Moser and Hirsch, 2016). Hence, rAAV vectors are emerged as ideal delivery approach due to their wide range of tropism, high transduction rate and very low immune response. In particular, the rAAV6 vector has been shown to provide more efficient and robust genome-editing in HSPCs than other delivery vectors shown in recent therapeutic potential application studies (De Ravin et al., 2017; De Ravin et al., 2016; Kuo et al., 2018; Moser and Hirsch, 2016; Schiroli et al., 2017). However, relative small pa ckaging capacity in rAAV6 has limited its use for delivering cassata larger more than 4.5 kb including the both homology arms. To improve the packaging capacity, Bak and Proteus (Bak and Porteus, 2017) have developed a dual-AAV6 donor vector system that enables site-specific integration of large transgene cassette up to 6.5 kb into primary T cells and HSPCs with long-term repopulation capacity. Overall, the conditions for delivery the components used in gene editing should always be optimised for each targeted gene to achieve most efficient targeting and minimum cytotoxicity. A comprehensive detailed protocol using CRISPR/Cas9 with rAAV6 as templet vehicle for HDR-targeted editing in HSPCs has been published by Bak and Daniel recently (Bak et al., 2018), which could be also served as a guide for implement gene editing technique for other nucleases Improve the HDR Unlike NHEJ pathway which occurs throughout the cell cycle, the HDR event is restricted in S/G2 phases of cell cycle which makes the HDR process much less efficient than NHEJ (Gutschner et al., 2016; Heyer et al., 2010). Therefore, inhibiting nuclease activity at G1/M phases and resting cells at S/C2 phases may improve HDR efficiency. The concept has been experimentally tested by Gutschner and colleagues (Gutschner et al., 2016). In which, the hGemCas9 system is generated by incorporating the human geminin domain which allows the nuclease to be ubiquitinated and degraded by APC/Cdh1 complex in G1 and late M phase, therefore leading to increased hGemCas9 activity in S/G2 phases. Using this cell-cycle-tailored hGemCas9 system, Gutschner et al have achieved an increased rate of HDR up to 1.87 fold compared to wild-type Cas9 in cell lines. A further development based on this approach was published recently by Lomova et al. (Lomova et al., 2018). In their study the hGemCas9 was used in c ombination with a cell synchronization compound RO-3306 which functioning in transiently arresting cells at S/G2 phase via inhibiting CDK1(Vassilev et al., 2006). It was shown from LomovaÃ¢â¬â¢s study that the ratio of HDR/NHEJ was increased to four-fold on human CD34+ cells compared to the controls in vitro, and a significant improvement of edited HSPCs in immune-deficient mice. The improved HDR has also been achieved by directly inhibiting the NHEJ pathway through targeting DNA ligase IV, a key enzyme in the NHEJ pathway, using the inhibitor Scr7 (Hu et al., 2018; Maruyama et al., 2015). Although high increased efficiency in HDR has been achieved in human cell lines and cancer cells, so far, there has been no published data of using Scr7 on human HSPCs. The assessment off-target sites Although ideal engineered nucleases would have singular genome-wide specificity, unintended off-targets can occur, particularly at loci with homologous to the intended targeting site. Several the off-target detection methods have been used in HSPCs gene editing studies. An early developed assay is based on using the silico prediction off-targets sites that have degree of similarity to the on-targets, and then followed by targeted sequencing (Fine et al., 2014) (Hsu et al., 2013). This initially developed method is still mostly used in the HSPCs editing studies as it is more practicable assay. However the fundamental limitation with this approach is it is not designed to identify off-target sites in an unbiased manner as the sites that not fit the computational criteria will not be discovered, To achieve unbiased off-target detection, the cell based genome-wide assays have been developed. On of such assay used in HSPCs editing studies is Integrase-defective lentiviral vector (IDLV) capture assay, which was designed to capture IDLV into sites of nuclease-induced DSBs. Then clustered sites of integrations are recovered by linear amplification-mediated PCR (LAM-PCR) and mapped using high-throughput sequencing (Gabriel et al., 2011). Although the IDLV capture can directly identify DSBs in living cells, it is relatively insensitive, owing to its low absolute integration efficiencies that require positive selection to overcome (Gabriel et al., 2011). And the assay may have high background due to event of random integration IDLVs into cellular genomes even in the absence of nuclease-induced DSBs (Gabriel et al., 2011). Whole genome sequencing (WGS) has been proposed as an unbiased method for defining engineered nuclease specificity. Although this method is useful for the analysis of single-cell clones (Veres et al., 2014), it lacks sensitivity, particularly for those low frequencies off-target in a population cells (Tsai and Joung, 2014). With existing deep sequencing technology, it is impractical to perform WGS on millions of cellular genomes, and it is inadequate to confirm the off-target sites at Therapeutic potential of HSPC gene editing Non-homologous end joining-based strategy NHEJ DNA repair pathway triggered by engineered nucleases is the active random repair process, leading to the alteration of nucleotide sequencing at the specific site via in-frame deletions, insertions. Sine it is not involved in harnessing the HDR machinery, it has become a viable genome editing option for correcting gene mutations. Two HSPC targeted loci, chemokine coreceptor 5 (CCR5) and BCL11A, have received the most early attention as their potential therapeutic benefits via NHEJ process. The concept of editing CCR5 was intrigued by the report that the transplantation of a donor HSCs with a naturally occurringÃ CCR5Ã mutation confers a loss of detectableÃ HIV-1 RNA and proviral DNA in a HIV patient (Hutter et al., 2009). Holt et al. first published the report of the successful disrupting CCR5 using the ZFNs (Holt et al., 2010). In their study, NSG mice transplanted with ZFN-modified HSPCs underwent rapid selection for CCR5(-/-) cells when challenged with CCR5-tropic HIV-1, showed significantly lower HIV-1 level compared to the controls. Several studies publishes later have also demonstrated the feasibility of CCR5 disruption in HSPCs that lead to resistance to HIV infection in vivo model (DiGiusto et al., 2016; Li et al., 2013; Xu et al., 2017). Among them, DiGiusto et al. conducted a preclinical study to assess efficacy and safety of the ZFN-based CCR5 disruption in HSPCs on the clinical-scale delivering CCR5-specific ZFN-mRNA to normal adult HSPCs. In which, they demonstrated effective biallelic CCR5 disruption of 40-60% in liquid culture cells, and in up to 72.9% of modified colony forming units from edited HSPCs. The edited HSPCs preserved long-term multiple lineage potentialÃ in vivo with no demonstrated potential for tumorigenesis or leukemagenesis (DiGiusto et al., 2016). Based on this, further safety and feasibility studies are ongoing in subjects infected with HIV-1 ([emailprotected]). Targeting HSPCs genomic locus BCL11A via NHEJ gene editing has been developed for potential treatment of the ?-hemoglobinopathies, which are inherited monogenic blood disorders due to the mutations in ?-globin gene causing either Thalassemis (abnormal haemoglobin production) or sickle cell disease (SCD) (abnormal haemoglobin tetramer) (Steinberg, 1999). The observed fact of that the severity of both conditions can be ameliorated by the induction of Fetal haemoglobin (HbF) (Collins et al., 1995) led to discover the BCL11A transcription factor as a repressor for HbF (Bauer and Orkin, 2015), and BCL11A erythroid-specific enhancer, GATAA in association with fetal-to-adult haemoglobin switching (Canver et al., 2015), which could be targeted for inducing HbF in HSPCs for potential treatment of those conditions. To this end, Bjurstom et al. conducted the genome editing strategy to disrupt the BCL11A exon2 in HSPCs using the engineered nucleases ZFNs, TALENs or CRISPR-Cas9 (Bjurstrom et al ., 2016). It was shown in their study that the ZFNs gave rise to more allelic disruption in theÃ targetedÃ locus which is associated with increased levels ofÃ HbFÃ in erythroid cells derived from nuclease-treated CD34+Ã cells in vitro. However, a low level of disruption in the BCL11A gene in bone marrow (4%) was observed after engraftment into NSG mice. Using the ZFNs approach Chang et al. performed study to compare targeted disruption of theÃ BCL11A, either within exon 2 or at the GATAA motif (Chang et al., 2017). It was shown from their study that the allelic disruption of GATAA not only give rise to robustÃ long-termÃ engraftmentÃ leading to elevated level ofÃ HbFÃ expression in erythroidÃ cells, but also prevent the adverse effect of erythroid enucleation seen in theÃ BCL11A exon2 ablation. Using same strategy, a comprehensive preclinical study has been carried out in HSPCs from adult donors and two patients with ?-Thalassemia Major (Psatha et al., 2018 ). The modification of GATAA motif in mobilizedÃ CD34+Ã cellsÃ from ?-thalassemia patients resulted in a readily detectable increased ?-globin with a preferential increase in G-gamma, leading to an improved phenotype that likely to give a survival advantage for maturing erythroidÃ cells. A phase 1/2 clinical trial for correcting the ?-thalassemia phenotype by genomeÃ editing is currently being evaluated by the same group. Homologous recombination based strategy In larger majority genetic blood diseases, the homologous directed repair strategy is required for correcting genotype, with delivering exogenous DNA template. The process is much more challenging than NHEJ-based gene editing due to its low efficiency, particular in targeting primitive HSPCs. However, the promising progress in targeted integration in HSPCs for some PIDs has been made in recent years. Interleukin-2 receptor common gamma-chain (IL2RG) The first attempt using the ZFNs for gene knockin in HSPCs was demonstrated by Genovese et al. (Genovese et al., 2014). In this study, two genomic loci, AAVS1 Ã¢â¬Å"safe harbourÃ¢â¬ or a mutational hotspot of IL2RG were targeted with a GFP cassette delivered with IDLV vector. Although there was 24-26% indels found in the ZFN target sites, only 5% GFP+ colonies were found in colony-forming cells (CFU) assay. At 8 weeks after transplantation edited CD34+ cells into NSG mice, the frequency of 2% GFP+ cells were found among primitive and committed progenitors in the BM. To improve gene targeting efficiency, Genovese et al. tailored the culture condition by extending cell activation time making them more permissive for the editing molecules, and by adding the compounds into the culture to preserve the stemness in primitive HSPCs (Genovese et al., 2014). The modified protocol indeed gave rise to significantly increased GFP+ cells (?2-fold) in primitive cell population in vitro and also in vivo in long-term engrafted HSPCs. Using improved the ZFNs protocol Genovese et al. performed the IL2RG gene correction in CD34+ cells derived from SCID-X1 patient with delivering IDLV vector consisting of the exons 5-8 IL2RG cDNA and a PGK-GFP cassette flanked by homologous sequences. In which, they found 3% GFP+ cells in primitive HSCs and up to 11% GFP+ in committed progenitors in liquid culture. The CFU assay yield 3 GFP+ colonies out of 100 scored, with a myeloid progeny colony showed reconstituted normal IL2RG protein expression. The data from this study highlighted the problem with targeting primitive HSCs for homologous recombination. A recent development in targeting integration of IL2RG has been demonstrated by same group (Schiroli et al., 2017). In order to establish therapeutic potential of a gene correction strategy for the treatment of SCID-X1, a humanise SCID-X1 mouse model was used to evaluate efficacy and safety of the edited HSPCs in a preclinical setting. To i mprove editing efficiency, they made the modification on the ZFN mRNA by incorporating the base analogs to prevent recognition by cellular sensors that associated with the activation of the interferon-responsive genes by exogenous RNAs. This modification results in a significant reduced cytotoxicity caused by in vitro electroporation of the ZFN mRNA, leading to high HDR (25%) in CD34+ cells derived from a SCID-X1 patient. By changing to use AAV6 as donor DNA vehicle following the ZFN mRNA electroporation, they achieved up to fivefold higher HDR-mediated gene editing in the most primitive CD34+ CD133+ CD90+ cells over the IDLV vehicle approach. It was also demonstrated in this study that optimised clinical relevant protocol is transferable to the clinic scale, showing reproducible editing efficiency even in a large scale 2.5107 HSPCs. More importantly, the edited cells preserved long-term engraftments in NSG mice, showing an average 12% HDR in HSPCs at 23 weeks end point, which excee ded the threshold (10%) of functional HSPCs required for fully reconstitute immune function at a standard transplant dose established in the their study (Schiroli et al., 2017). The off-target assay did not detect significant amounts of modification above the threshold of sensitivity in any of the off-target sites identified previously by genome-wide screening for the ZFN set (Gabriel et al., 2011). Based on these data, it would be interesting to see if the optimised protocol could lead to adequate editing efficiency in HSPCs derived from the SCID-X1 patient, which could paves the way to translation HSPCs gene editing into the therapy. X-linked chronic granulomatous disease (X-CGD) Two recent studies published by De Ravin et al. presented the promising results on the targeted integration of CYBB gene encoding gp91phox for the treatment of X-CGD (De Ravin et al., 2017; De Ravin et al., 2016). Their initial study (De Ravin et al., 2016) was based on the ZFNs targeted integration of transgene into a genomic Ã¢â¬Å"safe habourÃ¢â¬ AASV1 with the aim to overcome insertional mutagenesis by the viral vector gene therapy, where 3 X-CGD patients underwent the gene therapy developed myelodysplasia due to the integration at MDS-EV11 locus (Stein et al., 2010). De Ravin et al. carried out extensive experiments firstly to explore the optimised conditions in clinical relevant approaches for delivery of the ZFNs, and AAV6 delivery of donor construct containing promoter-less Venus marker cDNA into the intron 1of the PPP1R12C gene at AAVS1 locus. The results from their study showed up to 58% Venus-positive HSCs in vitro and 6Ã¢â¬â16% human cell marking were observed follow ing engraftment into NSG mice. Using their optimised approach, they then targeted HSPCs derived from X-CGD/gp91phox patients with donor constructs containing either a promoter less gp91phox (2A-2A-gp91), or gp91phox driven by a synthetic MND promoter (MND-91). Although the both approaches showed a similar targeted integration efficiencies (~15% gp91phox expression), a robust functional correction through MND promoter, rather than the endogenous PPP1R12C promoter was obtained with significant high MFI of gp91expression and DHR oxidase activity in edited HSPCs in vitro. At 8 weeks following transplantation of edited HSPCs into NSG mice, the MND-91 and 2A-2A-gp91 corrected HSPCs grafted average 3.7Ã ±4.2% and 10.7Ã ±4.2% of human CD45+ cells respectively from bone marrow gp91expressing cells. Since the gene therapy corrected cells in X-CGD patients do not entail a selective advantage, the question is if the level of reconstituted gp91expressing cells achieved in this study would be sufficient for the disease phenotype correction. Nerveless, the data presented in the study has provided the first promising alternative approach in correction of X-CGD. However, long-term efficiency in vivo still remain to be established, and the safety issue of disrupting PPP1R12C gene encoding for a phosphatase in stem cells also need to be determined. In a later study led by the same group (De Ravin et al., 2017), De Ravin et al. have achieved the targeted correction of the point mutation C676T X-CGD using CRISPR/Cas9 and delivering single strand oligo nucleotide (ssODN). The C676T mutation, accounted for 6% of X-CGD patients, occurs at the exon 7 of CYBB gene resulting in a premature stop codon and an inactive gp91phox protein. Following experiments to optimise the targeting CYBB 676 locus in normal CD34+ cells, they achieved level of HDR editing efficiency even within primitive (CD34+CD133+CD90+) HSPCs at ~30%, which is high than any previously reported. In CD34+ cells derived from CYBB 676 patient, they achieved targeted repair of 20% of HSPCs and restored gp91expression to 31% in myeloid cells differentiated from edited HSPCs, which resulted in restoration of the function of NADPH oxidase activity and superoxide radical production. Analysing of transplantation of gene-repaired X-CGD HSPCs into NSG mice at 8 and 20 weeks, they demonstrated not only improved stable human engraftment and corrected CYBB alleles, but also the production of functional mature human myeloid and lymphoid cells for up to 20 weeks. The off-target sequencing analysis on computationally predicted off- target sites in edited CD34+ cells from the patient revealed one single indel (3 bp) at the RP11-454H19.2 gene at a high read depth 1,200,000x, but not at 10,000 read depth. However, one single indel observed in the uncorrected healthy control CD34+ HSPCs, indicating that this could be due to amplification/sequencing errors at high level of coverage. Whole-exome sequencing at 800Ãâ" coverage of corrected patient CD34+ HSPCs also failed to detect any off-targets. Using same approach, De Ravin et al. have tried to correct a second X-CGD patient with CYBB 676 mutation (De Ravin et al., 2017). Although the gene repair efficiency was achieved in a similar level to the patient 1 in vitro, a less than 50% of the gene repair rate was observ ed after transplantation into NSG mice. This has highlighted the necessity of careful validation of editing condition at every level to achieve a consistent outcome. Nerveless, this study presented a viable approach in correction of a missense mutation in HSPCs by targeted integration that restore gene function under the control of the genes endogenous promoter. X-linked hyper-IgM syndrome (XHIM) XHIM is a primary immunodeficiency due to mutations in CD40 ligand gene (CD40L) expressed on the activated T cells. The mutated CD40L fail to bind CD40 on B cells which affect immunoglobulin class switch recombination that represented by the absence of IgG, IgA, IgE with normal to elevated IgM. XHIM patients are susceptible to bacterial infection, with development of autoimmunity and malignancies in some X-HIM individuals (Hayward et al., 1997; Levy et al., 1997). XHIM can be treated by allogenic HSCT, but has been associated with some sever site effects. Although the experimental gene therapy using viral vector in XHIM mouse model showed the correction of immune defect, the mice developed abnormal lymphoproliferation due to unregulated expression of the gene from ectopic genomic loci (Brown et al., 1998; Sacco et al., 2000). Therefore, using gene editing tools in targeted integration of XHIM gene under control of its endogenous promoter has become an optimal alternative approach for treating the disease. Using the TALEN as targeted gene editing approach Hubbard et al. have first demonstrated the feasibility in restoration of normal expression of CD40L and rescued IgG class switching in XHIMÃ patientÃ T cells (Hubbard et al., 2016). A later study by Kuo et al. developed the both TALEN and CRISPR/Cas9 platforms to achieve site-specific editing of a human CD40L cDNA, at the 5Ã¢â¬â¢UTR of the gene allowing bypassing all known disease-causing mutations in XHIM (Kuo et al., 2018). The both approaches were tested in T cells derived from XHIM patient. Although the TALEN approach resulted in CD40L expression at the baseline in unstimulated cells, an up-regulated CD40L expression to 20% was detected upon anti-hCD3/anti-hCD28 immune stimulation which is comparable to stimulated T cells from healthy donors. The corrected XHIM T cells demonstrated a normal receptor-binding activity to recombinant chimeric CD40-muIg. The data highlighted that a proportionally small n umber of gene-corrected T cells in XHIM may be sufficient to allow enough class-switching to ameliorate the disease. In CRISPR/Cas9 treated XHIM T cells, high rates of targeted gene integration was attained with restore physiologically-regulated CD40L expression and function. In targeting CD34+ cells from healthy donor, Kuo et al. have shown that both platforms gave rise to a similar level of allelic disruption rate in samples from 8 biological replicate, 4 PBSC donors (29.1 Ã ± 7.8% with TALEN, average 33% with CRISP/Cas9). A relative high targeted gene integration rate was observed in CRISP/Cas9 treated cells, particular when gRNA and Cas9 protein delivered as RNP (to 20.8 Ã ± 6.6%). By adding the adenovirus helper protein that co-introduced as mRNA during electroporation with TALENs or CRISPRs, a 2-fold enhanced gene modification was achieved. However, this augment effect was not observed in engrafted NSG mice in vivo. Following transplantation of edited cells into NSG mice at 12-20 weeks, the targeted gene integration was detected in the bone marrow from 80% of mice, with integration rates ranged from 0.3% to 22%, a mean of 4.4% across all treatment groups. The analysing of thymus from engrafted mice showed 60% mice had thymic reconstitution, With frequency of engraftment trending higher in those analysed at 5 months compared to 3 months post-transplant. The off-target activity was not detected based on silico predicted off-target sites for both TALENs and CRISPR in K562 edited cells. However, using IDLV capture approach in TALENs edited K562 cell, three off-target loci (OT1, OT2 and OT3) were observed. High-throughput sequencing of off-target sites in HSPCs and K562 cell treated with TALENs mRNA demonstrated statistically significant gene disruption at OT1 in HSPCs, and OT2 in both cells. However, there was no off-target site identified in CRISPR treaded cells using another cell based assay GUIDE-seq, which was designed recently with a high sensitivity for detecting the off-target sites mutagenized by Cas9-gRNA (Tsai et al., 2015). Taking together, CRISPR approach showed some advantages over TALENs in targeting integration of XHIM gene. Overall, this study paves an important step toward to developing a curative therapy for XHIM through site-specific gene correction. The major hurdles in HSPCs gene editing Despite the genome editing holds tremendous promise for the developing novel gene therapy, HSPCs targeted editing is still in its infancy, and many issues regarding this new technology are remained to be addressed before translating it into safe clinical application. One major hurdle facing in HSPCs targeted editing is low efficiency, particularly in vivo following transplantation of edited cells, where the engrafted cells and frequency of edited cells decline significantly within 8 to 12 weeks and continuously decline in prolonged period. This suggests the Ã¢â¬Å"realÃ¢â¬ long-term HSPCs either have failed to undergo genome editing due to their quiescence and more resistance to homologous recombination, or they have been damage by DSBs due to exposure to nuclease and lost their self-renew property underwent apoptosis.
Monday, May 25, 2020
Beryllium is the element that is atomic number 4 on the periodic table. It is the first alkaline earth metal, located at the top of the second column or group of the periodic table. Beryllium is a relatively rare element in the universe and not a metal most people have seen in pure form. It is a brittle, steel-gray solid at room temperature. Fast Facts: Atomic Number 4 Element Name: BerylliumElement Symbol: BeAtomic Number: 4Atomic Weight: 9.012Classification: Alkaline Earth MetalPhase: Solid MetalAppearance: White-Gray MetallicDiscovered By:Ã Louis Nicolas Vauquelin (1798) Element Facts for Atomic Number 4 The element with atomic number 4 is beryllium, which means each atom of beryllium has 4 protons. A stable atom would have 4 neutrons and 4 electrons. Varying the number of neutrons changes the isotope of beryllium, while varying the number of electrons can make beryllium ions.The symbol for atomic number 4 is Be.Element atomic number 4 was discovered by Louis Nicolas Vauquelin, who also discovered the element chromium. Vauquelin recognized the element in emeralds in 1797.Beryllium is an element found in beryl gemstones, which include emerald, aquamarine, and morganite. The element name comes from the gemstone, as Vauquelin used beryl as the source material when purifying the element.At one time the element was called glucine and had the element symbol Gl, to reflect the sweet taste of the elements salts. Although the element tastes sweet, it is toxic, so you shouldnt eat it! Inhalation beryllium can cause lung cancer. There is no cure for beryllium disease. Interestingly, not everyon e who is exposed to beryllium has a reaction to it. There is a genetic risk factor that causes susceptible individuals to have an allergic inflammatory response to beryllium ions.Beryllium is a lead-gray metal. It is stiff, hard, and nonmagnetic. Its modulus of elasticity is about a third higher than that of steel.Element atomic number 4 is one of the lightest metals. It has the one of the highest melting points of the light metals. It has exceptional thermal conductivity. Beryllium resists oxidation in air and also resists concentrated nitric acid.Beryllium is not found in pure form in nature, but in combination with other elements. It is relatively rare in the Earths crust, found at an abundance of 2 to 6 parts per million. Trace amounts of beryllium are found in seawater and air, with slightly higher levels in freshwater streams.One use of element atomic number 4 is in the production of beryllium copper. This is copper with the addition of a small amount of beryllium, which makes the alloyÃ six times stronger than it would be as a pure element.Beryllium is used in x-ray tubes because its low atomic weight means it has a low absorption of x-rays.The element is the main ingredient used to make the mirror for NASAs James Webb Space Telescope. Beryllium is an element of military interest, since beryllium foil may be used in the production of nuclear weapons.Beryllium is used in cell phones, cameras, analytical lab equipment, and in the fine-tuning knobs of radios, radar equipment, thermostats, and lasers. It is a p-type dopant in semiconductors, which makes the element critically important for electronics. Beryllium oxide is an excellent thermal conductor and electrical insulator. The elements rigidity and low weight make it ideal for speaker drivers. However, expense and toxicity limits its use to high-end speaker systems.Element number 4 is produced by three countries at present: the United States, China, and Kazakhstan. Russia is returning to beryllium pro duction after a 20-year break. Extracting the element from its ore is difficult because of how readily it reacts with oxygen. Usually, beryllium is obtained from beryl. Beryl is sintered by heating it with sodium fluorosilicate and soda. The sodium fluoroberyllate from sintering is reacted with sodium hydroxide to form beryllium hydroxideÃ Beryllium hydroxide is converted to beryllium fluoride or beryllium chloride, from which beryllium metal is obtained by electrolysis. In addition to the sintering method, a melt method may be used to produce beryllium hydroxide. Sources Haynes, William M., ed. (2011). CRC Handbook of Chemistry and Physics (92nd ed.). Boca Raton, FL: CRC Press. p. 14.48.Ã Meija, J.; et al. (2016). Atomic weights of the elements 2013 (IUPAC Technical Report). Pure and Applied Chemistry. 88 (3): 265Ã¢â¬â91.Weast, Robert (1984).Ã CRC, Handbook of Chemistry and Physics. Boca Raton, Florida: Chemical Rubber Company Publishing. pp.Ã E110.