This review covers studies which exploit triazole-modified nucleic acids in the range of chemistry and biology to medicine. The 1,2,3-triazole unit, which is obtained via click chemistry approach, shows valuable and unique properties. For example, it does not occur in nature, constitutes an additional pharmacophore with attractive properties being resistant to hydrolysis and other reactions at physiological pH, exhibits biological activity , and can be considered as a rigid mimetic of amide linkage. Herein, it is presented a whole area of useful artificial compounds, from the clickable monomers and dimers to modified oligonucleotides, in the field of nucleic acids sciences. Such modifications of internucleotide linkages are designed to increase the hybridization binding affinity toward native DNA or RNA, to enhance resistance to nucleases, and to improve ability to penetrate cell membranes. The insertion of an artificial backbone is used for understanding effects of chemically modified oligonucleotides, and their potential usefulness in therapeutic applications. We describe the state-of-the-art knowledge on their implications for synthetic genes and other large modified DNA and RNA constructs including non-coding RNAs. The natural nucleic acids, DNA and RNA, are exquisitely suited to store and propagate genetic information ,2. NucleIn the world of science, the key role for regulating life processes is ascribed to proteins. Since the genetic information about them is encoded in 2′-deoxyribonucleic acid (DNA), it found itself on the podium of fame. An unappreciated ribonucleic acid (RNA) stood in its shade—a silent hero with many faces. From the central dogma of molecular biology, it can be seen that the flow of genetic information, which takes place from DNA through RNA to protein, involves a molecule of RNA, and exactly the messenger RNA (mRNA). It follows that the mRNA molecule is coding RNA in transcription, because it rewrites information from DNA about proteins. A further stage is yet involved: ribosomal RNA (rRNA) and transfer RNA (tRNA). The first one acts as a scaffold, and the second one is a courier in the delivery of amino acids that are linked into long chains to form a protein. So, in translation, these two RNAs belong to the group of ncRNAs as they do not encode any protein sequence ,11. We ke.g., small nuclear RNA (snRNA) or small nucleolar RNA (snoRNA) . T. Tet al.7 and 8, in which two thymidines are linked by triazole, have been synthesized by reacting AZT or 5′-azidothymidine with 3′-O-propargylthymidine in excellent yields. Generally, 1% CuSO4 catalyst is enough to carry out this reaction. The reaction proceeds to completion at ambient temperature within 12 h. All novel thymidine analogues with a broad scope of modifications have been examined for their antiviral activity, but they have been found to be inactive against 13 viruses.Because of the important role of AZT in chemotherapy scientists are constantly looking for its valuable analogues. Also in this case, an inestimable support is given by click chemistry ,55,79. Uet al. published some model studies towards the synthesis of thymidine oligonucleotides with triazole internucleosidic linkages via Cu(I)-promoted ligation strategy . T. T187]. in vivo. Moreover, the resultant synthetic ribozyme with a triazole backbone mimic at the catalytic site saves its catalytic activity. This is also the first demonstration of transcription through a heavily modified DNA backbone linkage and suggestion that click-ligated DNA could be useful for the direct synthesis of biologically active RNA and proteins. Careful design has given rise to biocompatible triazole linkage that is treated as a normal one by DNA and RNA polymerases, and the gene containing triazole linkages has been shown to function in E. coli. This is how these scientists have pioneered a chemical method of linking DNA strands that is tolerated by living systems  cycloaddition, so-called click chemistry, has just enabled an intensive development of this chemistry area. The main goal of this work was to present efficient routes to design nucleic acids and their components dedicated for gene expression control. The use of gene silencing agents is a topic of intense research because these molecules can act as inhibitors of any target genes, expression of which causes diseases, among others viral infections, cancer growth, and inflammation. As naturally occurring ONs suffer from degradation due to endo- and exonucleases, modifications have become necessary to enhance the stability and improve pharmacokinetic properties from a therapeutic point of view. Modified nucleosides and nucleotides continue to attract significant focus in medical applications as building blocks for gene silencing, such as antisense, antigen, and RNA interference. The third generation of nucleic acids therapeutics—newest and most promising with enhanced binding affinity and biostability—are PNAs, LNAs, tcDNA, and CeNA.Fundamental knowledge from many fields, including chemistry, structural biology, cell biology, and medicine, has opened up the possibility of development of novel strategies against cancer and other diseases. This review highlights the contribution of click chemistry to drug development, but also to labelling and imaging for better visualization. The click chemistry covers chemical synthesis of small molecules as well as oligonucleotides by the bio-orthogonal ligation process and their application as drugs and tools in molecular medicine. The intention of this article is to provide both inside and inspiration in the field and stimulate further research to develop new chemical approaches. Thus, click reaction is one of the most popular interdisciplinary reaction that bridges the gap between chemistry and biology.


Refugee resettlement offices are the first point of contact for newly arrived refugees and play a significant role in helping refugees acclimate and settle into life in the United States. Available literature suggests that refugee women are vulnerable to poor sexual and reproductive health (SRH) outcomes, including sexually transmitted infections and HIV infections as well as adverse pregnancy outcomes, but little is known about the role that refugee resettlement offices play in supporting refugee women’s SRH. This study examines the capacity and interest of resettlement offices in providing SRH information and referrals to newly arrived refugees.N = 15) of refugee resettlement agencies (RRAs) who responded to the survey provide materials about SRH to clients, and 49% (N = 49) incorporate sexual health into the classes they provide to newly arrived refugee clients. Moreover, 12% (N = 12) of responding RRAs screen clients for pregnancy intention, and 20% (N = 20) directly refer to contraceptive care and services. This study is limited by the response rate of the survey; no conclusions can be drawn about those offices that did not respond. In addition, the survey instrument was not validated against any other sources of information about the practices of refugee resettlement offices.The research team conducted an online survey of staff members at refugee resettlement offices throughout the US in 2018 to determine (1) available SRH resources and workshops; (2) referrals to and assistance with making appointments for SRH and primary care appointments; (3) barriers to addressing SRH needs of clients; and (4) interest in building the capacity of office staff to address SRH issues. The survey was created for this study and had not been previously used or validated. Survey data underwent descriptive analysis. A total of 236 resettlement offices were contacted, with responses from 100 offices, for a total response rate of 42%. Fifteen percent  care services.No research has been done to date regarding the extent to which refugee resettlement offices are screening women for reproductive health needs, providing SRH education, or referring to medical providers for SRH care.Researchers conducted a survey of all of the refugee resettlement agencies in the United States to assess their practices regarding screening, education, and referrals for SRH needs. The survey also assessed perceived barriers to providing SRH screening, education and referrals, and respondents interested in improving the capacity of their office to provide these services.The study found that while nearly half of responding offices provide some SRH-related education through classes or workshops to newly arrived refugees, few provide educational materials or patient referrals for care.Responding offices cited lack of time and competing priorities as major barriers to providing SRH information and referrals to clients.Recently resettled refugee women are generally not receiving SRH screening, education, and referrals from refugee resettlement offices.Resettlement offices could better serve the SRH needs of refugee women. Conflict and crisis have dire consequences on women and girls’ sexual and reproductive health (SRH) and rights. Women and girls affected by conflict often have limited access to SRH care and are particularly vulnerable to unintended pregnancies, which may lead to high rates of unsafe abortion, maternal mortality, stillbirth, and perinatal mortality –3. WhileRefugee resettlement is undertaken by a public–private partnership between the US government and nonprofit organizations. There are currently 9 organizations with a network of local affiliates across the country that have cooperative agreements with the government to provide resettlement services. The initial services—those provided in the first 3 months after arrival—include housing, food, job placement, and medical care, and are funded by the Department of State’s Reception and Placement Program . At the To better understand how these agencies support the SRH needs of their clients, Advocates for Youth (AFY), a nonprofit organization that supports efforts to improve the SRH of adolescents and young adults, jointly conducted a survey with the International Rescue Committee (IRC), a nonprofit organization that responds to the world’s most severe humanitarian crises, helps people to survive and rebuild their lives, and is one of the 9 refugee resettlement agencies in the US. This effort is part of a broader AFY project to increase awareness of, access to, and use of contraception among refugee young adults. The findings from this survey may help lay the groundwork for strengthening the capacity of refugee resettlement agencies to integrate SRH services into programming and support refugees’ ability to plan for their futures by planning their pregnancies and families.This project was designed and conducted by AFY, with the assistance of students from Harvard’s T.H. Chan School of Public Health and staff from the United States’ national office and the Atlanta, Georgia, office of the IRC. The survey instrument addressed 4 key areas: (1) available SRH resources and workshops; (2) referrals to and assistance with making appointments for SRH and primary care appointments; (3) barriers to addressing SRH needs of clients; and (4) interest in building the capacity of office staff to address SRH issues. The survey consisted of 29 yes/no or multiple-choice questions; all survey data were qualitative. The survey was created expressly for this study and was not previously used or validated. The survey included an introduction to the study, explaining how the data were intended to be used. The survey may be reviewed in supplemental information, under the document title http://www.unhcr.org/en-us/us-resettlement-agencies.html), the research team identified 210 unduplicated non-IRC refugee resettlement offices. Survey participants from each of those offices were recruited through direct phone or email outreach, with the goal that the survey be completed by one staff member familiar with the full range of services available at each office. In May and June of 2018, all 210 non-IRC offices were contacted by email or phone, a maximum of 3 times. Once an office was known to have responded to the survey, no further contacts were attempted for that office. The survey was conducted using SurveyMonkey, an online platform.The survey was conducted in 2 stages, first with non-IRC resettlement offices and later with IRC offices. The survey was conducted in this manner due to a concurrent collaboration between AFY and the IRC; IRC management requested to reach out to their own affiliates themselves, rather than having AFY contact them via phone or email, as was the protocol for other resettlement agency offices. Using the website of the United Nations High Committee on Refugees  on SRH for clients . Of the Forty-one percent (37) of offices offer workshops that cover SRH topics. Among those offices, 51% (18) offer workshops in a single session, and 11% (4) offer a multi-workshop SRH series. Forty-nine percent (17) also include SRH content in cultural orientation courses. Forty percent (14) of offices reported conducting classes at least 4 to 6 times per year. The most common topics covered include: family planning/birth control; the reproductive health system and how pregnancy occurs; and how to access clinical reproductive health services. Sixty-nine percent (24) of workshops are always or usually separated by gender, and 82% (28) are voluntary for clients.Only 17% (15) of 86 responding offices have a health educator on staff. Only 12% (10) of agencies surveyed always or usually screen female clients upon intake for whether they want to become pregnant in the next year.Among the offices that responded, 52% (44) reported developing partnerships with other organizations to provide SRH workshops. These other organizations included medical clinics and providers and local departments of health.N = 76) reported being aware of nearby clinics where clients can get SRH services, but only 20% (17) reported that they refer clients to a clinic specifically for reproductive health services, including contraception and preconception care. However, when asked if the offices provided any additional assistance to clients who wished to obtain reproductive health services, 74% (59) of respondents reported providing assistance with making appointments; transportation to clinics; assistance obtaining medications; and assistance obtaining health insurance. Forty-four percent (35) of offices reported assisting their clients with having interpretation services during a clinical visit.Almost all offices  of respondents reported either “yes” or “maybe” when asked if they thought their office would be interested in increasing its capacity to provide SRH information and referrals to clients . Those wRefugee resettlement offices are typically refugees’ first points of contact with the social services and supports that are available to them upon arrival in the US. They assist newly arrived refugees with a number of primary needs, including housing, food assistance, cultural orientation, healthcare, and employment—needs which are clearly important to refugees’ ability to establish a new life in this country and to become self-supporting. This study examines the extent to which resettlement offices are addressing the SRH needs of newly arrived refugees, which could be accomplished by a nonmedically trained staff person who asks about clients’ desire to become pregnant, use of contraception, and whether or not she has any questions or concerns about her sexual or reproductive health.The findings from this survey document that only a minority of resettlement offices are directly helping refugee women and couples plan for their futures by planning their pregnancies and families, or linking them to SRH services. For example, only 15% of responding agencies provide written SRH information to clients, and only 5% had posters or signs providing this information. Only 41% of offices provide SRH content in classes or workshops, and just slightly more than 25% provide information specifically about family planning and contraceptive methods. While the majority of agencies refer clients for primary care and intend that refugees seek reproductive healthcare through that resource, only a small percentage of agencies refer clients to clinical resources specifically for reproductive healthcare. These findings highlight an oversight in the provision of care for refugee women, often times leaving the burden of seeking to understand, request, and access SRH services solely to women themselves. These findings point to a gap in services for refugee women, a gap that may have a detrimental effect on those women’s ability to set and reach their goals in a new community, such as educational, career, and well-being goals.Refugee resettlement agencies cited a number of barriers to providing SRH information and referrals to clients, including the presence of multiple competing priorities, a lack of culturally appropriate sexual health materials to give to clients, and clients’ own attitudes and cultural beliefs about reproductive health. Some agencies felt that addressing reproductive health was outside the scope of resettlement services, while other agencies reported lack of time and resources as the key barrier to providing this service. However, having an unintended or mistimed pregnancy would be detrimental to a family’s ability to achieve economic self-sufficiency, so attending to clients’ SRH needs is a necessary part of supporting their resettlement.The majority of respondents reported that their agency would or might be interested in improving their capacity to provide SRH information and referrals. The most commonly cited needs for offices to expand this capacity were written materials in multiple languages developed specifically for refugee clients and staff training on how to talk to clients about SRH. These findings point to the lack of on-site technical capacity at resettlement agencies to accurately and effectively formulate and share this information with women.For this study, IRC affiliate offices were recruited through IRC’s national headquarters, whereas non-IRC offices were recruited without the participation of their sponsoring agency, which likely explains the differential response rate between non-IRC and IRC resettlement offices. The high response rate among IRC offices makes it likely that the survey results accurately reflect the practices and resources available at all IRC offices. The same cannot necessarily be said of the non-IRC offices; with the lower response rate from non-IRC offices, study authors cannot definitively assert that the responses are representative of all resettlement offices. Also, the survey instrument was not validated against other sources of information regarding the practices of refugee resettlement offices. Additionally, while the survey did assess which topics were covered in written materials and posters, too few respondents answered these questions to allow for any meaningful discussion of material content. Finally, this study examines the provision of SRH information and education from the perspective of service providers, and not the experiences of refugee women themselves.Recognizing that resettlement agencies may lack the capacity to meet the SRH needs of their clients due to limited resources, the following recommendations may be helpful for integrating SRH services into existing resettlement service delivery.RRA staff inform and educate clients about a wide range of issues needed for their resettlement journey to live healthy and empowered lives. Staff can make a significant impact on refugee youth and women’s ability to make informed decisions and advocate for what they need and want in healthcare systems by offering precise information; presenting information in languages and formats that are accessible and appealing; and delivering information in ways that are nonjudgmental and compassionate. The easiest way to do this is to provide written materials in the form of handouts and brochures given to clients and posters prominently displayed in the office, including restrooms. This information should be available in the languages most commonly spoken by an agency’s clients. Workshops focused on birth control, planning a family, and seeking reproductive healthcare are also an important mechanism for educating clients. If RRAs do not have the capacity to have free-standing workshops, they can incorporate information and education into cultural orientation sessions or English language classes. The information refugee youth and women receive can support them in their practice of preventive SRH behaviors.One of the best ways to engage clients in seeking SRH information is to assess their pregnancy intention. This refers to people’s stated ideas about whether and when to be pregnant with first or subsequent pregnancies. Asking refugee women and youth about whether they would like to be pregnant within the next 6 months can help RRA staff connect clients to the information, resources, and services they need and desire, including prenatal, abortion, and contraceptive care. Yet only 12% of offices said that they asked this simple question. These questions may be integrated into standard intake process, or incorporated as part of other screenings, e.g., mental health and gender-based violence.For clients who do not want to become pregnant in the next year, referral to contraceptive services is a vital next step; however, while most agencies were aware of a service provider in their area, few made the direct referral. Staff should become familiar with local service provides in order to facilitate communication about client needs, including interpretation. This can put the client at greater ease before their appointment.Education, screening, and referral are good first steps but are often not sufficient to meet the range of pregnancy and contraceptive care needs that refugee women and youth have. Collaboration and partnerships between SRH providers and refugee resettlement agencies can fill important gaps and benefit both agency staff and clients. Partnerships can take many forms and agencies should choose the relationship that best helps clients overcome common challenges, such as being able to afford services; scheduling appointments through a live person and not an automated line; securing transportation to appointments; having interpretation during the appointment; and arranging for follow-up with the clinic. Good partnerships can offer reciprocal information sharing so that each partner can have a deep understanding of the services the other provides and how to access these services and reciprocal learning, so lessons learned are shared and documented and become the basis for the future direction of the partnership. Ideally, partnerships will also allow agencies to leverage funding in ways that can improve and expand contraceptive care and related services.Finally, the authors believe that RRAs are in a unique position to build the capacity of contraceptive care providers in their areas to better serve the needs of refugee women and youth. This important work will most likely take place in healthcare facilities with which RRAs have already established some degree of relationship, be it through referrals or partnership. RRA staff can offer insights about the refugee populations in their area that can help healthcare providers offer client-centered, trauma-informed, culturally and linguistically responsive care to refugee women and youth.Findings from this survey may assist with laying the groundwork for strengthening the capacity of US RRAs to integrate SRH services into programming and to support refugees’ ability to plan for their futures and to have intended pregnancies and families. The current landscape of capacity, established in part through this survey, will inform the cross-organizational effort by IRC and AFY to pilot programs aimed at increasing access to SRH education and services for refugee clients. Further, additional research into the experiences of refugee women themselves would contribute greatly to an understanding of their needs for service provision.S1 Survey(PDF)Click here for additional data file.S1 Data(PDF)Click here for additional data file.S1 Checklist(PDF)Click here for additional data file.


Mycobacterium tuberculosis, ADP-ribosylation of thymidine has emerged as the mode of action of a toxin-antitoxin system named DarTG, with the resultant products perceived as DNA damage by the cell. On the other hand, mammalian DNA damage sensors PARP1, PARP2 and PARP3 were shown to ADP-ribosylate phosphorylated ends of double-stranded DNA in vitro. Additionally, TRPT1 and several PARP enzymes, including PARP10, can add ADP-ribose to the 5’-phosphorylated end of single-stranded RNA in vitro, representing a novel RNA capping mechanism. Together, these discoveries have led to the emergence of a new and exciting research area, namely DNA and RNA ADP-ribosylation, that is likely to have far-reaching implications for the fields of DNA repair, replication and epigenetics.ADP-ribosylation is a chemical modification of macromolecules found across all domains of life and known to regulate a variety of cellular processes. Notably, it has a well-established role in the DNA damage response. While it was historically known as a post-translational modification of proteins, recent studies have shown that nucleic acids can also serve as substrates of reversible ADP-ribosylation. More precisely, ADP-ribosylation of DNA bases, phosphorylated DNA ends and phosphorylated RNA ends have been reported. We will discuss these three types of modification in details. In a variety of bacterial species, including  Th. Th2 posin vitro . There ain vitro ,51. No e2.2Mycobacterium tuberculosis and enteropathogenic Escherichia coli (EPEC), were found to express an operon encoding a toxin-antitoxin (TA) system consisting of a macrodomain and another undefined protein. Structural analysis suggested that this unknown protein could be a highly-divergent PARP-like enzyme . Ad. Ad66]. aviruses ,84. Thisaviruses .4.27GpppN mRNA cap recruiting eIF4E to induce translation initiation [+ moiety is attached to the RNA 5’-end, which has been shown to promote RNA decay in mammals [RNA modification by TRPT1 and PARP10 rendered the oligonucleotide substrates resistant to CIP treatment, indicative of an RNA capping mechanism ,68. Thisitiation . An inte mammals .Multiple results suggest that PARP10-mediated modification of RNA ends could have an immune function. Firstly, PARP10 expression was shown to inhibit VEEV translation and replication ,84. FurtUpon being added on viral RNA, the modification could be recognized by immune factors, having a role in the initiation of the immune response. Additionally, PARP10 inhibitory effects on viral translation could be a direct result of RNA ADP-ribosylation whereby the ADP-ribosylation cap would prevent translation. This hypothesis implies the existence of a mechanism preventing PARP10 from targeting cellular RNA. It has been observed that the RNA recognition motif (RRM) domain contributes to PARP10 catalytic activity . This su5in vivo due to technical challenges, there is strong evidence that ADP-ribosylation is a widespread nucleic acid modification across all domains of life. This suggests that ADP-ribosylation of DNA and RNA could both contribute to the well-characterised physiological effects of the modification, as well as reveal its new cellular functions. Similarly to the recent discoveries of HPF1 and serine ADP-ribosylation [ADP-ribosylation can no longer be solely regarded as a PTM. Several ARTs targeting DNA and RNA in a reversible manner have now been identified including DarT for the MARylation of thymine bases, PARP1−3 for the modification of phosphorylated DNA ends, and PARP10, PARP11 and PARP15 for the MARylation of phosphorylated RNA ends. While these modifications have not yet been detected sylation ,29,30, u10.13039/100010269Wellcome Trust (grant numbers 101794 and 210634), 10.13039/501100000268BBSRC (BB/R007195/1), 10.13039/100014008Ovarian Cancer Research Alliance (Collaborative Research Development Grant #813369) and 10.13039/501100000289Cancer Research UK (C35050/A22284).The authors report no declarations of interest.


The integration of untargeted metabolomics and transcriptomics from the same population of cells or tissue enhances the confidence in the identified metabolic pathways and understanding of the enzyme–metabolite relationship. Here, we optimised a simultaneous extraction method of metabolites/lipids and RNA from ependymoma cells (BXD-1425). Relative to established RNA (mirVana kit) or metabolite  single extraction methods, four dual-extraction techniques were evaluated and compared (methanol:water:chloroform ratios): cryomill/mirVana (1:1:2); cryomill-wash/Econospin (5:1:2); rotation/phenol-chloroform (9:10:1); Sequential/mirVana (1:1:3). All methods extracted the same metabolites, yet rotation/phenol-chloroform did not extract lipids. Cryomill/mirVana and sequential/mirVana recovered the highest amounts of RNA, at 70 and 68% of that recovered with mirVana kit alone. sequential/mirVana, involving RNA extraction from the interphase of our established sequential solvent addition and shaking metabolomics-lipidomics extraction method, was the most efficient approach overall. Sequential/mirVana was applied to study a) the biological effect caused by acute serum starvation in BXD-1425 cells and b) primary ependymoma tumour tissue. We found (a) 64 differentially abundant metabolites and 28 differentially expressed metabolic genes, discovering four gene-metabolite interactions, and (b) all metabolites and 62% lipids were above the limit of detection, and RNA yield was sufficient for transcriptomics, in just 10 mg of tissue. Integrated omics techniques can improve the understanding of mechanisms through which a cellular phenotype is generated, where each technique allows insight into cell regulation and function at a different but inter-related level of the genome ,2. The mCombining metabolomics and transcriptomics data from the same cells or tissues would allow a much better holistic understanding of how the cell generates the aberrant metabolome of a disease such as cancer, how the cell compensates for variability and evolutionary dynamics within the tumour microenvironment, and how the metabolome feeds back signals to the cell ,8,9. AccPublished methods typically extract RNA and metabolites from different regions of a given tissue specimen, which limits integration of datasets as neither the cell genotype nor phenotype is necessarily homogenous throughout tissue, particularly in malignant cancerous tissue ,12,13. SThree simultaneous RNA/metabolite extraction methods exist, optimised for plant tissue, microbes and human cell lines ,16,17. RA recently published method used an RNA lysis buffer to extract metabolites, and the cryomilled lysate was split into equal sized fractions for metabolomics and transcriptomics, which prioritises RNA extraction and quality but is not an RNA-first method, and does not overcome the problem of limited amounts of tissue, and so is not a simultaneous extraction method . The aimn = 2–4; methanol:water:chloroform ratios are shown in brackets): cryomill/mirVana method (1:1:2) [, sequential solvent addition and shaking (1:1:3). The total ion chromatogram of the extracts in both zwitterionic-polymer–hydrophilic interaction liquid chromatography-liquid chromatography-mass spectrometry  and reverse phase-liquid chromatography-tandem mass spectrometry  analyses showed adequate separation and smooth peaks  , cryomil (1:1:2) , cell ex (1:1:2)  and sequ (1:1:2) , which wth peaks . The totth peaks , indicatp < 0.05), which may have been due to the washing step employed, ensuring all water-soluble metabolites were concentrated in the aqueous phase free from lipids , whereas cryomill/mirVana and cryomill-wash/Econospin lipid profiles were very different  as expecTM miRNA Isolation Kit  (Transcriptomics Reference Method). All the extraction methods produced RNA of sufficiently high purity (A260/A280 ratios of approximately 2.0) and quality for downstream applications such as transcriptomic microarrays and RNA sequencing, since Roume et al. recommended an RNA integrity number (RIN) threshold of 7.0 [TM miRNA isolation kit similar to the positive control that may account for the better recovery observed.All the extraction methods were compared to a standard method for RNA extraction using the mirVanad of 7.0 ,17, whicd of 7.0 . HoweverIn conclusion, cryomill/mirVana was able to extract many metabolites with higher abundance, and cryomill/mirVana and sequential/mirVana recovered both high-quality RNA and a high RNA yield compared to the positive control and enabled both metabolomics and lipidomics analysis for a greater coverage of the metabolome.As a proof-of-concept, the selected dual extraction sequential/mirVana method was then applied to study the difference in metabolism and transcription induced by acute cellular stress, specifically by starving cells of serum. Serum starvation was chosen, as deprivation of cellular nutrients leads to an abrupt growth arrest in mammalian cells, characterised initially by RNA expression changes and metabolite level changes. Thus, comparing metabolomics and transcriptomics between serum-deplete versus serum-replete brain cancer cells offers a rapid means to verify this dual extraction method on a genome-wide level.2X 0.814, R2Y 0.995) and the predictive ability (Q2) of the model was much greater than 0.40  [2X 0.880, R2Y 0.998) with Q2 = 0.994; therefore, the model is validated, and degree of overfitting is minimal. Ninety-seven lipids significantly altered in relative abundance could discriminate the treatment from the control cells (vide infra), and as leukotrienes and prostaglandins are signalling molecules, this indicated increased paracrine signalling in the absence of serum [Multivariate analysis using PCA and OPLS-DA showed that serum-starved and serum-replete samples were statistically discriminated . PCA shoal model ) and app= 0.979) ,34. Ther= 0.979) ,36,37. Tol cells . The claof serum  , extracting metabolites, lipids and RNA. The metabolites and lipids were analysed by LC-MS and LC-MS/MS, and the RNA was measured by Nanodrop.All metabolites identified in the tumour of patient 15/243 were detected in 10, 20 and 40 mg samples . The genThe number of lipid groups detected in the tumour (lipids grouped according to their summed fatty acids), increased as the mass of tissue increased. Two hundred lipid groups were extracted from 10 mg of tissue, 258 were extracted from 20 mg and 325 from 40 mg of tissue . Peak arThe RNA mass extracted from tissue roughly doubled from 10 (7.6 µg RNA) to 20 (14.7 µg) to 40 mg (36.5 µg) tissue, which suggests that the yield of RNA is proportional to the amount of tissue used. The purity of the RNA was measured as 260/280 and 260/230 ratios: 10 , 20 , and 40 mg . The yield and purity of RNA were suitable for consideration of transcriptomics analyses including RNA sequencing, from a minimum of 10 mg of tissue.Accession numbers have not yet been obtained—they will be provided before publication.6 of BXD-1425 cells were detached using 1×Trypsin-EDTA, counted from a sub-confluent, log phase T175 flask and centrifuged to remove the medium. The cells were then re-suspended in PBS (1 mL in a 2 mL tube) and centrifuged at 305 rcf for 5 min at room temperature. The supernatant was removed and the remaining cell pellets were flash-frozen in liquid nitrogen and stored at −80 °C for the analysis.BXD-1425 cells were selected as a model in this study due to their in vitro stability and rapid growth rate . BXD-142mirVana kit: Extraction of RNA was performed according to the manufacturer’s protocol for the mirVanaTM miRNA Isolation Kit , with 500 µL lysis/binding solution being used and the RNA being eluted in 50 µL of nuclease-free water . The extracted RNA was stored at −20 °C.Extraction of metabolites was performed using a Metabolomic Reference Method for standard metabolites extraction . Cold meFour different dual extraction methods of metabolites and RNA were investigated and designated as: cryomill/mirVana , cryomilTM miRNA Isolation Kit as outlined in mirVana kit without cryomilling [The procedure was followed as stated elsewhere, including omilling . Note thThe procedure was followed as outlined by Valledor et al. using the Mixer Mill 301 at 25 Hz with cooled autoclaved-sterilized milling balls (5 × 2 mm + 2 × 5 mm), and a thermal shaker for the RNA pellet at 300 rpm for 30 min .C described by Vorreiter et al. was followed starting from resuspension in PBS [The protocol n in PBS . The RNAThe extraction procedure started with using sequential solvent addition and shaking for the extraction of metabolites, followed by the extraction of RNA using the mirVana kit from the solid interphase between the aqueous and the organic phases.TM miRNA Isolation Kit, using 500 µL of lysis/binding buffer. The mixture was transferred to an Eppendorf tube after adding the homogenate additive and leaving on ice. Total RNA was eluted into 2 × 50 µL of RNase free water and stored at −80 °C.BXD-1425 cells (human ependymoma cell line) were grown in DMEM with 10% FBS in T75 flasks until 70–80% confluent. A complete media change was made to either DMEM (serum-starved treatment) or DMEM with 10% FBS (serum-replete control), with four replicates produced. An experimental period of 28 h was chosen based on a previous study, where although an increase in VEGF-promotor activity from 3–24 h post-serum starvation was detected by a promoter-reporter construct, it was not until after 24 h that an increase in VEGF mRNA expression was noticeable by Northern blot , whereasIn all methods, the aqueous and the organic phases were separated, the solvents were evaporated using the Jouan Centrifugal Evaporator  at room temperature, and the extract residues were stored at −80 °C. For LC-MS, the aqueous and the organic extracts were reconstituted in 100 µL of methanol (4 °C) and 100 µL of isopropanol (4 °C), respectively, and centrifuged at 13,000 rcf, 4 °C for 10 min. Then, 70 µL from each was transferred into HPLC vials and stored at −80 °C until analysis; in addition, 20 µL from each sample were mixed as a pooled quality control (QC).TM miRNA Isolation Kit, using 500 μL lysis buffer and eluting into 2 × 50 μL of RNase free water.The study utilised tissue derived from a paediatric brain tumour patient treated at the Queen’s Medical Centre, Nottingham. The study was approved by the National Research Ethics Committee (NRES Committee East Midlands) (Reference Number 11/EM/0076). Tissue samples were cut from the ependymoma tumour of patient 15/243 in a sterile Petri dish over dry ice. Samples were homogenised using a Stuart Homogeniser SHM1 1116 in methanol  inside 2 mL tubes over dry ice. Chloroform  was added and vortexed well. Water  was added and vortexed well. Samples were centrifuged at 13,000 rcf for 10 min at 4 °C. Parts of the upper phase (100 μL) and lower phase (200 μL) were collected into 1.5 mL tubes. The upper phase was centrifuged (as before but for 5 min) and the solvent transferred to a HPLC vial. The lower phase solvent was evaporated using the Jouan Centrifugal Evaporator at room temperature (25 min) and the residue was resuspended in isopropanol , then centrifuged (as before) and the solvent transferred to a HPLC vial. The 2 mL sample tube was centrifuged again (as before) and the remaining solvent removed carefully to leave the solid interphase. RNA was extracted from the interphase using the mirVanaExperimental samples were randomised through the sequence. In addition, the pooled QC was run after every six samples to assess the instrument performance. Solvent blanks were run prior and subsequent to samples.m/z 70–1400 range at 50,000 resolution. The probe and capillary temperature were maintained at 150 and 275 °C, respectively. The following settings were used: sheath gas 40, auxiliary gas 5, and sweep gas 1, balanced AGC target. For positive mode ionization: spray voltage +4.5 kV, capillary voltage +40 V, tube voltage +70 V, skimmer voltage +20 V. For negative mode ionization: spray voltage −3.5 kV, capillary voltage −30 V, tube voltage −70 V, skimmer voltage −18 V.Metabolites were separated using an Accela UHPLC system coupled to an Exactive Orbital trap MS  as previously described ,49. Metam/z 150–1500 range at 70,000 resolution. The probe and capillary temperature were maintained at 412.5 and 256.25 °C, respectively. The following settings were used: sheath gas 47.5, auxiliary gas 11.25, and sweep gas 2.25, AGC target 3 × 106. The spray voltage was set to +4.0 kV or –4.0 kV. Data-dependent tandem MS/MS (ddMS2) spectra were produced on the 5 most intense ions at any one time at a resolution of 17,500, with AGC target 1 × 105, injection time 50 ms, range 200–2000 m/z, normalised collision energy 30, isolation window 1.0 m/z, intensity threshold 1.6 × 105, dynamic exclusion 10 s ).Lipids were analysed using a Dionex UltiMate 3000 HPLC system . The method was modified based on a method previously described . Lipid et-score), to process the data from the serum-starved to serum-replete validation experiment and the human tissue samples. Metabolites and lipids were identified with four levels of confidence; level 1 (L1) identification was based on matching the accurate masses, MS/MS fragmentation and retention times of the detected metabolite peaks with those of authentic standards which were co-analysed with the samples under identical experimental conditions, level 2 (L2) identification was based on matching the accurate masses and retention times  of the detected metabolite peaks with those of the authentic standards  or matching accurate masses and MS/MS spectra with compounds in a library when data were taken under the same acquisition parameters, level 3 (L3) identification was carried out when the predicted retention times or predicted MS/MS spectra or both were employed due to the lack of standards, and level 4 (L4) identification was based on unambiguously assigned molecular formulas, but insufficient evidence exists to propose possible structures [LC-MS raw metabolomics data from samples, blanks and QC samples were processed with XCMS for untargeted peak-picking , and peaructures . The ideructures ,55,56.TM miRNA isolation kit and each of the metabolite/RNA dual extraction methods.RNA was analysed using a Nanodrop  followed by a Tapestation instrument  to analyse the quantity and quality of RNA recovered by the mirVanaTM S arrays . All steps of sense cDNA synthesis, fragmentation and hybridization were performed according to the manufacturer’s protocol .For transcriptomics analysis of serum starvation samples, the RNA was diluted to 600 ng/µL (concentrations determined by Nanodrop) and DNA was removed using the Turbo DNA-free kit . The purified RNA was diluted to 100 ng/µL (concentrations determined by Tapestation) for a Transcriptomics Microarray. QC presented RIN of 9.7–10 . Whole-genome transcriptome analysis was conducted by hybridizing three biological samples of total RNA for treatment and four biological samples of total RNA for control to the Clariomt-test and/or a one-factor ANOVA were computed to check for the significance of the metabolite/lipid fold changes [p-values were then adjusted using the Benjamini–Hochberg false discovery rate to account for multiple testing problem  [For LC-MS analysis, cell growth rate differed between serum-deplete and serum-replete samples, with 2.38 ± 0.57 million and 3.61 ± 0.40 million cells after 28 h, respectively, and therefore, the serum starvation result was normalised to the number of cells per sample . Student’s  changes . The resificant) . SIMCA-P2 transformed and quantile normalisation using the robust multi-array average (RMA). Filtering was performed to remove genes where expression did not differ from that in controls and genes where hybridization signals fell below the threshold for reliable detection using Partek Genomics Suite. The list of genes of interest comprised genes upregulated or downregulated by at least one-fold with Benjamini–Hochberg adjusted p-value < 0.05.Microarray gene expression profile data were generated as CEL files and subjected to analysis by the Partek Genomics Suite 6.6 software . Quality Control (QC) metrics were checked by examining surface defects, hybridization, labelling, and a ratio of the 3′ probe set to the 5′ probe set (3′/5′ ratio) to provide the quality of the microarray data. The values were logp-value < 0.05 as all entered genes and metabolites were statistically significant, so no further filtering was required [Multi-omics data integration of metabolites and transcripts was performed through pathway-based network analysis using Cytoscape (v.3.4.0) with MetScape 3 (v.3.1.3) plugin, applying a minimum fold change threshold of one and a required . NetworkA dual extraction method, sequential/mirVana, was developed and verified as a superior method for the extraction of both metabolites/lipids and RNA from a brain cancer cell line and primary brain tumour tissue and has potential for other applications. Simultaneous extraction of metabolites/lipids and RNA from the same sample set provides a consistent quality of the metabolome and RNA transcripts, and their biological relationship is adequately maintained and hence a better integrative metabolomics-transcriptomics can be obtained. The method was applied to distinguish between serum-starved and serum-replete cells on both the transcriptional and metabolic level after an acute period of perturbation and the data could be integrated to enhance the biological interpretation. This method will facilitate omics integration in a clinically relevant manner.


The sustainability of human resource management (HRM) is the basis for an organization’s future growth and success. This study aims to investigate achieving HRM sustainability in universities. We use a quantitative research method design to investigate the factors that affect HRM sustainability at universities. The study was conducted during the spring and summer of 2020 at Iranian state universities. As the study’s statistical population included 2543 employees, a sample size of 334 employees was calculated using the Cochran formula. A questionnaire with 32 statements based on a 5-point Likert scale was used to collect the data, which were analyzed using PLS3 software. The findings show that human resource practices, social factors, psychological factors, employer branding, and economic factors have positive and significant effects on HRM sustainability at universities. Findings indicate that it is essential to consider the implementation of adequate HRM practices and related socio-economic and psychological supports for HRM sustainability in universities that can lead to the competitiveness of the higher education institutions such as universities. Human resources (HR) are regarded as the central pillar of an organization’s competitive advantage . FurtherEnhert et al.  argued tHence, HR sustainability is a set of skills, motivation, values, and trust created to avoid detrimental environmental effects by adopting justice, development, and welfare . SustainThese days, most organizations are faced with the lack of encouraged and expert employees. Therefore, it is essential to examine the concept of HRM sustainability based on the “triple-bottom-line” approach . ConsequAlthough sustainability in HEIs has been studied recently, to the best of our knowledge, HRM sustainability in universities has yet to be addressed adequately . DisterhSustainable HRM is important for universities that compete globally, and examining the factors that affect sustainable HRM for university staff is regarded as the main challenge . MoreoveOn the other hand, universities have internal goals such as knowledge creation, skills development, motivation, and trust-building. In addition, universities have some extra-organizational goals such as creating an active and successful workforce for society to fulfill the purposes of welfare, health, commitment, justice, long-term development, and creating a welfare society. HEIs play a significant role in achieving sustainability because universities spread sustainability principles through education, research, and communication with stakeholders. Their principal goal is to increase and strengthen awareness, knowledge, ability, and values to build a sustainable and fair system based on educational and academic freedom, where new ideas are nurtured, and up-to-date solutions are sought to create a sustainable lifestyle.Since there is a shortage of research on HRM sustainability and its dimensions in universities, this study aimed to investigate this research area to fill the theoretical gap in the literature. The major contribution of this study to the existing literature is the examination of HEIs’ perspectives on sustainable HRM. The findings of this study recommend that HEIs should be aware of the significance of this issue. It is also noteworthy that environmental and organizational factors can lead to HRM sustainability in universities. Since it is a relatively unexplored area of research, this study intends to investigate the relationship between these factors based on data collected from three state universities in Iran . Consequently, investigation of sustainable HRM from the HEIs’ perspective is regarded as the novelty of this study.Sustainability is an essential principle for HRM and contributes to achieving organizational goals and performance, which reduces harmful effects on stakeholders and maximizes organizational outcomes . It alsoHR and HR strategies have been identified as essential factors for organizational success . HR sustHR strategies must satisfy both employee interests and stakeholder’s needs. The interactive approach incorporates a broader range of values and activities related to collective concerns  and explHypothesis 0 (H0).Various Factors Have Different Impacts on HRM Sustainability at Universities.Organizational factors are among the most fundamental variables that affect HRM sustainability. According to the International Standardization Organization’s (ISO) model, organizational sustainability identifies opportunities, changes, and trends in the external environment ISO/CD9004 , promotiMoreover, HRM can contribute to organizational sustainability in the following five aspects : organizManager cooperation, as a dimension of HR, plays a crucial role in an organization’s employee sustainability and job satisfaction through employee performance appraisals . TherefoHypothesis 1 (H1).HR practices have a significant effect on HRM sustainability.Social sustainability and occupational changes are crucial processes that affect the economy . Social Job opportunities are crucial for social sustainability. Beyond providing income, employment allows people to experience social well-being through interactions with others . The desHypothesis 2 (H2).Social factors have a significant effect on HRM sustainability.Psychological factors, including anxiety, job attitude, and job-related stress, can cause changes in well-being   and dealing with work-related stress have been observed in psychological pressure or stress . TherefoHypothesis 3 (H3).Psychological factors have a significant effect on HRM sustainability.Employer branding refers to a set of efforts for communicating with employees and establishing the organization as an excellent place to work. As a competitive advantage, power can promote growth and development . A brandQualified faculty members and employees can generate outstanding intellectual capital that influences the organization’s performance and other outcomes to survive in this competitive environment . There iThis discussion leads to the following hypothesis:Hypothesis 4 (H4).Employer branding has a significant effect on HRM sustainability.Sustainable development refers to the long-term and systematic exploitation of natural resources, given that we should also preserve these resources for future generations. Such development enables countries to experience economic and social improvements without destroying their environmental resources . OrganizIn classical endogenous growth theory, human capital plays a central role in technological advancement and sustainable economic growth  (Xu and Hypothesis 5 (H5).Economic factors have a significant effect on HRM sustainability.Organizational workplace politics are an inevitable fact, and failing to deal with them realistically will predictably be detrimental to the organization . WorkplaHypothesis 6 (H6).Workplace politics have a significant effect on HRM sustainability.Finally, HRM sustainability can be described as the most complex challenge in the HR field. HRM theories, models, systems, and processes have shifted from a profit-driven motivation to a simultaneous three-way objective called the triple bottom line (TBL), which includes expanding social, environmental, and economic outcomes and addresses social, environmental, and economic issues . HRM susThis descriptive-correlational study is applied in terms of its developmental purpose and quantitative data collection. The purpose of applied studies is to develop applied knowledge in a specific field. The study’s statistical population included 2543 employees of three state universities in Iran, namely Shiraz, Yazd, and Isfahan Universities. The managers of the sample organizations declared the necessity of HR sustainability to avoid destruction, reduce electricity intake, and protect the environment. In addition, these universities achieved high ranks based on Greenmetric World University Ranking (UI Greenmetric World University Ranking) in 2020 and 2021. It was also easier for authors to access these three universities because of their locations, particularly during the COVID-19 pandemic. As structural equation modeling was used for the analysis, the sample size should be 5 to 10 times the number of questionnaire items. Therefore, based on Cochran’s formula, 334 employees were selected. Based on the analysis of the quantitative data, 73% of the respondents were male, and 27% were female; besides this, 78% held a Ph.D. degree, and 22% held a Master’s degree. Moreover, 23% of the participants were single, and 77% were married. Finally, 17% of the respondents had up to 5 years of experience, 63% had between 5 and 10 years of experience, and 20% had over 10 years of experience. Since there were no standard questionnaires on this topic, a researcher-made questionnaire  was deveMoreover, informed consent forms were distributed among these employees to indicate their voluntary participation in the project, and they were assured that the collected data would only be used in this study. They were also free to withdraw from the project at any time without any adverse consequences. In this study, HRM sustainability is considered the dependent variable, and organizational and environmental sustainability are considered the independent variables. Smart PLS3 software, which does not require a normal distribution, was used to analyze the data . It is nThe mean-variance index was extracted to determine convergent validity, and the root means index of the extracted variance was used to check for divergent validity. The obtained root mean values of the extracted variance are higher than 0.5, which indicates that the variables have divergent validity.Based on the results obtained using the Smart PLS 3 software  the first hypothesis was confirmed. The second hypothesis, reflecting the positive and significant effect of the social components on the sustainability of HRM, was confirmed as the obtained t-statistics value for this relationship was 2.768. The third hypothesis refers to the significantly positive effect of the psychological components on HRM sustainability. It was approved because the obtained t-statistics value for this relationship was 2.968. The fourth hypothesis refers to the significantly positive effect of employer brand on the sustainability of human resource management, which was confirmed because of the obtained t-statistics value of 2.536. The fifth hypothesis, highlighting the effect of economic components on the sustainability of human resource management, was however rejected since the obtained t-statistics value (1.115) is lower than the critical value (1.96) at the 95% confidence level. The sixth hypothesis concerning the direct effect of political components on the sustainability of human resource management was approved because the obtained t-statistics value was 2.488.2 coefficients was used to examine the intensity of relationships between dependent structures. R2 is regarded as a measure explaining the exogenous variables’ influence on an endogenous factor; accordingly, 0.19, 0.33, and 0.67 indicate weak, medium, and robust R2 values, respectively. In this study, the criterion for HRM sustainability was 0.847, so the structural model showed a good fit at the robust level. The results are shown in The RThe results of 2 criterion proposed by [The Qposed by ,74, deteposed by . In thisThe overall model contains all calculations and measures of the structural model. Hence, the overall fitness of the model can be confirmed based on the GOF criterion using 0.01, 0.25, and 0.36 to reflect poor, medium, and robust GOF values. The GOF value for the model is 0.897, indicating that the overall fit of the research model is robust.The standardized root means square residual (SRMR) was implemented as another approximate model fit criterion. The values of 0.05, 0.08, and an SRMR value lower than 0.10 indicate an acceptable fit for the overall model as proposed by ,76,77 reFinally, t-statistics were employed to examine hypothetical relationships amongst variables. Six sub-hypotheses were developed to assess the primary hypothesis. As shown in This research was designed to explore the factors affecting HRM sustainability in universities. The current literature shows that much attention has been paid to sustainability during the past two decades, which has led to an increase in sustainability-related measures. Universities are influential in societies; they play a central role in developing science, technology, innovation, justice-orientation, talent development, empowerment, and wealth-creation while strengthening the spirit of cooperation and responsibility and fostering an environment for thinkers, researchers, and professionals. They also enhance and disseminate new ideas to promoting individual and social life in a suitable environment for free intellectual thought and scientific critique. Therefore, HRM sustainability in universities plays a vital role in achieving those noble goals.The findings regarding the first hypothesis show that components of HR practices had a significantly positive impact on HRM sustainability. The respective findings are consistent with results of other studies by ,37, indiProvided that the staff acts with satisfaction and determination to develop the organization, university empowerment is developed through strategic development planning, supporting research and innovation, and developing future activities, research, and professional skills. Increasing employees’ independence and motivation enhances learning and creativity opportunities at the workplace. Furthermore, the university staff’s responsibilities and ethical obligations have led to adherence to principles and regulations. Sustainable talent development is achieved through the direct experience of acquiring knowledge and skills. Unique mechanisms such as succession planning, motivation techniques, teamwork, training, coaching, specialized seminars, self-development, knowledge sharing, hands-on learning, and networking can enhance talent and ultimately create HRM sustainability at the university level.The second hypothesis refers to the positive and significant impact of social components on HRM sustainability. This result is consistent with research by ,15,23,43The third hypothesis refers to the significantly positive effect of the psychological components on HRM sustainability. This result is consistent with previous studies by , highligMeeting employees’ psychological needs provides the basis for survival, well-being, and workplace advancement, where the work environment is compatible with individuals’ abilities and capabilities. This can be done by holding in-service training sessions, workshops, and similar activities that increase creativity within the organization, which aids in development and advancement while strengthening and influencing job growth opportunities. Furthermore, these factors can affect HRM sustainability and lead to growth, development, and achievement of academic goals.The fourth hypothesis refers to the significantly positive effect of employer brand on HRM sustainability. Thus, the collected data support this hypothesis. These findings are consistent with , who staThe fifth hypothesis refers to the effect of economic components on HRM sustainability. This result is inconsistent with the findings of . AuthorsThe sixth hypothesis refers to the direct effect of political components on HRM sustainability. This result corroborates those of ,67, who According to the results of this study, HRM sustainability at HEIs is a relatively new paradigm. It should be investigated along with other valuable and practical components of environmental, socio-cultural, and human resource practices, as well as organizational, psychological, economic, and political factors at different levels . Furthermore, dynamics in temporal dimensions (short-term and long-term) should be highlighted because interactions and relationships between different levels are needed to achieve the goals. Objectives should be achieved using different organizational resources so that society’s existing needs do not endanger the future and control negative feedback. Applying sustainability principles in HRM practices also provides workers with enduring social, economic, and physical well-being. Due to the development of universities, long-term goals inside and outside universities are achieved by creating a balance between work and life.Given that stakeholders, policymakers, and consumers have realized that certain activities may have adverse environmental effects, HEIs need to consider the sustainability of human resources to ensure their long-term success. In addition to acting as an efficiency measure, sustainable HRM can support self-sustained development processes. Regardless of inadequate evidence in the literature regarding a clear definition of HRM sustainability and the lack of emphasis on this issue, this study shed some light on the meaning of sustainability and its relationship with HRM.Despite all of its contributions, the present study cannot be without limitations. For instance, some of the university employees were reluctant to participate due to their conservative personality or biased responses; hence, the generalizability of the findings of this study are called into question. Moreover, there are different factors and characteristics regarding sustainable HRM in various cultures that make it difficult to generalize the findings to all the societies. Consequently, other researchers are recommended to conduct similar studies in different universities or different cultures using the same model. Furthermore, it is suggested to identify and investigate other intra- and inter-organizational outcomes in sustainable HRM in different universities. Given its dynamic process, sustainability does not refer to rigidness. Accordingly, it is essential to conduct further studies to explore sustainability in HRM.


The intention behind the Research Topic on RNA Biology of Microorganisms was to provide a broad view of the roles of RNA in the physiology of microorganisms, rather than focusing on specific functions. This purpose was mostly accomplished, since the published articles cover subjects from: (i) the life cycle of RNA transcripts, particularly of mRNAs, from birth to degradation; (ii) the various canonical and non-canonical roles of tRNAs, including translation and control of protein fate, as well as influencing genome variability; and (iii) regulatory non-coding RNAs: their identification by novel experimental strategies, the role of redundant RNA versions and the relationship between sRNAs of pathogenic bacteria with host RNAs. Putting all this information together, sheds light on the interplay of events that occur since the birth of an RNA, its distribution in the cell to accomplish its specific roles and its fate upon changes in environmental conditions. In the next paragraphs we briefly describe the contribution of each article to this global view of the biology of microorganisms.Wang and Arstimovitch describe in their review article that NusG plays a number of roles in different cellular contexts, from the classical transcription antitermination of untranslated RNAs by counteracting Rho function on naked RNAs, through stimulation of transcription by avoiding backtracking of RNA polymerase, slowing down of transcription by halting RNAP at certain sequences and connecting RNAP to the ribosome during translation, to recruiting the ribosome to some mRNAs with weak ribosome binding sites. Paralogs of NusG are widely distributed in nature playing roles on specific genes using certain residues at conserved domains (NGN and KOW), while making similar contacts with RNA polymerase through conserved residues.Several transcription factors regulate the binding of RNA polymerase to specific transcription promoters, among them NusG, a general transcription factor that is highly conserved in all organisms. Irastortza-Olaziregi and Amster-Choder, a detailed description of the strategies employed by bacterial cells to ensure association between transcription and translation in the apparently not physically separated milieu that encompasses the genetic material and cytoplasm. Physical coupling by direct or indirect interactions (bridged by proteins like NusG) between transcribing RNA polymerase and translating ribosomes are discussed. Lo and behold. Other strategies, such as the control of transcription rate by interaction of (p)ppGpp with RNA polymerase to coordinate transcription and translation, the localization of transcribing DNA to certain locations in the cell away from the bulk nucleoid (at the inner membrane or the surface of the nucleoid) where translation can take place, are also described. However, an important part of the article is devoted to the analysis of emerging data which promote the idea that uncoupled transcription-translation occurs in the context of the apparently non-separated nucleoid-cytoplasm organization of bacterial cells. Several different evidences reveal that this can happen by several tactics, e.g., ribosome free transcription of 5'UTRs, translation-independent localization of mRNAs to different locations in the cell, and faster transcription than translation in B. subtilis. A number of strategies, including association of RNA binding proteins  with the 5′ ends of mRNAs or sRNAs, as well as formation of secondary structures (riboswitches), might aid in the protection of free mRNAs in the cytoplasm against ribonucleases together with delaying translation until an mRNA reaches the proper location. Other proteins that are probably involved in uncoupled transcription-translation are RNA chaperones (Hfq and ProQ).In a review article presented by Vargas-Blanco and Shell gives a detailed description of the many different mechanisms that give rise to degradation of RNAs, particularly under stress conditions. It is accepted that steady state of RNAs is a balance between synthesis and degradation. Exo- and endo-ribonucleases are responsible for the degradation of RNAs, and their activity can be modulated in several ways to interfere with the RNA substrates, for example, sRNAs and RNA-binding proteins can block the access of RNases. Also, the chemical nature of the 5'end  can influence the access of exonucleases. Moreover, secondary structures can also interfere with the access of nucleases. At the 3'end, the formation of poly (A) can facilitate the action of exonucleases. As a general rule, under stress RNAs are stabilized, but their cellular level decreases. This apparent contradiction might be explained by lowering transcription under these conditions.The review published by Setlow and Christie reviewed the data obtained concerning the existence of RNAs in B. subtilis spores, as well as other spore-forming bacteria. As opposed to previous assumptions, evidence for the existence of RNAs in spores has been obtained for 1,800 different mRNAs, with no more that 50 mRNAs present in more than 1 copy/spore in the population, with most of them present in <10% of the population. The majority of these mRNAs and rRNAs present in spores are fragmented, and more fragmentation is observed with time post-sporulation. No evidence for the participation of these RNAs in translation after spore germination has been reported, except for malS mRNA, coding for malic enzyme, which might provide substrates for the synthesis of ATP. Hence, the data obtained thus far suggest that the main role of RNAs in spores is to provide nucleotides for the synthesis of new transcripts during germination.Arias et al., the authors report that replacement of non-optimal glycine codons by optimal counterparts in the Cdc13 cyclin gene completely impairs the proliferation of cells. Overexpressing the tRNA decoding the non-optimal codons in wild type cdc13 strains gave rise to severe defects in cell division, accompanied by an increase of Cdc13 aggregation, without affecting the levels of mRNA. The authors propose that the presence of rare glycine codons in cdc13 plays a role in adjusting the translation efficiency of Cdc13 mRNA to a level competent with proper folding of the protein to guarantee its functionality.Three articles describe different ways by which transfer RNAs (tRNAs) play unorthodox roles in translation, as well as new roles in processes other than translation. In the article by Krahn et al. points to non-canonical structures formed by tRNAs. The classical and well-known secondary structure of tRNAs resembles a clover leaf, in which the acceptor stem, D loop arm, anticodon arm, variable arm and TφC arm are conserved between all organisms. However, some variations of this conserved structure have been shown to accomplish specific roles in the charging and translation of specific amino acids that are decoded by specialized termination codons. Examples, provided by selenocysteinyl, pyrrolysyl-tRNA, and others, show that these tRNAs are aminoacylated by canonical amino acids, which are later transformed into the derivatives that are incorporated to proteins. These tRNAs show extended acceptor, TφC and D stems. These modification are relevant for their recognition by the enzymes that modify the amino acids, as well as by the specialized translation elongation factors that allow recognition of the specific coding termination codons, thus facilitating the incorporation of these amino acids into proteins. Special emphasis is placed on the deviation of mitochondrial tRNAs from canonical structures, where different versions are found, including the lack of either the TφC or the D arms, or both, in tRNAs that are still functional in nematodes.A review article by Guimarães et al. They depict the other roles, beside translation, of tRNA and their genes (tDNAs). The authors first describe the participation of transcription factor TFIIIC, one of the RNA polymerase III general transcription factors, which transcribes tDNAs (and 5S rRNA) in eukaryotes by recognizing internal transcription promoters. The transcription machinery helps in the recruitment of architectural chromatin proteins, such as condensin and adhesin, that help limiting the spreading of heterochromatin into the active euchromatin. A second case described by the authors is the effect of tRNAs on genome stability. The tDNAs are known as sites where R loops are formed, particularly when replication collides with transcription of tDNAs. These R loops are precursors of genome instability. Additionally, tDNAs in yeast, as well as in bacteria, are prone to insertion of transposable elements (retrotransposons). Besides the insertion of these genetic elements, they create a framework for duplications or deletions of segments of the genome within inserted genetic elements. Another interesting effect of tRNAs on genome stability and evolution occurs in yeast when tRNA pools are altered, generating variability in the decoding capacity of certain tRNAs, imposing consequences on proteome stability. In the long run, these effects cause instability of chromosomes, particularly in regions encoding stress response genes as adaptive evolutionary events.Other dimensions of tRNAs utility are highlighted in the review article by Li et al., and by El Mouali et al. describe studies of duplication of regulatory RNAs and their roles in different states of the bacterial life. The first example is that of the two copies of 6S RNAs (6S-1 and 6S-2) from Bacillus thuringensis that are co-transcribed from an operon, whose transcript requires processing. The deletion of the gene encoding 6S-1, and not 6S-2, affects bacterial growth in stationary phase, as well as spores production. The role of the 6S-2 is not known yet. The second example is that of the two copies of the RNAs, CsrB, and CsrC, that regulate the function of CsrA, the protein controlling translation of mRNAs of the carbon storage regulon. These regulatory RNAs, existing in a number of bacteria, bind to CsrA, titrating the protein and thus avoiding its interaction with GGA motifs in the target mRNAs. While CsrB is constitutively expressed in Salmonella, CsrC is regulated at the transcription level by the master carbon regulator CRP-cAMP. It is repressed at the logarithmic phase with the contribution of Spot42, an sRNA that is also controlled at the transcription level by CRP-cAMP.During the last couple of decades, a vast number of publications described the identification of non-coding RNAs as molecules that regulate gene expression. In this Research Topic, four articles, all reporting original research, expose different aspects of the regulatory role of non-coding RNAs. The articles by Bar et al. describes a new computational approach, which uses machine-learning-based algorithm for the prediction of novel sRNAs, to define previously unknown sRNAs found by the recently developed RIL-sec technology for identifying sRNA that bind to the Hfq chaperone. Currently, around 2,800 sRNAs in E. coli have been found to interact with Hfq. The ~1,000 identified in this work are encoded by different regions in the genome, either from intergenic regions or as part of the sense or antisense coding sequences in mRNAs transcripts.Novel technologies for global analysis of RNAs have uncovered a vast variety of small non-coding RNAs (sRNAs) that play diverse functions in the regulation gene expression, mostly at the post-transcriptional level by binding to target mRNAs and altering their fate, both in bacteria and in eukaryotes. First identified as curiosities decades ago and later, due to advancements in bioinformatics and genome sequencing, many RNAs were predicted and identified in intergenic regions. Recent development of RNA-seq-accompanied methodologies have made possible the identification of many more sRNAs, in both intergenic and within coding regions of various organisms. Different approaches for isolating and identifying sRNAs in non-coding and coding regions of genomes have been developed. Their interaction with certain proteins has made it feasible to isolate and identify bound sRNAs and their regulated target mRNAs, permitting the prediction of their function in the cell. The article by Legüe et al. sheds light on the interaction between the model worm C. elegans and bacterial pathogens. The authors report that chronic exposure to the pathogen induces diapause formation (PIDF) in C. elegans. This defense mechanisms is transgenerational, that is, it is inherited by the worm and can be recalled upon exposure to pathogens a few generations forward. This mechanism requires the RNA interference machinery of the worm and sRNAs from the pathogenic bacteria. The authors used bioinformatic analyses of transcriptomic data from holobionts (bacteria and worm) to predict intergenerational and transgenerational interactions of sRNAs from both organisms. Their data points at pairing of RNAs from the two organisms, RNAs with similar sequences from the two organisms and eukaryotic sequence motifs found in bacterial RNAs. Surprisingly, chemical modification of tRNAs emerged as crucial events controlling the formation of the defensive form of the worm and, hence, relevant for the PIDF.Interest in the interaction between commensal bacteria and their host(s) has increased in recent years, mainly due to the analysis of the human microbiome. The article by The topics described in this Research Topic expose the many faces, roles and occurrence of RNA molecules. These studies show that the dynamic changes in mRNAs level toward a steady state concentration, which allow the cell to balance gene expression, is accomplished at the level of transcription, finely tuning the activity of RNA polymerases, but also by controlling the decay of RNAs. Also shown is the interplay between mRNAs and ribosome in bacteria, which is not limited to coupled transcription-translation, but is also relevant for uncoupled processes that allow adaptation to specific cellular requirements. Transfer RNAs, for a long time regarded as vehicles for transporting amino acids to the ribosome during translation, are emerging as active regulators of gene expression, making codon usage a tool for responding to specific physiological events, as well as molecules that play roles in evolutionary processes at the level of genome stability. The diversity of small non-coding RNAs continues to unveil, and their ability to play important regulatory roles is found not to be constrained to particular organisms, but to extend to the interaction of bacteria with their eukaryotic hosts. Together, these articles bring many exciting findings that sheds new light on bacterial RNA biology.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.This work was supported by Grants No. 1274/19 from the Israel Science Foundation (ISF) founded by the Israel Academy of Sciences and Humanities to OA-C, DST-SERB (order no. EMR/2016/002247), Govt of India to RB, National Institute of General Medical Sciences R35GM136213 to JL, and Fondecyt, Chile 1190552 to OO.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Non-coding RNA (ncRNA), released into circulation or packaged into exosomes, plays important roles in many biological processes in the kidney. The purpose of the present study is to identify a common ncRNA signature associated with early renal damage and its related molecular pathways. Three individual libraries  were prepared from each hypertensive patient  for ncRNA sequencing analysis. Next, an RNA-based transcriptional regulatory network was constructed. The three RNA biotypes with the greatest number of differentially expressed transcripts were long-ncRNA (lncRNA), microRNA (miRNA) and piwi-interacting RNA (piRNAs). We identified a common 24 ncRNA molecular signature related to hypertension-associated urinary albumin excretion, of which lncRNAs were the most representative. In addition, the transcriptional regulatory network showed five lncRNAs  and the miR-301a-3p to play a significant role in network organization and targeting critical pathways regulating filtration barrier integrity and tubule reabsorption. Our study found an ncRNA profile associated with albuminuria, independent of biofluid origin  that identifies a handful of potential targets, which may be utilized to study mechanisms of albuminuria and cardiovascular damage. Hypertension is a multifactorial disease that affects cardiovascular and renal systems ,2, and pNon-coding RNA (ncRNA) species comprise more than 90% of all transcripts and have attracted increasing attention in a broad range of biological processes over the last decade ,6,7. NcRAmong these macromolecules, the role of miRNAs in the kidney has been studied extensively, and preliminary evidence indicates that they may regulate progression of glomerular and tubular diseases ,14,15,16NcRNAs can be released into circulation bound to RNA-binding proteins or packaged into extracellular vesicles (EVs), such as exosomes, and may function as paracrine effectors in the crosstalk between different cell types in the kidney ,25. ReceOur aim was to identify a combined signature of various ncRNA biotypes in liquid biopsy, independent of biofluid origin, in urine, plasma or exosomes from hypertensive patients with albuminuria, using high-throughput sequencing analysis, which may more closely reflect the overall biology of underlying early damage than use of single markers. Finally, we constructed an lncRNA–miRNA–mRNA regulatory network with the ncRNA signature combining bioinformatics and correlation analyses associated with development of UAE in hypertension.The study population included 48 essential hypertensive subjects, 22 subjects with increased UAE and 26 normoalbuminurics (non-UAE). General patient characteristics and antihypertensive medication are shown in Small RNA-sequencing single-end technology was used to detect RNA types in the three different biofluid fractions  from hypertensive patients with or without increased UAE. When we analyzed all mapped reads as a whole, we observed that the most frequent RNA biotypes in proportion were piRNA with 38%, miRNA with 32% and miscellaneous RNA (miscRNA) with 16%. This last group included Y-RNA and Vault-RNA, where Y-RNA represented 99% of mapped reads. LncRNA represented 63% of other mapped read groups . In addiWe next sought to analyze the proportion of RNA biotypes present in each of the three biological biofluids, finding miRNA to be the predominant biotype with mapped reads in urinary exosome fraction, representing approximately 65% of total mapped reads in both hypertensive patient types (with or without UAE). Nonetheless, piRNAs were also the most representative RNA type in both patient groups, with 40% in plasma exosomes and 51% in plasma . Of the p-value < 0.05), 4336 were found in urinary exosomes (U-Exo), 4645 in plasma exosomes (p-Exo) and 1415 in plasma samples , miRNA  and piRNA  A. Next, Diverging bar charts showed the fold-change expression of the 24 common ncRNAs in each biological fraction C. The maThe ten DE lncRNAs and two miRNAs established in the molecular signature were selected and potential predicted target mRNAs were identified, creating the common DE lncRNA–miRNA–mRNA network . Hub nodTo further clarify the biological roles of the common short fragments of protein-coding DE RNA in the three biofluids from hypertensive patients with UAE, we performed the gene set Over-Representation Analysis (ORA) with the following findings: 144 common transcripts in the three biofluids and four networks related to pathogenesis of hypertension and presence of UAE were identified, showing a central node with more than six edges.The first network identified is associated with transforming growth factor (TGF)-signaling and includes SMAD3, WNT7B, BMP6 and PDGFRB proteins. The second important network is associated with kidney urinary concentration mechanisms, such as kidney water reabsorption, salt reabsorption and K/Cl cotransporter . The third network is linked to modulation of MAPK signaling, including YWHAQ, PRKAG3, PRKAB2 and TRIB2 proteins. Finally, the last network mediates membrane trafficking with YWHAQ, GRIP1, RAB3IL1, HOOK1 and CYTH1 proteins involved . In addiFinally, we generated a new interaction network joined to the lncRNA–miRNA–mRNA targets interaction network , with thIn the present study, we identified a 24-ncRNA signature associated with albuminuria in hypertension, independent of biofluid, which is common to urinary exosomes, plasma exosomes and circulating in plasma, containing predominantly lncRNA. We also constructed a transcriptional regulatory network (common lncRNA–miRNA–mRNA targets) and predicted the target genes. We found five lncRNAs  and one miRNA (miR-301a-3p) with significantly higher node degree and topological network values compared with the other nodes, implying that these hub RNAs are essential in network organization and are potential key regulators controlling UAE development in hypertension-related RNA network. In addition, we used Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis to assess the enriched biological functions regulated by the ncRNA signature, most of them implicated in mechanisms inducing renal damage.A key feature of the present study was the strategy used. The vast majority of previous studies have identified a unique ncRNA biotype, mainly miRNA, associated with hypertension renal damage in a specific biological fraction ,31, wherAnalyzing DE ncRNA in hypertensive patients with or without UAE, the three most representative ncRNA biotypes were lncRNA, miRNA and piRNA in all three biofluids. Considerable research has been conducted over recent years into the molecular mechanisms of hypertension-associated renal pathology; however, most previous studies have focused mainly on protein-coding genes or miRNAs ,34,35,36The common 24-ncRNA profile related to albuminuria in hypertension was composed mostly of lncRNA, followed, in order, by pseudogenes, snoRNAs, miRNAs and piRNAs. Ten DE lncRNAs were identified and in the RNA network constructed, five of these showed the highest node degree and significant role in interacting with ncRNA targets, serving as hub nodes  joined to miRNA-301a. The GO and KEGG pathway analyses assessing biological functions enriched in response to albuminuria highlighted several pathways: modulators in the renal epithelial–mesenchymal transition , renal fibrosis , endocytosis  activity) and ECM constituent (SOCC activity and laminin binding). Accumulating experimental evidence indicates that these enriched pathways have always been involved in renal impairment. As an example, Chaudhari et al. emphasized SOCC as a crucial regulator of ECM synthesis and deposition by glomerular mesangial cells, its dysregulation being implicated in the pathogenesis of mesangial and interstitial fibrosis in diabetic nephropathy ,39,40,41Additionally, we predicted a common protein-coding gene network for the three biofluids, finding among the nodes with highest degree several transcripts known to be associated with albuminuria development and progression of kidney damage in hypertension, such as: SMAD3, WNT7B, BMP6 and PDGFRB  ; YWHAQ, The major goal of this study was to identify a combined signature of various ncRNAs, such as lncRNAs, miRNAs and piRNAs, independent of biofluid origin, which may more closely reflect the overall biology of underlying early damage in hypertension than use of single markers. Another highlight was to assess the ncRNA targets to construct a regulatory network and identify the hub nodes that play an important role in network organization. These findings provide insights into the mechanisms involved in the architecture of the glomerular filtration barrier and renal tubular reabsorption and provide potential targets for treating hypertension-associated albuminuria. As discussed above, these data are supported by literature evidence in association with renal impairment. Hence, experimental validation of these findings, identifying precise cellular sources and mechanisms underlying common ncRNAs, are expected to set a benchmark for early renal damage research. Finally, further research using larger and independent cohorts is warranted to confirm the ncRNA signature found.In summary, our study found an ncRNA profile associated with albuminuria, independent of biofluid origin , that targets critical pathways of filtration barrier integrity, tubule reabsorption and vascular endothelial function, suggesting an important role for ncRNA signature in hypertension-associated early renal damage and cardiovascular risk progression. Further experimental studies should be performed to demonstrate the utility of these candidates as promising therapeutic targets in albuminuria and widen opportunities in comprehensive renal damage research.n = 21) and 22 patients without persistent elevated urinary albuminuria (UAE) (≥30 mg/g urinary creatinine) [This was an observational case-control study which included 21 patients with essential hypertension (atinine) . All hypFresh first morning urinary samples (100 mL) were collected in sterile containers, and human blood samples were collected in EDTA tubes and centrifuged to separate the plasma fraction. All samples were processed within one hour after reception to isolate the exosomal component, as explained below.Exosomes were isolated from urine (Exo-U) and plasma (Exo-P), using a protocol based on sequential ultracentrifugation. Exosome pellets were characterized by qNano Gold instrument , transmission electron microscopy and western blot. Detailed protocols are explained in our previous study authored by Perez-Hernandez J et al. .Total RNA was extracted from exosomes using the Total Exosome RNA and Protein Isolation kit ; RNA was obtained using the miRNeasy mini kit  from plasma samples. Quantification of total RNA, quality and size distribution were analyzed by capillary electrophoresis  with the RNA 6000 Pico chip.Single-patient libraries were prepared from 2 μL of total RNA from each condition  using CleanTag Small RNA library preparation kit , following a small RNA library preparation protocol optimized to very low input samples, as previously described . Librarihttp://hannonlab.cshl.edu/fastx_toolkit, accessed on 13 October 2021), removing adapters, low quality reads and nucleotides. Alignment was made with STAR v2.7.3a [Data quality control of the raw data was conducted with FastQC v0.11.8 . Subsequ v2.7.3a , followi v2.7.3a . Sorted  v2.7.3a , Rsamtoo v2.7.3a  and Geno v2.7.3a  and piRN v2.7.3a . We therThe count matrix was included in a DGEList object using the edgeR Bioconductor package . The metp-values were adjusted using Benjamini–Hochberg method. p < 0.05 was considered statistically significant. The edgeR Bioconductor package was used for all statistical analyses [The contrasts between hypertensive patients with (UAE) and without albuminuria (non-UAE) were determined by performing a negative binomial generalized log-linear model to analyze the read counts for each gene, adjusted for sex. The analyses . The graanalyses  or VennDanalyses , as apprhttp://rtools.cbrc.jp/LncRRIsearch, accessed on 13 October 2021) [The targets for lncRNAs were predicted using LncRRIsearch, a web server for comprehensive prediction of human and mouse lncRNA–lncRNA and lncRNA–mRNA interaction (er 2021) . The tophttp://www.targetscan.org/vert_72/, accessed on 13 October 2021) [http://mirdb.org/, accessed on 13 October 2021) [https://mirtarbase.cuhk.edu.cn/~miRTarBase/miRTarBase_2022/php/search.php, accessed on 13 October 2021) [In the case of miRNA-targets, three web-based tools were used: TargetScan (er 2021) , miRDB (er 2021)  and miRTer 2021) . The selhttp://www.webgestalt.org/, accessed on 15 October 2021), using GO and KEGG databases [Gene set Over-Representation Analysis (ORA) was performed in WEB-based GEne SeT AnaLysis Toolkit (atabases . The proatabases . All bioatabases . In this