Little is known about the extent to which Health Extension Programme (HEP) has played its role to increase service uptake among young girls. This study aims to estimate the status of young girls’ sexual and reproductive health (SRH) services utilisation in rural Ethiopia and to examine the role of health extension workers (HEWs) in this regard.A cross-sectional study.A community-based study among all nine regions of Ethiopia.Nine hundred and two young girls aged 15–24 years were included in this study.We used data from the national HEP assessment, collected from March to May 2019. Multilevel binary logistic regression was used to investigate the association between exposure to HEP and SRH services utilisation of young girls and we reported an adjusted OR with a corresponding 95% CI as measure of the degree of associations.Only 19.18% (95% CI 16.74% to 21.89%) of young girls used SRH services with significant regional variability . Exposure to HEP , knowing about the availability of HEP services  and having good trust in HEWs  and other sociodemographic factors were significantly associated with increased SRH services utilisation.SRH service utilisation.Although the overall SRH service utilization of young girls in rural Ethiopia was very low, HEWs have a great contribution to improving service utilization of young girls through strong health education provided during home visits, school visits and at health posts. More investment along this line has the potential to improve service uptake among young girls. Encouraging HEWs to build trust among this segment of the population and creating awareness of SRH-related services is crucial to improv service uptake. The study was done at country level, which is more representative.It did not assess the attitude of health extension workers about sexual and reproductive health (SRH) delivery.Recall bias that may have underestimated the magnitude of SRH services.We could not assess the effect of cultural factors as a barrier for using SRH service qualitatively.Youths between 15 and 24 years of age account for more than 1.2 billion people globally with 90% of them living in low-income and middle-income countries.4Having healthy youth leads to improved outcomes for a family, society and a nation as a whole. Hence, investing in their health has the potential to break the cycle of poverty through increased productivity, decreased health costs and improved social capital.9Previous studies in Ethiopia showed that the SRH knowledge of young girls and their SRH service utilisation is very low, which might worsen their SRH problems.3 11Cognizant of these facts, the Ethiopian government launched a Health Extension Programme (HEP) in 2003 as a community-based programme to increase the quality and access to health services in order to prevent disease, promote healthy behaviours, and improve knowledge and service utilisation.During the data collection, Ethiopia was administratively divided into nine regions and two city administrations. Each region is hierarchically divided into zones, districts and then kebeles. A kebele is the smallest administrative unit expected to serve an average of 5000 people, and a district is the lowest budgeted centre of the government. More than 95% of the kebeles in the country have HPs. HPs are categorised under the primary level of Ethiopian health system tiers, constructed within each kebele and staffed on average by two HEWs. Each HEW is expected to serve 1500–2500 people and there are nearly to 40 000 HEWs in more than 17 000 HPs in Ethiopia.HEWs are high school graduates with an additional 1 year of education that enables them to be engaged in health education and delivery of FP methods and selected curative services. They are expected to spend 75% of their time in outreach services  and 25% on curative services at the HP. For the success of the HEP programme, HEWs are supervised at least weekly and monitored by the nearby primary healthcare unit health professionals.13A cross-sectional national survey was conducted using a multistage sampling method to evaluate the Ethiopian rural HEP, and for this study, data were extracted from this survey. The survey covered all the nine administrative regions of the country and data collection was conducted from March to May 2019. In total, 62  districts were covered in the assessment by using appropriate methodological flow. Within each district three kebeles were randomly selected and then within each selected kebeles, 34 households were randomly selected from the total list of households in the kebele. A total of 902 young girls between the ages of 15 and 24 years from a total of 6324 rural households were included in the HEP programme evaluation assessment and we have used these data for analysis. The detailed methods and sampling procedures are described elsewhere.17During the HEP programme evaluation assessment, we used a structured questionnaire adapted from different literature.No patients involved.This is a dichotomous outcome variable that refers to the status of a young girl in using at least one of SRH services: (A) attending health education on SRH issue (during a home visit or at school or during HP visit) or (B) FP utilisation . The variable assumes a value of ‘1’ if a given young girl used at least one of the SRH services and ‘0’ if she did not use any.This is the exposure variable that we used to investigate the role of HEP in enhancing SRH service utilisation of young girls. We measured this variable as follows: young girls who meet HEW for any service at a home visit, school visit or HP visits within a 1-year period prior to data collection time were labelled as ‘exposed to HEW’ or else they were labelled as ‘not exposed’.Age, educational status, marital status ), source of information for FP, region and wealth index of the household .This is a composite variable constructed from seven perception-related questions, intended to measure a young woman’s attitude towards HEWs. Those young girls who answered strongly agree or moderately agree to all seven perception-related questions were considered to have ‘good trust in HEWs’ and those who answered strongly disagree or moderately disagree were labelled as ‘poor trust in HEWs’.Young girls who know at least two SRH-related HEP packages/services  were considered as having an ‘adequate knowledge’ and the other young girls were considered as having ‘inadequate knowledge’.young girls who know at least one modern FP method  and implants) were labelled as ‘knowledgeable about modern FP methods’, and those who were not able to list any one of the five methods were considered as ‘not knowledgeable about modern FP methods’.This is another composite variable that used responses from nine questions. A young girl who gave at least one correct answer from a total of nine HIV transmission-related questions was labelled as having ‘knowledge of HIV transmission’ and if she did not give a correct answer for any of the nine HIV transmission-related questions, she was considered as ‘not knowledgeable about HIV transmission’.We used Stata V.14 for the analysis and reported frequencies, percentages, mean and SDs as descriptive summary measures of key variables. We used multilevel binary logistic regression to model the odds of using SRH services. Due to the multistage cluster sampling procedure, individual young girls were nested within kebeles and then within regions. We examined the effect of the individual-level variables, and the region-level variables using two-level logistic regression modelling. During analysis, the characteristics of young girls were taken as individual level (level 1), and regions were taken as level 2. The region was considered as level two to understand regional variability which is useful for future intervention.Based on this model, the intraclass correlation coefficient (ICC) was calculated to evaluate whether the variation in the odds of using SRH services was primarily within or between regions. Both the crude OR and the adjusted OR (aOR) with their corresponding 95% CI were reported as measures of strength of association.A total of 902 young girls were interviewed, yielding a 100% response rate. The mean age of the respondents was 17.48 years (SD = ±2.27), 816 (80%) young girls were in the age range of 15–19 years, 170 (18.82%) had no formal education and 779 (86.36%) were never married .Only one hundred and seventeen (12.97%) young girls knew at least one method of FP and 97 (10.75%) young girls were knowledgeable about modes of HIV transmission and prevention. Based on answers of strongly agree or moderately agree to attitude-related questions, 651 (76.77%) young girls had a good perception of the HEW services being friendly to young’s FP needs, 602 (71.92) self-reported good trust in HEW and 732 (86.73%) had a good perception of HEP services. Three hundred and forty-nine (38.69%) young girls recommended an increase in the frequency of home visits by HEWs .National-level SRH services utilisation of young girls was 19.18% (95% CI 16.74% to 21.89%). Among the 244 young girls that live in pastoralist areas, only 29 (11.90%) used SRH services. Similarly, among 658 young girls that live in agrarian areas, 144 (21.9%) used SRH services. There is a significant amount of variation in the status of SRH utilisation across the region accounting for 17.16% of the total variability .SRH services use among young girls was significantly higher for girls who had exposure to HEP through HEWs’ home visits, HEWs’ school visits, or during HP visits (70.5%) as compared with girls who did not have such exposure (37.6%).Young girls who were exposed to HEW were three times more likely to use SRH services than girls who were not exposed to HEW .Other factors that are associated with increased SRH services utilisation are knowing SRH-related services are available at HPs , having good trust in HEW , being in the age range of 20–24 years  and being ever married  compared with their counterparts .In this study, the status of SRH service utilisation of young girls was very low, however, it was increased when they were exposed to HEP through health education provided by HEW. Exposure to HEWs, knowing about HEP service availability, and having trust in HEW were significantly associated with increased SRH service utilisation among young girls. While older age and being ever married also were significantly associated with increased SRH service utilisation, educational status of young girls, wealth index of the household, perception about SRH service delivery by HEW, and perception of the youth-friendliness of HEWs were not significantly associated with SRH service utilisation of young girls.SRH service utilisation of young girls was relatively lower compared with the findings of previous studies in Ethiopia.13One of the HEP packages was designed to increase youth SRH service utilisation through health education during home visits, school visits and HP-based service provision.Adequate knowledge of available components of HEP is also significantly associated with increased service utilisation among young girls. Among different components of SRH-related HEP packages, most of the respondents recalled health education and FP services. This may reflect the differential focus given to FP education by HEWs during service delivery. Studies in Ethiopia and Uganda showed that knowing the place of SRH service increases service utilisation.Confidentiality is one of the major ethical codes in medicine and SRH service utilisation requires a similar level of confidentiality because of its sensitivity.33 34In line with a previous Ethiopian study, conducted at Baher Dar, young girls between the ages of 20 and 24 years were more likely to use SRH services than girls aged 15–19 years.8Although the findings of the Ethiopian Demographic Health Survey showed that many young girls are engaged in sexual intercourse before marriage, the issue of SRH is culturally sensitive and socially unacceptable for unmarried girls which may decrease service utilisation among young girls.35Some limitations of this study include the survey’s failure to assess the attitude of HEWs about SRH delivery to unmarried young girls which, as just discussed, likely makes young girls less likely to use SRH services from HEP. Other limitations include recall bias on the part of young girls surveyed which might have underestimated the magnitude of SRH services used by these girls. Further, the survey used both female and male data collectors; using male data collectors for this kind of study may have affected the magnitude of SRH service utilisation reported. This study also did not ask about barriers that hinder HEWs from increasing youth service utilisation. Additionally, we did not perform a qualitative study to assess the effect of cultural barriers such as existing norms, beliefs and gender-related barriers on SRH service using. However, using nationally representative data is one of the important strengths of this study.This study demonstrates that SRH service utilisation of young girls in rural Ethiopia is very low. Our findings also indicate that HEWs have a significant contribution to improving SRH service utilisation of young girls through health education during home visits, during school visits and at HPs. However, more effort might be required to significantly increase the exposure of young girls to HEP services to increase service uptake. Encouraging young girls to build trust in HEWs and make young girls know what SRH-related services are available at the HP is the first step towards improving service uptake. HEWs need to give special focus on younger girls and never married young girls to increase their service uptake.


The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) system is widely used as a genome-editing tool in various organisms, including plants, to elucidate the fundamental understanding of gene function, disease diagnostics, and crop improvement. Among the CRISPR/Cas systems, Cas9 is one of the widely used nucleases for DNA modifications, but manipulation of RNA at the post-transcriptional level is limited. The recently identified type VI CRISPR/Cas systems provide a platform for precise RNA manipulation without permanent changes to the genome. Several studies reported efficient application of Cas13 in RNA studies, such as viral interference, RNA knockdown, and RNA detection in various organisms. Cas13 was also used to produce virus resistance in plants, as most plant viruses are RNA viruses. However, the application of CRISPR/Cas13 to studies of plant RNA biology is still in its infancy. This review discusses the current and prospective applications of CRISPR/Cas13-based RNA editing technologies in plants. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems are found in prokaryotes such as archaea (~80%) and bacteria ~40%) . CRISPR 0% . CRISClass 1 Cas systems comprise types I, III, and IV with diverse variants and effector complexes, whereas class 2 Cas systems consist of types II, V, and VI with an effector module containing a single multifunctional protein . A typicA single multifunctional Cas13 effector protein contains two higher eukaryotes and prokaryotes nucleotide-binding domains (HEPN) that provide RNase activity. The Cas13 protein, when associated with crRNA, forms an RNA-guided RNA targeting complex to recognize and cleave ssRNA targets. Based on Cas13 phylogeny, features, and functional characterization, this system is further classified into six subtypes: VI-A , VI-B , VI-C , VI-D (Cas13d), and recently, VI-X (Cas13X) and VI-Y (Cas13Y) ,18,19,20Leptotrichia shahii (Lsh) . Ho. HoBergeuirement . Cas13b Fusobacterium perfoetens (FpeCas13c).Cas13c is not as functionally characterized compared to other types of Cas13. However, the known characteristics, such as the presence of DR on the 5′ end of crRNA and the spacer length 28–30 nt), are similar to that of Cas13a and Cas13d  19,28].,28.19,28–30 nt, aRuminococcus flavefaciens XPD3002 (CasRx/RfxCas13d) is a prominent homolog for multiple organisms [Cas13d is the smallest of Cas13a-d and is one of the most efficient type VI systems for RNA targeting ,22 Tabl. The Casrganisms ,30,31. Irganisms . Type VIType VI CRISPR/Cas systems provide various applications in various organisms through different RNA technologies such as RNA interference, RNA detection, RNA editing, and RNA targeting ,37,38. CTurnip Mosaic Virus (TuMV) RNA genome, targeting its helper component proteinase (HC-Pro) and coat protein (CP) sequences in Nicotiana benthamiana and Arabidopsis thaliana, respectively [potato virus Y (PVY), tobacco mosaic virus (TMV), southern rice black-streaked dwarf virus (SRBSDV), and rice stripe mosaic virus (RSMV) in various plants [RNA interference (RNAi) is an innate antiviral immunity mechanism that has been successfully used to combat various plant viruses. Nevertheless, the availability of such antiviral strategies is still limited to specific virus groups. Many viruses readily mutate and have developed different counter-defense mechanisms, leading to the rapid emergence of new antiviral approaches. The recently developed CRISPR/Cas13 is a promising tool for engineering plant immunity against a broad range of RNA viruses that constitute a majority of plant viruses a. Althouectively ,41  that binds to target RNA but lacks endonuclease activity , allowinAlternative splicing is a process regulating gene expression in which exons of a gene are spliced to form multiple alternative transcripts. The CRISPR/Cas13 system can regulate RNA splicing pathways by introducing point mutations into the 5′ and 3′ ends of a splice donor site (GU) and a splice acceptor site (AG), respectively, resulting in mis-splicing and losing targeted splice variants. Konerrman et al.  demonstrAgrobacterium sp. strain CP4 (CP4 EPSPS), in a mixture of soybeans (The target RNA recognition and binding abilities of CRISPR/Cas13 provide means for its application in various functional and diagnostic studies. Previously, Abudayyeh et al.  harnessesoybeans . This plCRISPR/Cas technology has become a powerful genome-editing tool due to its applications in various fields for genetic alterations, disease diagnostics, and crop improvement. For the past decade, the DNA targeting CRISPR/Cas9 system has evolved into a remarkable tool for its implementation in various DNA technologies such as gene knockout, gene activation, gene editing, gene therapy, and DNA detection in multiple organisms ,65,66,67In addition, targeting non-coding RNA such as long non-coding RNA, microRNA, and circular RNA in plants using CRISPR/Cas13 can become a promising tool to study their role in plants. Previously, targeting non-coding RNA in plants was performed using CRISPR/Cas9 at the DNA level to find their roles in plant growth, development, and stress responses ,70,71,72Though CRISPR/Cas13 can be used to target ssRNA in many organisms, the RNA targeting and RNA binding in Cas13 have constraints, some of which might limit its use. One major limitation is the collateral activity of Cas13 that causes non-specific RNA cleavage ,77,78. TRNA targeting and editing in plants using CRISPR/Cas13 systems can provide great insights into plant transcriptomic studies. CRISPR/Cas13 application in plants has mainly been to impart tolerance against plant viruses. However, it is also emerging as a prominent tool to target mRNA, circRNA, and other non-coding RNAs. In addition, nucleic acid detection using CRISPR/Cas13 is gaining attention in many recent studies for its high specificity, especially in viral diagnostics. Though Cas13 has a few limitations such as a PFS requirement and non-specific collateral activity affecting its editing efficiency, orthologs such as LwaCas13a, PspCas13b, RfxCas13d can be used to avoid the PFS preference, and collateral RNA cleavage of Cas13 has not been observed in plants yet. Therefore, based on current applications and future perspectives, CRISPR/Cas13 systems can potentially emerge into a robust RNA targeting platform in plants, providing novel opportunities in modern agricultural applications.


The increase in degenerative diseases involving articular cartilage has pushed research to focus on their pathogenesis and treatment, exploiting increasingly complex techniques. Gene expression analyses from tissue are representative of the in vivo situation, but the protocols to be applied to obtain a reliable analysis are not completely cleared through customs. Thus, RNA extraction from fresh samples and specifically from musculoskeletal tissue such as cartilage is still a challenging issue. The aim of the review is to provide an overview of the techniques described in the literature for RNA extraction, highlighting limits and possibilities. The research retrieved 65 papers suitable for the purposes. The results highlighted the great difficulty in comparing the different studies, both for the sources of tissue used and for the techniques employed, as well as the details about protocols. Few papers compared different RNA extraction methods or homogenization techniques; the case study reported by authors about RNA extraction from sheep cartilage has not found an analog in the literature, confirming the existence of a relevant blank on studies about RNA extraction from cartilage tissue. However, the state of the art depicted can be used as a starting point to improve and expand studies on this topic. Nucleic acid extraction has become a routine procedure in molecular biology, leading the way to the characterization, isolation, and manipulation of the main molecular components of cells and organisms. It is known that purification of intact RNA is the primary step of many molecular biology techniques, including PCR, but RNA is a very labile molecule extremely sensible to degradation by RNases ubiquitously present. Considering these aspects, a well-performed technique of RNA extraction able to provide a high-quality, undegraded, and abundant product is needed. In addition, the tissue from which the nucleic acid must be extracted also plays an important role in the quality of the final extraction product. In the orthopedic field, there are many technical difficulties deriving from handling connective tissues composed primarily of a tough matrix with very few cells, such as bone, cartilage, ligaments, tendons, or intervertebral discs, which are hypocellular and resistant to tissue disruption . Due to It follows that the analysis of these cell types in their complete and physiological microenvironment would be appealing since it would provide a better representation of gene expression profiles in vivo . ObtainiIn a previous work of ours, we defined and validated a method useful to extract RNA from osteoblasts starting from the fresh calcified human bone samples, focusing on obtaining a suitable RNA quality in terms of purity and integrity, as well as identifying a set of reliable reference genes . The preSince a large variety of genes and their products are known to play a role in the pathophysiology of various osteoarticular diseases, even more it follows that a large amount of pure RNA is needed to perform all the possible investigations. The gradual rise of the age of the worldwide population is necessarily associated with the increase in several joint diseases, which OA is the most common. It involves up to 15% of the adult population ,13, so rFor these reasons and based on increasing incidence of degenerative diseases involving articular cartilage fraction and the related interest on this issue, we wanted to deepen our knowledge on the techniques for extracting RNA from cartilage and assess their differences through a review of the literature whose results are described below. Finally, our personal experience was reported.DNA and RNA extraction mainly follows protocols with standardized reagents, many of which are available in commercial kits. www.pubmed.com (accessed on 1 July 2022) and www.webofknowledge.com (accessed on 1 July 2022). All publications from the start of scientific literature to June 2022 were included. Additional studies that were not found by our initial search were identified analyzing the reference lists from the included articles.Literature data searching was performed on 2 databases: https://www.zotero.org (accessed on 1 July 2022) program, in order to delete duplicate papers, a careful selection of them was made on the basis of title, abstract, and, in most cases, by reading the section “Materials and Methods”. The criterion for which the selection of the articles to be included in the review was based on the study of the different cartilage extraction methods from intact tissue, including animal studies and clinical studies.The research topic, rather specific in the technical details to be identified and rather general in its applicability at the same time, prompted the authors to perform research with a “broad” strategy. More in detail, the following key terms, and their combinations were used: “RNA AND (extraction OR isolation) AND cartilage”, “RNA AND extraction AND cartilage”) on both databases. The limits set were the English language, reviews, and other manuscript forms. Given the relative novelty represented by molecular biology, no publication date limits have been entered. Additional studies that were not found by our initial search were identified by analyzing the reference lists from the included articles. After submitting the entire list obtained to the public reference manager Of the 433 retrieved papers resulting from literature research carried out on PubMed and Web of Science Core Collection databases, only 15% referred to RNA extraction from mature cartilage tissue. Among them, only few papers report references on quality and purity assurance and not all specify the results related to these parameters.Tissue collection and, mostly, the subsequent storage conditions represent the first critical step for the isolation of suitable-quality RNA. For this reason, precautions are needed to be carry out to diminish RNA degradation. The most ideal tissue specimen is one that carries a complete unaltered representation of the in vivo tissue, as well as the perfect procedure is one that immediately processes the fresh samples for RNA extraction ,23,24,25However, logistic problems can cause a delay in sampling, compromising the quick tissue processing for RNA isolation, making it necessary to rapidly snap-freeze the clinical samples and store them at −70/80 °C for several days ,27,28. Llater™ solution, and stored at −4 °C [later™ Solution, and RNA clean-up kits after isolation, did not improve the quality of the obtained RNA [2, cooled in 2-methyl-butane, and stored at −70 °C until use [The majority of the studies that performed RNA extraction from fresh cartilage samples, straightaway frozen the tissue in liquid nitrogen before performing the extraction procedure immediately after ,41,42,43at −4 °C  or −80 °at −4 °C  minimiziat −4 °C . Howeverined RNA . Differentil use .After sample storage at −80 °C, thawing represents another delicate but controllable step to the RNA isolation. In fact, cell membranes are disrupted by freeze-thaw cycles and this process could degrade RNA transcripts due to the intrinsic RNase activity, which is present in a variable extent in all tissue extracts. Avoiding RNA degradation represents a major challenge along the chain of RNA extraction; in vivo during surgery, ex vivo during specimen transport to the laboratory as well as during thawing and manipulation after snap-freezing .Regarding this first step, literature data agree on the importance of few precautions: a quick treatment of specimens to inactivate degrading enzymes; fresh unfixed tissue is better than chemically fixed tissue; short period of storage yields better quality and quantity of nucleic acid; nucleic acid preservation is enhanced by thawing the tissue as quickly as possible .The term “homogenization”, in general, describes the breakdown of tissue structure to form a homogeneous suspension or emulsion of cellular fragments through a mix of mechanical actions such as maceration or crushing or by chemicals such as detergents or organic solvents. Due to the high amount of intercellular matrix that hinders the extraction of RNA from the cells, immersed and hidden in the matrix itself, a homogenization step is essential, to pulverize the tissue.Different approaches are available for tissue homogenization, including rotor-stator homogenization, bead-milling homogenization and grinding with a mortar and pestle. They are basically mechanical approaches in which cartilage tissue can also be disrupted in different working buffers. In order to exemplify, we decided to refer to these approaches as “mechanical” in order to distinguish them from those methods that use liquid nitrogen during the entire procedure or in a part of it, referred to as “criopulverization”.The traditional and easier approach to pulverize from cartilage is represented by a chilled pestle and mortar . Similar® and homogenized it with a dispersing instrument (ULTRA-TURRAX®), that enables to work at high circumferential speeds even with small rotor diameters [Several authors utilize homogenization instruments such as MagNA Lyser (Roche) ,47,53 aniameters . Conversiameters . Similariameters ,59,60. Miameters ,61. Eveniameters . The detParticular instruments such as a freezer mill, a microdismembrator or, simply, a frozen mortar and pestle can be used for this purpose . The mos® or Tri Reagent to the samples, thus accelerating the entire process [The microdismembrator, similarly, is suited for fast homogenization of problematic samples, previously frozen in liquid nitrogen. It works with beads, but not in nitrogen immersion. In particular, Mikro-Dismembrator S  with a maximum frequency of 50 Hz is especially well suited for fast homogenization of samples frozen in liquid nitrogen ,51,62,68 process ,51,62. A process . Table 3®” RNA extraction method, first described by Chomczynskj and Sacchi [Nowadays, a number of approaches to RNA extraction are available, which are basically a variation of the most common and oldest RNA extraction technique: the AGCP extraction, commonly referred to as the “TRIzold Sacchi  , TriReagent (Sigma-Aldrich), ToTALLY RNA (Ambion), and FastPrep Pro (Q-biogene). Many authors utilize salts such as CsCl or cesium trifluoroacetate C2CsF3O2 o CsTFA) in order to separate RNA from DNA or proteins  to easily purified total RNA, often from approximately 10 to 1000 mg of cartilage tissue ,66,76,77Purity and integrity of RNA are critical elements for the overall success of RNA-based analyses. Due to its high instability, fragments of RNA commonly can occur in a sample, compromising the results of downstream applications ,80.Different quality control methods are commonly used, even if, to date there is no real consensus on the best one . Table 5260: A280 and A260: A230 values), based on the ability of nucleic acids to absorb UV light at a wavelength of 260 nm. Quantity and quality assessment using a UV/VIS spectrophotometer should be performed at multiple wave lengths: 240 nm (absorption of possible contaminants), 260 nm (specific for nucleic acids), 280 (specific for proteins) and 320 (absorption of possible contaminants). A major advantage of the system is the very low sample consumption of 1–2 μL, which is especially important when using precious materials such as human biopsy. A ratio of absorbance at 260 and 280 nm (A260: A280) greater than 1.8 is usually considered a suitable indicator of RNA purity [260 measurement can be compromised by the presence of genomic DNA leading to an over-estimation of the actual RNA concentration.Spectrophotometry technology, such as NanoDrop™, provides information on RNA quantity as well as purity , equipped with a stainless impactor. After 10 min of precooling in liquid nitrogen, pulverization was carried out by three cycles at the impact frequency of 15 cps (2 min each cycle) and a cooling step of 2 min between the cycles. All tested extraction methods involved resuspending the sample in TRIzol® reagent immediately after cryopulveritazion of the tissue and then incubated overnight at 4 °C. The subsequent extraction step was performed by the addition of chloroform in a volume equal to one-fifth of the TRIzol®. To obtain a cleaner product, the mixture of sample- TRIzol® was distributed in several conical tubes, avoiding exceeding 1 mL for each tube, before the addition of chloroform.Our approach involved pulverization of frozen sheep articular cartilage samples (n = 5), followed by five different extraction methods. Specifically, all the samples were removed with a scalpel from the femur head, weighed (average weight: 139.2 mg), and immersed in RNAIn order to isolate RNA from its dense PG-rich ECM, salts such as NaCl and NaOAc, in addition to glycogen, were used in three out of five methods , with some little modifications between them.Method N.1: The aqueous phase harvested after centrifugation at 12,000 rpm (at 4 °C for 15′) was then added to 20 μg/μL glycogen, 1.2 M sodium chloride (NaCl), and 0.8 M sodium acetate (NaOAc). RNA precipitation was performed by the addition of isopropanol, 10′ of incubation at RT, and 30′ of centrifugation at 12,000 rpm at 4 °C. Finally, the obtained RNA was twice washed with 75% cold ethanol and dried under the hood.Method N.2: Method 2 is identical to 1, apart from the addition of a second TRIzol®/chloroform extraction step immediately after the collection of the aqueous phase. More in detail, a volume of TRIzol® equal to twice the harvested aqueous phase was added. After 15′ of incubation at RT, the procedure continued with the addition of chloroform as in Method 1;Method N.3: Similar to Method 2, it included twice the passage of extraction by TRIzol®, but the second aqueous phase harvested was added to glycogen and a double volume of isopropanol. Different from other methods, it followed an incubation at −20 °C overnight followed by RNA extraction similar to what was described in Method N.1 but with only one wash in 75% Ethanol;Method N.4: This method, also known as “clean up”, is characterized by further steps at the end of Method 3, aimed at a better purification of the extract. The RNA obtained by Method 3 was added to a mixture consisting of glycogen, NaOAc, and isopropanol in deionized H2O , then incubated 60′ at −80 °C and centrifuged. After washing with 75% ethanol, the sample was dried and resuspended in H2O. Finally, 10′ at 60 °C should increase the RNA resuspension;Method N.5: It represents the combination between the TRIzol® method and the one based on the use of columns. More precisely, the aqueous phase obtained by TRIzol®/chloroform was added with glycogen and 75% ethanol in equal volume, then loaded on a commercial mini spin column (Qiagen RNeasy Mini Kit).Isopropanol was used as a precipitation solution in all the methods except for Method 5, where purification was carried out on mini spin column chromatography using the manufacturer’s instructions (Qiagen RNeasy Mini Kit). In all the methods tested, 75% Ethanol was used to wash the pellet and RNase-free water to resuspend the samples.260:A280 and A260:A230 values). It shows that the A260:A280 values for each sample were all approximately 2 (average of 1.78), regardless of the method used, with the best result obtained for method 2. However, great variability was seen in A260:A230 values. The lowest A260:A230 values were found in the RNA samples obtained using methods 1 and 5, even if it does not correspond to the lowest concentration’s values. The best RNA values per sample’s weight were obtained using methods 1, 3, and 4. It is worth noting that higher yields were obtained by applying methods 1 and 4, which, differently from Method 3, implied salts use, suggesting their key role in RNA extraction. Regarding Method 2, a better A260:A280 value resulted in a lower RNA yield. In our experience, Method 5 seems to be not suitable.Despite the experience of our laboratory in the extraction of RNA from many musculoskeletal cells, the present review arose from the practical need to extract RNA from fresh cartilaginous tissue. This need is owned by insufficient knowledge in the literature due to the complexity of handling such connective tissues, which are both hypocellular and resistant to tissue disruption.®, RNeasy, TRIzol®/RNeasy, and RNAqueous extraction methods. Concerning the methods of choice for cartilage homogenization, they concluded that the methods should be chosen since the subsequent isolation approach will be used. They argued that milling associated with TRIzol® reagent and RNeasy resulted in significant RNA degradation and, consequently, in false positive results in gene expression. Conversely, the milling did not negatively influence RNA yield and quality. Similarly, Adams et al. [® protocol performed on snap-frozen samples crushed with mortar and pestle and homogenized by Polytron sonication with a modified version. In particular, obtained human articular cartilage samples were firstly minced with a scalpel and incubated into culture media. Then, chondrocytes were isolated from the surrounding proteoglycan-rich matrix by a chemical digestion with trypsin in rotation with 5.0 mm glass beads, followed by an incubation in collagenase and then lysis in TRIzol® [In this regard, an important issue is the use of specialized equipment such as microdismembrator or freezer mills to process the fresh cartilaginous tissue. However, such instruments could not always be available in all laboratories. Furthermore, these methods typically use columns for RNA extraction and purification, obtaining suitable RNA quality but poor yield . Even whs et al.  compareds et al.  have ides et al.  instead s et al.  proposed TRIzol® . Regardi TRIzol® . For thi TRIzol® . Again r TRIzol® . These dIntegrity and purity of RNA is an essential critical prerequisite for gene expression analysis, especially if diagnostic, therapeutic, or prognostic conclusions will be drawn, as degraded RNA represents a limit for obtaining meaningful and realistic results . It is n260: A230 does not always predict success in downstream applications. This is due to the fact that A230 is often constant for nucleic acid purified using a specific kit, while the amount of RNA can vary, resulting in a decreased A260: A230 ratio. Some disadvantages of using absorbance are the lack of specificity and sensitivity. In fact, the method is not capable of distinguishing between dsDNA, RNA, or ssDNA, so the amount of genomic DNA present in an RNA preparation cannot be determined by absorbance. Furthermore, contaminants that absorb around 260 nm can contribute to the final absorbance value, overestimating the nucleic acid concentration. Finally, since single nucleotides can also contribute to the reading, changes in RNA integrity are not reflected in the measurement [260: A280 ratio of RNA from tissue is generally lower than that isolated from cells  due to the presence of “contaminating” extracellular matrix components [260: A280 ratio, it is also important to remove any insoluble materials. For this reason, describing his preferred method, Adams et al. suggest centrifuging and precipitating the RNA suspension with acetic acid and ethanol after CsTFA centrifugation [260: A280 ratio that can affect the final measurement.It is worth noting that the Asurement . Furthersurement . In addimponents . To obta260: A280 results, in our experience, we obtained an average value of 1.78. This value is lower than the optimal value of 2 for this parameter, but it is in line with what was reported by Aigner et al., and it could be due to the presence of extracellular matrix components, which act as contaminants. Furthermore, it does not differ much from the values reported by other authors (Regarding our A authors .Also, with regard to gel electrophoresis evaluation, it is often reported as a further assessment of RNA integrity. Up to now, it is still questionable if we can use the 28S/18S ratio or the RIN, both based on the quantity and quality check of the ribosomal sub-units, to make a definite statement on the mRNA quality, which is the final target in qRT-PCR. Based on structural differences alone, it might be expected that the stability of mRNA differs from rRNA. Santiago et al. affirm that mRNA integrity corresponds more closely to 28S than to 18S integrity , while MIn conclusion, this paper does not claim to find an ideal method for extracting RNA from cartilage tissue, but it provides an overview of studies that have treated the topic. From these studies, only in a small part dedicated exclusively to the methodological investigation, the researcher can draw ideas for the experimentation that must be carried out.If the complexity of the topic, still too little debated, does not allow clear and definitive conclusions to be drawn, it is still useful to have available the papers that deal with it and the considerations that arise from it.


Canalicular efflux transporters, such as BSEP and MRP2, are crucial for the removal of BA/BS to the bile. Basolateral influx transporters, such as NTCP, OATP1B1/1B3, and OSTα/β, cooperate with canalicular transporters in the transcellular vectorial flux of BA/BS from the sinusoids to the bile. The blockage of canalicular transporters not only impairs the bile flow but also causes the intracellular accumulation of BA/BS in hepatocytes that contributes to, or even triggers, liver injury. In the case of BA/BS overload, the efflux of these toxic substances back to the blood via MRP3, MRP4, and OST α/β is considered a relief function. FXR, a key regulator of defense against BA/BS toxicity suppresses de novo bile acid synthesis and bile acid uptake, and promotes bile acid removal via increased efflux. In drug development, the early testing of the inhibition of these transporters, BSEP in particular, is important to flag compounds that could potentially inflict drug-induced liver injury (DILI). In vitro test systems for efflux transporters employ membrane vesicles, whereas those for influx transporters employ whole cells. Additional in vitro pharmaceutical testing panels usually include cellular toxicity tests using hepatocytes, as well as assessments of the mitochondrial toxicity and accumulation of reactive oxygen species (ROS). Primary hepatocytes are the cells of choice for toxicity testing, with HepaRG cells emerging as an alternative. Inhibition of the FXR function is also included in some testing panels. The molecular weight and hydrophobicity of the drug, as well as the steady-state total plasma levels, may positively correlate with the DILI potential. Depending on the phase of drug development, the physicochemical properties, dosing, and cut-off values of BSEP IC Membrane transporters comprise two large superfamilies, the solute carrier (SLC) superfamily and the ATP-binding cassette (ABC) transporter superfamily. The SLC superfamily typically facilitates the influx of substrates, but as most SLCs are exchangers, they may also operate as efflux transporters . MechaniSome transporters are better known for their roles in mediating the absorption–distribution–metabolism–excretion (ADME) of xenobiotics, including drugs . HoweverSome hepatocytic transporters, on the other hand, have mostly been investigated for their roles in the homeostasis of a special class of physiological substrates: bile acids and bile salts. The impaired homeostasis of bile components—bilirubin being another classical example —may leadDrug-induced liver injury (DILI) is the leading cause of drug development termination and drug withdrawal ,8. The pATP8B1, the PFIC type 2 with mutations in ABCB11/BSEP, and the PFIC type 3 with mutations in ABCB4/MDR3. Interestingly, children with different types of PFIC display a different spectrum of symptoms: failure to thrive is common in PFIC1, elevated ALT is common in PFIC2 (BSEP deficiency), and thrombocytopenia is common in PFIC3 (MDR3 deficiency) [Cholestasis is defined as decreased bile flow. Depending on the cause and site of the disturbance, cholestasis may be classified as intrahepatic if the dysfunction  is intrinsic to the liver or extrahepatic if the extrahepatic portion of the biliary tree is obstructed. At the level of clinical laboratory findings, according to the Council for International Organizations of Medical Sciences (CIOMS) criteria, liver injury is designated as cholestatic if (i) serum alkaline phosphatase (AP) activity is elevated to >2-fold of the upper limit of normal, or (ii) the alanine amino transferase (ALT) serum activity to AP serum activity ratio (ALT/AP) is ≤2 . The inciciency) .SLC10A1), organic anion transporting polypeptide 1B1 (OATP1B1)/solute carrier organic anion transporter 1B1 (SLCO1B1), and OATP1B3/SLCO1B3. Canalicular transport is mainly mediated using a bile salt export pump (BSEP)/ABCB11 and multidrug resistance-associated protein 2 (MRP2)/ABCC2. MRP3/ABCC3 and MRP4/ABCC4 provide the sinusoidal efflux function. The heterodimeric organic solute transporter (OSTα/β)/SLC51A/B is a facilitative transporter and may mediate transport into as well as out of the hepatocyte [Bile acids (BA) synthesized de novo in the liver undergo amidation to form bile salts (BS), also called monovalent bile acids. Most bile acids are then conjugated with glycine in healthy humans and taurine in rodents. Both BA and BS can undergo Phase II metabolism to yield sulfated or glucuronidated BA/BS. The Phase II conjugates of monovalent BS are also called divalent bile salts. The transport of BA/BS into hepatocytes is mediated by the sinusoidal influx transporters sodium taurocholate co-transporting polypeptide (NTCP)/solute carrier 10A1  membrane of hepatocytes . NTCP ism and an unchanged Vmax for taurocholate [The significance of NTCP was clearly demonstrated in a study on knockout mice that displayed hypercholanemia mainly composed of conjugated BS. Several variants described in humans ,29 showeSLC10A1 knockout mice and p.S267F humans [A case report described three children with hypercholanemia persisting beyond 1 year who were shown to be homozygotes for this mutation . The nonF humans .In vitro drug inhibition studies have almost exclusively used taurocholate (TC) as the probe substrate, and the NTCP variants show striking differences in the transport of taurocholate and rosuvastatin ,29. In hOf note, NTCP interacts with the scaffolding protein four-and-a-half LIM-domain 2 (FHL2) . The expOATP1B1 and OATP1B3 are influx transporters localized in the sinusoidal membrane of hepatocytes. Unlike NTCP, OATP1B1 and OATP1B3 are broad substrate specificity transporters with many drug substrates , which iOATP1B1 is the main transporter mediating sodium-independent BS uptake ,43, but SLCO1B1 polymorphisms on fasting total BA plasma levels have been contradictory [Slco1a/1b knockout mice [Although reports about the effect of adictory ,52, elevout mice . Follow-out mice .ABCB11, causes PFIC-II [BSEP plays a pivotal role in the canalicular transport of BA/BS . In huma PFIC-II . Interes PFIC-II . This is PFIC-II .The chemical inhibition of BSEP leads to decreased canalicular efflux and accumulation of BS in the hepatocyte . Unlike In hepatocytes, MRP2 is expressed in the canalicular membrane. MRP2/Mrp2 has been linked to cholestasis as a reduction in bile flow was seen in rats upon loss-of-function mutations or the chemical inhibition of Mrp2 ,62. HoweAbcc3 knockout mice [Abcc3 knockouts [MRP3 is a major efflux transporter expressed on the sinusoidal membrane of hepatocytes . It is gout mice ,75. LiveIn patients with obstructive cholestasis due to gallstones, MRP3 expression was increased 3.4-fold and 4.6-fold at the mRNA and protein levels, respectively , which iMRP4 is expressed on the sinusoidal membrane of hepatocytes . MRP4 haSLC51B) deficiency presents with features of cholestasis [OSTα/β is a complex that functions solely in a heterodimeric form . It is blestasis .In mice, however, Ostα/β may also mediate the uptake of BA as knocking out Ostα provided protection in the obstructed liver. It appears that in the Ostα knockout mice, the compensatory upregulation of the Phase II enzymes (Ugt1a1 and Sult2a1) and basolateral efflux pumps Mrp3 and Mrp4 led to increased efflux and a subsequent increase in the renal elimination of the Phase II metabolites of BA/BS, thus lowering the hepatic BA/BS load . The redA delicate balance of BA/BS, phospholipids, and cholesterol is key to the physicochemical stability of bile. The impaired homeostasis of phospholipids interferes with the excretion of BS and thus precipitates cholestasis. An important cooperation between the two major phospholipid translocases has been shown in a study where overexpression of MDR3 was toxic to HEK293T cells, and this toxicity was counteracted by the concomitant overexpression of the ATP8B1–CDC50A complex [The multidrug resistance protein 3 (MDR3) is a phosphatidylcholine floppase. The carriers of functionally defective variants of MDR3 have altered phosphatidylcholine/BS ratios and may develop cholelithiasis, intrahepatic cholestasis in pregnancy, or PFIC-3 . MDR3 haATP8B1 translocates phosphatidylserine from the outer leaflet to the inner leaflet. Consistent with its function as an aminophospholipid flippase, the treatment of Atp8b1-deficient rat hepatocytes with CDC resulted in focal canalicular membrane disruption and the luminal accumulation of NBD-phosphatidylserine . An impaHepatocytes maintain BA homeostasis by regulating the expression of rate-limiting enzymes that mediate BA synthesis and/or modulating the levels of BA transporters . An abunActing through FGF4R located in the sinusoidal membrane of hepatocytes, FGF19 represses the expression of CYP7A1, the rate-limiting enzyme of BA synthesis . FXR is The other key regulatory nuclear hormone receptor is the pregnane X receptor (PXR) which induces the expression of sulfotransferase SULT2A1 which, in turn, extensively sulfates BA/BS . PXR agoIt has been proposed that BA accumulation triggers adaptive and adverse responses . AdaptivCholestatic, but not non-cholestatic, drugs have recently been shown to inhibit the amidation and sulfation of BA in HepaRG cells . A 24 h Bile acid toxicity affects multiple organelles including the mitochondria, the endoplasmic reticulum (ER), and endosomes/lysosomes. Oxidative stress is one of the adverse responses inflicted on hepatocytes upon BA accumulation, and mitochondria are a major source of oxygen radicals . Unlike 2+ which triggers ROS production [Endoplasmic reticulum (ER) stress is also accompanied by the generation of reactive oxygen species (ROS) because the accumulation of misfolded proteins in the ER leads to the leakage of Caoduction ,126. Impoduction . This maoduction , and theoduction .Whereas native BA/BS are broadly considered the most relevant probes to be used in vitro, fluorescent substrates, BA analogs, and unrelated compounds alike have been employed in both vesicular and cellular assays ,131,132.The BA/BS uptake by NTCP, OATP1B1, and OATP1B3 has been studied routinely in cell lines overexpressing the respective recombinant uptake transporter ,43. OtheFor efflux transporter studies, membrane vesicle assays are the method of choice. Vesicular transport studies on bile salt transport by all major transporters including BSEP, MRP2, MRP3, and MRP4 have been published ,138,139.It is generally accepted that interference with BS transport is a key causal factor in cholestasis. Major publications from both the industry and the International Transporter Consortium have recommended the testing of BSEP inhibition. The relevant papers with suggested cut-off values ,148,149 ss/IC50 values use 0.05 and 0.1 as cut-offs [50/Chep,inlet,u < 26  may also serve as an indicator [plasma values are still only predicted and may thus be unreliable, risk assessment can be based solely on IC50, and different groups have suggested cut-off values of 25 [50/Cmax,total ≤ 1 and the safe zone for BSEP inhibition starting at IC50/Cmax,total > 100 [Studies referring to Ccut-offs ,144,145,ndicator . For comes of 25 . Aleo etal > 100 . In otheal > 100 ,108.ss/IC50 ≥ 0.1 has been considered a significant contributor to cholestasis and was suggested to be included in decision-making [ss/IC50 ≥ 0.1) BSEP inhibition [Whether in vitro inhibition assays for other bile acid and bile salt transporters may add value to DILI risk assessment is a matter of debate. Although the overall effect of the inhibition of influx and efflux transporters is challenging to quantify, initial integrative models have been published ,152. Inhn-making . Inhibitn-making ,154. Conn-making . Meanwhihibition .50 < 20 µM. However, whether the substrate transport was specific to MDR3 remained a concern [The genetic impairment of MDR3 function leads to PFIC-3, and the inhibition of MDR3 by antifungals has been linked to cholestasis ,156. The concern , and tes concern .Drugs may not only contribute to DILI by inhibiting bile salt efflux. Other mechanisms that may precipitate or aggravate hepatotoxicity include the blockade of FXR-mediated adaptive responses ,158 or t50 ≤ 25 µM threshold has been suggested [50 ≤ 50 µM may be a reasonable cut-off [ss,plasma/BSEP IC50 ≥ 0.1, testing for the inhibition of other relevant hepatocyte bile acid transporters may be needed [For compounds in the discovery stage, a BSEP ICuggested . In the  cut-off ,148. Fore needed .


In recent years, with the increasing incidence of colorectal cancer (CRC) and its high fatality rate, CRC has seized the attention of the world. And liver metastasis, as the main cause of death of CRC, has become the leading cause of treatment failure in CRC, especially metachronous liver metastasis, have caused patients who underwent bowel resection to experience multiple tortures.Metachronous liver metastasis has severely affected the quality of life and prognosis of patients. Therefore, in this review, we discuss risk factors for metachronous liver metastasis of CRC, which is the premise for effective intervention for CRC patients who suffer metachronous liver metastasis after undergoing surgery, as well as the signaling pathways associated with CRC.The occurrence of metachronous liver metastasis is closely related to histology-based prognostic biomarkers, serum-based biomarkers, tumor microenvironment, pre-metastatic niche, liquid biopsy and tissue-based biomarkers. Further research is required to explore the risk factors associated with liver metastasis of CRC. Due to globally increasing morbidity and mortality, more and more attention has been paid to colorectal cancer (CRC). According to GLOBOCAN 2020 statistics, CRC ranks the third most common malignancy in incidence, with more than 1.9 million new cases, whereas the second in cancer-related deaths . The medA large number of studies have demonstrated that patients with liver metastasis at the initial diagnosis have a poorer prognosis than those with metachronous liver metastasis , 12. EveLymph node status has been recognized as a prognostic factor for CRC. It is generally believed that primary tumors metastasize to local lymph nodes first, and then metachronous distant metastasis occurs through lymphatic system . UnexpecIn addition, most previous studies have suggested that the difference between metachronous and synchronous liver metastasis relied on location, size and differentiation of the primary tumor , 22. NevIn 2001, Vermeulen et al.  proposedCEA, a tumor-associated antigen expressed on the surface of cancer cells originated from endoderm, is a structural protein of cell membrane. In CRC, CEA-positive rate is no less than 90% . CEA hasRegardless of different sites of recurrence or metastasis, CEA was closely related to the liver , 35, 36.As far as we know, CEA is eliminated in the liver, so as long as the metabolic function of the liver is impaired, a high level of serum CEA may present even in benign diseases , 44. ForA retrospective analysis including 425 patients who diagnosed with relapsed or metastatic CRC reported that elevated CEA serum level was related to synchronous (but not metachronous) metastasis of metastatic colorectal cancer (mCRC) with poor prognosis . HoweverIn recent years, combination of CEA with hydroxylated collagen peptide in urine has improved the sensitivity of detecting liver metastasis , 51. TheInflammatory bowel disease (IBD), a chronic non-specific intestinal inflammatory disease, is a common precancerous lesion with 10–18% chance of developing CRC –55. In tMoreover, circulating inflammatory markers are associated with aggressiveness of CRC and may serve as predictors of metachronous liver metastasis , 60–62. Chemokines are chemotactic cytokines specifically bound to G-protein-coupled receptors, which can promote migration and colonization of inflammatory cells (such as white blood cells) towards tumor sites . In CRC,As for tumor microenvironment in the liver, the density of mononuclear inflammatory cells infiltrating in primary tumor can be in proportion to that in metastatic lesion . AlthougIn recent years, liquid biopsy has gradually become a new alternative strategy to traditional biopsy, through real-time dynamic analysis of tumor composition. To minimize tumor heterogeneity in terms of space and time, liquid biopsy may overcome limitations of traditional biopsy. The most common liquid biopsy relies on circulating tumor cell (CTC) and circulating tumor DNA (ctDNA) –93. ctDNDispersal of a small number of tumor cells differs from metastasis. Metastasis can occur when highly heterogeneous mutation is initiated and a large number of tumor cells are disseminated. The number of metastatic tumor cells is actually very small and clusters of tumor cells are more likely to metastasize than individual one , 99. CTCAt present, selection of targeted therapy and evaluation of drug sensitivity vs. resistance can be achieved according to gene mutation landscape in combination with signaling pathways. Different gene mutational blueprints present in primary tumor and metastasis during tumor development and progression . FurtherRAS gene family Fig. , includiBRAF Fig. , a compoMargonis et al.  claimed BRAF V600E mutation positively correlates with MSI, which is caused by loss of DNA mismatch repair (dMMR) expression , 132. SiPIK3CA  is an important signal transduction factor downstream of EGFR Fig. , with muAmong HER family members, besides EGFR, HER2, HER3 and HER4 also play an important regulatory role in the physiological functions of cells and the pathogenesis of solid tumors. HER2, in particular, is currently recognized as an oncogenic driver and has been proven to be one of the causative genes of breast cancer. The poor prognosis of breast cancer is associated with HER2/neu protein overexpression due to HER2/neu gene amplification, which is similar to colon cancer, and HER2 amplification is used in the treatment of CRC as one of the mechanisms of cetuximab resistance , 137. SaIn recent years, the expression of not only HER2, but also HER3 in CRC has gradually attracted attention. One study examined the expression status of HER3 in mCRC patients and concluded that there was a moderate correlation between HER3 expression in primary tumors and liver metastasis in CRC, and there was no difference in the expression of HER3 in synchronous and metachronous liver metastasis. Controversially, Styczen et al.  enrolledAt present, whether HER3 can be used as a predictor of metachronous liver metastasis is still in the initial stage of research. The high consistency of HER3 in primary tumors and liver metastasis provides a greater possibility for it to be a high-quality predictor.Clinically, the depth of primary tumor (T) invasion and lymph node (N) involvement have become indispensable indicators for predicting prognosis of CRC. In addition, tumor differentiation, site, venous or lymphatic invasion, as well as molecular biomarkers are associated with recurrence and prognosis of CRC –147. TheAlthough liver metastasis has been emphasized in CRC therapy, due to technical limitations, individual differences, and tumor heterogeneity, micro-metastasis is difficult to detect at the time of initial diagnosis. With the development of individualized treatment/precision medicine, adjuvant therapy may enable early diagnosis of liver metastasis. Currently, there is no consensus on defining synchronous or metachronous metastasis. Engstrand et al.  includedTumors are of polyclonal origin, which harbor spatial heterogeneity  and temporal heterogeneity (variation in kind or arrangement of components across time) –152. GenAt present, MAPK pathway is the major target for CRC liver metastasis treatment. Patients with metachronous liver metastasis usually receive chemotherapy or targeted therapy. Therefore, for patients who suffer from metachronous liver metastasis as demonstrated by imaging or symptoms, genetic mutation landscape, derived from various primary tumors or driven by secondary targeted therapy-induced mutations, is impossible to verify. Especially after receiving systemic chemotherapy for patients with no distant metastasis after operation, occurrence of metachronous metastasis and timing of metachronous metastasis will influence prognosis. In addition, targeted therapy may modify primary tumor tissue and serological indicators, and cause artificial interference to liver metastasis, and such error is unavoidable.In conclusion, biomarkers and gene expression associated with metachronous distant metastasis of CRC can be reflected by primary tumor. To monitor patients after primary tumor resection can help early detect distant metastasis, especially liver metastasis. These biomarkers predict metachronous liver metastasis, so that patients' survival rate and quality of life may be improved. Furthermore, more mechanistic research is required to explore the progression of CRC and what factors accelerate the occurrence of metachronous distant metastasis.


An essential step in restricting HIV infectivity by the antiviral factor APOBEC3G is its incorporation into progeny virions via binding to HIV RNA. However, the mechanism of APOBEC3G capturing viral RNA is unknown. Here, we report crystal structures of a primate APOBEC3G bound to different types of RNAs, revealing that APOBEC3G specifically recognizes unpaired 5’-AA-3’ dinucleotides, and to a lesser extent, 5’-GA-3’ dinucleotides. APOBEC3G binds to the common 3’A in the AA/GA motifs using an aromatic/hydrophobic pocket in the non-catalytic domain. It binds to the 5’A or 5’G in the AA/GA motifs using an aromatic/hydrophobic groove conformed between the non-catalytic and catalytic domains. APOBEC3G RNA binding property is distinct from that of the HIV nucleocapsid protein recognizing unpaired guanosines. Our findings suggest that the sequence-specific RNA recognition is critical for APOBEC3G virion packaging and restricting HIV infectivity. Interaction between APOBEC3G and RNA is critical for its antiviral function. Here, the authors report four co-crystal structures of rhesus macaque APOBEC3G and RNA, demonstrating unpaired AA and GA dinucleotide motifs are preferentially recognized. Inside HIV-1 viral producer cells, a critical step before hA3G lashes antiviral activity against HIV-1 is its incorporation into progeny viral particles via binding HIV RNA22. Upon arriving at recipient cells, packaged hA3G deaminates HIV-1 negative cDNA during viral reverse transcription, causing massive hypermutation in the viral genome. In addition, hA3G shows a certain level of deaminase-independent HIV restriction activity30, which is proposed to be through affecting HIV reverse transcriptase (RT) function or/and direct binding to viral RNA to interfere with viral cDNA replication.Human APOBEC3G (hA3G) is a member of apolipoprotein B mRNA editing enzyme catalytic polypeptide like (APOBEC) family31. Multiple RNA binding residues of A3G have been identified previously by various approaches41. However, how A3G can selectively bind G-rich and A-rich RNA sequences to promote its virion packaging is unclear.hA3G has two cytidine deaminase domains in tandem, the N-terminal CD1 (or NTD) and C-terminal CD2 (or CTD). The ability to bind nucleic acids and form oligomerization is attributed mainly to CD1/NTD (referred to as CD1 hereafter), while deaminase activity is performed by CD2/CTD (referred to as CD2 hereafter). RNA crosslinking CLIP-seq data show that hA3G and two other human APOBEC family members, APOBEC3F (hA3F) and APOBEC3H (hA3H), display preferential binding to G-rich and A-rich RNA sequences that are not scanned by ribosomes during translation42. Despite some commonly shared interfaces at CD1-CD2 boundaries, all these three reported forms show different CD1-CD2 domain arrangements with significant differences in CD1-CD2 bonding contacts. So far, the precise domain arrangement when rA3G is bound with RNA or DNA is unknown.Poor solubility and the tendency to form large aggregates have been two major obstacles to A3G structural study. However, wild-type and soluble A3G variants of individual domains and soluble variants of the full-length A3G of human and primate origin yield multiple structures, revealing atomic arrangements of various forms of A3G. Specifically, there are three previously reported full-length structures of A3G variants containing no oligonucleotide  or a deoxy-cytidine dinucleotide bound near the CD2 Zn-center (PDB ID: 6WMA)47. Our structure-guided mutational studies using both rA3G and hA3G suggest that the sequence-specific RNA-recognition mechanism is critical for A3G virion packaging and restriction against HIV infectivity.In this study, we obtained the co-crystal structures of a primate rhesus macaque APOBEC3G (rA3G) binding to several types of RNA substrates, resulting in monomeric rA3G-ssRNA and dimeric rA3G binding to 5′- or 3′-overhangs on both ends of a double-stranded RNA (dsRNA). Surprisingly, all these structures reveal a unique CD1-CD2 domain arrangement of rA3G when in complex with the RNA molecules. The structures also illustrate a molecular mechanism for rA3G to specifically recognize unpaired adenine dinucleotide (AA) RNA, and, to a lesser extent, GA (but not AG) dinucleotide RNA. This differs from the RNA binding property of the HIV nucleocapsid protein (NCp7), which targets unpaired guanosines139CQKRDGPH146 to 139AEAG142)41 and designated this construct as rA3GR8 in this study. We found that rA3GR8 expressed in Escherichia coli binds to bacterial RNA that can be degraded to various degrees by RNase treatment and can be partially dissociated from rA3GR8 during purification41. As a result, fractions of various amounts of RNA-bound or RNA-free rA3GR8 can be purified. An attempt to crystallize the purified rA3GR8 fraction with bound RNA was unsuccessful, possibly due to heterogeneous species of co-purified RNA.We tried to obtain the complex structure of A3G binding to RNA to understand how A3G interacts with RNA and how such RNA interactions mediate oligomerization and virion packaging. We successfully produced a soluble variant of rA3G carrying a replacement of N-terminal domain loop 8 (R8-RNA complexes in vitro using the purified RNA-free fraction of rA3GR8 and short synthetic RNA. Preliminary RNA binding tests using an RNA molecule comprised of a dsRNA with both 5′ and 3′ overhangs yielded a pronounced peak shift by size-exclusion chromatography (SEC). As a result, three types of RNA were designed to evaluate sequence-specific RNA binding: dsRNA with a short 5′ or 3′ overhang  at 5′ and 3′ overhang (RNA1-GG and RNA2-GG) showed KD ranging from 170 to 250 nM with more than tenfold less affinity. The RNA containing pyrimidine dinucleotides CC or UU at 5′ overhang or 3′ overhang (RNA1-CC/UU and RNA2-CC/UU) showed even less affinity, with KD values around 400 nM. The least favored were ssRNA with pyrimidine dinucleotide sequences (RNA3-CC and RNA3-UU). The ssRNA also displayed the most dramatic difference in KD value between AA and GG/CC/UU sequences. Collectively, these results indicate that rA3G has a strong preference for unpaired AA dinucleotides in RNA.We decided to reconstitute rA3GR8 binding to ssRNA , a nucleic acid chaperone that prefers unpaired guanosines (Gs) in RNA47 and is critical for the highly selective HIV RNA packaging process49. A purified RNA-free fraction of the NCp7 protein tagged by an N-terminal glutathione S-transferase  prefers AA dinucleotide RNA by analytical size-exclusion chromatography (SEC)  to 8 bp (dsRNA8). Additionally, RNA1-AA was modified to 5′ U1U2A3A4-dsRNA8-C13C14U15U16U17U18 with 5′ and 3′ overhangs on both strands  was used in the co-crystallization study (designated as rA3GR8/E259A). After multiple attempts, diffracting crystals of three rA3GR8/E259A-RNA complexes were obtained, and their structures were determined  on each AA dinucleotide-containing overhang 50. Lacking sufficient protection to RNA bound with rA3G is consistent with observations that rA3G-RNA is more sensitive to RNase treatment than A3H-dsRNA during recombinant protein purification.Each rA3Gang Fig. . Averagextal was determined as a monomer with one rA3GR8/E259A molecule binding to one ssRNA molecule at 2.10 Å   Fig.  and +90°3X) Fig. , c. The 2 that is comparable to the buried interface area in the different apo forms. Surprisingly, the interface identified herein overlaps with the previously described CD1 h6-h6 apo-rA3G dimer interface41, thus, the RNA-bound form precludes the h6-h6 mediated dimerization seen in the apo form. About 12 amino acid residues from CD1, two residues from the linker region, and 17 residues from CD2 are mapped to the interface  is used here as an example to depict the detailed interactions between rA3G and the AA (or A5A6) dinucleotide in the 5′-U4A5A6-3′ core sequence  and the 3′-end A as 3′A (or 3′A6) hereafter.The arrangement of CD1 and CD2 in the RNA-bound structures bridges a positively charged electrostatic potential surface area near the zinc-catalytic center of CD2 and a largely positive charged surface area of CD1 Fig. . Compari6 in the AA (or A5A6) dinucleotide is bound to an aromatic/hydrophobic “cave”, a pocket outside but right next to the zinc-center of CD1, forming strong hydrophobic interactions with the solvation energy effect of −2.47 kcal/mol  and loop 7 , fitting 3′A6 base snugly into the pocket. Furthermore, OP1 of U7 phosphate oxygen (at the 3'-end of 3'A6) forms hydrogen bonds with the side chains of three tyrosine residues Y59, Y124, and Y125 , indicating that 3′-phosphate is critical in RNA binding, and three tyrosine residues have somewhat overlapping RNA binding function. In silico analysis reveals that placing a guanine nucleotide inside the pocket would generate a clash between the C-2 amino group of the guanosine and the aromatic ring of Y125. Additionally, the C-6 carbonyl group of the guanine base is highly unfavorable when in proximity (3.0–3.1 Å) to the two backbone carbonyl groups of the residues P25 and L123  dinucleotide is bound to a groove conformed by the CD1 and CD2, also forming strong hydrophobic interactions with the solvation energy effect of −3.09 kcal/mol . K270 of CD2 makes contacts through hydrophobic packing with 5′A5 via its aliphatic side chain. Unlike the tight interaction between the cave-like pocket and the 3′A6, the shape of this groove could snugly fit with a purine base (A5 or G5) with similar hydrophobic stacking interaction, but a much weaker fit with a smaller pyrimidine base (U5 or C5). However, a carbonyl group (C-6) of guanine (G5) at the equivalent position is unfavorable next to the carbonyl group of F268. This could explain the observed rA3GR8 binding preference for the sequence motif 5′-A5A6–3′ over 5′-G5A6–3′ . S28 uses its side chain and the main chain to form two hydrogen bonds with U4 2′-OH present only in RNA (bond lengths 2.8 and 3.1 Å), suggesting that S28 may specify RNA interaction. The S28 side chain and main chain also form two hydrogen bonds with U4 sugar (O3′) and base (O2) (bond lengths 3.5 and 3.1 Å). These RNA-specific interactions of R24 and S28 are consistent with the published mutational data, showing R24A and S28A reduced RNA binding34. I26 and L27 provide hydrophobic contacts with U4. Additionally, the NH2 group of K270 is 4.7 Å away from the U4 phosphate backbone, which has the potential to form a weak hydrogen bond interaction directly or through a water molecule.Umol Fig. . R24 not5A6-3′ dinucleotides within the sequence 5′-U1U2U3U4A5A6U7U8U9U10-3′, forming exceptionally strong hydrophobic interactions via the two adjacent surface features: the pocket on CD1 (for 3′A6) and the CD1-CD2 interface groove (for 5′A5). Furthermore, 14 hydrogen bonds form between the sequence motif 5′-U4A5A6-3′ and the rA3G residues located on the CD1 loops 1, 3, 7, and CD2 (F268). Lastly, rA3G binds to the rest of the ssRNA (U7/U8/U9), forming additional seven hydrogen bonds  as a positive control. Under similar crystallization conditions as those produced diffracting co-crystals of RNA3-AA with rA3GR8/E259A, only RNA3-GA with rA3GR8/E259A co-crystallization was successful. Failure to form crystals with ssRNA containing the AG and GG dinucleotide motifs under the experimental conditions is likely caused by their weak binding affinities with rA3GR8/E259A, and, thus, lack of stable complex formation. These results also confirm that rA3G favors the purine dinucleotide sequence motifs AA/GA over AG/GG.To further gain structural basis into rA3G GG Fig. , co-crysR8/E259A was determined as a monomer with one rA3GR8/E259A molecule binding to one ssRNA molecule at 1.83 Å , much-reduced hydrophobic interaction was found with 5′G5, as the solvation energy effect was −0.74 kcal/mol. Further analysis shows that the groove allows the shift of 5′G5 (vs 5′A5) so that the reoriented 5′G5 avoids clashes of its carbonyl at the C-6 position with the F286 main-chain carbonyl group and uses its N1 to form a hydrogen bond with the F286 main-chain carbonyl  and in a negative environment at the C-terminal end of an alpha helix. These factors can explain the less favorable binding of 5′G5 than 5′A5.The complex structure of the RNA3-GA with rA3G3 Å Fig. . Six nucsly Fig. . While t8). Even though the bulk of two individual dsRNA8 situated at two different locations on the rA3G surface, the unpaired AA dinucleotides on the 5′- or 3′-overhang of each dsRNA8 is the common key to be recognized by rA3G  3′ to the bound AA core (5′-A3A4-3′) is hovering over the surface of CD1-loop 3 (58VYP60). The side chain of the loop 3 residue Y59 shows no clear electron density at 1 sigma contour level, suggesting it doesn’t make strong contact with the RNA.In the 5′-overhang dsRNA co-crystal structure, dsRNARNA Fig. . A C-G b8 and rA3G, resulting in a relatively rigid structure with defined electron density for the dsRNA. The dsRNA8 has a G-C base pair (G8-C1) 5’ to the bound AA ssRNA core (5′-A9A10-3′)  making five hydrogen bonds with S28 through its base/sugar moiety (bond lengths 2.5−3.7 Å), including two that are guanine base specific . Additional five hydrogen bonds are present between the side chains of R24/N176/N177 and the phosphate backbone of C5, G6, G7, and G8 of the dsRNA (bond lengths 2.8−3.7 Å). Contacts with the phosphate backbone and the base G8 in the dsRNA region enhances the overall stability of the dsRNA8 structure.In the 3′-overhang dsRNA co-crystal structure, there are multiple contacts between dsRNA3′) Fig. , with G826ILS28) or loop 7 (124YY125 and 126FW127). These residues are all critically involved in binding to the AA dinucleotides in the rA3G structures and have been reported by multiple previous biochemistry studies to affect RNA association and dimerization in human A3G40. Both mutant 4 and 5 carry two mutations from the CD2 domain, F268A and K270A, intended to disrupt the observed interaction with the 5′A of the AA dinucleotide at the CD1-CD2 interface. Mutant 5 also carries additional CD1 mutations, Y154R/P179E, to disrupt their contacts with CD2 loop 3 as in the RNA-bound conformation. Thus, mutant 5 containing F268A/K270A plus Y154R/P179E mutations is intended to disrupt the binding of the 5′A by CD2 residues and, at the same time, weaken the CD1/CD2 interface. Mutant 6 carries three mutations on CD1 h6, L184E, A187E, and T188E, intended to disrupt the CD1/CD2 interface of the RNA-bound form.We tested the virion packaging and HIV-1 restriction by the wild-type rA3G and hA3G and their mutants using HIV-1 (ΔVif) in cell culture Fig. . These rThe results show that virion packaging is most defective in rA3G mutants 1, 2, and 3 Fig. , which a31.To examine the RNA binding affinity of mutants 1–6 of rA3G, we performed RNA binding assays comparing the same concentration gradient of purified RNA-free rA3G mutant proteins binding to AA dinucleotide RNA Fig. . MutantsR8/E259A, in complex with AA and GA dinucleotide-containing RNA molecules in the form of ssRNA and dsRNA with 5′- or 3′-overhangs. These structures reveal a molecular mechanism of rA3G specifically recognizing and binding unpaired 5′-AA-3′ and 5′-GA-3′ dinucleotide sequences. Hydrophobic interactions are the main driving force as rA3GR8/E259A monomer binds 3′A via a highly specific hydrophobic/aromatic cave-like pocket on the non-catalytic domain CD1 and 5′A or 5′G in a hydrophobic groove formed at the CD1-CD2 interface identified in all four RNA-bound rA3G structures in this study. Comparison of rA3G binding features for the ssRNA AA dinucleotide with that of modeled hA3G structure reveals a set of identical residues interacting with the AA dinucleotide RNA sequence  reveals additional similarities and differences. In both cases, there are symmetric short ssRNA overhangs on both ends of the dsRNA interacting with the respective proteins, suggesting their critical role in RNA-assisted dimer formation. Moreover, at least one unpaired base in each overhang is captured by a similar aromatic/hydrophobic pocket on a monomer. However, the polarity of the bound ssRNA is opposite when considering the similar hydrophobic pockets in both rA3G and A3H binding to the ssRNA. Second, rA3G has more extensive interactions with the 3-nt ssRNA that can be at both 5′- or 3′-overhang, with CD2 of rA3G participating in binding to one of the two adenine bases. A3H interacts with 2-nt ssRNA on 5′-overhang. Third is that rA3G mainly interacts with one strand (the AA dinucleotide-containing strand) of the dsRNA, while A3H shows significant contact with both strands of dsRNA.Previously reported A3H dimer structures also show dimerization through the bound RNA without any protein-protein contact55. In this study, our structural and biochemical data explain why rA3G specifically favors unpaired 5′-AA-3′ over 5′-GG-3′ or any other dinucleotides. The 3′A binds to the hydrophobic pocket of rA3G as the major anchoring point, and the 5′A binds to the CD1-CD2 interface groove to enforce the anchoring. Our data also reveal that, while the CD1 pocket can only bind the 3′A, the interface groove for the 5′A can also bind a 5′G with less favorable binding energy but cannot bind 5′C/5′U well. Our structural data, together with our biochemical data, suggest that rA3G has the strongest affinity for RNA containing unpaired 5′-AA-3′ (KD of 10 to 17 nM), a lesser affinity for 5′-GA-3′ , much less affinity for 5′-AG-3′ , and even lower affinity for 5′-GG-3′ (KD of 1500 nM).An extensive body of prior hA3G research has shown the promiscuous nature of RNA binding with a preference for G-rich and A-rich sequenses56. This bias toward A nucleotide in the ssRNA regions becomes even more profound in the viral RNA structure as determined by SHAPE, reaching 47.5% for unpaired A, while having only 21.3, 19.2, and 11.9% for unpaired U, G, and C, respectively57. While unpaired Gs are likely associated with viral RNA chaperons58 such as HIV NC, unpaired AAs are not known to be tightly sequestered by the HIV NC or other viral proteins. Therefore, a preference for unpaired AA sequences in RNA could be advantageous for A3G as a host restriction factor. Viral RNA with multiple numbers of unpaired and accessible AA  dinucleotides can result in dimerization and multimerization of bound rA3G on RNA. Such “aggregation” of A3G to the enriched AA dinucleotides on viral RNA could serve as a potential mechanism for selective A3G encapsidation into HIV virions. Further study can focus on in vivo validation by reducing or increasing the unpaired AA dinucleotide content in the HIV-1 genome using synonymous mutagenesis and investigating the impact on A3G HIV-1 virion packaging and HIV restriction. With improved protein sample quality of human and other primates A3G, one can conduct structural studies related to sequence-specific nucleic acid recognition to uncover biochemical properties and functional mechanisms of human and primate A3G proteins, and other double domain APOBECs.HIV-1 genomic RNA  is known for having a higher percentage of adenine (A) (36.2%) while having a low cytosine (C) (17.6%)AGE34493) carries a replacement of N-terminal domain loop 8  was constructed in the pET-sumo vector (Thermo Fisher) and generated a sumo fusion that carries an N-terminal 6xHis tag and a PreScission protease cleavage site41. Briefly, rA3G expressing E.coli Rosetta™(DE3)pLysS cells were cultured at 37 °C in LB medium supplemented with 50 μg/ml kanamycin until OD600 reached 0.3, the growth temperature was then lowered to 16 °C, and E. coli cells were further cultured. When OD600 reached 0.7–0.9, protein expression was induced by IPTG. E. coli cells were further cultured at 16 °C overnight and harvested by centrifugation . Cell pellet was lysed in buffer A  by sonication. Cell supernatant was obtained after centrifugation  and the 6xHis sumo rA3GR8 fusion was captured by Ni-NTA agarose gravity-flow chromatography and eluted with 0.5 M imidazole in buffer B containing 0.25 M NaCl . Ni elution fractions were concentrated and switched to buffer B containing 0.5 M NaCl and 0.1 mM EDTA. RNase A (1 μg/μl) and RNase T1 (2 units/μl) were included in the concentrated Ni elution fraction and left overnight in a cold room. PreScission protease was then added for 2 to 3 h to remove the sumo tag, followed by the initial size-exclusion chromatography (SEC) step using Superdex 200 pg (HiLoad™ 16/600) column equilibrated in buffer B with 0.5 M NaCl on an AKTA Pure chromatography system. The peak fractions of the cleaved rA3G were pooled, concentrated, and passed through a HiTrap 5 ml Heparin HP column using the binding buffer (buffer B with 0.5 M NaCl) and elution buffer (buffer B with 2 M NaCl). The final size-exclusion chromatography polishing step was done on a Superdex 200 pg (HiLoad™ 16/600) column equilibrated in buffer B with 0.25 M NaCl. Peak fractions were pooled, concentrated to 7−9 mg/ml, and stored at −80 °C until use. Sequences of all mutant constructs were verified by Sanger sequencing. Mutant proteins were purified using the same protocol. The final polishing step by SEC was omitted if the absorbance ratio (A260/A280) of the protein sample after heparin column purification was less than 0.6, indicating the protein sample was largely free of RNA. Each protein sample was quantified by NanoDrop with the absorption coefficient correction and verified by 12% SDS-PAGE  was synthesized (Thermo Fisher) and cloned into an Escherichia coli expression vector pGEX6P1 as an N-terminal glutathione S-transferase and nucleocapsid fusion (GST-NC). The sequence of the GST-NC construct was verified by Sanger sequencing. Expression of GST-NC or GST in the E. coli XA90 strain was carried out following the expression protocol for rA3G41. Cell pellet of GST-NC or GST was lysed in buffer A. Cell supernatant of GST-NC or GST was obtained after centrifugation . GST-NC or GST was captured by Glutathione agarose gravity-flow chromatography and eluted with 40 mM reduced Glutathione in buffer B containing 0.5 M NaCl. Elution fractions were concentrated, and a similar RNase treatment as described above was carried out with the GST-NC sample. The initial size-exclusion chromatography (SEC) step for GST-NC or GST was carried out using Superdex 200 pg (HiLoad™ 16/600) column equilibrated in buffer B with 0.5 M NaCl. The peak fractions of GST were pooled, concentrated, and stored at −80 °C. The peak fractions of GST-NC were pooled, concentrated, and switched to buffer B with 0.25 M NaCl. It was further purified with HiTrap 5 ml Heparin HP column using binding buffer (buffer B with 0.25 M NaCl) and elution buffer (buffer B with 2 M NaCl). Peak fractions were pooled and concentrated. The final size-exclusion chromatography polishing step was carried out on Superdex 200 Increase 10/300 GL column equilibrated in buffer B with 0.25 M NaCl. Peak fractions were pooled, concentrated to 4−5 mg/ml, and stored in −80 °C until use. The absorbance ratio (A260/A280) of the purified GST-NC was 0.53 and the purified GST was 0.51, as measured by NanoDrop. Purified GST-NC and GST were verified by 12% SDS-PAGE . dsRNA with overhangs was prepared by mixing an equal volume of the two RNA strands and annealed by heating to 95 °C for 5 min, followed by slow cooling to room temperature.RNA oligonucleotides were purchased from Integrated DNA Technologies (IDT) and the sequences are listed in Supplementary Table TM TyphoonTM Biomolecular Imager  was used to visualize gel images.Formation of annealed dsRNA with overhangs using FAM labeled RNA or unlabeled RNA was verified by 20% native gel electrophoresis (acrylamide:bis-acrylamide ratio of 19:1) in 0.5X TBE buffer   mutation was purified using the same protocol as described above. rA3G-RNA complex was prepared by mixing rA3G (4 mg/ml) with RNase inhibitor and then added to the ssRNA with a molar ratio 1 to 1 , or to the annealed dsRNA with overhangs with molar ratio 2 to 1 . After incubation on ice for 1 h, precipitation was removed by centrifugation . Initial screening was carried out by sitting-drop vapor diffusion method using ARI Crystal Gryphon Robot (ARI) and crystallization screening kits (Qiagen and Hampton Research) at 18 °C. Crystallization hits were optimized by hanging drop vapor diffusion method at 18 °C. The diffraction-quality crystals of RNA1-AAxtal complex were obtained with the reservoir solution consisting of 1.8 M Na/K phosphate pH 6.9; crystals of RNA2-AAxtal complex were obtained with the reservoir solution consisting of 0.1 M KCl, 0.1 M Tris-HCl pH 8, and 12% PEG 2000 monomethyl ether; crystals of RNA3-AAxtal complex were obtained with the reservoir solution consisting of 0.22 M Na2SO4 and 20% PEG 3350.rA3GR8/E259A and RNA3-NNxtal where NN = AA, GA, AG, or GG were carried out in parallel by hanging drop vapor diffusion method at 18 °C. Guided by the crystallization conditions that produced diffracting co-crystals of rA3GR8/E259A and RNA3-AAxtal, we used a three-by-three screening grid with a reservoir solution combination of 0.18/0.20/0.22 M [Na2SO4] and 18/20/22% [PEG 3350]. Diffraction-quality crystals of rA3GR8/E259A and RNA3-GAxtal complex were obtained with the reservoir solution comprised of 0.18 M Na2SO4 and 20% PEG 3350, while no crystal was observed with RNA3-AG and RNA3-GG. We carried out another crystallization screening for RNA3-AG/rA3G and RNA3-GG/rA3G using the PACT suite (Qiagen) with 96 defined chemical solutions. One hit was identified, but it turned out to be the same as the hit for the apo-rA3G.Co-crystallization trials of rA3G2. X-ray diffraction data were collected at the Advanced Photo Source  beamlines 23ID-B and 23ID-D, and at the Advanced Light Source  beamline 5.0.3. Data were processed and scaled using HKL-2000 or automated data processing in JBluIce. Initial phase information was obtained by molecular replacement method with PHENIX using the crystal structures of the rA3G N-terminal domain (PDB 5K81) and the hA3G catalytic domain (PDB 3IR2) as search models. The structural model was refined using PHENIX59 and modified with COOT60. RNA was built manually in COOT and further refined using ERRASER61. Data collection statistics and refinement parameters are summarized in Table 63. Buried surface areas were calculated with QtPISA66. Structure images were prepared with PyMOL .Crystals were transferred to the synthetic mother liquor supplemented with suitable amounts of glycerol for cryoprotection and flash-cooled in liquid Nn PDB 5K8 and the TM TyphoonTM Biomolecular Imager  was used to visualize gel images. ImageQuant TL  was used for image quantification. Dissociation constant KD was calculated using GraphPad Prism version 8.4.3 for Windows, GraphPad Software, San Diego, California USA, www.graphpad.com. Three independent experiments were carried out for each RNA molecule.RNA with 6-FAM at 10 nM was titrated by rA3G in 20 μl reaction volume containing 50 mM HEPES pH 7.5, 250 mM NaCl, 1 mM DTT, 0.4 units/μl RNase inhibitor, and 10% glycerol. Reaction mixtures were incubated on ice for 10 min and analyzed by 8% native PAGE at 4 °C. An acrylamide:bis-acrylamide ratio of 29:1 was used in preparing 8% native gel for overhang dsRNA complexes, and a higher acrylamide: bis-acrylamide ratio of 72.5:1 was for ssRNA complexes. AmershamRNA binding was assayed using gel filtration. Samples of rA3G protein alone (1.7 or 3.3 μM), RNA alone (1.7 or 3.3 μM), or rA3G-RNA mixture at 1:1 molar ratio, were applied to a Superdex 200 Increase 10/300 GL column (Cytiva) in buffer B with 0.25 M NaCl and 0.4 units/μl RNase inhibitor. Elution profiles of relevant samples were superimposed, and peak positions were compared.41. HEK293T cells were cultured in DMEM supplemented with 10% FBS and Penicillin (100 U/mL)/Streptomycin (100 μg/mL) and maintained at 37 °C, 5% CO2. The cells were co-transfected with 500 ng of pHIV-1 LAI ΔVif ΔEnv (NIH AIDS Reagent Program), 180 ng of pMD2.G , and between 60−200 ng of pcDNA3-N-terminus-1xFLAG tagged A3G expressing plasmid to achieve similar expression levels of A3G wild-type and mutants, using Lipofectamine™ 3000 Transfection Reagent (Thermo Fisher). Empty pcDNA3 was used to equalize the amount of transfected plasmid DNA. The medium was changed after 24 h and virus-containing supernatant was collected 24 h after the medium change. The supernatant was filtered through a 0.45 μm PVDF filter (Millipore). To determine viral infectivity, TZM-B1 reporter cells (NIH AIDS Reagent Program) were plated at 1 × 104 cells per well of a 96-well plate and infected with virus normalized by p24 levels . After 48 h of infection, the cells were washed with PBS, and the levels of infectivity were measured by colorimetric detection with a Pierce™ β-galactosidase assay reagent (Thermo Scientific) using a spectrophotometer . The relative viral infectivity was quantitatively compared under the condition of No A3G as 100%.The Single-cycle replication assays with the VSV-G pseudotyped HIV-1 LAI ΔVif ΔEnv were performedg at 4 °C for 30 min. The transfected HEK293T cells (cell lysates) and the precipitated virions were lysed using 1x RIPA buffer (Sigma). Western blot analysis was performed with anti-FLAG M2 mAB  to determine A3G levels in lysates and virions, respectively. For each internal loading control, α-tubulin  was used for cell lysates and p24  was used for virions. Cy3-labeled goat-anti-mouse mAb  was subsequently used as a secondary antibody. Cy3 signals were visualized using Typhoon RGB Biomolecular Imager  and quantitatively measured with ImageQuant TL  to show normalized A3G levels.Filtered virions from the Single-cycle assays were precipitated with NaCl  and PEG-6000  at 4 °C for 6 h and centrifugated at 3724×Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary