N = 55) and Vietnam (N = 43). The main purpose of transportation was to transport duck flocks between rice paddies used for scavenging. Trucks were commonly utilized for transport in both countries , while boats were only used in Vietnam . Transporters in Vietnam moved larger flocks and traveled over longer distances. Deaths of ducks due to diseases were reported in both countries . Throwing away of carcasses was the primary method of disposal of dead birds in Indonesia , but was not practiced in Vietnam (p < 0.001), while more transporters in Vietnam  buried carcasses compared to Indonesia . Consumption of carcasses , sale of dead ducks  and processing of ducks for fish feed  was conducted in Vietnam, but not in Indonesia. Vehicles were predominantly cleaned in rivers and stored outside in Vietnam, while cleaning and storage was usually conducted in houses/garages in Indonesia. In conclusion, we identified management practices that potentially impact transmission of avian pathogens, such as HPAI virus. In Indonesia, unsafe management practices were related to multipurpose usage of transport vehicles and disposal of birds in the environment, while in Vietnam, they were related to the mixing of birds during transport, the processing of dead carcasses and the storage and cleaning of transport vehicles.Highly pathogenic avian influenza (HPAI) virus is endemic in Indonesia and Vietnam, where “moving” duck production is commonly practiced. Questionnaire surveys were conducted with transporters of “moving” duck flocks in Indonesia ( Highly Pathogenic Avian Influenza (HPAI) H5N1 virus is endemic in Vietnam and Indonesia and has caused substantial human and poultry losses . From 20From 2004 to 2019, 22.5 and 20.0% of the global HPAI outbreaks in poultry occurred in Indonesia and Vietnam, respectively , while iDuck farming is an important sector of the poultry industry in Indonesia and Vietnam. Duck management is classified into “stationary” and “moving” duck production, with stationary flocks allowed to graze around the village vicinity and secured at night near village houses, while moving flocks are moved between areas of recent rice harvests and kept in confinement overnight close to the daytime scavenging locations –10. MoviThe objectives of this study were to (1) identify management factors during transport of moving duck flocks in Indonesia and Vietnam that could potentially be associated with an increased risk of avian pathogen dissemination , and to (2) compare and contrast differences in movement, management and biosecurity practices implemented by duck flock transporters in Indonesia and Vietnam.Previous research conducted with moving duck flock owners in Indonesia and Vietnam in 2008 and 2009 described the HPAI H5N1 infection status of ducks and the movements elected by owners for their duck flocks (Data collection was conducted in Indonesia by veterinarians from the Disease Investigation Centre (DIC) in Wates, Yogjakarta and in Vietnam by veterinarians from The Regional Animal Health Centre VI, Ho Chi Minh City using an interview process using local languages. All interviewers were trained in data collection.The study design for this research was reviewed and approved in Indonesia by the Disease Investigation Centre (DIC) in Wates, Yogjakarta; and in Vietnam by the Regional Animal Health Centre VI, Ho Chi Minh City. Data collection for this study was conducted in accordance with the accepted survey guidelines for surveillance activities of both organizations.Questionnaires were developed in English and later translated into the national languages  in order to capture potential associated risk factors associated with spread of avian pathogens such as HPAI virus during the transport of duck flocks: type of transport utilized; number of flocks (and ducks) transported per time period; age of ducks transported; number of duck flocks combined in a transport load; other poultry species transported; number of farms visited to obtain one transport load; cleaning and disinfection before and after transport; location where the transport vehicles were stored, cleaned and disinfected; distance and duration of transport; management of ducks before departure, during transport and after arrival in scavenging area; contact of transported ducks with other poultry and other animals; frequency of transporting ducks; raising of ducks at home by people loading and transporting poultry; transport of items  together with ducks; experiences of sickness or deaths of ducks during transport; disposal of dead ducks; and occurrence of health problem in people loading and transporting ducks. Thus, the questionnaires included a mixture of closed and open-ended questions. Copies of the questionnaires are provided in the Pilot testing of the questionnaires was performed with two transporters in both countries before conducting the main survey to identify any problems, misunderstandings or to discover additional risk factors of interest that should be surveyed. The questionnaires were updated accordingly. The total number of questions in the questionnaire was 39, with identical questions being used in Indonesia and Vietnam.tabplot- in Stata was used to visualize the frequency of responses provided on a 4-point Likert scale. The total number of survey responses for each response category were compared between Indonesia and Vietnam using the Fisher's exact test. To facilitate the utilization of the Fisher's Exact Test for data analysis, Likert scale groups “very important” and “important” were combined into a category “importantly” and Likert scale groups “not important” and “not conducted” were combined into a category “not importantly.” Similarly, Likert scale groups “common” and “sometimes” were combined into a category “commonly” and Likert scale groups “seldom” and “not conducted” were combined to a category “infrequently.” The non-parametric Mann–Whitney U-test was used to compare ordinal and not normally distributed continuous variables between Indonesia and Vietnam.Data analysis was conducted in SPSS  and STATA . Descriptive analysis included the calculation of frequencies, means, medians and range values. The command –A total of 114 transporters of moving duck flocks were interviewed, with 16 transporters being excluded from the analysis as they provided incomplete information in the questionnaire. Thus, 98 transporters provided completed responses to all questions and were analyzed in detail, consisting of 55 transporters from Indonesia and 43 from Vietnam .p = 0.58). Although not statistically significant, transporting of ducks to markets was less common in Indonesia , while transport of ducklings to and from hatcheries was more common in Indonesia. Items transported together with ducks include chickens, other birds, feed, and eggs (p = 0.09). Additionally, 40.0% of transporters from Indonesia indicated that they “commonly” transport eggs together with ducks, but this was either seldom or not practiced in Vietnam (p < 0.001)  .p = 0.0014), and transport of non-animal related items , with the most common items transported being building materials (p < 0.001), with farming being the most common activity in both countries , while boats were the most common mode of transport in Vietnam , but were not utilized in Indonesia (p < 0.001). Motorbikes were used by one transporter in each country. Almost all transporters (99.0%) utilized only one type of transport.Trucks, boats and motorbikes were used for transportation . Trucks Of the 70 transporters, who provided data on truck designs, 95.7% (67/70) of trucks were not covered or closed. Trucks had between 1 and 4 levels, with the majority of them  having three levels. Boats were generally open  and had between 1 and 3 levels, with 85.7% (24/28) of them having three levels. Neither of the two motorbikes was covered.p < 0.001). Vietnamese transporters indicated more frequent exposure of their vehicles to wild birds compared to Indonesian transporters .Respondents from Vietnam were more likely to store their transport vehicles outside  compared to Indonesia .The volume of transport and distance traveled in Indonesia and Vietnam are shown in p < 0.001 and p < 0.001, respectively). About 34.5% (19/55) of transporters in Indonesia collected and 53.7% (29/54) delivered moving ducks to village areas compared to 58.1% (25/43) and 65.1% (28/43) in Vietnam , highlighting that within-village scavenging areas are more common in Vietnam.The locations from where ducks were collected from and transported to by transporters are shown in Collection and delivery of ducks to markets was uncommon for both Indonesia and Vietnam, with about 11% and < 5% of transporters in Indonesia and Vietnam, respectively, “commonly” conducting this practice.p = 0.17) did “commonly” return with other ducks.Characteristics of return journeys are outlined in A higher proportion of transporters in Vietnam compared to Indonesia provided care to birds during transport. In Indonesia, water and feed was provided to ducks by 13.0% (7/54) and 7.4% (4/54) of transporters, respectively, compared to 88.4% (38/43) and 65.1% (28/43) of transporters, respectively, in Vietnam. Spraying of birds with water was conducted by 5.6% (4/54) of transporters in Indonesia and 32.6% (14/43) of transporters in Vietnam, while rest stops for ducks were provided by 11.1% (6/54) of transporters in Indonesia and 18.6% (28/43) of transporters in Vietnam.Out of 41 transporters providing information on the number of duck deaths per truck load in Indonesia, the mean number  of duck deaths per load was 2.4 , while for Vietnam, out of 16 transporters providing information, the mean number  of duck deaths per truck load was 3.4 . For 26 transporters with boats from Vietnam who provided data, the mean number  of duck deaths per load was 2.6 .p = 0.11). Injuries occurred in similar frequencies during transport in Indonesia and Vietnam (p = 0.84). In contrast, transporters from Vietnam more frequently experienced death of ducks due to dehydration .Transporters reported the causes of death as “disease,” “dehydration,” “injury,” “other,” and “unknown.” Deaths of ducks during transport due to diseases were observed by transporters in both countries at similar frequencies (p < 0.001). Sale of dead ducks (p = 0.006), processing of ducks for fish feed (p = 0.034), giving ducks to neighbors (p < 0.001), and household consumption of ducks (p < 0.001) were all practiced in Vietnam, but not in Indonesia . For Vietnam, “other” management practices also included return of carcasses to duck owners , while 9.3% (4/43) of transporters provided carcasses to para-veterinarians.p = 0.10). However, the most common location for cleaning transport vehicles in Indonesia was inside houses or garages , but this was less common in Vietnam . Car washes were more utilized by Indonesian transporters compared to Vietnamese transporters .Cleaning and disinfection of transport vehicles in the water of nearby rivers was common in both countries  (p = 0.0067), while transporters from Vietnam more “commonly” used disinfectant on loading surfaces compared to Indonesia (p < 0.001).The most common cleaning practices, i.e., removing feces from loading surfaces and washing loading surfaces with water were similar for both countries (ctively) . HoweverN = 54) and Vietnam  indicated that they did not experience any health problems (p = 0.58).Information was also obtained about whether transporters had experienced health problems while loading and transporting ducks. Almost all of transporter respondents from Indonesia , thus proving potential opportunities for dissemination of avian pathogens if surface areas of transport vehicles were not properly cleaned and disinfected  or indirect (if cleaning is not conducted properly between transports session) spread of HPAI virus , 17, 20.infected , 17, 23.infected , 23, 24.In general, collection and delivery of ducks to markets was not very common for transporters in either Indonesia or Vietnam. This indicates that the collection of ducks and delivery to markets is not in their domain, and is more likely conducted by middle men or traders, who have their own vehicles and collect moving ducks from scavenging areas or from farms. Similar observations were made by Meyer et al.  and MeyeDiffering from the findings of Henning et al.  and MeyeTransporters from Indonesia more frequently experienced deaths of ducks due to disease during the journeys, while transporters from Vietnam more frequently experience deaths of ducks due to dehydration. This difference may be explained by the fact that Indonesian transporters had more opportunities of direct and indirect contact between ducks as birds were sourced and delivered to a wider range of locations; while transporters from Vietnam generally traveled longer distances resulting in potentially longer stressful periods for ducks.With regards to the disposal of duck carcasses, guidelines from international organizations recommend burial, composting, incineration, rendering or landfill disposal as they are effective in mitigating virus spread and minimizes public health and environmental effects , 29, 30.Additionally, it also presents a public health risk if untreated river water is consumed by people, especially given that around 4% of households in Indonesia rely on rivers as their main water supply –34. InteNone of the transporters involved in this study was using Personal Protective Equipment (PPE), during handling of ducks or cleaning of transport vehicles. Previous research highlighted that lack of awareness and training, but also that low income influences the under-use of protective equipment in developing countries .It is difficult to determine if the cohort of transporters recruited into the study is representative of the spatio-temporal distribution of transporters in both countries. This is due to limited literature describing nationwide spatio-temporal distribution of moving duck flocks in both Indonesia and Vietnam and the non-existence of a sampling frame of transporters for both countries. Government lists of moving duck farmers, let alone transporters of moving duck flocks do not exist in either country. The snowball sampling strategy used in this study was the only methodology that allowed us to overcome this problem. It has been previously noted that duck farming is widespread in West and Central Java in Indonesia; and highly concentrated around the Mekong Delta region in Vietnam , 36, 37.The use of survey questionnaires introduced biasness of various forms: recall bias, as the journeys may have been conducted many months earlier resulting in errors in recording journey characteristics and parameters; and social desirability bias, such as that transporters may be unwilling to disclose past experiences of ill-health after handling ducks; and that they may erroneously reported a greater frequency of disinfecting their transport vehicles than in reality. However, we used well-trained interviewers in this study and we are confident that the information we collected is reliable.Finally, the data summarized here were collected in 2009. Some might argue that there could be changes to the duck industry since then. However, based on the value chain analysis elucidated by Meyer et al. , our desIn conclusion, while HPAI is endemic in both Indonesia and Vietnam, known risk factors that perpetuate HPAI in duck farming differ in importance between the two countries. This is due to dissimilarities in the duck farming industries between the two countries that impact the mode of transport used, movement patterns, disposal methods, and cleaning and disinfection approaches. While practices associated with higher biosecurity risk in Indonesia are related to the multipurpose usage of transport vehicles and the disposal of birds in the environment, unsafe practices in Vietnam relate to the potential mixing of birds during transport, the processing of dead carcasses and the storage and cleaning of transport vehicles.The original data presented in this study are included in the article's Ethical approval for interviews with human participants in this study was not provided because the study design for this research was reviewed and approved in accordance with local legislation and institutional requirements in Indonesia and Vietnam. In Indonesia, this was conducted by the Disease Investigation Centre (DIC) in Wates, Yogjakarta; and in Vietnam by the Regional Animal Health Centre VI, Ho Chi Minh City. Data collection for this study was conducted in accordance with the accepted survey guidelines for surveillance activities of both organizations. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.JH, JM, LTV, and DY: design of the research study. JH, LTV, and DY: data collection. SWSK and JH: data analysis. SWSK, JH, and JM: development of manuscript. All authors contributed to the manuscript and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This technology platform enables GEP using small RNA fragments. After optimization of the protocol, RNA of high quality and sufficient quantity was obtained from FF CNB in 92% of samples. For the remaining 8% of cases, FFPE material prepared by the pathology department was used for RNA extraction. Both resulting RNA end products are compatible with the nanoString nCounter® technology.The use of gene expression profiling (GEP) in cancer management is rising, as GEP can be used for disease classification and diagnosis, tailoring treatment to underlying genetic determinants of pharmacological response, monitoring of therapy response, and prognosis. However, the reliability of GEP heavily depends on the input of RNA in sufficient quantity and quality. This highlights the need for standard procedures to ensure best practices for RNA extraction from often small tumor biopsies with variable tissue handling. We optimized an RNA extraction protocol from fresh-frozen (FF) core needle biopsies (CNB) from breast cancer patients and from formalin-fixed paraffin-embedded (FFPE) tissue when FF CNB did not yield sufficient RNA. Methods to avoid ribonucleases andto homogenize or to deparaffinize tissues and the impact of tissue composition on RNA extraction were studied. Additionally, RNA’s compatibility with the nanoString nCounter Gene expression profiling (GEP) has proven a valuable strategy to advance our understanding of the molecular landscape and drug resistance mechanisms of several cancer types, including breast cancer (BC) ,18,19,20+ , human epidermal growth factor receptor 2+ (HER2+) ERBB2-amplified, and ER−, progesterone receptor− (PR−) and HER2−  BC [With respect to BC, GEP is increasingly performed in routine practice, guiding patient management . GEP hasasal) BC . This haasal) BC . Moreoveasal) BC ,24. Signasal) BC .A major challenge to GEP is the sampling and preparation of biological material, i.e., the RNA required for analysis . In turnWe set up a protocol for RNA extraction from FF CNB BC samples. This protocol is made publicly available through this publication and pays attention to tissue homogenization and avoidance of RNases. We showed that the extraction of high-yield and high-quality RNA was feasible for >90% of FF CNB samples and that FFPE samples served as an adequate back-up source for RNA extraction in cases where RNA extraction failed.n = 1; cancer of the vulva, n = 1; colon carcinoma, n = 2, and BC, n = 88) and FFPE tumor samples  were obtained using a 16 G × 100 mm or 18 G × 100 mm core needle biopsy instrument from patients who were treated at Universitair Ziekenhuis Brussel (UZ Brussel) from December 2017 to January 2020 and who gave informed consent. Fresh CNB were collected in 50 mL tubes  containing 5 mL RNAlaterTM solution . Samples were stored at 4 °C for a maximum of 1 month before further processing. The project followed the Helsinki Declaration and was approved by the ethics council of the UZ Brussel (2017/344 and 2017/400).Fresh core needle tumor samples  resulting from this work is provided in the laterTM solution . To ensure emersion of the biopsy in the RNAlaterTM solution, the 50 mL tube was shortly centrifuged in an Eppendorf centrifuge  at 375 relative centrifugal force (rcf) for 1 min at 4 °C. All the following steps were performed on ice and in a horizontal laminar flow cabinet  that was thoroughly cleaned with RNase ZAP decontamination wipes . These wipes were further used to clean all necessary equipment. The weight of the CNB was determined. First, a 2 mL Safe-Lock Eppendorf tube  was labeled with the biopsy code and weighed using an analytical scale . A Kimberly-Clark KimWipe disposable tissue  was placed on the surface of the laminar flow cabinet. RNase-free disposable forceps  was placed on this tissue. The 50 mL tube containing CNB was inserted into the laminar flow cabinet on ice. Next, the CNB was transferred using the RNase-free disposable forceps to a sterile Petri dish . To ensure efficient homogenization, CNB that were longer than 1 cm were cut in two with a sterile disposable scalpel  using the same RNase-free disposable forceps to stabilize the tissue sample. CNB were transferred with the RNase-free disposable forceps to the 2 mL Safe-Lock Eppendorf tube. This tube was weighed again and the weight of the CNB was calculated by subtracting the weight of the empty tube from the weight of the biopsy-containing tube. The CNB was snap frozen by immersion of the biopsy-containing tube in liquid nitrogen. Snap-frozen samples can be stored at −80 °C.Fresh CNB were taken under ultrasound guidance and immediately transferred to a properly coded 50 mL tube  containing refrigerated 5 mL RNAThe 2 mL Safe-Lock Eppendorf tube containing the snap-frozen CNB was re-inserted into the horizontal laminar flow cabinet, and a 5 mm stainless-steel bead  was added using a single-bead dispenser . Next, the Eppendorf tube was inserted into a pre-cooled adapter  and subsequently inserted into the TissueLyser II instrument  for the first homogenization step (beating method) for 30 s at 30 Hertz. In case not all the tumor tissue was homogenized, the tumor tissue was repositioned at the bottom of the 2 mL Safe-Lock Eppendorf tube under sterile conditions using the RNase-free disposable forceps. This homogenization step was repeated for a maximum total time of 2 min (4 cycles). Next, lysis buffer consisting of RLT buffer containing 0.3718M β-mercaptoethanol  provided in the Qiagen RNeasy kit  was added according to the manufacturer’s protocol . Subsequently, the 2 mL Safe-Lock Eppendorf tube containing the (homogenized) BC CNB in lysis buffer was incubated overnight at 4 °C. After 14–16 h of incubation at 4 °C, the 2 mL Eppendorf tube was transferred to a vortex instrument  and vortexed for 1 h at 4 °C. In case the tumor tissue was not completely dissociated, the lysate and remaining tissue was transferred to a 50 mL centrifuge tube and homogenized using the TissueRuptor II dissociator until the desired degree of homogenization was obtained . Next, the probe was detached and subsequently rinsed with lysis buffer . The lysate in the 50 mL centrifuge tube was briefly spun using the Eppendorf centrifuge. Finally, the lysate was transferred to a new 1.5 mL Eppendorf DNA LoBind tube  and used as starting material for subsequent total RNA extraction. Total RNA extraction was performed using the Qiagen RNeasy kit  according to the manufacturer’s instructions. The eluate was reloaded on the column to obtain a higher RNA yield.The SOP resulting from this work is provided in the ® 6AI Vacuum Infiltration Processor, Alphen aan den Rijn, The Netherlands). Tumor biopsies were fixed using 10% formalin for 1.5 h at 35 °C and dehydrated by immersing the tissue in different concentrations of ethanol for 4.5 h at 35 °C. Next, xylene was used as a clearing agent for 2 h at 35 °C. Finally, samples were paraffin embedded at 58 °C for 3 h.FFPE tumor biopsies were generated using the Sakura instrument  was prepared by removing the previous blade and by deparaffinating the microtome using deparaffinization clean lab solution . Gloves were renewed and sprayed with RNase-ZAP solution . Once the gloves were air dried, the work area was cleaned using RNase-ZAP solution and forceps were placed on a Kimberly-Clark KimWipe disposable tissue. The microtome and forceps were rendered RNase free by cleaning the zones of the microtome that came in contact with the slide using RNase-ZAP wipes and KimWipes soaked with NaOH, Milli-Q water, and finally 100% EtOH. After the instrument was air dried, we mounted a new blade  and repeated the RNase-free cleaning steps. RNase-free macrodissection was initiated on mounted FFPE blocks and the desired curls were collected in a 1.5 mL Eppendorf DNA LoBind tube. Total RNA extraction was performed using the Qiagen RNeasy FFPE kit  according to the manufacturer’s instructions. Deparaffinization of the FFPE curls can be performed using the deparaffinization solution (Qiagen) or the heptane/methanol method without impact on downstream RNA applications. The eluate was reloaded on the column to obtain a higher RNA yield.A Qubit 4 Fluorometer and the Qubit RNA HS Assay Kit  were used to assess the RNA yield. Absorbance at 260 and 280 nm was evaluated using a NanoPhotometer Classic . Samples were run on an Agilent 2100 bioanalyzer using the RNA 6000 nano  or pico  kit and the eukaryotic total RNA program. The bioanalyzer electropherograms were analyzed using Agilent 2100 Expert Software to determine the RNA size distribution, the RNA integrity number (RIN) value, and the DV200 values (percentage of RNA fragments with a length >200 nucleotides).® technology. Samples were hybridized according to the manufacturers’ recommendations using the nCounter® Human PanCancer Immune Profiling Panel. Absolute counts were quantified by the nCounter digital analyzer . Raw counts were normalized using the RUVSeq method adjusted for nanoString nCounter® gene expression analysis as described by Bhattacharya et al. [RNA from FF (50 ng) and FFPE (100 ng) BC CNB were compared in terms of detection of gene expression variation using nanoString nCountera et al. . Princip® v8. Box plots indicate the median value and the upper and lower quartiles. Whiskers were drawn according to the minimum–maximum method, with an exception for the normalization of raw counts, for which whiskers were drawn according to the Tukey method. When applicable, significance levels were calculated using an unpaired, two-tailed Mann–Whitney test in case two groups were compared or the Kruskal–Wallis test in case of multiple groups. Statistical significance was determined as p < 0.05. Asterisks in the figures signify a statistically significance difference as follows: *, p < 0.05, **, p < 0.01, and ***, p < 0.001.Spearman correlations and graphical data representations were done using GraphPad PrismWe optimized the procedure to isolate RNA from FF CNB in light of an ongoing phase I clinical trial in BC patients in which we perform GEP to study the immune activating capacity of intratumoral delivery of a proprietary mRNA drug, referred to as TriMix . We hypoWe performed chemical lysis using the lysis buffer provided in the SV total RNA isolation kit of Promega on human colon, breast, and kidney tumor CNB. To that end, CNB were stored for 24 h at 4 °C in a Trizol-free lysis buffer in order to subsequently extract RNA using an easy-to-perform, non-toxic method. It was empirically determined that lysis was incomplete, as there was remaining residue. The extraction of RNA did not yield sufficiently high concentrations to quantify the RNA or assess its quality (data not shown). Therefore, we shifted towards evaluating a mechanical disruption protocol.Different protocols for mechanical disruption of tissues are available . These mWe extracted RNA in 81 out of 88 samples (success rate of 92%) with a yield of 5572 ng on average  and TNBC (n = 3) subtype was small in comparison to the luminal A and B subtypes (n = 65). Therefore, it is prudent not to draw strong conclusions, although we provide n = 60) or invasive lobular BC (ILC) (n = 7) based on histology. Only one patient was referential for invasive papillar BC, invasive mucinous BC, and intraductal papilloma histological subtype  and therefore not included in the graph. p-value of 0.0618 suggested a potential trend that the tissue composition could affect the RNA yield.We did not find a positive correlation between the amount of extracted RNA and the weight of the FF CNB A, which In 8% of cases the extracted RNA from FF BC CNB was insufficient in yield, purity, and/or integrity. In a clinical setting, FFPE specimens are generated routinely for histopathology purposes and entail large amounts of clinically relevant data. Therefore, we evaluated whether sufficient RNA could be extracted from FFPE samples when RNA extraction from FF BC samples fails.® assay, we consulted input parameter recommendations. Since a maximum input of 40 µm slides is suggested, we used two sections of 20 µm thickness as input, ensuring a higher percentage of intact cells in larger sections and efficient deparaffinization, and subsequently followed the Qiagen RNeasy FFPE kit specifications for RNA extraction. We extracted sufficient amounts of RNA (success rate of 100%) with a yield of, on average, 848.13 ng (range of 122.32 to 1720.4 ng) to use in downstream nanoString nCounter® GEP analysis for all seven samples .To further guarantee the quality of the RNA extracted from FFPE specimens, it was of utmost importance to avoid ribonuclease (RNase) contamination during the RNA isolation process. We first optimized and validated a protocol for microtome sectioning in an RNase-free manner . In func samples A, but th samples A. The A2 samples B. Furthe samples C,D. This samples C,D compa samples D,E. Thes samples D. Notablived RNA . In addi® technology. Due to the various efforts made in the field of molecular biology, required input levels have remarkably decreased; only 50 ng of RNA input (derived from FF CNB) is required to perform a nanoString nCounter® assay, which is a tenfold decrease compared to the microarray analysis described in 2002 by Ellis et al. [Most studies focus on the use of clinically available FFPE specimens as starting material to perform downstream GEP . Exceptis et al. . This aln = 12) by analyzing expression of 770 genes included in the Human PanCancer Immune Profiling panel using nanoString nCounter® technology. The results confirmed that RNA derived from both sample types is suited for downstream nanoString nCounter® GEP analysis [® GEP. Our conclusion is consistent with Wimmer et al., who saw highly reproducible and concordant gene behavior in FF and FFPE microarrays, supporting the validity of using two sample types for GEP [To ensure correct GEP analysis, we first assessed different characteristics  of the extracted RNA from FF as well as FFPE samples and integrated this as standard control prior to GEP. Since formalin fixation introduces crosslinks between molecules and modification of RNA by formaldehyde, which can impair downstream assays, or the presence of gDNA contamination can affect the accuracy of GEP, it is important to evaluate RNA expression to predict cellular biological functions of the extracted RNA . Therefoanalysis ,56. We oanalysis . To counanalysis . Samples for GEP . As the  for GEP . Notably® GEP. This optimized RNA extraction protocol resulted, in the case of FF BC CNB, in 92% successful RNA extractions. For the remaining 8%, FFPE specimens served as a back-up plan; we were also able to extract sufficient RNA to perform downstream GEP.Most studies focus on the use of clinically available FFPE specimens for downstream multigene analysis. Exceptionally, FF CNB are available. However, comprehensive protocols with a high level of detail that are easy to use and allow an unexperienced researcher to successfully extract RNA from precious clinical samples are not yet available. Therefore, we provided a standardized approach for FF as well as FFPE core needle sample processing and downstream RNA extraction compatible with nanoString nCounterDifferent parameters such as (1) starting material, (2) sample handling, (3) requirements of downstream assays, and (4) lab equipment determine the choice of RNA extraction method. We observed that the critical parameter for successful RNA extraction starting from FF CNB is complete dissociation of the tumor CNB. For an RNA extraction procedure starting from FFPE specimens, RNase contamination is a major wrongdoer. More specifically, FFPE specimens are more prone to fragmentation, and therefore it is highly recommended to counteract further degradation of the RNA by macrodissecting FFPE specimens in an RNase-free manner. Therefore, we provided standardized operating procedures to optimally extract RNA starting from FF or FFPE tumor specimens. In both scenarios, we maximized RNA output from minimal input. Moreover, we validated the use of RNA derived from FF as well as FFPE for GEP by characterizing different quality parameters as functionality.Advantages of this protocol: This protocol allows RNA to be extracted from small, difficult-to-lyse FF biopsies in 92% of cases and from FFPE-generated specimens—both compliant with downstream nanoString GEP.Limitations of this protocol: Specific lab equipment is required because homogenization is a success-determining factor.Time considerations of this protocol: The basic protocol concerning tissue homogenization and RNA extraction from FF CNB can be completed in two days. Homogenization, overnight lysis, and the vortex step for 1 h are the most time-consuming steps. The duration of the RNase-free macrodissection protocol is dependent on the number of samples and the number of times the procedure needs to be repeated; for one FFPE specimen, this work takes another 30 min in addition to the macrodissection itself.® GEP.Based on these results we were able to provide an experimental workflow of RNA isolation of FF and FFPE core needle BC biopsies. Both RNA derived from FF and FFPE BC CNB are valid sources to be used as starting material for downstream nanoString nCounterK.T. holds a patent for dendritic cells electroporated with tumor antigen mRNA and TriMix (WO2009/034172). No other potential conflicts of interest were disclosed by the other authors.


Following the discovery of nucleic acids by Friedrich Miescher in 1868, DNA and RNA were recognized as the genetic code containing the necessary information for proper cell functioning. In the years following these discoveries, vast knowledge of the seemingly endless roles of RNA have become better understood. Additionally, many new types of RNAs were discovered that seemed to have no coding properties (non-coding RNAs), such as microRNAs (miRNAs). The discovery of these new RNAs created a new avenue for treating various human diseases. However, RNA is relatively unstable and is degraded fairly rapidly once administered; this has led to the development of novel delivery mechanisms, such as nanoparticles to increase stability as well as to prevent off-target effects of these molecules. Current advances in RNA-based therapies have substantial promise in treating and preventing many human diseases and disorders through fixing the pathology instead of merely treating the symptomology similarly to traditional therapeutics. Although many RNA therapeutics have made it to clinical trials, only a few have been FDA approved thus far. Additionally, the results of clinical trials for RNA therapeutics have been ambivalent to date, with some studies demonstrating potent efficacy, whereas others have limited effectiveness and/or toxicity. Momentum is building in the clinic for RNA therapeutics; future clinical care of human diseases will likely comprise promising RNA therapeutics. This review focuses on the current advances of RNA therapeutics and addresses current challenges with their development. For years it was believed that DNA was transcribed into RNA and that this RNA (messenger RNA) was then translated into a protein; however, this all became more complicated when RNA interference (RNAi) was discovered ,2. RNAi Most drugs currently on the market are either small molecules or proteins. Small molecule-based drugs are commonly competitive inhibitors of their target proteins, while protein-based drugs are commonly used to bind to target proteins, replace non-functional target proteins, or supplement for an inadequate amount of a target protein ,7,8. A sRNAs as therapeutic agents have been vastly studied over the past few decades, as they are more cost-effective and easier to develop than traditional small molecule- or protein-based therapeutics . As of nBefore RNAs were used therapeutically, there were many challenges that had to be overcome in order to make them feasible treatment options for human diseases. For example, nucleic acids are negatively charged and do not passively cross the hydrophobic lipid barrier of the cell. Further, exogenous RNAs are degraded rapidly by RNases once they are injected into the host. Finally, some exogenous RNAs cause an immune response that hampers the translation of the target protein or causes the development of a toxic cell environment. Luckily, scientists over the past couple of decades have substantially overcome these barriers with the use of many unique delivery methods, such as nanoparticles that protect the RNA and enable cell-specific delivery of the therapeutic agent.mRNA is coding RNA that is transcribed using genomic DNA as a template and serves to encode proteins . mRNAs amRNAs that encode either adjuvants or antigens have also been proposed as possible vaccine candidates that may be able to prevent many diseases. In 1993, Martinon et al., reported that there was induction of anti-influenza cytotoxic T lymphocytes following immunization with liposome-complexed mRNA-encoding influenza virus nucleoproteins in a murine model . This waCurrently, there are two mRNA vaccines that are FDA approved and one that is in clinical trials. The first mRNA vaccine approved by the FDA was BNT162b2 . This vaAlong with the two FDA-approved mRNA vaccines, there are two additional vaccine candidates. The first is CVnCoV, which is currently in Phase III clinical trials . This vaCFTR) gene. This mutation leads to the buildup of mucous in many organs, especially the lungs . . 120]. TTTR mRNA leading to mRNA degradation and decreased TTR protein expression . . 176]. A# of nucleotides individual sequences that can be tested simultaneously [Aptamers are generated using the systematic evolution of ligands by exponential enrichment (SELEX) method. This procedure uses a randomized library that contains ~4aneously . While aaneously . This laAptamers can act through three main mechanisms of action: (1) aptamers that are specific to a particular cell type can deliver other therapeutic agents to the target tissue or cells; (2) aptamers can act as an agonist and thus functionally activate their target molecules; (3) aptamers work as antagonists and block the interaction of molecules in pathways associated with disease development .Pegaptanib was the first-ever FDA-approved aptamer and is used for the treatment of neovascular age-related macular degeneration. This disease is characterized by retinal degeneration, causing vision loss . IncreasDefibrotide was approved by the FDA in 2020 for the treatment of hepatic veno-occlusive disease/sinusoidal obstruction syndrome . This caNOX-A12 is an RNA aptamer that is in clinical trials for the treatment of various types of cancers, for example, pancreatic cancer, colorectal cancer, and multiple myeloma ,183,184.NOX-E36 has completed Phase I clinical trials for the treatment of diabetes and albuminuria (NCT01547897). Increased HbA1c and albumin/creatinine ratio (ACR) are hallmarks of these conditions. NOX-E36 has been shown to reduce these levels in patients with diabetes and albuminuria  by bindiThere are other aptamers in clinical trials for various human diseases that are composed of DNA. For example, AS1411 is a DNA aptamer for the treatment of cancers such as acute myeloid leukemia . HoweverRNA molecules are multi-functional and are extremely versatile. State-of-the-art studies have demonstrated considerable promise for the clinical use of RNA therapeutics to treat and prevent human diseases. Further, RNA therapeutics are relatively cheaper, easier, and faster to develop than traditional protein- and small molecule-based drugs. RNA therapeutic approaches vary in how they treat different clinical conditions. For example, siRNAs are highly specific with only one mRNA target; therefore, they are good for the treatment of diseases where pathologies are caused by the alternations of only one single gene. However, miRNAs have the virtue of targeting multiple mRNAs; consequently, they are suitable for the treatment of diseases in which various pathologies and/or alternations of many genes are involved. The current challenges for RNA therapeutics include: (i) Cell specificity—ideally, the best RNA therapeutic molecule would have on-target cell specificity without off-target and undesired on-target effects. (ii) Cell-specific delivery agents—one of the most significant challenges in RNA therapeutics is efficient and stable delivery of the molecule to the cell type of interest and being functionally active to perform their role.Clinical trials should focus heavily on early study design to prevent possible adverse outcomes such as acute toxicity. Additionally, this early study design should focus on in vivo functional assays rather than in vitro functional assays alone. Further, the most critical step in RNA therapeutics development is to compare the clinical outcomes in their ability to fix the mechanistic parameters. To accomplish this goal, the RNA therapeutic candidates must be rigorously examined, particularly for their immune tolerance, pharmacokinetics, and pharmacodynamics. However, RNA therapeutic agents are likely developed based on the cellular and molecular mechanisms underlying pathologies of the diseases; thus, these molecules are placed in a prime position for future clinical trials. The current knowledge gaps warrant a modern approach to better understand the pathologies at the cellular and molecular level that will enable us to tackle the therapeutic approaches to treat the disease, not only improving the symptomology, but also fixing the exact cause.While there are currently challenges to RNA therapeutic development, unprecedented interdisciplinary approaches and promising developments in modern science, along with improved early study design for clinical trials, will overcome these obstacles in the foreseeable future. This will provide substantial hope for the clinical utility of RNA therapeutics for different disease conditions and lead to a better quality of life for millions of patients.


Results show that the presence of UNC-33L does not rescue the defective dauer phenotype in unc-33(mn407) mutants. However, UNC-33L significantly rescued premature death and uncoordinated locomotion in young unc-33(mn407) adults. The degree of UNC-33L-mediated rescue was less noticeable as the nematodes aged, denoting that both age and the presence of UNC-33L interact in the production of the phenotypes.Herein, we tested the ability of UNC-33L to rescue dauer formation, lifespan, and locomotion defects of  C. elegans have a short life cycle that consists of a larva molting into a reproducing adult by passing through 4 larval stages . Under favorable conditions, the nematode will go through all 4 larval stages . On the other hand, if conditions become unfavorable, the nematode will shift into dauer formation right after the L2 phase (L2d). During this alternative life cycle, C. elegans become stress-resistant, developmentally arrested, and long-lived (Albert and Riddle 1983). In addition, they undergo morphological remodeling that allows them to adapt to harsh conditions (Cassandra and Russell 1975). Dauers develop a thickened cuticle, undergo body constriction, and cell compaction. The thickened cuticle inhibits environmental toxins, such as detergent from affecting internal organs . Once conditions become favorable, the nematode will exit out of the dauer phase and continue on to become a reproducing adult.unc-33 mutants are unable to enter this alternative life cycle, and the reasoning behind this is unclear. The C. elegans UNC-33 protein is the ortholog of human microtubule-binding protein CRMP2 . UNC-33/CRMP2 plays a role in microtubule organization and axon-dendrite sorting to establish an organism’s nervous system . In the absence of UNC-33 polypeptides, nematodes present with uncoordinated locomotion and defects in axonal elongation . In humans, studies have found CRMP2 defects in patients with Alzheimer’s and other age-related neurodegenerative diseases .Although wild-type (WT) nematodes can enter this dauer phase, et al. 2007; Rajawat and Bossis 2008). Evidence collected from the study of aging suggests that the continuous removal of dysfunctional organelles and the synthesis of new ones allow for a delay in the aging process, gleaning that there are mechanisms extending longevity and inhibiting aging. Further exploring the aging mechanisms is the objective of this study.Aging is a biological phenomenon that occurs in all life forms due to the decline and deterioration of cells and organs. Aging cells and organs progressively accumulate dysfunctional cellular components, are subject to oxidative damage, and have a decline in protein turnover rates and housekeeping mechanisms  mutants. UNC-33 codes for three alternatively spliced isoforms UNC-33L (Large), UNC-33M (Medium), and UNC-33S  with each isoform varying at the N terminus . unc-33(mn407) mutants contain a 500 bp deletion in the unc-33 ORF and produce none of the alternatively spliced isoforms of UNC-33 . In previous studies, Maniar and colleagues found that UNC-33L was able to fully rescue uncoordinated locomotion, egg-laying defects, and mislocalization of RAB-3::mCherry and SAD-1::GFP in unc-33(mn407) mutants, suggesting that UNC-33L is sufficient to restore neuronal activity to WT levels in the unc-33 mutant background . To this end, we hypothesize that the introduction of UNC-33L in an unc-33 null mutantwill result in a rescue of the dauer defective phenotype, lifespan, and age-dependent locomotion.To analyze the effects of the unc-33(mn407);UNC-33L transgenic nematodes were unable to produce dauers (6.74% +/- 0.025%), a phenotype characteristic of unc-33(mn407) mutants (3.23% +/- 0.026%). Meanwhile, dauer formation was scored as 96.68% +/- 0.011% in daf-7(e1372) mutants and 73.14% +/- 0.081% in WT nematodes (unc-33(mn407); UNC-33L transgenics and unc-33(mn407) mutants . However, pairwise comparisons between the remaining strains resulted in statistically significantly different results as follows: unc-33(mn407); UNC-33L vs WT p-value ≦0.0001; daf-7(e1372) vs unc-33(mn407)p-value ≦0.0001; unc-33(mn407); UNC-33L vs daf-7(e1372) p-value ≦0.0001, WT vs unc-33(mn407)p-value ≦0.0001;and WT vs daf-7(e1372) p-value ≦0.0001. These results suggest that the addition of UNC-33L was not sufficient in rescuing the defect in dauer formation of the unc-33(mn407) mutants as scored by the 1% SDS protocol.To test the rescue of the dauer defective phenotype, we treated nematodes with 1% SDS and assessed dauer formation. Analysis of our results show that ematodes . Examinaunc-33 mutants and found a partial rescue denoted by a p-value ≦0.0001 in the pairwise comparison of unc-33(mn407) vs unc-33(mn407); UNC-33L transgenics using a Kaplan-Meier test. Analysis of WT vs unc-33(mn407); UNC-33L pairwise comparison had a p-value ≦0.0001. The Kaplan-Meier estimates of 50 percent mortality were 12 days of adulthood for WT, 8 days of adulthood for unc-33(mn407); UNC-33L transgenics, and 5 days of adulthood for unc-33(mn407) mutants. Additionally, examinations of lifespan assays showed that animals expressing UNC-33L had death rates similar to WT during days 1-5 of adulthood. Following day 5 of adulthood, unc-33(mn407); UNC-33L nematodes showed an increase in death rates, resulting in a phenotype intermediate between WT animals and unc-33(mn407) mutants  vs unc-33(mn407); UNC-33L transgenics . Synchronized populations of worms were cultivated on NGM agar plates containing OP50 SDS Protocol:daf-7(e1372) which were kept at 25 ºC for 5 days. After the 5-day starvation period was reached, animals were resuspended with 1 mL DI water and transferred into 1.5 mL tubes. Worms were centrifuged for 1 minute at 9221 x g and pellet worms were treated with 1 mL of 1% SDS for 20 min at room temperature on an orbital shaker. After the 20 minute incubation, worms were centrifuged for 1 minute at 9221 x g and the supernatant was removed. Pelleted worms were washed 3 times using 1 mL of M9 buffer and centrifuged for 1 minute at 9221 x g at 20ºC after each wash. After the last wash, 100uL of pellet worms were transferred onto an NGM plate with no bacteria and the percent survival was quantified. Alive animals were picked one by one and then burned to ensure adequate scoring.To assess dauer formation, we examined 200 starved worms per strain, per trial. Animals were starved for 5 days at 20 ºC, with the exception of Statistical Analysis:et al. 2011). The locomotion data was interpreted using two-way ANOVA after determining normality via the Shapiro-Wilks test and homogeneity of variance via the Levene test.All data analysis was performed using R. For dauer formation, hypothesis testing was evaluated using one-way ANOVA after determining normality via the Shapiro-Wilks test and homogeneity of variance via the Levene test. A Post-Hoc Tukey’s test was used for pairwise comparisons. Lifespan assays were analyzed via Kaplan-Meier Survival Analysis using Oasis programming (Yang unc-33(mn407), and CB1372 daf-7(e1372) nematodes were obtained from the Caenorhabditis Genetics Center.N2 (WT), SP1382 unc-33(mn407); kyEx3861 , was obtained from Cori Bargmann’s Lab . Strain CX13244 was created by microinjecting a plasmid driving the expression of an UNC-33L cDNA under the regulation of the pan-neuronal tag-168 promoter.Strain CX13244 kyIs445 [PVD::mcherry::RAB-3 + PVD::SAD-1::GFP];


Nuclear localization signals (NLS) are generally short peptides that act as a signal fragment that mediates the transport of proteins from the cytoplasm into the nucleus. This NLS-dependent protein recognition, a process necessary for cargo proteins to pass the nuclear envelope through the nuclear pore complex, is facilitated by members of the importin superfamily. Here, we summarized the types of NLS, focused on the recently reported related proteins containing nuclear localization signals, and briefly summarized some mechanisms that do not depend on nuclear localization signals into the nucleus.Video AbstractThe online version contains supplementary material available at 10.1186/s12964-021-00741-y. One of the characteristic features of eukaryotic cells are membrane-bound functional organelles such as the nucleus, mitochondria, golgi apparatus, and others, which are surrounded by cytoplasm. For cells to function normally, organelle proteins synthesized in the cytoplasm must be selectively and efficiently transported into their destination compartments where they can exert their physiological functions . ConsequUnlike proteins bound to the endoplasmic reticulum or mitochondria, whose N-terminal targeting signals are often cleaved after arrival at their destination organelle, nuclear localization signals remain intact and can be located at almost any part of the protein sequence, indicating the possibility of multiple rounds of nucleocytoplasmic transport .A nuclear localization signal (NLS) was firstly identified through the analysis of mutants of simian virus 40 (SV40), whose NLS is composed of seven amino acids, Pro-Lys-Lys-Lys-Arg-Lys-Val (PKKKRKV) . NLS seqIn recent years, NLS were widely used in cancer treatment and viral infection prevention –15, and 126PKKKRKV132, with five consecutive positively charged amino acids (KKKRK). Interestingly, studies have shown that its reverse sequence has no nuclear transport function -(X)2–5-PY [PY-NLS is characterized by 20–30 amino acids that assume a disordered structure, consisting of N-terminal hydrophobic or basic motifs and C-terminal R/K/H(X)esidues) . Two subesidues) . CollectX)2–5-PY , where X263FGNYNNQSSNFGPMKGGNFGGRSSGPY289, which includes a hydrophobic region (273FGPM276) required for its nuclear localization. The NLS of heterogeneous nuclear ribonucleoprotein 1 (Hrp1) closely matches the consensus of bPY-NLS (Table 511HRR513) and C-terminal R525 (X)2-5PY motif are necessary and sufficient for nuclear import, while also being required for receptor binding and protein function, respectively [Human heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is known as hPY-NLS due to its sequence 66TLLLRETMNNLGVSDHAVLSRKTPQPY92 at the N terminus of the human cytomegalovirus protein UL79. This NLS very closely resembles a hPY-NLS, containing the C-terminal PY-core portion of the consensus sequence preceded by stretches of hydrophobic amino acids [639PGKMDKGEHRQERRDRPY656), among which the hydrophobic region 639PGKM 642 and C-terminal R652(X)2-5PY motif are essential. Each of the mutations R652A, P655A and Y656A could destroy the ability of the PY-NLS to direct EWS protein to the nucleus, similarly to what was observed when the whole PY-NLS was deleted [Wang et al. discovered the PY-NLS sequence no acids . A doublno acids , and is However, a large number of ncNLS do not have a regular characteristic structure, including those of PTHrP (parathyroid hormone-related protein) , Pho4 p.161RKHKTNRKPR170) and NLS2 (216NRRAKAKR223) are independently insufficient for robust nuclear localization when attached to green fluorescent protein (GFP) [159RNKKKK164), and the other from the RARα (486RKVIK490), the NLS of the RARα portion in NLS-RARα is more favorable for the nuclear localization of NLS-RARα [In addition to the discussed cNLS and ncNLS, there also exist other types of special NLS, some of which have been studied in more detail. These include: (a) The putative NLS has basic amino acid sequence composition characteristics of nuclear localization signal. After verification, some of them have nuclear localization function, while others do not. For example, a PY-NLS-like sequence was predicted to be encoded by residues 259–306 of poly(A) binding protein nuclear 1 (PABPN1). This putative NLS is dominated by basic amino acids, but has no nuclear positioning function . (b) A sin (GFP) . HoweverNLS-RARα , 45.s and importin βs, based on their structural and functional features [s protein family only contains 6 members, namely, importin α1, importin α3, importin α4, importin α5, importin α6 and importin α7 [A schematic model for nuclear protein import through NPC illustrates how can a complex biological function can be generated by a spatially and temporally organized cycle of interactions between cargoes, carriers and Ran GTPase . NLS-depfeatures . In mammortin α7 , 49. Theortin α7 . Ran funortin α7 . Becauseortin α7 . This coortin α7 , 53.s contain two conserved domains, including the central HEAT domain , protein phosphatase 2A (PP2A), and TOR1) and the importin β N-terminal domain (IBN) [Classical NLS on cargo proteins are recognized by the importin α subunit, which in turn is recognized by the importin β subunit. The resulting cNLS-cargo-importin α-importin β1 trimer is then imported into the nucleus through a series of steps . This prin (IBN) . The cenin (IBN) , 56. Thein (IBN) , 57. Thein (IBN) . This obin (IBN) .Table 2PThe cNLS-mediated protein transport mechanism can be conveniently divided into four steps: assembly of the cargo-carrier import complex in the cytoplasm, translocation through the NPC, import-complex disassembly in the nucleus, and importin recycling .Step 1: It has been demonstrated that more RanGDP protein is concentrated within the cytoplasm, and the cNLS of the cargo proteins are bound by the adaptor protein importin α, which is subsequently recognized by the carrier importin β1 to form a cNLS-cargo-importin α-importin β1 trimer. In the absence of importin β1, “NLS-like” sequences of the N-terminal IBB domain form an intramolecular bond with the NLS-binding site, inhibiting the interaction between importin α and the cNLS-cargo .Step 2: Importin β1 does not directly interact with the cNLS-cargo but acts to direct importin α toward the NPC . The numStep 3: Once the trimer complex is inside the nucleus, RanGTP binding causes a conformational change in importin β1, which releases the IBB region of importin α. This autoinhibitory domain, together with Nup50 and CAS, facilitates cNLS dissociation and delivery of the cNLS-cargo in the nucleus .Step 4: After dissociation, importin α is exported from the nucleus by CAS in conjunction with RanGTP. The importin β1-RanGTP complex returns to the cytoplasm, where the GTP is hydrolyzed, releasing RanGDP from the importin, ready for reuse in the next round of transport  s superfamily. Vast numbers of proteins are transported into and out of the nuclear by approximately 20 species of importin βs superfamily nucleocytoplasmic transport receptors [The early studies on cNLS revolutionized the field, but it quickly became apparent that many nuclear proteins do not contain classical monopartite (MP) or bipartite (BP) NLS, and must either use alternate entry mechanisms or piggyback on cargo proteins that do contain a classical NLS . One exaeceptors .s-dependent cargo proteins without a PY-NLS frequently use several importin βs-mediated nuclear import pathways [s. It was shown that stimulated ERK directly interact with importin β7, while JNK and p38 form a trimer complex with importin β7 or importin β9 together with importin β3 and the kinase to facilitate the stimulated nuclear translocation of the latter [In general, PY-NLS-containing cargo proteins seem to be specifically imported by importin β2 . By contpathways . Viral, pathways . The stie latter . This nuIt is worth noting that some proteins do not have NLS, but can use independent nuclear transport mechanisms. One example is a specific armadillo repeats of the β-catenin protein, a key mediator of Wnt signaling . The β-cSimilarly, Lyst et al. found that the nuclear protein MeCP2 (Methyl-CpG binding protein 2) may pass through the nuclear pore complex in its NLS-independent manner and import proteins, which contains two sequence-specific DNA binding motifs, AT-hook1 and methyl-CpG binding domain (MBD). Among them, an intact MBD is sufficient for nuclear localization and then be retained in the nucleus due to its affinity for DNA .s. Bannister et al. found that the binding of Rch1 ) to importin β1 was increased approximately three to fourfold after it was acetylated at Lys22 [A number of specific mechanisms precisely regulate nuclear transport in response to a variety of signals. Post-translational modification (PTM) of signaling molecules through phosphorylation/dephosphorylation is the best-understood mechanism through which nuclear transport is regulated by many different kinases/phosphatases , 76. Durat Lys22 .Another regulatory mechanism that controls nuclear transport relies on the binding of NLS-containing cargo proteins to specific cytoplasmic or nuclear factors that serve to anchor or retain them in cytoplasmic or nuclear compartments . In the s mediated nuclear import. One example is that C9orf72 arginine-rich dipeptide repeat proteins (DPRs) can interact with importin βs, disrupt its cargo loading, and inhibit nuclear import of importin βs, importin α/β1, and cargoes in permeabilized mouse neurons and HeLa cells, in a manner that can be rescued by RNA [Moreover, a recent report demonstrated that arginine-rich dipeptide repeat proteins (DPRs) bind directly to importins and mediate importin condensation in a concentration- and repeat length-dependent manner . This ind by RNA , 85..). The study of NLS can help reveal the nuclear transport mechanism of human and viral proteins [NLS-mediated protein import into the nucleus is an important part of nuclear and cytoplasmic information exchange in cells. At present, there have been examples of the efficiency of cross-linking peptide modification with nuclear localization signal content as vectors for intranuclear DNA delivery for the gene delivery into non-dividing cells . Becauseproteins . Also, iproteins . Althoug


The interaction of sisomicin with CUG RNA repeats is characterized by the change of fluorescent signal, which is initially minimized by covalently incorporating the fluorescein into the RNA bases and later increased upon ligand binding. The results show that sisomicin can bind and stabilize the folded RNA structure. We demonstrate that this new fluorescence-based binding characterization assay is consistent with the classic UV mT technique, indicating its feasibility for high-throughput screening of ligand-RNA binding interactions and wide applications to measure the thermodynamic parameters in addition to binding constants and kinetics when probing such interactions.Pathogenic CUG and CCUG RNA repeats have been associated with myotonic dystrophy type 1 and 2 (DM1 and DM2), respectively. Identifying small molecules that can bind these RNA repeats is of great significance to develop potential therapeutics to treat these neurodegenerative diseases. Some studies have shown that aminoglycosides and their derivatives could work as potential lead compounds targeting these RNA repeats. In this work, sisomicin, previously known to bind HIV-1 TAR, is investigated as a possible ligand for CUG RNA repeats. We designed a novel fluorescence-labeled RNA sequence of r(CUG) Pathogenic microsatellite RNA expansions are toxic and have been associated with many untreatable neurodegenerative diseases , includiTherapeutics that eliminate or neutralize these RNA repeats hold great promise for the treatment of many neurodegenerative diseases since they are fatal without the treatments available today. Different strategies have been developed targeting these pathogenic RNA repeat sequences including antisense oligonucleotides (ASOs) ,13,14,1510 as the RNA template to simulate disease-causing CUG repeat expansions in DM1  as a pos in DM1 A, we appmT alone provides very limited conformational information for these studies, thus requiring the incorporation of other techniques to improve the overall mechanistic understanding, e.g., UV optical measurement assisted by a fluorescence-based assay for the study on RNA three-way multibranch junctions [λmax 3600 M−1cm−1 and quantum yield-water 0.68) was used to introduce the bulge perturbations showing improved baselines. Another strategy involving the concepts of a molecular beacon (MB) or fluorophore/quencher pair [The second part of our work is focused on characterizing the interaction between sisomicin and the FAM modified CUG RNA template. We were particularly interested in the thermal stability of this interaction and designed a fluorescence thermal melting assay. Conventional UV unctions . Similarunctions . Despiteunctions ,42,43,44unctions , 2-aminoλmax 75,000 M−1cm−1 and quantum yield 0.9) into the CUG repeated sequence for better sensitivity and avoided high salt conditions with more accurate binding characterizations. Our assay adopted an indirect strategy on the accurate mT acquisition by verifying the existence of two regions for the dissociation of the sisomicin-RNA complex at the actual mT. The detailed binding characterization was conducted at 20–65 °C, showing that sisomicin stabilized the folded RNA structure upon ligand binding and, furthermore, these results were confirmed by CD spectra. We carefully compared our fluorescence mT data with traditional UV mT results and explained the discrepancy between them in detail. By applying the Van ’t Hoff plot to study the dissociation of the RNA-ligand complex at 20–65 °C, we demonstrated that the new approach of mT measurement is consistent with the traditional UV optical melting technique. This implies that our fluorescence thermal denaturation assay is suitable for broad applications to probe RNA-ligand interactions, comprehensive binding characterization, measurements of the thermodynamic parameters, and determining binding constants and kinetics. This binding characterization assay can also be applied for identifying ligands targeting other microsatellite RNA expansions with high throughput fashion when corresponding modified RNA repeat sequences are employed as templates and targets.However, these strategies are not desirable for probing the interactions of small molecules targeting RNA repeats due to their high salt conditions and low sensitivity to detect fine RNA conformational changes caused by small molecule binding. The novel fluorescence melting assay that we have developed highlighted the covalent incorporation of 6-FAM IV-1 TAR , could af 928 nM C.d was calculated as 945 nM through a sigmoidal fitting. These results are consistent with the aminoglycoside comparison data conducted at room temperature in As our microplate assay is more suitable for drug screening operated in a high-throughput fashion, further mechanistic investigations on ligand binding towards RNA repeats require a more sensitive technique in terms of fluorescence measurement. We then selected sisomicin as the model ligand and performed a detailed binding characterization of CUG RNA repeats on a fluorometer using fluorescence titration, which is similar to our fluorescence thermal melting assay except at room temperature only. The fluorescence titration experiment was carried out with sisomicin targeting annealed FM1 CUG × 10 RNA repeats. Sisomicin additions were prepared with different concentrations (1.5 µL) to achieve equivalent molar ratios as follows: 0, 1, 4, 8, and 16. 6-FAM has an emission maximum of 517 nm, which was not affected by the Raman scattering peak at 580 nm in water solution samples shown in mT) studies to characterize the thermal stability of the FM1 CUG RNA template and its interaction with sisomicin. Different CUG repeats 10 as r10, and FM1 r(CUG)10 as r10FAM, were synthesized to conduct the thermal stability and structural comparisons. It has been demonstrated that the RNA double helix upon binding with a ligand can usually increase the mT value, indicating enhanced stability of the RNA structure [mT behavior from r10 (r10 and r10FAM): a 4-degree jump to 64 °C at L/M = 1, followed by a decrease back to 62 °C as L/M increased, suggesting that the annealed r7 strand might possess a different secondary structure from the r10 ones.Continuing with the sisomicin as the titrating ligand, we performed UV-melting temperature  RNA concentration; (b) RNA modification; and (c) number of CUG repeats. For factor (a), roughly tripling r10FAM concentration from 1.5 µM to 5 µM led to a slight increment on mT by less than 2 °C, and normalized melting curves remained overlapped in most cases, indicating that RNA concentration effect is trivial in the experimental conditions, and it does not affect the thermal denaturation behavior of RNA. For factor (b), covalently coupling fluorescein in between bases C and U at the 5th CUG repeat in r10 introduced a perturbation to the local structure and caused a small shift of the melting curve towards the left and a decrease of 2 °C on mT. The results in mT upon transitioning from r7 to r10. However, we observed that instead of a higher mT, r10 showed a lower mT in all cases, although r7 was on the left side of r10 in each UV melting plot. This indicates the existence of different secondary structures between the two strands: r7 may form a normal duplex containing 14 GC base pairs; while r10 forms a hairpin with a four-nucleotide hairpin loop and 9 GC base pairs in its stem, which would align with our self-folded hypothesis of the r10 sequence in the scheme of The variety of RNA repeat templates and different RNA concentrations used in thermal denaturation studies enable us to examine the following three factors on mT results inspired us to go for a fluorescence thermal denaturation study on the interaction of sisomicin-RNA repeats using FAM-modified r(CUG)10. Before proceeding further, we carefully examined the factors that could affect the fluorescence readings under the elevated temperature conditions for this thermal denaturation study. UV mT experiments suggest that FM1 CUG × 10 possesses a hairpin secondary structure. Initially, the self-folded hairpin secondary structure could hydrophobically embed the fluorescein and the overall fluorescence is minimized. Upon ligand binding at room temperature, the conformational change of the ligand-RNA complex gets the fluorophore exposed to the solution gradually along the binding process, leading to an observation of a binding curve based on the fluorescence readings. At elevated temperatures, along with conformational change due to the sisomicin-RNA binding, the FAM containing RNA is expected to have some thermodynamic change such as thermal motion/rotation. This effect can come into play and make a significant impact on fluorescence intensity, which will in turn affect the data accuracy towards the sisomicin-RNA interaction study. We designed a fluorescence stability test for annealed FM1 CUG × 10 at 20–80 °C to assess the latter factor. The temperature was increased from 20 to 80 °C at an increment of 5 °C and each temperature was held for totally 10 min . As shown in The above UV 10 RNA repeats were used as a control to be compared with annealed FM1 r(CUG)10 RNA repeats having hairpin secondary structures. Results with denatured and annealed RNA repeats are shown in mT data points were plotted and mT values were calculated through sigmoidal fittings for annealed FM1 CUG × 10 in mT of 52.2 °C to be compared with a UV mT value of 55.8 °C in mT study). According to the results in mT curve in mT data in mT value . Similarly, the fluorescence mT curve in mT value of 54.9 °C with sisomicin addition at L/M = 1, increasing 2.7 °C from the initial value. However, the rest of the plots in mT with L/M > 1, contradicting the trend in UV mT experiments. To explain this inconsistency, we need to introduce a new parameter below to discuss the stability of annealed FM1 RNA structure.To probe more insights into the sisomicin-RNA interaction, we further carried out a thermal melting study employing fluorescence titration technique at 20–65 °C. Denatured FM1 r(CUG)In this study, the fluorescence intensity of RNA samples  generally increases with temperature under the experimental conditions. Here, we define ∆F as the maximum change of fluorescence intensity  in each graph in In As discussed in our UV optical melting study, binding with a ligand can enhance the stability of the RNA structure. Surprisingly, even denatured CUG repeats showed some binding with sisomicin, suggesting that the denatured repeat sequence still possessed some random structure—possibly a random loose coil with FAM being concealed from exposure. At an L/M ratio of 1, ∆F was minimized bearing a sharp change from the highest ∆F with no sisomicin added, implying that the denatured RNA structure only had approximately one site per RNA molecule. Increasing sisomicin concentration would increase some other interactions  and make the sisomicin-RNA random coil structure get slightly less stable or less compact. However, ∆F remained at ~9000, showing that the structure did not change much with L/M > 1 and was more stable than the case with no sisomicin added. For annealed CUG repeats, the most stable state of ligand-RNA structure was reached at an L/M ratio of 4, which is consistent with the self-folded hairpin structure having approximately four or five binding sites . Without ligand added, annealed CUG repeats exhibited higher stability with a smaller value of ∆F because they possessed some secondary structure in comparison with denatured CUG repeats. At the L/M ratio of 1, its stability did not change much as ∆F increased slightly. When the L/M ratio reached 4, the binding sites of the annealed RNA structure were saturated and ∆F was minimized, indicating under these conditions the complex of annealed CUG repeats binding with sisomicin reached the most stable state. When L/M > 4, the complex had some conformation change: FAM got slightly more exposed and the stability decreased from the most stable state to become close to the state when L/M = 1.Results of ligand concentration effect on binding at different temperatures were plotted in d values were calculated using sigmoidal fitting. In the case of multiple binding sites , sigmoidal curve fitting worked very well. However, sigmoidal curves fit poorly in the single binding site case (denatured CUG repeats), suggesting possible non-specific binding and/or multilayer adsorption domination when ligand was added continuously beyond the single binding site saturation. As Kd is the dissociation equilibrium constant, thermodynamic parameters of the dissociation of sisomicin with annealed CUG repeats were calculated using the Van ’t Hoff equation to plot lnKd vs. 1/T in mT very close to those values at L/M = 0 and 1 in 10 (in mT = 55.8 °C). This provides a new method to determine mT. Moreover, the presence of two regions indicates that the dissociation of the RNA-ligand complex would become more energetically favorable beyond mT.Shown in mT inconsistency in mT results, ∆F was minimum at L/M = 4, meaning that the sisomicin-RNA complex should achieve the most stable state with a higher calculated mT on the fluorescence mT plot. Below 65 °C, thermal motion/rotation of FAM is negligible for annealed CUG RNA repeats, so it is true that the sisomicin-RNA complex is most stable at L/M = 4 as ∆F is the minimum. To explain the mT disagreement, keeping in mind that the thermal melting is a dynamic process and mT is defined as the temperature at which half of the nucleotide strands are in the random coil or single-stranded state. At the melting temperature mT, FM1 CUG × 10 RNA has two states: annealed self-folded hairpin structure and random coil/single-stranded structure with a molar ratio of 1:1. At this point, although the denatured half nucleotide strands are in coil/single-stranded state, the hydrophobic structure of FAM in the sequence is preserved, that is, the exposure degree of FAM to the solution remains unchanged, which is supported by the data in mT curve in mT of 52.2 °C at L/M = 0. Sisomicin addition can stabilize the self-folded RNA hairpin structure. As previously discussed with our denatured CUG × 10 RNA data, the random coil/single-stranded structure can still have one binding site for small molecules. Hence, a calculated higher mT was achieved for the fluorescence mT curve in mT curve of the sisomicin-RNA complex becomes sigmoidal and gives a lower mT. Continuing to increase L/M, the folded RNA hairpin structure is already saturated, meanwhile, non-specific binding and/or multilayer adsorption on the coil/single-stranded RNA structure become dominant and significantly reduce the fluorescence intensity from these RNA structures, resulting in the overall fluorescence intensity on the fluorescence mT curves at L/M = 8 and 16 even started to decrease at 55 and 50 °C, respectively. The interaction and conformation changes of the sisomicin-RNA complex at mT explain the existence of two regions for the dissociation of the RNA-ligand complex and validate this new mT determination approach.In return to the discussion of the mT provides mT value as a good thermal stability indicator and other useful information such as thermodynamic parameters; however, it is not very sensitive at probing RNA-ligand interactions. Our fluorescence thermal melting assay, on the other hand, can provide all this information, and it is also very sensitive for studying RNA-ligand binding. By introducing a new parameter ∆F, it can depict the dynamic binding process and point out that the most stable state for a RNA-ligand complex is when all its binding sites are just occupied. Although our fluorescence experimental data back up this statement, unfortunately it only stands as a reasonable proposal if we do not have substantial structural information to support it. Usually, simulation results can work as useful structural supporting information to fulfill this assignment, however we would like to solve this issue from an experimental basis if possible. Circular dichroism (CD), as the best known technique to measure the effects of polarized light on chiral biomolecules such as nucleic acids, can give detailed structural and enantiomeric insights on both small molecule and macromolecular structures and has been widely used to provide profound secondary structure information on nucleotides [Conventional UV leotides . We usedleotides ,47,48. WTraditionally, thermal melting of hairpin nucleotides is considered having only two states: folded and unfolded. However, the existence of multiple states has been suggested and observed in some literature ,50,51, emT technique, the fluorescence thermal melting assay provides a new approach to determine Tm with extensive binding characterization such as probing thermal stability and measuring thermodynamic parameters, binding constants, and kinetics. Our assays can also be useful for the possible structural optimization of lead compound’s derivatives through differentiating fluorescence changes caused by adding/modifying some functional groups in the compound structure. This combined with our future structural and simulation studies would have great potential to speed up the drug discovery process.The biggest challenge on thermal stability studies of RNA-ligand interactions using fluorescence spectroscopy is how to differentiate fluorescence conformational changes due to ligand binding from those caused by thermal melting. We introduced a new parameter of ∆F to overcome this obstacle, showing that ∆F can work as a stability indicator and reach its minimum when all RNA binding sites are occupied by ligands. Then, we used CD as a complementary technique providing structural supporting information and verified this finding. In conclusion, we have reported two sensitive and efficient fluorescence assays based on a novel fluorescence-labeled CUG RNA sequence designed to address two major tasks/challenges in drug discovery towards pathogenic CUG RNA repeats. The microplate assay can be easily developed into a high-throughput screening assay for identifying lead ligands targeting disease-causing RNA expansions, offering both convenience and flexibility in terms of HTS and binding characterization. Consistent with the classic UV Native and FAM-modified RNA oligonucleotides in ® Synergy H1 hybrid multi-mode reader as the fluorescence readout operated with settings as follows: excitation at 485 nm; emission at 520 nm; and gain at 100. The RNA template employed in the assay was prepared by heating FAM modified r(CUG)10 RNA repeats at 95 °C for 5 min, followed by slow cooling to room temperature for ~2 h. Wells loaded with 100 µL of 200 nM annealed RNA repeats were subject to a 1-µL ligand addition in different concentrations  generating ligand to RNA molar ratios—L/M at 0.5, 1, 2, 4, 8, and 16. The RNA sample plates were scanned by the microplate reader before and after ligand addition to calculate fluorescence changes. Experiments were performed in triplicate with three accumulations. Binding modeling with sigmoidal data fitting  or annealed  form was loaded in a 200 µL cuvette and subsequently titrated by addition of 1.5 µL ligand in different concentrations . Each concentration of the mixture was studied by heating and cooling samples at a range of 20 to 65 °C with an increment of 5 °C and a 2 min hold time at each temperature. Excited at 485 nm, fluorescence emission spectra were recorded in a range of 500–600 nm with a 3 nm slit width. Raw spectra were smoothed with the Lowess 11 pt method and fluorescence readings at the wavelength of 520 nm were used for data analysis to be consistent with our fluorescence microplate assay. Experiments were carried out at least in triplicate. Data fitting was performed with SigmaPlot software.CD experiments were performed on a Jasco-815 CD spectrometer at either room temperature or specific temperatures utilizing a Peltier temperature controller. A typical 5 µM RNA sample annealed in nuclease-free water  was prepared in 200 µL volume and loaded in a 350 µL quartz cell with a 1 mm path length. In a heated CD titration experiment, a prepared RNA sample was subsequently titrated by equivalent sisomicin to generate ligand to RNA molar ratios—L/M at 0, 1, 4, 8, and 16. Then, each concentration of the mixture was studied at 20, 40, and 60 °C, respectively, by heating and cooling the sample at 1 °C/min with a 2 min hold time for each temperature. CD spectra were collected at a scanning speed of 100 nm/min from the wavelength of 300 to 200 nm and with 1.0 nm bandwidth and 1.0 s digital integration time. All raw CD spectra were smoothed with the Savitzky–Golay 25 pt method. There was no baseline correction needed for samples prepared in nuclease-free water, and experiments were carried out at least in triplicate.


Pospiviroidae have single-stranded circular RNA genomes that adopt a rod-like or a quasi-rod-like conformation. These genomes contain a central conserved region that is involved in replication in the nucleus through an asymmetric RNA–RNA rolling-circle mechanism. Members of the family Pospiviroidae lack the hammerhead ribozymes that are typical of viroids classified in the family Avsunviroidae. The family Pospiviroidae includes the genera Apscaviroid, Cocadviroid, Coleviroid, Hostuviroid and Pospiviroid, with >25 species. This is a summary of the ICTV Report on the family Pospiviroidae, which is available at ictv.global/report/pospiviroidae.Members of the family  Pospiviroidae have circular single-stranded RNA genomes of a few hundred nucleotides. They may assume rod-like or quasi-rod-like conformations containing a central conserved region (CCR) and a terminal conserved hairpin (TCH) or a terminal conserved region (TCR) (Members of the family on (TCR) , Fig. 1 in vivo) are repeatedly transcribed into oligomeric complementary (−) RNAs. Such intermediates serve as templates for generating oligomeric (+) RNAs that are cleaved by a host enzyme of the RNase III class. The termini of the resulting linear monomers are ligated by the host DNA ligase 1 to generate the mature circular viroid RNA [Avsunviroidae, the (−) oligomeric RNAs of members of the family Pospiviroidae are not cleaved and do not generate the corresponding circular forms.Replication is nuclear and mediated by DNA-dependent RNA polymerase II, which is redirected to use RNA templates through an asymmetric RNA–RNA rolling-circle mechanism. Circular RNA molecules of (+) polarity (by convention the most abundant strand roid RNA . In contictv.global/taxonomy. Demarcation of genera is based upon the type of CCR and the presence of a TCH or TCR (Pospiviroid, such as potato spindle tuber viroid, share the same CCR and have a TCR. Most infect herbaceous hosts, mainly solanaceous species. Hostuviroids, such as hop stunt viroid, share the same CCR and have a TCH, except for members of the species Dahlia latent viroid, which have a TCR instead of the TCH. Hop stunt viroid has a wide natural host range, while dahlia latent viroid is restricted to Dahlia spp. Cocadviroids, such as coconut cadang-cadang viroid, share the same CCR and have a TCH. Some members infect monocotyledons, while others can only infect dicotyledons. Apscaviroids, such as apple scar skin viroid, share the same CCR and have a TCR. Apscaviroids mainly infect woody plants. Coleviroids, such as Coleus blumei viroid 1, share the same CCR and have a TCR or a TCH. The natural host range of coleviroids is restricted to species in the genus Coleus.Current taxonomy: H or TCR , as wellH or TCR . SpeciesPospiviroidae: ictv.global/report/pospiviroidae.Full ICTV Report on the family


NREP) as a prognostic biomarker and therapeutic target for GC. We assessed the clinical relevance, function, and molecular role of NREP in GC using bioinformatics analysis and experimental validation. Our results showed that in GC, NREP overexpression was significantly associated with a poor prognosis. Our findings also suggested that NREP may be involved in the activation of cancer-associated fibroblasts and the epithelial–mesenchymal transition (EMT), with transforming growth factor β1 mediating both processes. In addition, NREP expression showed a positive correlation with the abundance of M2 macrophages, which are potent immunosuppressors. Together, these results indicate that NREP is overexpressed in GC and affects GC prognosis. Thus, NREP could be a prognostic biomarker and therapeutic target for GC.The identification of biomarkers and effective therapeutic targets for gastric cancer (GC), the most common cause of cancer-related deaths around the world, is currently a major focus area in research. Here, we examined the utility of Neuronal Regeneration Related Protein ( (A) Study Flow chart, (B) Diagram depicting the regulation mechanism of NREP in the tumorigenesis of gastric cancer. Gastric cancer (GC) is the third most prevalent cancer globally . Both peIn this era of individualized and precision medicine, molecular targeted therapies and immunotherapies are developing very rapidly, and they have shown great promise in the treatment of GC . AlthougThere has been recent progress in molecular targeted therapies for GC. Trastuzumab in combination with chemotherapy has been found to improve survival in human epidermal growth factor receptor 2-positive GC . HoweverNREP regulates the expression of TGF-β1 not only at the translational but also at the transcriptional level  and tumor-associated fibroblasts, enabling them to play a decisive role in tumor survival and progression. Signaling crosstalk between cancer cells and mesenchymal cells ultimately leads to an environment that supports tumor growth and metastasis. Recently, high NREP expression was observed around the rims of invasive human glioma tumors. Furthermore, NREP knockdown in human glioma cells (SF767) has been found to reduce their migration ability in vitro  latency-related protein, is an intracellular polypeptide  that is highly conserved across species and is expressed in the brain, smooth muscles, regenerated tissue, and malignant glioblastomas . NREP real level . NREP alneration . The molneration . During neration . Given tin vitro , 2017. HA complete list of reagents and antibodies is provided in 2.AGS (moderately differentiated GC cells), HGC27 (undifferentiated GC cells), GES-1 , and THP-1 cells (human monocytic cells) were purchased from the cell bank of the Chinese Academy of Sciences . MKN74 and MKN45 cells  were purchased from the Japanese Collection of Research Bioresources Cell Bank. Human MSCs were purchased from Cyagen Biosciences . GC and THP-1 cells were cultured in RPMI-1640 medium with 10% fetal bovine serum (FBS), and GES-1 cells and human MSCs were cultured in DMEM with 10% FBS. All cells were incubated at 37°C in 5% CO-actin (F): 5′-GCGTGACATTAAGGAGAAGC-3′; β-actin (R): 5′-ACGTCACACTTCATGATGG-3′; NREP (F):5′ TTGAGCGAATGCTACCAGAG-3′; and NREP (R):5′-AGGCG AGGCTACGGAAAG -3′.Quantitative RT-PCR was performed using the protocol provided in previous studies . Total RThe protocol for western blotting was based on previous studies . Target/The study’s protocol was approved by the ethics committee of the Jiangsu Province Hospital of Chinese Medicine, and informed consent was obtained from clinicians and patients (2019NL-166-02). GC tissue and the surrounding non-tumorous tissue  were collected during surgery from 30 previously treatment-naïve patients with GC at the Jiangsu Provincial Hospital of Traditional Chinese Medicine. Tumors were staged and graded using the 8th edition of the American Joint Committee on Cancer tumor-node-metastasis staging system . After eThe protocol used for Immunohistochemistry (IHC) was based on earlier studies . Images NREP. Viruses were designed, synthesized, and produced by GeneChem Corporation. Transfection was performed according to the supplier’s protocol. HGC27 and MKN74 cells were transduced with the recombinant lentiviruses using 2 μg/mL polybrene for 24 h. Subsequently, we identified stably transfected GFP-expressing cells using 1.5 μg/mL puromycin. We assessed NREP overexpression and knockdown as well as transduction efficiency using western blots.We used lentiviral vectors for overexpressing and knocking down We examined cell supernatants for TGF-β1 levels using the TGF-β1 Enzyme-linked immunosorbent assay (ELISA) Kit based on the given instruction manual. A microplate reader (BioTek Synergy HT) was used to examine optical density at 450 nm.We assessed the clonogenic ability of cells using a clone formation assay, as described previously . The num6 cell/mouse) were injected subcutaneously into the right armpit region of the mice (n = 6 per group). 7 days later, tumor formation was observed beneath the skin. The maximum (a) and minimum tumor diameter (b) were measured twice weekly. On day 15, the mice were euthanized and all tumors were collected. Tumor volume was calculated (V = 1/2ab2), and the growth curves of the subcutaneous xenografts were drawn.All animal experiments were approved by the ethics committee of the Jiangsu Province Hospital of Chinese Medicine (2021-5-062). Twenty-four 4-week-old male BALB/c nude mice were obtained from the Beijing Institute of Biomedicine . MKN74 cells transfected with sh-NREP, oe-NREP, and NC and control cells  were treated with phorbol 12-myristate 13-acetate  for 48 h to allow the induction of macrophage differentiation (5 cells/mL) from the control, knock-down (sh-NREP), overexpression (oe-NREP), and negative control (NC) groups were seeded onto 0.4-μM transwell inserts. For co-culture, the culture medium in the inserts with GC cells was removed and transferred to the top of the pates with differentiated THP-1 cells. After 48 h of further co-culturing, cells were obtained, and immunofluorescence staining was performed.THP-1 cells  and illustrated using GraphPad Prism 8.0 . All experiments were carried out at least thrice. ∗∗P < 0.01 and ∗P < 0.05 were considered statistically significant.Data were reported as the mean ± standard deviation. We used NREP in GC was first investigated using the TIMER and1 and GEPIA databases2 , and 95% confidence intervals (CIs) were obtained using the “forest plot” R package.Multivariate Cox regression analysis of TCGA-STAD data was used to identify whether NREP levels were analyzed based on various classification parameters, such as the T/N/M stage, pathologic stage, and histologic grade.The differences in NREP expression were examined using Kaplan–Meier survival analysis and log-rank tests. P values, HRs, and 95% CIs were obtained using log-rank tests and univariate Cox proportional hazards regression. Time receiver operating characteristic (ROC) analysis was applied to calculate the accuracy of prognostication based on NREP levels.Differences in survival based on NREP-lined expression and to explore their potential functions (NREP were identified based on TCGA-STAD data (criteria: |logFC > 3| and P < |0.05|). Subsequently, we conducted functional enrichment analysis of NREP and the identified genes using the Enrichr database 4 4 ; we visuSTRING)4 . In addihttp://www.gsea-msigdb.org/gsea/msigdb/index.jsp) was used for GO enrichment analysis  was performed using the Broad Institute GSEA software 3.0. The geneset “subset of GO (Gene Ontology)”  and Mixed Complementarity Problem (MCP)-counter algorithm from the TIMER web tool . Moreover, the analysis of TIMER and TCGA data also showed high NREP expression in GC (The Cancer Genome Atlas-Stomach Adenocarcinoma data showed that on in GC .NREP in GC tissues using data from the Gene Expression Omnibus (GEO) database. Data from the P < 0.01, ANOVA). NREP protein expression in GC was further verified using IHC data from The Human Protein Atlas. NREP was found to be primarily expressed in the cell membrane and cytoplasm , and no such relationship was observed for other clinical features. Further analysis of the prognostic value of NREP using TCGA-STAD data showed that the low-risk group had a longer duration of survival than did the high-risk group (P < 0.05). Additionally, according to TCGA-STAD data, the areas under the ROC curves of NREP expression for 1-, 3-, and 5-year OS were 0.589, 0.651, and 0.708, respectively, . NREP silencing also decreased tumor cell clone formation . GSEA for NREP revealed the potential role of NREP in “epithelial–mesenchymal transition” and “TGF-beta signaling.” Analyses using TIMER data revealed a positive correlation between NREP and TGF-β1 expression , MMP2 , MMP9 (R = 0.13 P = 0.01), and VIM . Further, NREP expression was found to show a negative correlation with CDH1 expression . Moreover, after TUNEL staining, no TUNEL-positive cells were detected in cells overexpressing NREP. In contrast, NREP knockdown and LY364947 treatment increased the number of TUNEL-positive cells . Subsequent single-cell-level analyses revealed that NREP was mainly expressed in fibroblasts, which are important players in EMT (The abundance of cancer-associated fibroblasts (CAFs) was found to be a potential prognostic factor in GC . Using ts in EMT .NREP overexpression in GC facilitates the conversion of MSCs into CAFs, we co-cultured MSCs with GC cells . Furthermore, we co-cultured MSCs with GC cells showing different levels of NREP expression for 4 days. Immunofluorescence staining revealed that α-SMA and Vimentin levels were up-regulated in the NREP overexpression group, whereas they were down-regulated in the sh-NREP group . Interestingly, ELISA revealed a 4-fold increase in the levels of TGF-β1 in the cell supernatant after MSCs were co-cultured with NREP-overexpressing cells . Consistent with our previous results, these findings also showed that a TGF-β inhibitor can decrease the ability of MSCs to differentiate into CAFs in a co-culture unit.Local and recruited MSCs are known to transform into CAFs at close proximity to tumor cells. To test whether GC cells . After tNREP overexpression on CAF recruitment through a transwell experiment. We observed that with an increase in NREP expression, the ability of GC cells to recruit CAFs was significantly enhanced  and CD163  expression , which are surface markers of M2 macrophages. Therefore, our findings indicate that NREP promotes M2 macrophage activation — a process that is considered strongly carcinogenic.During the development of GC, a large number of peripheral blood mononuclear cells are attracted to the tumor mesenchyme and transform into TAMs . PhenotyNREP is elevated in GC cells and tissues. High NREP levels are associated with some clinicopathological features of GC and a poor patient prognosis. Our results show that NREP may act as an important player in the complex gene regulatory machinery driving GC via processes such as EMT activation, CAF activation, actin cytoskeleton remodeling, and M2 macrophage infiltration, ultimately promoting tumor development. However, our study has a few limitations. First, in vivo experimental evidence was lacking, and there was insufficient clinical evidence. Therefore, more focused research is needed to elucidate the detailed biological functions and mechanistic roles of NREP in GC and to uncover the functional and regulatory niches of this gene.Taken together, our results demonstrate that The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the Ethics Committee of the Jiangsu Province Hospital of Chinese Medicine. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by the Ethics Committee of the Jiangsu Province Hospital of Chinese Medicine.J-pL and Y-hZ developed the experimental plan. Y-jL and S-hZ performed all experiments. S-hZ and Y-dH analyzed the data. Y-jL and J-pL wrote the manuscript. All authors have read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.