Parent–adolescent discussion on sexual and reproductive health (SRH) issues leads to increased awareness on reproductive health matters and reduces risky behaviors among adolescents and also contributes to negative SRH outcomes. The aim of this study was to assess parent–adolescent discussion on SRH issues and its associated factors in Sawla town, Southern Ethiopia.A community-based cross-sectional study was conducted among parents of 10–19-year-olds in Sawla town from March to April 2020. A simple random sampling technique was employed to select 697 study subjects. Data were collected using a pretested structured questionnaire, adapted from the 2016 Ethiopia Demographic Health Survey and other published literature. The questionnaire consisted of questions about socio-demographic factors, knowledge about SRH issues, attitudes towards SRH issues, and discussions between parents and adolescents concerning SRH issues and was administered by face-to-face interviews p-value < 0.05 and 95%CI for adjusted odds ratios (AOR) were used to declare the significance of the associations.Of the total participants, 168 (25.7%) respondents had reported discussion about at least two components of SRH issues in the last 6 months prior to the study. Multiple logistic regression model revealed that attending primary education , secondary education  and diploma and above , monthly income of above 2000 Ethiopian Birr , good knowledge and positive attitude  towards SRH issues were statically significant factors associated with increased parent–adolescent discussion about SRH.The occurrence of parent–adolescent SRH discussion is low in Sawla town. Educational status, average monthly income, knowledge and attitude on SRH issues were major factors affecting parent–adolescent discussion on SRH issues. Continuous capacity building on SRH issues for parents to increase the level of parental knowledge and attitude about SRH issues and sustainable advocacy should be provided about the importance of parent to the adolescent discussion. Moreover, it is important to support and scale up the adult learning program to decrease the level of illiteracy. The health threats for adolescents today are predominantly behavioral rather than biomedical and more of today’s adolescents are involved in health behavior with the potential for serious consequences. In most cultures, parents and family members are an influential source of knowledge, beliefs, attitudes, and values for children and youth. Open parent–adolescent discussion on sexual and reproductive health (SRH) issues often leads to increased awareness on reproductive health matters and reduces risky behaviors among adolescents and also is the base to reduce bad reproductive health outcomes. This study provides an overview of parent–adolescent discussion on SRH issues among parents in Sawla town in Southern Ethiopia. We found that only about one-fourth (25.7%) of the parents discuss at least two components of SRH issues with their children in the last 6 months prior to the study. Educational status, average monthly income, knowledge, and attitude on SRH issues were major factors for these disparities. Knowing the level of parent–adolescent discussion on SRH issues with their children and factors affecting the discussion may help concerned bodies to design Continuous capacity building and life skill training programs on SRH issues for parents and adolescents. Adolescence is a continuum of physical, cognitive, behavioral, and psychosocial change that is characterized by increasing levels of individual autonomy, a growing sense of identity and self-esteem, and progressive independence from adults . AccordiReproductive health implies that people are able to have a satisfying and safe sex life and that they have the ability to reproduce and the freedom to decide if, when, and how often to do so . SRH regParent–adolescent discussion on SRH issues remains a challenging subject of discussion in many traditional communities as the social environment in them still constrains the exchange of information on such issues .The results of adolescent sexual behavior are a huge burden both for the adolescent and society. The problem is not that adolescents are sexually active but rather that they have little preparation and guidance in developing responsible sexual behavior. Developmentally, adolescents reach physical maturity before they are cognitively able to appreciate the consequences of their behavior .An adolescent’s primary source of information regarding sexuality is his or her peer group, all of whom are experiencing and reinforcing the same behaviors. The family, the major fraternize of other behaviors, is not as powerful a force in shaping responsible sexual behavior because of parental discomfort with sex education and sexual discussions. Parents most often respond with their hopes that their children will grow into adulthood without unintended pregnancy or a sexually transmitted infection (STI). They sometimes follow this with the hope that their kids will never be a victim or perpetrator of intimate partner violence or other sexual abuse . This isAdolescents are exposed to various sexual and reproductive risks such as early sexual debut, unprotected sex, early pregnancy, school dropout and STI/HIV due to lack of communication with their parents. Worldwide, HIV/ is the leading causes of disease burden for young people (those aged 10–24 years) . Half thThe government of Ethiopia developed Adolescent and Youth Reproductive Health (AYRH) Strategy in 2006, implemented from 2007 to 2015 and revised from 2016 to 2020. The strategy encouraged adolescents and youth to utilize available health services. The strategy mention the importance of parent–adolescent communication on SRH issues, but the government has not made an effort to encourage or facilitate it .Even though discussion on SRH issues is a public health concern among adolescents, it has not received due attention in Ethiopia. The Parent–adolescent discussion and associated factors were not well studied in Ethiopia especially in Southern part. The previous studies done were school based and focused discussion on SRH issues only from adolescents’ point of view. Since this study was community based and from parents’ point of view, it will play major role in filling research gaps on this issue. Therefore, this study aimed to investigate the extent of parent–adolescent discussions on SRH and the parental factors that affect the likelihood of these discussions in Sawla Town, Southern Ethiopia.The study was conducted in Sawla Town, located in Southern nations and nationalities and People Regional State (SNNPR), Ethiopia. It is 310 km far from Hawassa, capital city of South Regional State in Ethiopia and 549 km away from Addis Ababa, the capital city of Ethiopia. Sawla has total population of 46,957, of which 51% are females. According to Ethiopian Health and Demographic Survey (EDHS) 2019, around 25.4%  adolescents were expected to live in the town. The town has 10 kebeles  with 9582 households; of these 3903 households have adolescents. Sawla has one General Hospital, one public health center, two government clinics, nine private primary clinics, two private medium clinics, two pharmacies and seven drug stores which provide maternal and other health services to the population. Only one health center provides the adolescent and youth friendly services [A community based cross-sectional study was conducted from March to April, 2020.The source population was all parents living in Sawla town who had children between 10 and 19 years (adolescent) of age. Parents who were unable to communicate or severely ill at the time of data collection were excluded from the study.Sample size was calculated using double population proportion estimation formula in EPI-INFO version 7 based on key variables taken from previous studies. The assumptions are 95% confidence interval, 80% power of test and ratio of exposed to unexposed = 1:1. The calculated sample size was 634. After adding a non-response rate of 10%, the final sample size became 697.A simple random sampling method was employed to select the households. The number of households included in each kebele, the smallest administrative unit, is determined in proportion to the total number of households in each kebele. A computer generated random numbers were used to randomly select households from the lists of households who have adolescents Fig. .Fig. 1ScData was collected using pretested structured questionnaire, adapted from EDHS 2016 and other published literature , 22. TheFace to face interviews were conducted by six experienced health professionals, supervised by public health officer from the town health office. Data collectors and supervisor were trained for two days through practical exercises.Parents who discussed at least two SRH issues  with adolescents in the last 6 months , 23.Those parents who scored above the mean of the sexual and reproductive health knowledge related questions , 24.Those parents who scored above the mean of the SRH knowledge related questions .Questionnaire was prepared in English language, translated to local language (Goffatho) and back translated to English by an expert for consistency. Questionnaire was pre-tested on 5% of the calculated sample size among parents from Galma Town which is not included in the actual study to ensure clarity of the questionnaire and then the necessary modifications and correction was made to standardize and ensure its validity. Adjustments in the sequence and wording were made based on the results of the pre-test. Questionnaire was checked for completeness on a daily basis. Incorrectly filled or missed questionnaire were sent back to respective data collector for correction. Principal investigator would recheck the completed questionnaires.The collected data was coded and entered in to Epi-Data Version 3.1  and analThe response rate was 94% (653 of 697 parents participated). The mean age of respondents was 42 years (standard deviation (SD) 6.60). Nearly 80% of the respondents were Christian 521) and ethnically Gofa (518) Nearly a quarter  of the respondents had no formal education while 34.8% (227) were civil servants. One hundred forty-five (22.2%) had monthly income of 1000–2000 Ethiopian Birr. The majority of respondents were married  and a parent; only 1.7% (11) were guardians  were considered to have good knowledge. Of the total 653 respondents, almost all knew about at least one contraceptive method, 641 (98.2%). When asked separately, majority knew about injectable (96%), condom (95.4%), pills (91%) and natural safe period (standard days method) (70.9%). Almost all (98%) participants knew at least one STI including HIV. Specifically, majority knew about HIV (98.3%) and Syphilis (89.9%) followed by Cancroid (66.5%). Almost all respondents knew at least one physical and behavioral change during puberty 649(99.4%). They mentioned breast development, onset of menstruation in females, change in voice in both sexes, refusal of parent command, feeling angry for minor reasons and interest towards opposite sex 99.1%, 98.3%, 96.6%, 77.3%, 73.2% and 71.7%, respectively  participants had positive attitude towards SRH issues. Majority of respondents 447(68.50%) strongly disagree accepting premarital sex, while 5 (0.80%) had no problem with having premarital sex. About the same percent of parents (55%) agreed that both girls and boys must remain a virgin until they are married. Of the total 653 respondents, 400 (61.3%) believed that parent to adolescent discussion on SRH issues can delay first sexual intercourse. Four hundred sixty-four (71.10%) of parents believed that if unmarried couples want to have sexual intercourse before marriage they must use condom. Of all, 442 (67.70%) believed that HIV and other STIs can be prevented by using condom during sexual intercourse. About half of the participants (332) reported that their culture forbids parent to adolescent discussion about SRH issues.The study showed that 485 74.27%) of the total participants did not have any discussion on SRH issues , sexual intercourse , contraception , condom use , STI  and unintended pregnancy  . Parents who have attended secondary education were nine times more likely to discuss SRH issues compared to those with no formal education . The odds of discussion on SRH issues among adolescents and their parents was 22.3 times more likely among parents who attended higher education (diploma and above) .Parents who reported a monthly income of above 2000 Ethiopian Birr were 2.4 times more likely to discuss SRH issues with their adolescents  compared to parents with a monthly income less than 1000 Birr.Parents who had good SRH knowledge were two-fold more likely to discuss SRH issues with their adolescents  than their counterparts. The odds of discussing SRH issues with their adolescents were 4 times higher among parents who had positive attitude than their counterparts   parents reported that they had discussion on two or more topics in the last 6 months prior to the study about SRH issues with their adolescent children. This finding is similar to findings from other different regions of Ethiopia like Awabel Woreda in East Gojjam/Western Ethiopia (25.3%), Harar 28.7%) and Benishangul Gumuuz (28.9%). Similar findings were reported from China  which sh.7% and BIn this study discussion about puberty was 8.3%, which is similar to previous study done in Ziway (10.6%) . HoweverOnly 7% participants in this study reported discussion on contraception. This is lower than reported by other studies done in different parts of Ethiopia, including in Debremarkos (25.9%), Harar (26.39%), Yirgalem (36.1%) and Bullen woreda of Benishangul Gumuuz (41.3%). The difference may similar to as reported above, being difference in measurement criteria , 31, 32.One fifth 21.1%) participants of this study discussed about STI and HIV/AIDS. This is lower than the study in Bullen woreda of Benishangul Gumuuz, Ethiopia (78.6%). This may be due to difference in study population and that they considered discussions between teachers, friends, sisters and brothers as parental discussion . Eightee.1% partiParents also indicated various reasons why they do not discuss sexual and reproductive health issues with their adolescents. Among the major reasons, cultural norms (54.2%) was mentioned which is in line with the study done in Ziway (54%) but greater than other studies done in Yirgalem, Harar and Bullen woreda of Benishangul Gumuuz , 31, 32.Thirty-six percent of the respondents do not discuss due to lack of communication skills. This result is consistent with many studies done in different parts of Ethiopia and from abroad , 33, 34 Majority of the participants had good knowledge about SRH issues (72.4%) . This is greater than the studies done in different parts of Ethiopia like, Harar (67.38%) and Debremarkos (57.3%). This difference may be due to time gap between the studies which may have resulted in improvements in available resources , 23.The result of multiple logistic regression models revealed that parents who had no formal education were less likely to discuss about SRH issues when compared to those parents who had primary education , secondary education  and diploma and above .This result is consistent with different previous studies , 33–36. Parents who reported a monthly income of above 2000 Ethiopian Birr were 2.4 times more likely to discuss SRH issues with their adolescents  compared to parents with a monthly income of less than 1000 Birr. This is similar to the study done in Harar . ParentsThis study is community based and conducted from parents’ perspective unlike many researches done from adolescent perspective. However, the study was based on self-reporting and responses might be affected by social desirability bias because of the sensitive nature and cultural barriers for reporting. Since the study design was cross sectional, cause and effect relationship could not be established. The presumption that discussion is positive, when in reality, some discussions can be quite negative and harmful is limitation of the study. Another limitation is that only adults were interviewed. It would have been interesting to get the adolescents' perspective and see if it aligned with the parents.As a primary socializing agent, and as live model, parents are very important in shaping the current and the future lives of their adolescents. Indeed, this study provides important programmatic implications for amending policies and programs which address cultural and social factors that influence parental discussion about SRH issues.Different evidences suggest that parent to adolescent discussion on reproductive health issues is the basis for safe and healthy transition to adulthood. However, in this study even if parents had good knowledge and positive attitude, the proportion of discussion about sexual and reproductive health is low. This is bounded by different cultural taboos, fear of discussion and lacks of communication skill and belief that talking about sex will initiates sex. Parents who attended primary education had better chance of discussing on SRH issues with adolescents. Those with higher monthly income had discussed on SRH with adolescents. Similarly, those with good knowledge and positive attitude also had discussed about SRH with their adolescents than their counterparts. We recommend provision of continuous capacity building on SRH issues for parents to increase the level of parental knowledge and attitude about SRH issues and sustainable advocacy should be provided about the importance of parent to adolescent discussion. Moreover, it is important to support and scale up the adult learning program to decrease the level of illiteracy. In the next AYRH Strategy update education for parents on SRH issues and skills development for empowering them to have SRH discussions with their adolescents. Another recommendation would be to engage health providers with the tools and job aids to help them discuss the importance of parent–adolescent communication on SRH issues, and including activities around this topic in donor-funded AYRH projects in Ethiopia. Finally, research is needed to fully understand the issues relevant to social meanings and beliefs that were major reason for low discussion between parent and adolescent.


N = 68) and RIN ranging between 7.0 and 8.6, which was unprecedented using standard RNA purification protocols. This study confirmed the reproducibility of the workflow through repeated trials at different postnatal time points and across the distinctive ILMs. High-throughput diagnostics from 90 RNA samples indicated no sequencing alignment scores below 70%, validating the extraction strategy. Significant differences between the standard and experimental conditions suggest circumvented challenges and broad applicability to other skeletal muscles. This investigation remains ongoing given the prospect of therapeutic insights to voice, swallowing, and airway disorders. The present methodology supports pioneering global transcriptome investigations in the larynx previously unfounded in literature.Challenges related to high-quality RNA extraction from post-mortem tissue have limited RNA-sequencing (RNA-seq) application in certain skeletal muscle groups, including the intrinsic laryngeal muscles (ILMs). The present study identified critical factors contributing to substandard RNA extraction from the ILMs and established a suitable method that permitted high-throughput analysis. Here, standard techniques for tissue processing were adapted, and an effective means to control confounding effects during specimen preparation was determined. The experimental procedure consistently provided sufficient intact total RNA ( To perform these roles, the ILMs control VF movement via complex neuromuscular activation3. Upon contraction, the posterior cricoarytenoid (PCA) abducts the VFs, opening the glottis for swallowing and airflow during active and passive breathing3. In contrast, the thyroarytenoid (TA) muscles within the adductor complex adduct the VFs, a requisite of normal phonation4. Each set of muscles is innervated by ipsilateral branches of the recurrent laryngeal nerve (RLN). Following nerve injury, newly-formed axons from the proximal stump, directed in part by guidance cues, grow back to the denervated ILMs in an aberrant fashion. This non-selective reinnervation can result in synkinesis and VF paralysis with significant patient morbidity9. Effective treatment of nerve paralysis to re-establish normal VF function will require the restoration of the pre-injury innervation pattern. Further, therapeutic manipulation of the guidance cues expressed by the ILMs remains essential for rehabilitation. Evaluating gene expression in these muscles before and after RLN injury will provide the insight necessary to identify high-value targets for further research and therapeutic intervention. To this end, comprehensive RNA-Sequencing (RNA-Seq) of the distinctive ILMs will permit an unbiased, large-scale transcriptome assessment to identify unknown factors governing VF movement.Vocal fold (VF) motion is regulated by the intrinsic laryngeal muscles (ILMs) that facilitate glottic activities integral to daily life, including voicing, swallowing, and breathing10. Whole-transcriptome RNA-Seq provides a broader, more precise characterization and distribution of mRNA transcripts compared to low-throughput methods ; thus, its technology warrants highly undegraded RNA samples11. Until recently, insufficient RNA recovery hindered transcriptome investigations in skeletal muscles, given their characteristically high metabolic activity12. In addition, the challenges of RNA extraction from post-mortem skeletal muscles are also significant across species. For instance, human post-mortem skeletal muscle analysis previously provided substandard RNA quality, summarized as RNA integrity numbers (RIN) ranging between 1.6 and 7.613. This compromised RNA integrity restricted the downstream assessment to a low-throughout application and perturbed the RT-qPCR performance. In contrast, living tissue samples following human muscle biopsy yielded between 456 to 676 ng total RNA and 7.9 ± 0.6 RIN12. Unfortunately, compared to other soft tissue types, the distinctive ILMs have not been amenable to RNA-Seq due to differences in cell characteristics14. Efforts to recover sufficient RNA from the distinctive ILMs have only been documented in low-throughput profiling techniques and histological studies due to their lower requisites for RNA quality22. In this regard, fundamental knowledge gaps apropos the ILM innervation patterns reflect the unresolved challenges of high-quality nucleic acid input for bulk RNA-Seq. While efforts to sequence RNA from the skeletal muscles have emerged in recent literature, the methods described are unadoptable to this study, as they do not address the challenges of extracting high-quality RNA from the distinctive ILMs. In the rat, there is increasing evidence of bulk RNA-Seq using skeletal muscle in the hindlimb muscles24. However, given their greater abundance of intrinsic RNA compared to the small ILMs, the proposed extraction strategies are insufficient for the ILMs as they do not account for the limited RNA content. A Parkinson’s study performed by Lechner et al. (2021) assessed the RNA-Seq expression analysis from the rat TA. Although, their sequencing strategy enabled the input of low-throughput RNA extracts25. This method, commonly known as TruSeq, requires a small sample size and has unique RNA specifications unamenable to other bulk RNA-Seq technologies26. Other studies demonstrated successfully high-throughput RNA analysis from tissued excised from the human and rabbit larynxes29. Unfortunately, they sampled all the soft tissue associated with the VFs, including VF mucosa and respiratory epithelium, rather than isolated ILM alone. Further, RNA extraction procedures described in a recent whole-genome profiling study from the bat cricothyroid muscles yielded variable and substandard RNA in the distinctive ILMs30. Despite these studies, there remains a lack of publicly available data describing a functional and reliable extraction method from the distinctive ILMs for general RNA-Seq use.Minimal RNA degradation is paramount to reliable downstream analysis in any gene-profiling study, especially in robust sequencing techniques30. Other methodological strategies for RNA purification, such as nucleic acid extraction via magnetic beads, were not applied here due to a lack of evidence of its use in rat skeletal muscles. Our evaluation of the diverse commercial protocols indicated that relevant challenges of high-quality RNA extraction from rat ILMs are likely due to the muscles’ small size, unique cellular content, and fiber composition31. Other critical variables also influenced the quality of the RNA, namely, the condition of the tissue or the impact of degradation during processing steps. The vulnerability of RNA in rat ILMs is in contrast to findings that characterize skeletal muscle mRNA as highly stable relative to the liver, brain, and adipose tissue14. This suggested that variable RNA stability across species, tissue types, and organ layers may require modified extraction strategies according to the specimen and sample size. To that extent, this study established a systematic, high-quality RNA extraction method from isolated postnatal rat ILMs that permitted RNA-Seq. By developing a suitable technique to minimize native and non-native derived degradation, enhanced RNA recovery was maintained. Identifying an ideal weight model also limited the impact of total tissue weight during handling steps. These findings demonstrate the critical factors of RNA isolation techniques that permit high-quality transcriptome analysis.The present study aimed to identify and resolve threats to consistent, high-quality RNA recovery in rat ILMs. Previous whole-genome analyses in the rat ILMs and other skeletal muscles utilized spin-column technology or reagents to separate the nucleic acids. Hence, similar protocols were evaluated in this investigation260/A280 ratio), concentration (ng/µL), and total RNA yield (ng); while electropherogram  analysis provided RIN values for quality control (QC) assessment. RNA extraction using the following kits was performed according to the manufacturer’s instructions unless otherwise stated. In compliance with RNA-seq recommendations, this study aimed to generate extracts with greater than 500 ng Total RNA, 1.98 A260/A280 ratio, and 7.5 RIN. Qualifications for bioanalyzer assessment included sufficient total RNA recovery (> 200 ng), high purity , and medial thyroarytenoid (MTA) intrinsic laryngeal muscles. This study quantified the impact of mitigation strategies to establish optimal tissue processing conditions  provided a cost-effective means for performing RNA isolations ; however, the amount of recovered RNA was low (247.76 ± 107.72 ng) and often did not meet the 200 ng requirement for RNA-Seq application (Table pt = 2), prompting both an increase in total RNA output  and a depreciation in the overall A260/ A280 ratio  that were not statistically significant. Additionally, substantial variations in the quality of samples across assays discouraged the continued use of this protocol (Table The ns Table . This copt = 1) resulted in a high variance in the A260/A280 ratio (3.60 ± 2.92) and amount of recovered RNA (94.07 ± 68.61 ng) such that extracts performed in the same batch were inconsistent and rarely qualified for further processing (Table pt = 2) resulted in a statistically significant difference in the overall A260/A280 ratio (P < 0.0001), the increase in total RNA yield (P > 0.99) was nonsignificant (Table The RNeasy Micro Kit (Qiagen) was performed under identical conditions for tissue processing as previously stated Table . The advQuick-RNA, P = 0.05; RNeasy, P = 0.004) (Table 260/A280 ratios (1.54 ± 0.10) in the recovered extracts, a 96.8% incidence (61 out of 63 samples) of phenol contamination across assays, and substantially longer procedure time than column-based methods; however, this procedure regularly provided RNA yields that exceeded the RNA-Seq sample requirements (Table P > 0.99) remained nonsignificant; however, this method provided the only uncontaminated samples in this trial. In addition, a slight reduction in the ratio of chloroform and prolonged incubation in isopropanol (~ 20 min) resulted in an individual RNA extract (1 out of 16 samples) with spectrophotometer measurements suitable for further processing . Bioanalyzer assessment indicated a 7.0 RIN; however, these changes failed to provide comparable results beyond this sample.Trizol reagent (Invitrogen) yielded significantly higher total RNA quantities using individual ILMs compared to the results achieved using the spin-column kits  relative to the former method  and significantly increase the overall purity  and spin-column RNA purification (RNeasy) protocols to assess the moderation of purity and contamination. This technique did not reduce the detection of impurities (100% phenol contamination) or significantly increase the A260/A280 ratios (P > 0.99) and thus further trials were discontinued  though this change was not statistically significant. This method provided several samples with spectrophotometer measurements that warranted bioanalyzer assessment despite the lower total RNA yield (Table The combination of the guanidinium thiocyanate solution (TRIzol) and PureLink RNA Mini Kit (Invitrogen) protocols resulted in enhanced RNA quality Table . Phase s260/A280 ratios (2.31 ± 0.54) throughout this investigation  the resultant A260/A280 ratios using this spin-column (Aurum) kit did not extend below 1.96. Though most extracts were inapplicable for meaningful RNA-seq analysis, this kit provided no invalid RIN values  or two pooled ILMs  would improve RNA integrity yield, respectively. While the applied changes significantly increased the RIN (P > 0.0001), these changes were insufficient to promote protocol systemization or consistent recovery (Table The Aurum kit (Bio-Rad) provided the most favorable Aatios 2.3 ± 0.54 tatios 2.3 ± 0.54 tatios 2.3 ± 0.54 tatios 2.3 ± 0.54 tatios 2.3 ± 0.54 tlater), which hinders RNase activity through precipitation and metal chelation10, significantly increased the RIN in the overall model . Electropherogram peaks showed general depreciation of RNA molecules and insufficient total RNA recovery using standard tissue handling procedures . Alternatively, the addition of RNase-inhibiting reagents caused more distinct ribosomal RNA peaks and increased the RIN . Low 28S/18S ratios (below 2.0) also revealed the vulnerability of RNA during isolation steps; however, the reliability of the 28S/18S ratios as an indication of relative degradation is unclear . Beyond its indispensable inhibitory properties, overnight incubation in RNase-inhibiting reagent also promoted the hardening of the muscle fibers, which increased the labor required to affect cell lysis.Stabilization of endogenous RNA by ribonuclease (RNase) inhibiting reagents improved RNA integrity compared to the standard method Fig. a. The apres Fig. b–d, leftDeveloping an effective cell lysis method for adult rat ILMs required a systematic assessment of manual and mechanical homogenization strategies. Manual techniques involved physical breakdown via disposable pestle in specialized RNase-free homogenization tubes. Mechanical rotor–stator homogenization (TissueRuptor II) provided high-speed, automated tissue breakdown through replaceable probes. Finally, an integrated lysis method that incorporated both strategies was also adopted.P = 0.33). Across global experiments (pt = 3), the integrated homogenization method increased the recovery of RNA (887.59 ± 216.18 ng) and demonstrated significant differences compared to the manual  and mechanical methods   and the rotor–stator lysis methods  in the overall model  and evaluating the results from varying disruption times across several assays. These investigations allowed the present study to systematize the experimental procedure by identifying the upper and lower limits that ensured considerable intact RNA yields given the tissue characteristics. Table The evaluation of total RNA recovery attributable to the span of rotor–stator use . A significant increase in RNA yield was observed in the PCA (P = 0.03) and MTA (P = 0.003) muscles, while the changes observed in the LTA were not significant  and the overall model  were evaluated to determine the optimal number of muscles per sample to isolate sufficient quantities of RNA for sequencing  resulted in sufficient RNA quantities in most (88.9%) but not all cases , and MTA muscle (1121.35 ± 555.19 ng), which was not readily observed using fewer pooled muscles  RIN values from 7.0 to 8.6 (7.8 ± 0.4) with no RNA yields below 250 ng  indicated substandard extraction for high-throughput sequencing technology. This reagent was also discouraged due to the observed frequency of phenol contamination, which perturbs the RNA concentration and hinders downstream application35. Thus, attenuating the risk of phenol contamination was of utmost importance given the high binding affinity of RNA to oxidized quinones in the presence of alcohol36. Previous laryngeal muscle studies using this reagent to extract RNA from the rat model have only performed low-throughput analysis39. In selecting an appropriate kit, this study valued a ~ 2.0 A260/A280 ratio and a low variation between samples in consideration of the influence of batch effect during RNA-Seq analyses40. The chosen kit provided the most favorable extracts with A260/280 ratios higher than 1.96, indicating successful DNase digestion. Consistent data void of chemical contamination across all assays also encouraged the use of this protocol. Though the standard method did not result in high RNA yields, it was the most practical complication to mitigate relative to other tested kits.Comparisons of the purification procedures suggested that standard tissue preparation techniques, as per the respective protocol guidelines, do not account for the variability or sensitivity of starting material. Moreover, the shortcomings of the tested spin-column kits were likely related to the assumed standardized preparation of animal tissue rather than a reflection of the effectiveness of the column-based systems. The low Apt = 3). Compelling evidence in other rat model studies has demonstrated the feasibility of successful RNA-Seq analysis using the Aurum Total RNA Fatty and Fibrous Tissue Kit41. Due to the greater column capacity (100 mg) of the aforementioned kit, its application can be assessed in future studies.A limiting factor of the preferred kit was its inability to consistently provide sufficient total RNA without pooling muscle fibers  as external factors that mediate alterations to tissue transcriptomes45. Conversely, previous research has challenged the alterations to RNA integrity following prolonged ischemia finding no correlation between RNA quality and the PMI in human skeletal muscle and colonic epithelial tissue46. Without a clear consensus in the literature, the present study mitigated degradation during the PMI by minimizing the total procedure time and employing RNase-inhibiting reagents45. This study applied RNase-inhibiting reagent (RNAlater) to the dissected ILMs to limit degradation. Overnight incubation of the muscles in cold RNase-inhibiting reagent established the immobilization of active hydrolytic contributors during freeze/thaw cycles. ILM dissection from fresh tissue rather than after incubation in the stabilizing reagent allowed for efficient separation. Storing individual ILMs also increased the surface area exposed to the RNase-inhibiting reagent. Alternatively, incubating the whole larynx overnight would likely result in partial or fragmented muscle removal. Given the elevated RIN values and low 28S/18S ratios, this study concluded that the stability this reagent provided was not absolute. Previous methodological studies that assessed the efficiency of RNase-inhibiting reagents at different time points demonstrated reduced RIN after 24 h, justifying an overnight incubation47.Assessment of recovered RNA revealed the significant influence of tissue preparation procedures, where RNA degradation remained inevitable upon organismal death until the development of the lysate. In downstream assessments, degradation, depicted by the loss of short genes and the changes to longer genes in the mRNA transcript, perturbs the innate gene profileLimitations using RNase-inhibiting reagents were related to their effectiveness over time. Namely, overnight storage in RNase-inhibiting reagents followed by prolonged storage at − 80 °C was not assessed for changes in RNA integrity. Overnight storage was complicated by its propensity to cause column clogging and hole formation due to hardened ILMs that deemed several samples (23 out of 48) unusable Table . Finally48. The advantages of the developed method were four-fold. First, using an integrated homogenization strategy significantly increased the yield of RNA recovery per assay, allowing most extracts to qualify for RNA-Seq. Second, the ideal weight model permitted a systematic approach for optimal RNA extraction void of excessive inter-and intra-variability across the global experiments. Third, the conceptualization of the unique cryogenic disruption module provided an effective means for tissue disruption void of chemical and biological contaminants. Fourth, the experimental method reduced the incidence of column clogging and hole formation, making all recovered RNA applicable for bioanalyzer assessment , pooling three ILMs per sample reflects both the minimum and the maximum amount needed for optimal RNA extraction of adult rat ILMs 100% of the time. Suitable RNA recovery is achievable using two ILMs, but only 88% of the time. A limitation of the proposed pooling strategy is the results may not accurately reflect population variation in gene expression levels55. This age-related study indicated that more resilient fibers in the PCA and TA muscles, relative to the cricoarytenoid muscle, are attributable to their inherent function. These observations suggested that the load required for the breakdown of the PCA is greater than that of the TA muscles due to their increased adaptive resistance and fiber content. Evaluating various purification strategies implicated degradation as particularly adverse for ILMs due to their small muscle fiber cross-sectional area and volume, ergo limited RNA abundance57. Previous quantitative PCR analysis of abductor (PCA) and adductor (LTA and MTA) muscles also demonstrated the significant vulnerability of the cellular components of these muscles to damage during RNA isolation procedures13. In short, for optimal RNA extraction from rat ILMs, substantial force is needed to affect cell lysis; however, too much can compromise intact RNA . The Institutional Animal Care and Use Committee of Columbia University Medical Center approved the animal use protocol. Under a 12 h dark/light cycle and ambient temperature (21 °C), Sprague–Dawley rats were placed in group housing with ad libitum access to food and water. Young adult rats (~ 300 g) (n = 482) were distributed in sets of triplicate treatment groups according to their sex (3 rats per group). The procedures and their modifications for the optimized protocol are described as follows:40. The specimen was then transferred to a sterile petri dish and rinsed in 2 mL of RNAlater , which served as a stabilizing agent. After aspirating excess solution from the petri dish, the specimen was placed onto a sterile surface for dissection via a Leica S8 APO compound microscope . The esophagus and pharynx were carefully excised from the posterior aspect of the harvested tissue before extracting the right and left laryngeal abductor muscles (PCA). The aryepiglottic folds were then removed to provide access for dissection of the right and left adductor muscles (LTA and MTA) with fine-tip forceps. ILMs from the rat larynx were less than 5 mm in length, and individual muscle bellies weighed between 2 and 13 mg. Rather than storing the whole larynx in the reagent after rinsing, per other optimized protocols for RNA purification, individual ILMs were immediately incubated in 1 mL of RNAlater following dissection. Samples were then placed at 4ºC overnight and then processed the following day.Adult rats were euthanized by a lethal injection of ketamine and xylazine before laryngeal muscle dissection. Once euthanized, 70% ethanol served as a skin disinfectant before harvesting the larynx. A midline incision was performed in the neck. Following retraction of submandibular glands and extralaryngeal muscles, the larynx was identified and removed en bloc with the proximal trachea, esophagus, and pharynxQuick-RNA MicroPrep , RNeasy Micro Kit , TRIzol Reagent , and the Aurum Total RNA Mini Kit . In addition, the following procedures were evaluated: TRIzol protocol using a half volume of all reagents, Trizol in combination with RNeasy Mini kit (Qiagen), Trizol plus PureLink RNA Mini Kit , and RNAlater in sequential integration with the Aurum kit (Bio-Rad). RNA purification was followed according to the kit instructions, and deviations from the recommended preparation of the starting material are reported in the Results section. Protocol modifications to the Aurum kit and Trizol protocols facilitated the optimization of RNA purification. Here, parameters related to homogenization duration, disruption method, number of tissues per tube, and volume of reagents were modified.The following kits were tested for the evaluation of the most effective RNA isolation: ZR Zap  for removing ribonuclease contamination. Other surfaces and instruments were also sprayed during all laboratory processes. The Aurum Total RNA Mini kit allowed for the most optimal and consistent extraction of high-quality RNA. For the prescribed procedure, tissues were removed from stabilizing reagent and briefly dabbed on a paper towel to remove excess solution, according to the manufacturer’s guidelines. Samples were pooled (pt = 3) according to muscle type, position (right or left), and gender. Similar laryngeal muscle tissues were combined in respective BioMasher III tubes , with the filters removed, to facilitate manual grinding and lyophilization in liquid nitrogen via the disposable pestles. Before the physical breakdown of muscle tissue, the pooled samples were weighed to determine the appropriate duration of mechanical homogenization to yield cell lysates with significant intact RNA. After pooling, the calculated median total weights were 50 mg, 40 mg, and 20 mg for PCA, LTA, and MTA, respectively. An apparatus for cryogenic disruption was assembled using medium binder clips and BioMasherIII tubes . RNA integrity, the value of which serves as the final determinator of efficacy for RNA-Seq analysis, was assessed using an Agilent 2100 bioanalyzer. The RNA integrity number (RIN) calculated using this software presents a range of values from 1.0 to 10.0—indicating complete degradation and intact RNA, respectivelylater and cryogenic disruption on RNA quality, two-way ANOVA followed by Bonferroni’s multiple comparisons test was computed. The assessment of homogenization techniques and pooling strategies were facilitated by standard two-way ANOVA followed by Dunnett’s multiple comparisons tests to assess RNA quantity and purity that resulted from cryogenic disruption. Linear regression analysis was performed to demonstrate the relationship of contributing factors in the ideal weight model. The data were tested for normality via Q-Q plot and regression analysis. Finally, data in all supplemental figures were computed by two-way ANOVA followed by Tukey’s post-hoc tests. The present study considered P < 0.05 as the level of statistical significance. .The present study expresses all data as mean ± SD unless otherwise stated. All statistical analyses were performed using GraphPad Prism 9.3.1 . Ordinary one-way ANOVA followed by Dunnett’s multiple comparisons tests were performed to compare the experimental results to the recovery of RNA using currently available protocols. A repeated analysis of variance followed by Tukey’s post-hoc tests allowed for individual comparisons of the commercial extraction methods. To examine the effect of RNASupplementary Figures.


Advanced non-squamous non-small cell lung cancer (NS-NSCLC) patients without driver gene mutations are usually treated with immune checkpoint inhibitors (ICIs) plus pemetrexed as maintenance therapy after first-line ICIs plus 4–6 cycles of pemetrexed/platinum. Some patients in the real world receive ICIs monotherapy as maintenance therapy. No clinical study has compared the efficacy and safety of ICIs with or without pemetrexed as maintenance therapy.We performed a retrospective study analyzing clinical data of patients with NS-NSCLC who were diagnosed in Zhejiang Cancer Hospital from September 2018 to May 2021 and received maintenance therapy after 4–6 cycles of ICIs plus pemetrexed/platinum. Patients were divided into ICIs plus pemetrexed group and ICIs monotherapy group. Progression Free Survival 1 (PFS1) and PFS2, defined as the interval from the date of initial treatment and maintenance therapy to the date of systemic progression/death or the last follow-up, respectively.P = 0.979). Among patients with PD-L1 TPS < 1%, the median PFS1 was worse with ICIs monotherapy . Among patients with PD-L1 TPS ≥50% or 1–49%, the median PFS1 in both groups was not statistically significant . Results for median PFS2 were similar to median PFS1, with statistically significantly different only in patients with PD-L1 TPS < 1% (P = 0.008). The 2-year survival rates of the two groups were similar . The incidence of fatigue was significantly higher in the ICIs plus pemetrexed group (P = 0.023).A total of 120 patients received ICIs with or without pemetrexed as maintenance therapy. Eighty-two patients received ICIs plus pemetrexed as maintenance therapy, and 38 patients received ICIs monotherapy. There were no statistically significant difference in median PFS1 between the ICIs monotherapy group and ICIs plus pemetrexed group (12.00 months vs. 12.07 months, ICIs with or without pemetrexed can be used as maintenance therapy after first-line ICIs plus 4–6 cycles of pemetrexed/platinum in patients with advanced NS-NSCLC based on PD-L1 expression. The incidence and mortality of lung cancer are still among the top malignant tumors globally . Non-smaMaintenance therapy in the National Comprehensive Cancer Network (NCCN) guidelines refers to using at least one of the agents given in the first line, beyond 4–6 cycles, in the absence of disease progression . In patiImportantly, no clinical studies have investigated the difference in the efficacy and safety of ICIs with or without pemetrexed as first-line maintenance therapy in NS-NSCLC. We conducted a retrospective study to investigate whether ICIs combined with pemetrexed as maintenance therapy have clinical benefits in patients with advanced NS-NSCLC. We also stratified patients for their PD-L1 expression to achieve precise benefit.All patients were diagnosed with NS-NSCLC in Zhejiang Cancer Hospital from September 2018 to May 2021. Inclusion criteria: 1) First-line use of ICIs plus with pemetrexed/platinum (cisplatin or carboplatin) for 4–6 cycles; 2) Complete response, partial remission or stable disease after 4–6 cycles of induction chemotherapy; 3) Eastern Cooperative Oncology Group Performance Status (ECOG PS) 0–1; 4) Complete baseline clinical data, including diagnostic age, gender, smoking, ECOG PS, intrathoracic metastasis status, liver metastasis status, bone metastasis status, brain metastasis status, programmed cell death-ligand 1 (PD-L1) expression and Adverse events. Exclusion criteria: 1) Pathologically diagnosed as squamous cell carcinoma; 2) EGFR, ALK, and ROS1 genes are positive; 3) ECOG PS 2–4. This study was approved by the Ethics Committee of the University Cancer Hospital of the Chinese Academy of Sciences . It was carried out under the Declaration of Helsinki.Patients received 4–6 cycles of ICIs plus pemetrexed/platinum (cisplatin or carboplatin) in the induction phase. Patients who achieved complete remission (CR), partial remission (PR), or stable disease (SD) after induction entered the maintenance period with the patient’s consent. Under normal circumstances, the attending physician would recommend pemetrexed combined with ICIs as maintenance therapy. If the patient refused to continue maintenance of pemetrexed or is intolerant to chemotherapy, ICIs monotherapy was used for maintenance therapy. According to the maintenance regimen, patients were divided into ICIs plus the pemetrexed group and ICIs monotherapy group. The type of ICIs used for NS-NSCLC was predominantly pembrolizumab (37.5%), with the remaining therapy including camrelizumab (23.3%), sintilimab (21.7%), tislelizumab (10.0%) and toripalimab (7.5%). All chemotherapy and ICIs were performed under the standard doses of NCCN guidelines. PD-L1 expression was evaluated using PD-L1 22C3 pharmDx . We used the Response Evaluation Criteria in Solid Tumors (RECIST) v1.1 to assess the response to each treatment. Before the analysis, two oncologists checked the efficacy. They assessed the tumor response according to RECIST v1.1 through chest computed tomography and/or brain magnetic resonance imaging (MRI) every 4–8 weeks. The objective response rate (ORR) is defined as the proportion of patients with CR plus PR. Progression-free survival 1 (PFS1) was defined as the interval from the date of initial treatment to the date of systemic progression/death or the last follow-up, whichever is the first to trigger the review of the event date. Progression-free survival 2 (PFS2) was defined as interval from the date of maintenance therapy to the date of systemic progression/death or the last follow-up. Overall survival (OS) was defined as the interval from the date of diagnosis of advanced NS-NSCLC to the date of death or the last follow-up. Safety assessments included physical examination, documentation of adverse events, electrocardiogram, and laboratory tests. Adverse events were graded according to National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE) version 5.0.p values < 0.05. SPSS 26.0 (IBM) and GraphPad Prism version 9.0 (GraphPad Software) were used for statistical analysis of data.Chi-square test or Fisher’s exact test were used to test the association between the treatment group of all patients and the clinical classification variables. Kaplan-Meier curves were used for univariate survival analysis. The Cox proportional hazards model was used to complete the univariate and multivariate survival analyses with a hazard ratio (HR) and corresponding 95% confidence interval (95%CI). Significance between groups was defined as From September 2018 to May 2021, 215 patients received first-line ICIs plus pemetrexed/platinum (cisplatin or carboplatin) at Zhejiang Cancer Hospital, and 123 (57.2%) patients entered the maintenance phase. Among them, three patients (1.4%) received pemetrexed as maintenance therapy, 38 patients (17.7%) received ICIs monotherapy as maintenance therapy, and 82 patients (38.1%) received ICIs plus pemetrexed as maintenance therapy  in the ICIs monotherapy group. The median number of ICIs maintenance cycles was 9  and pemetrexed maintenance cycles was 6  in the ICIs plus pemetrexed group.P = 0.979) .There were no statistically significant differences in median PFS1 between the ICIs monotherapy group and ICIs plus pemetrexed group      Fig. A. Simila08) Fig. B-D.Fig. P = 0.003). The detailed data is shown in Fig. A subsequent univariate analysis illustrated that the features such as Age, Gender, Smoking, Liver metastasis, Intrathoracic metastasis, Bone metastases, and ICIs plus pemetrexed or ICIs Maintenance were not independent influence factors PFS. The results of the multivariate analysis indicated that brain metastasis was an independent factor affecting PFS .Median follow-up was 23.20 months (95%CI 20.04–26.34) from the first day of diagnosis of advanced NS-NSCLC. As of December 31, 2021, 37 patients  had died in the two groups, and the 2-year survival rate of the ICIs monotherapy group and ICIs plus pemetrexed group were 66.7 and 69.5%, respectively. The difference was not statistically significant (P = 0.023). Decreased appetite , hematologic toxicity , pneumonia , peripheral edema  were also more likely to appear in the ICIs plus pemetrexed group, but the difference was not statistically significant. During maintenance, No patients in two groups had grade 5 AEs. 6 patients (15.8%) in the ICIs monotherapy group and 15 patients (18.3%) in the ICIs plus pemetrexed group experienced grade 3–4 AEs (P = 0.938).During the maintenance treatment of 120 patients, 60 patients  experienced 100 Adverse events (AEs), mostly grade 1 or 2 . The most common any-grade AEs were hematologic toxicity, pneumonia, and fatigue  [As we all know, the tumor PD-L1 expression level is considered a biomarker associated with the efficacy of ICIs –17. Howectively) . In our For safety, common adverse events in prospective studies of ICIs plus pemetrexed as maintenance therapy were hematologic toxicity, pneumonitis, hepatic function abnormality, and fatigue –6. The iThe relationship between chemotherapy and immunotherapy has not been fully explored. Chemotherapy acts synergistically with immunotherapy by upregulating the expression of the tumor antigen itself or MHC class I molecules that bind to the antigen to enhance tumor antigen presentation or enhance the intensity of effector T cell activity –21. In cThe Checkmate-9LA study of 2-cycle chemotherapy plus two ICIs suggests that short-course chemotherapy can exert a rapid anti-tumor effect and avoid the adverse events caused by long-term chemotherapy . A studyOur study has several limitations. First, the retrospective nature of our study may have influenced some outcomes, including the recording of adverse events. Second, the sample size of the ICIs monotherapy maintenance group in this study was small, mainly because the samples were from single-center. Third, the observation period is not long enough. Regarding OS, the number of events was immature. We would report it after follow-up. Finally, the discontinuation rate for pemetrexed in this study is noteworthy and may affect the generalizability of the results.In summary, our study showed that ICIs with or without pemetrexed can be used as maintenance therapy after first-line ICIs plus 4–6 cycles of pemetrexed/platinum in patients with advanced NS-NSCLC based on PD-L1 expression. In NS-NSCLC patients with PD-L1 TPS ≥1%, no statistically significant difference in efficacy was observed between ICIs monotherapy and ICIs plus pemetrexed as maintenance therapy; however, the efficacy of ICIs monotherapy maintenance was worse in patients with TPS < 1%. In the future, how to combine chemotherapy with immunotherapy to achieve optimal results and reduce adverse events warrants further reserch.


RNA is the key player in many cellular processes such as signal transduction, replication, transport, cell division, transcription, and translation. These diverse functions are accomplished through interactions of RNA with proteins. However, protein–RNA interactions are still poorly derstood in contrast to protein–protein and protein–DNA interactions. This knowledge gap can be attributed to the limited availability of protein-RNA structures along with the experimental difficulties in studying these complexes. Recent progress in computational resources has expanded the number of tools available for studying protein-RNA interactions at various molecular levels. These include tools for predicting interacting residues from primary sequences, modelling of protein-RNA complexes, predicting hotspots in these complexes and insights into derstanding in the dynamics of their interactions. Each of these tools has its strengths and limitations, which makes it significant to select an optimal approach for the question of interest. Here we present a mini review of computational tools to study different aspects of protein-RNA interactions, with focus on overall application, development of the field and the future perspectives. Central to their role in fundamental biological processes are RNA-protein interactions (RPI) that primarily involve modular RNA-binding proteins (RBP), although exceptions exist  and structure-based methods that use structure-derived features (such as solvent-accessible surface area or secondary structure) to make predictions. Walia et al., have reviewed tools for sequence-based methods  while stin vivo data and to identify deleterious genomic variants. With over 2000 citations, this scalable and modular pattern discovery method is a popular application of ML-based learning methods with important applications in predicting protein-RNA interactions   , CLIPZ (atabase)  and RBPDatabase) . Other matabase)  use a geTools like RBind (Binding sites on RNA) , NAPS n and PRIdProtein-RNA interactions can be predicted using structure-based information. These methods became possible as the number of protein-RNA complex structures deposited in the PDB increased in numbers. Here, either the availability of structures or structure predictions of protein, RNA or both as a complex have been employed to develop several approaches.16% of the tools to predict RPI have been developed based on the structure of a protein . InitialAnother popular method is PRNA  which emThe shape and geometry of RNA can significantly influence RBP binding. Zhang and co-workers developed DeepNet-RBP  which usGraphProt  is a fleetc.  were available in the Protein Data Bank (PDB) . Howeveretc. . Both teetc. . PRIME  of at least 2.0 kcal mol─1, are defined as hotspot residues . Given tab initio and empirical methods, modified electrostatic, van der Waals (vdW), and torsional parameters to accurately reproduce the energetics of nucleobase stacking, base pairing, and key torsional conformers. CHARMM modified its forcefield parameters to make it suitable for nucleic acids  simulations have been successfully used to characterize complex conformational transitions in proteins. In principle, MD simulation is a powerful tool for characterizing such conformational changes in RNA molecules, which depends on force-field parameters. AMBER force field  uses a cic acids . OPLS-AAic acids . Apart fic acids . Martiniic acids  helps inic acids  complex E. coli strains. Among the above databases, NPInter, PRD, RNAct, CLIPdb and RBPDB provide and extract information from experimental techniques. In future, an exhaustive interaction database which could store detailed and multi-dimensional information about an interaction entry, such as a binding region/motif, structure detection method, interactions, etc. would add more value to such efforts. Furthermore, the ability to predict potential RPI is based on availability of such catalogued information, making such databases necessary and valuable.Various databases provide comprehensive repositories of RNA-protein interaction data that have been gathered from experiments, literature and other databases and computational predictions. PRD , NPInterThis review provides a comprehensive survey of computational tools for the analysis of protein-RNA interactions. The availability of mathematical models and profiles, together with sensitive search algorithms, enable effective association of new genes into pre-existing families of RNA-binding proteins from mere sequence information. Repositories such as protein sequence families (PFAM)  and in-bRBPs are modular and may possess more than one RBD that are connected with flexible linkers or may occur as isoforms. Automatic tools are not available to provide accurate assignment of domain boundaries and nomenclature. Which of these domains bear sequence signatures of functionally important residues, how they co-operate in exhibiting function and their relative affinity for RNA still are open questions. Several proteins that contain RNA-binding domains also contain substantial stretches of disordered regions. Their structural and functional regulation are hard to capture and remain a treasure-house of knowns. Despite the choice of objective mathematical models, presence of spatial motifs of RNA-binding residues or RNA that acquire three-dimensional structure might escape detection. Some RBPs are known to bind both DNA and RNA and this might cause sufficient confusion in prediction since the results may be viewed as false positives.The success of computational algorithms is usually measured using statistical parameters such as accuracy, sensitivity and specificity. The performance of algorithms to recognize RNA-binding proteins is challenged by the absence of a comprehensive and reliable gold-standard to look upon. It is now accepted that different databases of RNA-binding proteins that use different high-throughput experimental approaches and platforms do not agree very well with each other . It woulFinally, RNA-binding proteins play important roles in diverse biological roles such as developmental processes and ageing. They are also amenable to functional regulation such as phosphorylation switches and expression levels. In future, meta-analyses are required to assimilate such functional information.


In 2007, a reform of Swedish primary healthcare began when some regions implemented enhanced patient choice in combination with free establishment for private providers. Although heavily debated, in 2010 it became mandatory for all regions to implement this choice system.The aim of this article was to review all published research articles related to the primary healthcare choice reform in Sweden, to investigate what has been published about the reform and summarise its first 15 years.A scoping review was performed to cover the breadth of research on the reform. Searches were made in Scopus, Web of Science and PubMed for articles published between 2007 and 2021, resulting in 217 unique articles. In total, 52 articles were included.The articles were summarised and presented in relation to six overarching themes: arguments about the primary healthcare choice reform; governance and financial reimbursements; choice of provider and use of information; effects on equity and access; effects on quality; and differences between private and public primary healthcare centres.The articles show that the reform has led to an increase in access to primary healthcare, but most studies indicate that the increase is inequitably distributed in terms of socioeconomy and geographical location. The effects on quality are unclear but several studies show that the mechanisms supposed to lead to quality improvements do not work as intended. Furthermore, from a population health perspective, it is time to discuss how such a responsibility can be reintegrated into primary healthcare and function with the choice system. Inspired by new public management, there has been an increasing focus on patient choice in Northern Europe and the Nordic countries since the beginning of the 1990s . In DenmThe political majority behind the reform claimed that a diversity of providers – which would compete with each other on the same terms for patients to get reimbursed – would enhance cost efficiency, improve access and stimulate quality improvements. Access and quality would improve because many patients would choose the provider with the highest quality and best access . Importaområdesansvar), employing a multidisciplinary workforce, typically with four to 10 general practitioners (GPs) and other professionals such as district nurses, physiotherapists, social workers, work therapists, psychologists and specialist nurses ation OR marketi[s/z]ation) AND . We included peer-reviewed studies published in the English language between 2007 and 2021. The year 2007 was chosen as a starting point because that was the first year a Swedish region introduced a PHC choice system. We also hand-searched the reference lists in the articles included in the final review.Supplemental material. We included articles that discussed the PHC choice reform or illustrated how the PHC choice reform has been implemented, has functioned, been governed and what effects it has had on a range of aspects, for example, access, equity and quality. We included qualitative and quantitative articles as well as theoretical and empirical articles. Articles that studied aspects of Swedish PHC without mentioning the PHC choice reform were excluded. The results are summarised and presented in relation to six overarching themes of relevance to the functioning and intended effects of the reform: the arguments about the PHC choice reform; governance and financial reimbursements; choice of provider and use of information; effects on equity and access; effects on quality; and differences between private and public PHC centres.Studies were selected by first merging the three database searches  and then removing duplicates manually: resulting in 213 unique articles. In addition, four studies were found through a screening of the references of the included articles. Thereafter, both authors read the title and abstract of these articles together to assess whether they should be read in full text: resulting in 85 articles. The authors read half of these articles each and in discussion with each other decided that 52 articles met the inclusion criteria see Supplemental material for more information about the included articles.Of the 52 included articles, 29 were quantitative, 20 were qualitative, two utilised a mixed method and one was a review article. Twenty of the included articles had a geographical scope that covered the entire country, whereas 26 articles covered between one and three regions, see Before the full introduction of the PHC choice reform in 2010, potential effects were discussed by researchers, predominantly by those who warned against negative effects. Fears were voiced that existing social inequities in access would be reinforced –15; thaRegarding the overall governability, in 2015 Saltman and Duran  noted thIn 2019, Glenngård  showed tIn the study by Glenngård from 2019 , the reiThe importance of the reimbursement systems was also illustrated by Winblad et al.  in a stuSeveral articles examine the effects of different reimbursement models related to the PHC choice reform. How diagnosis coding changed after an introduction of economic compensation based on the reported diagnoses was investigated in two articles. In 2012, Hjerpe et al.  showed tPay-for-performance (P4P) reimbursements in the Swedish PHC choice system is the topic of a few studies. In 2015, Ödesjö et al.  showed tIn 2007, when only a few regions had introduced PHC choice systems, Hjelmgren and Anell  found thThe study thus pointed to the importance of information when making a choice of provider (a supply-side factor). To what extent such information exists was investigated by Ranerup et al.  2 years Some studies focused on how individuals search for and use information when making a choice of PHC provider (demand-side factors). They all indicated that information is not used to a large extent. For example, Wahlstedt and Ekman  showed tThere is also some evidence in the literature about who searches for and uses information. Those using Internet-based information sources were younger, more educated, more often women and had better self-perceived health . FurtherRegarding access, Isaksson et al.  showed tThree studies have investigated GP visits. Analysing register data from the years 2007–2011 in Region Skåne, Beckman and Anell  showed tFurthermore, Isaksson et al.  found thTwo published articles aimed to evaluate the impact of the PHC choice reform on different types of healthcare quality. In 2020, Dietrichson et al.  found smFurthermore, in an ecological register study from 2021, Mosquera et al.  evaluateA few studies have investigated differences between private and public PHC centres, of which three studies investigated prescription patterns and/or perceived quality. The prescription of antibiotics was investigated by Maun et al.  and GranThe study by Maun et al.  also invThe Swedish PHC choice reform was intended to strengthen patients’ choice of provider and facilitate the establishment of private PHC centres. The idea was that a diversity of providers competing for patients and the accompanying public reimbursement would enhance cost efficiency, improve access and stimulate quality improvements. Critics, however, argued that the reform could impair equity because the regions would no longer be able to steer the location of PHC centres directly to areas with higher care needs, and because not everyone has the capacity to make informed choices. The review of published studies may help us answer whether the anticipated effects have been realised and to understand how the PHC choice reform’s mechanisms function, how PHC is governed and how PHC centres respond to different types of financial incentives. Overall, the results are consistent with previous research on patient choice, which has, however, mainly focused on hospital choice –63.With regard to governance, the reviewed studies suggest that the regions lost some of their governing capacity, that is, their traditional way of governing by way of planning and budgets. However, the transfer of power to the patients, who, with their choice of provider, allocate resources, was intentional and the studies show that the regions now use other governing and control measures such as financial reimbursements, dialogues and contracts. Furthermore, the regions try to regulate the PHC market to meet important values such as geographical equity and provider quality (e.g. through risk-adjusted reimbursements), but there are questions about how effective this type of regulation is. Several studies suggest that steering via clinical quality measures is underdeveloped , which is likely to be linked to the lack of systematically collected data on PHC quality in Sweden. Furthermore, the reviewed studies conclude that financial incentives are powerful and affect both the actions of PHC centres and professionals. All PHC centres respond to financial incentives but it seems as if private PHC centres respond to a greater degree.Regarding patients’ choices, the review suggests that people want the possibility to choose PHC provider. In addition, it seems as if having enough information is associated with the likelihood of making a choice, but at the same time, the reviewed studies indicate that people search for information about providers to a limited extent. In particular, it is rare that people search for information about the providers’ quality and they more often ask family and friends for advice  . HoweverTaken together, the studies of the effects of the PHC choice reform on access and equity show that access has increased, both the number of PHC centres and utilisation. It is, however, uncertain how these increases in access are distributed among different patient groups because the reviewed studies show somewhat contradictory results. Most studies, however, indicate that the PHC choice reform has had a negative impact on equity and that the increase in utilisation is greater among individuals with higher socioeconomic status, which is consistent with previous research . HoweverTo sum up its first 15 years, we can conclude the PHC choice reform has, to some extent, had the intended effects. Access has increased both in terms of utilisation and the number of PHC centres, and patients’ choice has been strengthened. In addition, some of the critique against the reform has been shown to be justified, in particular with regard to equity, in which several studies indicate that the increase in access foremost has benefitted socioeconomically stronger individuals. Regarding the reform’s effect on quality, the evidence is unclear but several studies show that the mechanisms supposed to lead to quality improvements do not work as intended. One problem is the lack of clinical quality information to guide patients’ choices, and another, that providers do not know when a patient has used the ‘exit’ mechanism. Furthermore, it is unclear whether popular providers are able or willing to expand. The high costs of expanding a PHC centre by acquiring new facilities and staff may limit the economic incentives to expand even for providers with high demand , and somAnother intended effect is that the PHC choice reform increased the number of private PHC centres. The studies indicate that private providers are more susceptible to financial incentives . Thus, the reimbursement models become a key component in steering PHC and can create positive as well as negative outcomes depending on their design. Policymakers must therefore analyse potential effects and be ready to change the reimbursement models if they find that the incentives lead to unwanted effects. What policymakers must also do is to support the development of PHC quality information that can be used in the choice of provider, and ensure the development of a tool or platform that can be used to compare PHC providers based on relevant quality indicators, and that supports those with reduced capacity to make a choice of provider.God och nära vård). It has also been argued that the PHC providers’ responsibility for population health within an area should be reintroduced. A commission suggested in 2017 (SOU 2017:47) that providers should engage in preventive and health promotion efforts in collaboration with civil society and municipalities. This reasoning is in line with the World Health Organization’s approach to PHC as ‘a whole-of-society approach’ and international calls for integrating PHC and public health [God och nära vård.It is important to note that although this review focused on a patient choice reform, the studies rather illustrate different aspects of the patient choice system in Sweden, which also incorporates free establishment (any willing provider). This means that a company that fulfils the requirements set up by a region is allowed to establish a PHC centre anywhere within the region, even in an area where there is an overprovision of PHC. Further regulation of providers and their establishments has been suggested, for example, making it possible for the regions to have different rules in areas with difficulties attracting providers (SOU 2019:42), and the abolition of the PHC choice system for older people with great care needs (SOU 2016:2). However, the challenges in Swedish PHC go beyond the PHC choice system (SOU 2019:42); for example, the general undercapacity in PHC  . Overallc health , 68. PotFinally, there are a few limitations to this review study. It is possible that we missed relevant studies, in particular on the effects on clinical quality and effects caused by different reimbursement models, by using the inclusion criterion that the studies framed their questions in relation to the PHC choice reform. Furthermore, we did not include grey literature such as reports produced by government agencies or the regions, in which it is possible that additional findings may be presented (e.g. regarding costs and efficiency). It is also important to bear in mind that for some of the investigated topics only a few studies have been published. We cannot draw the conclusion that an effect does not exist based on a lack of evidence of such an effect in only a few studies. In addition, we did not assess the quality of the included articles and some of them are based on data from only one or a few regions while others cover all of Sweden. More studies are needed that can provide evidence on how the PHC choice reform has affected quality and costs. To be able to perform studies of high quality, we must know more about the length and content of PHC centre visits. The included studies have all looked at the number of visits as a measure but what is included in these visits is unclear.Summing up its first 15 years, we can conclude that the PHC choice reform has, to some extent, had the intended effects. Access and patient choice have been improved and there are now more private PHC centres. There are, however, indications of negative effects on equity, but it is still unclear how quality has been affected. Importantly, it seems as if the mechanisms behind the quality-enhancing function of the PHC reform do not function as intended. To improve the PHC choice system, the reimbursement models need constant oversight, and it is also important that there is a development of comprehensive clinical indicators that can be used when choosing providers and in the regions’ steering of the PHC providers. Furthermore, it must be discussed how a responsibility for population health can be integrated in PHC and the choice system.Click here for additional data file.Supplemental material, sj-docx-1-sjp-10.1177_14034948221095365 for Fifteen years with patient choice and free establishment in Swedish primary healthcare: what do we know? by Mio Fredriksson and David Isaksson in Scandinavian Journal of Public Health


The hypoxia treatment significantly inhibited proliferation and induced apoptosis in GC-2spd cells. The RNA modification signatures of total RNAs (2 types) and differentially sized RNA fragments  were altered, and tRNA stability was partially affected. Moreover, the expression profiles of sncRNAs, such as microRNAs, tsRNAs, rsRNAs, and ysRNAs, were significantly regulated, and this might be related to the alterations in RNA modification and subsequent transcriptomic changes. We comprehensively analyzed alterations in RNA modification signatures in total RNAs, tRNAs (approximately 80 nt), and small RNAs (17–50 nt) as well as the expression profiles of sncRNAs and transcriptomes in hypoxia-treated GC-2spd cells; our data suggested that RNA modifications may be involved in cellular responses under hypoxic stress conditions and could provide a basis for a better understanding of the molecular mechanisms underlying male infertility.Hypoxia is a known stress factor in mammals and has been shown to potentially impair male fertility, which manifests as spermatogenic dysfunction and decreased semen quality. Studies have shown that RNA modifications, the novel post-transcriptional regulators, are involved in spermatogenesis, and hypoxia-induced alterations in RNA modification in testes and sperm cells may be associated with impaired spermatogenesis in mice. However, the molecular mechanisms via which RNA modifications influence spermatogenesis under hypoxic stress conditions are unclear. In this study, we generated a mouse Germ Cell-2 spermatid (GC-2spd) hypoxia model by culturing cells in a 1% O Hypoxia is a stress factor that elicits widespread physiological responses; however, these responses may induce maladaptive consequences. Hypoxic stress has been reported to be responsible for altered male reproductive function . The fin6A) was found to be dynamically regulated at different developmental stages of spermatogenesis in humans  and methyltransferase-like 14 (mettl14) may lead to spermatogonial stem cell depletion, inducing impairment in spermatogenesis  result in dysregulated spermatogenesis, low sperm count, poor sperm quality, impaired fertility  and incubated in an incubator containing 21% O2 and 5% CO2 . The CoCl2-induced hypoxia cell model is commonly used in research to evaluate the effects of hypoxia on cell proliferation and apoptosis as well as other deleterious effects of hypoxia  or transferred to a hypoxia workstation  containing 1% O2, 5% CO2, and 94% N2 at 37°C. Experiments were routinely performed in triplicate (n = 3).Germ cell-2 spermatids (GC-2spd) were purchased from the Conservation Genetics CAS Cell Bank  and cultured in DMEM  supplemented with 10% fetal bovine serum  and 1% antibiotics . GC-2spd cells were seeded into six-well plates at a concentration of 1 × 10 hypoxia . Here, a2 conditions. After 12, 24, 48, and 72 h of culturing, each set of cells was digested with trypsin and washed twice with phosphate-buffered saline  at room temperature. Cell counting was performed using an improved Neubauer hemocytometer .GC-2spd cells were prepared as described previously under normoxic, hypoxic, and CoCl2 for 24 h were stained using the PE Annexin V Apoptosis Detection Kit  according to the manufacturer’s protocol and analyzed using a flow cytometer and Kaluza software .GC-2spd cells subjected to hypoxic treatment for 48 h or treated with CoCl2 for 24 h were washed twice with ice-cold PBS and then lysed in ice-cold radio immunoprecipitation assay lysis buffer  supplemented with 1 mM phenylmethylsulfonyl fluoride  for 0.5 h to extract total cellular proteins. Protein concentrations were determined using an enhanced BCA protein assay kit . A volume of 20 micrograms of protein was separated by 10% SDS-PAGE  and transferred onto nitrocellulose membranes . Next, the membranes were blocked with 5% non-fat dry milk in TBS containing 0.1% Tween (TBST) for 1 h at room temperature and then incubated overnight at 4°C with rabbit anti-HIF-1α antibodies  or mouse anti-β-actin antibodies . Then, the membranes were washed with TBST and incubated with horseradish peroxidase-labeled goat anti-rabbit IgG  and horseradish peroxidase-labeled goat anti-mouse IgG  for 1 h at room temperature. The blots were visualized using an ultrasensitive enhanced chemiluminescence device . Band intensities were quantified using FluorChemHD2 software .GC-2spd cells subjected to hypoxic treatment for 48 h or treated with CoClTotal RNA was extracted using TRIzol reagent  according to the manufacturer’s protocol. TRIzol (1 ml) was added to a microtube containing GC-2spd cells and vortexed vigorously. Then, the samples were incubated at room temperature for 5 min. Next, 200 μL of chloroform was added to the samples, vortexed for 15 s, incubated for 10 min at 20–22°C, and then centrifuged at 12,000 × g for 15 min at 4°C. The aqueous phase was collected in a microtube and mixed with an equal volume of isopropanol. After gentle mixing and incubation at −80°C for 30 min, the mixture was centrifuged at 12,000 × g for 15 min at 4°C. The final RNA pellets were resuspended in RNase-free water for the determination of RNA concentrations using a UV spectrometer.Total RNA (3 μg) was separated by 7 M urea-denatured 15% PAGE and stained with the SYBR Gold solution . Small RNAs with sizes of 17–50 nt and 80 nt were excised from the gel and extracted as previously described  for the 6A was calculated as the molar concentration of m6A/the molar concentrations of m6A+ Am + A+ m1A+ I + Im + m1I.Purified RNA (50–100 ng) was transferred to a microtube and incubated with 5 U/ul alkaline phosphatase , 0.01 U/ul phosphodiesterase I , and 5 U/ul benzonase  at 37°C for 3 h. Then, 3 K devices with an omega membrane  were used to remove the enzymes from the mixture by centrifugation. Mass spectrometric analysis was performed using a Xevo-TQ-S mass spectrometer connected to an Acquity-UPLC I-class system  equipped with an electrospray ionization source. Except for pseudouridine which was analyzed in the negative ion mode, for the analysis of the other molecules, the MS system was operated in the positive ion mode using a multiple reaction monitoring (MRM) scan model. LC-MS/MS data were processed using MassLynx software (version 4.1). The percentage of each modified ribonucleoside was normalized to the total amount of quantified ribonucleosides with the same nucleobase. For example, the percentage of mPurified tRNA molecules (80 nt RNA fragments) were incubated at 37°C for 15 min in RPMI 1640-base systems: 15 ng of RNA in 9 μL of RPMI 1640 , 9 μL of RPMI 1640 containing 0.8% serum , or 9 μL of RPMI 1640 containing 0.0003 μL of RNase A/T1 . After incubation, the samples were immediately placed on ice, and RNA loading dye  was added to them. A volume of four microliters of the samples was run on a 15% native PAGE gel at 4°C. The gels were stained with SYBR Gold before imaging.tRNA-Gly: 5′-AATTCTACCACTGAACCACCCATGC-3’;tRNA-Ala: 5′-CGCTCTACCACTGAGCTACACCCCC-3’;5.8S- rRNA: 5′-TTCTTCATCGACGCACGAGC-3’.Northern blotting was performed as previously described . In brieThe membranes were sequentially washed with low-stringent buffer (2 × SSC with 0.1% (w/v) SDS), high-stringent buffer (0.1×SSC with 0.1% (w/v) SDS), and washing buffer (1×SSC) at 42°C. After washing, the membranes were incubated in 1 × blocking buffer  at room temperature for 3 h, and then anti-DIG antibodies  were added to the blocking buffer at a ratio of 1:10,000 and incubated for an additional 1 h. The membranes were washed four times with DIG washing buffer  for 15 min each time, rinsed with DIG detection buffer  for 5 min, and then incubated with the CSPD reagent  in the dark for 20 min at 37°C before being imaged using a ChemiDoc imaging system .https://github.com/junchaoshi/sports1.1)  were generated using an Illumina HiSeq 2000 device . Small RNA library construction, library quality validation, and RNA sequencing were performed by Personalbio small RNA service , as described in our previous article . Clean rorts1.1) . ExpressTranscriptome sequencing was performed by Personalbio . In total RNA, mRNA with a poly-A structure was enriched using oligo (DT) magnetic beads. Then, the mRNA was randomly interrupted, connectors were added to it, and cDNA fragments were synthesized by reverse transcription, using RNA as a template for library construction. Finally, PCR amplification was performed to enrich library fragments. After RNA extraction, purification, and library construction, the samples were subjected to double pair-end (PE) sequencing using the Illumina NovaSeq sequencing platform.p-value was less than 0.05.The raw sequencing data were filtered using Cutadapt ; then, hp < 0.05.Data are presented as mean ± SEM, and each experiment was repeated at least three times. Statistical analyses were performed using GraphPad Prism 8 . Data on proliferation, apoptosis, and RNA modifications in GC-2spd cells were analyzed by ordinary one-way ANOVA multiple comparisons or two-way ANOVA multiple comparisons; multiple comparisons were performed using Sidak’s or Dunnett’s test. Differences were considered statistically significant at 2 treatment groups  were significantly increased in GC-2spd cells  and 17–50 nt-sized RNA molecules (sncRNA) were excised and purified for the identification of RNA modification signatures . The levreatment . Aside freatment . It has f RNases . Furthernditions .3C, m5C, Gm, m1G, m2G, m22G, m7G, and m1A) were significantly increased in treated GC-2spd cells  were previously found to be altered and this affected tRNA stability in GC-2spd cells subjected to hypoxic stress. We thought it necessary to further investigate modifications and the expression levels of small RNAs (17–50 nt). The levels of nine types of RNA modifications  is a major hypoxia-induced transcriptional regulator that reflects the degree of oxygen deprivation , and si2.5; ). Both trivation . When cerivation . In thisHypoxia has deleterious effects on the male reproductive system ; however5C modification were upregulated. The presence of m5C in RNA has been shown to contribute to tRNA stability in mammals , and 17–50 nt-sized RNA fragments (sncRNA) in GC-2spd cells, and these might be responsible for changes in RNA stability and subsequent alterations in sncRNA expression profiles and the transcriptome under hypoxic conditions. Although the detailed mechanisms via which the alterations in RNA modification affect cellular responses to hypoxia stress need further research, data obtained in the present study offer a foundation for exploring the role of RNA modifications in the cellular responses of GC-2spd cells under hypoxic conditions and provide novel information on the molecular effects of hypoxia on male reproductive health.