S. aureus SCCmec elements that is composed of an A-family polymerase catalytic domain in complex with a small second protein that confers single-stranded DNA binding. Here we use new structure prediction methods in conjunction with sequence database searches to show that related primases are widespread among putative mobile genetic elements in the Bacillota. Structure predictions show that the second protein adopts an OB fold (common among single-stranded DNA binding (SSB) proteins) and these predictions were far more powerful than simple sequence comparisons in identifying its homologs. The protein-protein interaction surface varies among these polymerase—SSB complexes appear to have arisen repeatedly by exploiting partial truncations of the polymerase’s N-terminal accessory domains.Mobile genetic elements can encode a wide variety of genes that support their own stability and mobility as well as genes that provide accessory functions to their hosts. Such genes can be adopted from host chromosomes and can be exchanged with other mobile elements. Due to their accessory nature, the evolutionary trajectories of these genes can differ from those of essential host genes. The mobilome therefore provides a rich source of genetic innovation. We previously described a new type of primase encoded by  Despite sharing similar core chemistry, the priming and elongation of DNA synthesis are usually carried out by different enzymes, termed primases and polymerases. Delegating these activities to separate enzymes most likely facilitates regulation of replication and improves the accuracy of the products–considerations that are more important in the replication of chromosomes than in the replication of mobile genetic elements. Nearly all known primases can be divided into just two large, structurally unrelated families: the DnaG family, which has a toprim motif at its core, and the AEP family, which has an RRM (RNA recognition motif) at its core . An inteS. aureus) strains. SCCs share a conserved integration site and a core genetic locus. Central to the core locus is an operon encoding one or two site-specific DNA recombinases from the “large serine” family that can insert and excise the element  family of mobile genetic elements . SCCs ar element . Immedia element . Related element . In the  element . The Araelicases . They prde novo–that is, in the absence of a primer. The chemistry of joining the initial two nucleotides together is the same as the elongation step of adding a nucleotide onto the end of an existing primer: a 3’ hydroxyl attacks the alpha phosphate of a nucleotide triphosphate, displacing pyrophosphate. Therefore, the key differences between primases and primer-dependent polymerases must lie in the substrates and in their ability to utilize them: priming requires a single-stranded template and two (d)NTPs rather than a double-stranded primer-template duplex and a single (d)NTP. The modeling presented here suggests strategies that CcPol-MP may use to overcome barriers to de novo initiation.These findings raised the question of how DNA polymerases are normally prevented from initiating synthesis Here we apply the new structural modeling capabilities of AlphaFold to address how MP confers ssDNA binding activity, how the two proteins interact, how widespread CcPol-MP type primases are and how variable they can be . These qThe polymerase component of these new putative primases appears to have evolved from a DNA Pol I and to have undergone multiple truncation events that entailed evolving new interactions with the cognate SSB. DNA Pol I contains 3 overall domains: an N-terminal 5′ to 3′ exonuclease domain, a central 3′ to 5′ exonuclease domain, and a C-terminal DNA polymerase domain . The polOur goal was not to carry out a comprehensive survey, but rather to find a broad variety of CcPol-MP like protein pairs from different bacterial species. To do so we used a mixture of BLAST searches (of NCBI and UniProt databases) and webFlaGs , using amec type V and an SCCmer-like element) found in tandem in the same S. aureus strain, both of which we have demonstrated primase activity for [.The percent identity matrices in S. aureus which shared 79% identity. Only the geobacillus vulcani example lacked an SSB-containing ORF. Pairwise sequence identity among the SSB proteins ranged from 7% to 70%, with the majority of pairs sharing less than 20% identity. All of these examples are found in Bacillota, although we did not purposefully limit our searches to that group of bacteria. However, their niches vary widely, from humans  to pine litter (Paenibacillus pinistramenti), sea-salt pans  and an antarctic lake (Planococcus antarcticus).The ORFs examined were highly diverse in sequence . The premec, are inserted near the end of the rlmH gene by the action of site-specific recombinases from the “large serine” family. The inserted element is flanked by degenerate inverted repeats that include last few codons of rlmH. Most of the Pol-SSB pairs described here were similarly found downstream of rlmH, and for many we could also identify a putative 2nd end of the encoding element by searching for a match to the last ∼12 bp of rlmH. Similar to the composite SCC and pseudo-SCC elements found in staphylococci . Strains with the predicted empty sites for the relevant islands include Clostridioides difficile strain Z31 (GenBank: CP013196.1), Bacillus cereus strain EFR-1 (GenBank: CP064072.1) and Bacillus wiedmannii strain FSL P2-0415 (GenBank: LXFR01000008.1). Finally, we noted that most of these elements carry ORFs related to genetic conflict such as restriction-modification systems, and some ORFs with domains of unknown function similar to those found in the conserved recombinase locus of SCC elements , these five ORFs perform a different function.Five of the putative Pol proteins analyzed contained little more than a conserved central RRM motif with small, variable N- and C-terminal extensions . The RRM++—binding active site residues in the palm subdomain and additional conserved residues in the functionally important helix O of the fingers subdomain  co-purify, for most of the examples that included a full polymerase domain, AlphaFold predicted structurally plausible Pol–SSB complexes with low predicted alignment errors not only within each individual subunit but also for the relative placement of the two subunits, implying high confidence  a single-turn helix from the SSB docks into a similar location. Although these models appear to place the SSB rather awkwardly relative to the Pol domain, there is a flexible-looking linker between the very N-terminal domain and the 3′-5′ exonuclease that is likely to allow repositioning. The similarity of these latter five models, given that the polymerase proteins are no more than 55% identical to one another in sequence  rather than an N-terminal region for SSB interactions  pairs described here are encoded by mobile genetic elements. First, the CcPol-MP pair that initiated our interest in this family is found on the SCC family of genomic islands of S. aureus (including many of the methicillin resistance-carrying SCCmec elements that create MRSA strains). Second, they appear to occur sporadically, rather than universally, in any given species. Third, at least in all of the examples listed in The survey presented here shows that PolA-SSB type primases, which we previously discovered in certain via natural competence or conjugation, both of which result in a single-stranded incoming donor DNA    raises tde novo? The initial step in priming uses slightly different substrates than the elongation reaction: priming requires that the enzyme bind a single stranded template and two (d)NTPs, whereas for elongation, it must bind a primer-template duplex and one (d)NTP. The most relevant comparison between enzymes that can initiate de novo and ones that cannot is between the RNA polymerases (RNAPs) of bacteriophages such as T7 and the chromosomally-encoded bacterial DNA Pol I enzymes, all of which belong to the A family of polymerases  thumb subdomain. We also noted that most of the predicted interactions are mediated by the variable C-terminal tail of the SSB component, and that the chromosomally-encoded canonical bacterial SSB protein also uses a different C-terminal extension to bind other replication-related proteins (How did the PolA-SSB interaction evolve and why is it so variable? The N-terminus of the polymerase, which is found at the beginning of the 3-protein operon, appears to have been randomly truncated at different positions. Truncations in the middle of a folded domain would have left an exposed hydrophobic surface that the SSB may have been able to interact with: presumably weakly at 1st, then optimized through evolutionary selection. Only the proteins . Further


The critical shortage of healthcare workers, particularly in rural areas, is a major barrier to quality care for non-communicable diseases (NCD) in low-income and middle-income countries. In this proof-of-concept study, we aimed to test a decentralised model for integrated diabetes and hypertension management in rural Bangladesh to improve accessibility and quality of care.The study is a single-cohort proof-of-concept study. The key interventions comprised shifting screening, routine monitoring and dispensing of medication refills from a doctor-managed subdistrict NCD clinic to non-physician health worker-managed village-level community clinics; a digital care coordination platform was developed for electronic health records, point-of-care support, referral and routine patient follow-up. The study was conducted in the Parbatipur subdistrict, Rangpur Division, Bangladesh.A total of 624 participants were enrolled in the study (mean (SD) age, 59.5 (12.0); 65.1% female).Changes in blood pressure and blood glucose control, patient retention and patient-visit volume at the NCD clinic and community clinics.The proportion of patients with uncontrolled blood pressure reduced from 60% at baseline to 26% at the third month of follow-up, a 56%  reduction after adjustment for covariates. The proportion of patients with uncontrolled blood glucose decreased from 74% to 43% at the third month of follow-up. Attrition rates immediately after baseline and during the entire study period were 29.1% and 36.2%, respectively.The proof-of-concept study highlights the potential for involving lower-level primary care facilities and non-physician health workers to rapidly expand much-needed services to patients with hypertension and diabetes in Bangladesh and in similar global settings. Further investigations are needed to evaluate the effectiveness of decentralised hypertension and diabetes care. The study is among the first to evaluate a digital technology-supported, decentralised primary care model for integrated diabetes and hypertension management in a resource-constrained setting.Generated important preliminary effectiveness data on a decentralised model to rapidly expand primary care services to people with hypertension and diabetes.Enabled by digital tools, rich longitudinal data were collected to evaluate patient treatment outcomes and retention.The duration of the study is short, and generalisability is limited by a local sample drawn from a subdistrict in northern Bangladesh.Cardiovascular disease (CVD) and type 2 diabetes are major causes of premature death and disability. An estimated 10.5% (or 536.6 million) of adults are living with diabetes; CVD caused 18.6 million deaths worldwide in 2019.4Traditionally designed to provide basic episodic care with an emphasis on maternal and child health and infectious diseases, primary healthcare systems in LMICs are under-resourced and ill-prepared for the surging burden of CVD and other non-communicable diseases (NCD).13–16Although few examples exist for hypertension and diabetes care, decentralisation with task shifting (sharing) in HIV care has been identified as a viable strategy to rapidly scale up antiretroviral therapy in resource-limited settings.21Digital technologies offer the potential to improve access to and quality of healthcare in LMICs.23–25Little is known whether decentralising routine hypertension and diabetes services to lower-level primary care facilities and non-physician health workers is beneficial for enhancing care quality and accessibility in resource-poor settings and how digital technology can be harnessed to support the processes. In the present proof-of-concept study, we aimed to (1) generate preliminary data on the effectiveness of a novel digital technology-supported decentralisation of primary care diabetes and hypertension management in a rural subdistrict in Northern Bangladesh and (2) understand its potential future implications for the patient care pathway, the patient load in primary care facilities at a subdistrict and village level and patient retention.Like many other LMICs, Bangladesh is experiencing rapid demographical and epidemiological transitions. In recent years, CVDs have replaced infectious diseases and maternal and neonatal conditions as the leading causes of death in Bangladesh.29 30Bangladesh has a three-tiered  primary healthcare system in rural areas . The sub31Since 2011, the Government of Bangladesh has promoted improved hypertension and diabetes care through national multisectoral actions, most notably through the establishment of dedicated NCD-related healthcare delivery points (‘NCD Corners’) at subdistrict hospitals.33This proof-of-concept study was implemented in a newly initiated study NCD clinic at the Parbatipur subdistrict, in collaboration with three community clinics at Khorakhai, Jahanabad and Manmathapur. Parbatipur is a subdistrict of Dinajpur district, in the Rangpur Division of northern Bangladesh. Parbatipur had a population of 365 103 in 2011. The public primary healthcare system in Parbatipur consists of one Upazila Health Complex , 10 union health (sub)centres and 34 community clinics.The study is a single-cohort proof-of-concept study. Decentralised hypertension and diabetes care comprised screening, routine monitoring and dispensing of medication refills at village-level community clinics, as well as treatment initiation and titration at subdistrict-level NCD clinics. Patient flow in this model of care included community health workers motivating adults aged 40 years and above to get screenings for hypertension and diabetes at community clinics; once confirmed, patients received initial care at the study NCD clinic and subsequent follow-up at community clinics. At the follow-up visits, community healthcare providers perform a basic physical examination , blood pressure and blood glucose measurement), inquire about any new symptoms, assess CVD risk, emphasise the importance of medication adherence, provide advice regarding a healthy lifestyle and refill prescribed medications. Patients were referred back to the NCD clinic for medication titration and further evaluation if the treatment target was not met or any urgent symptoms or conditions were recorded.The study NCD clinic was staffed by one medical doctor and one paramedic. Both the doctor and the paramedic received a 4-day training on hypertension and diabetes management following the package on essential NCD interventions of the WHO. The paramedic had 1 year of postgraduate medical education. The doctor carried out a physiological examination and provided diagnostic and treatment services according to the Bangladesh National Protocol for Management of Diabetes and Hypertension, adopted from the WHO Package of essential NCD interventions.One community healthcare provider was employed to provide services in three community clinics (2 days per week in each community clinic). The community healthcare provider was supported by three newly recruited NCD community health workers (one for each community clinic). The community healthcare provider received a 1-year postsecondary education medical training. Community health workers were newly recruited from local communities with a minimum of secondary school education. The community healthcare provider and the community health workers received a 7-day initial training on behavioural risk factors of hypertension and diabetes, anthropometry, lifestyle counselling and follow-up, and received ongoing regular (weekly to monthly) supervision from the study NCD clinic medical staff and the local study investigators.The community healthcare provider and community health workers were responsible for administering written informed consent, registering participants and referring adults for screening, follow-up, medication adherence and health education/counselling in the community. They were regularly supervised by the medical doctor from the study NCD clinic and by the study investigators. The study NCD clinic and community clinics were equipped with the necessary medical, pharmaceutical and logistical supplies and equipment. Electronic health records were maintained by the study digital care coordination platform.Adults aged 40 and above were approached at their households and registered and referred by community health workers to be screened at the community clinics. Those with a history of hypertension or diabetes and who screened positive for hypertension or diabetes were referred to the study NCD clinic for further evaluation. The WHO non-laboratory-based CVD risk scores were calculated.Pregnant women and patients with life-threatening conditions were excluded from the study. Patients with very high blood pressure or those with acute symptoms of cardiovascular events were referred immediately to tertiary hospitals as per national protocol and were excluded from the analyses. Treatment decisions and patient management followed the Bangladesh national protocol for integrated management of hypertension, diabetes and cholesterol using a total cardiovascular risk approach .10.1136/bmjopen-2023-073743.supp1Supplementary dataThe development and deployment of a digital platform for primary care hypertension and diabetes care coordination in Bangladesh, online supplemental figures B–D). Briefly, the digital platform was developed in close collaboration with primary healthcare providers and policymakers through multiple stakeholder meetings and with extensive user-centric research to understand user needs. The system comprised an Android tablet-based application with dashboards to support task shifting/sharing of registering, screening, counselling and routine patient management. The application allowed community health workers and community healthcare providers to enter patient data, which was accessible to healthcare providers at subdistrict-level NCD clinics. With built-in algorithms, the application flags behavioural risk factors, prompts counselling requirements, highlights elevated levels of blood pressure/glucose and generates referral recommendations for confirmatory diagnosis and treatment. At the study NCD clinic, a paramedic performed additional examinations and updated patients’ data. The digital care coordination platform generated CVD risk scores based on WHO CVD risk non-laboratory-based charts (South Asia) and had all pertinent information needed by the medical doctor at the NCD clinic to make treatment decisions and management plans, which were then incorporated into the patient record. Patient dashboards were continuously updated, enabling healthcare providers at community clinics to follow-up with routine examinations, medication refills and counselling.A digital care coordination platform was developed to address challenges in delivering guideline-based decentralised care in primary healthcare settings. A detailed description of the development and deployment of the digital system is available in The present study relied on routinely collected data through the digital care coordination platform. Data were collected during screening and follow-up visits between 1 September 2021 and 28 February 2022. Self-reported sociodemographic characteristics , health behaviours  and medical history  were collected by interviewer-administered questionnaire installed on tablets. Height was measured with stadiometers , and weight was measured with a digital weighing machine (Tanita UM70). Blood pressure was measured with an automatic digital monitor (Omron HEM 7120) following standard operating procedures. Capillary blood was used to measure fasting/random blood glucose with glucometers (Accu-Chek Active).The primary outcome measurements were changes in the proportion of people with uncontrolled conditions from baseline to follow-up. Patients were considered to have uncontrolled hypertension if they had SBP ≥140 mm Hg and DBP ≥90 mm Hg and uncontrolled diabetes if they had FPG ≥7.0 mmol/L or RPG ≥11.1 mmol/L. Mean changes in blood pressure and blood glucose from baseline were considered secondary outcomes.Patient retention was assessed as the incidence of dropouts for patients enrolled. Dropout was defined as no recorded visit for ≥2 months. Intervention-related adverse events were documented as safety measurements. This study was approved by the Institutional Review Board of BRAC University (IRB reference number 2020–009-IR). Written informed consent was obtained from all participants.A purpose-built database was developed to facilitate the collection, secure storage and appropriate sharing of study data, including the reporting of clinically relevant results to individual participants. The database was commissioned under the supervision of the Chief Investigator and the respective site Principal Investigator. Personal and clinical data were separated by pseudonymisation to enhance data security. Appropriate authentication and access control mechanisms were implemented. Data were stored securely both locally and in a cloud-based infrastructure. All personal identifiers were removed before analysis.2) and community clinic location . The analyses were repeated for the subset of patients who had uncontrolled hypertension or diabetes at enrolment.Descriptive statistics stratified by patient type  were reported. Changes in the proportion of patients with an uncontrolled condition were analysed with generalised estimating equation (GEE) Poisson regression with robust variance. Follow-up visits were included as binary indicators in the regression model to capture the change in blood pressure and blood glucose control relative to the baseline. GEE Poisson is widely used for repeated binary outcomes as it provides consistent estimates of the relative risk and is more stable than the GEE binomial model.As secondary outcomes, changes in SBP and DBP in each of the five visits from baseline were assessed with linear mixed-effects models among those with a diagnosis of hypertension (with or without diabetes). Changes in mean fasting and random blood glucose were not assessed given the limited number of repeated measurements . We assessed factors associated with loss to follow-up with Poisson models. A logarithmic time to drop out was incorporated into the incidence ratio estimation. Follow-up time was rounded to a roughly 1-month interval to aid better interpretation. Data management and analyses were conducted using STATA MP v14. Statistical significance was determined by two-sided hypothesis tests (p<0.05).Sensitivity analyses were conducted to assess the potential bias introduced by sample attrition with inverse probability weighting. The inverse probability weighting approach weighted the analysis by the inverse of the predicted probability of dropping out of the study. A conditional logit model with the same set of covariates described above was used to predict the probabilities. In addition, we restricted our analyses to patients with at least three follow-up visits to test whether the results were sensitive to better balancing sample composition for timing of enrolment and follow-up.Before the study, we conducted formative research, including a qualitative assessment to explore technology readiness and acceptability of services among health providers and a quantitative assessment of the readiness of health facilities to deliver NCD services. Multiple meetings with primary care physicians, NCD nurses, community health workers and local health authorities were organised to understand the current care pathway and to identify gaps in usual care. Informal discussions with patients regarding their experience with the current model of care were conducted. Patients were not involved in the analysis or interpretation of the data, nor did they contribute to the writing of the manuscript.Of the 2232 people approached, 2208 (98.9%) consented to participate in the study. Among those who consented, 2008 people attended the screening of hypertension and diabetes at the community clinics, of whom 829 were found to have possible hypertension and/or diabetes and were referred to the study NCD clinic for further evaluation . From thAmong the 549 patients with hypertension, the age-adjusted and sex-adjusted proportion of people with uncontrolled blood pressure decreased from 60% (95% CI 56% to 64%) at baseline to 26% (95% CI 19% to 33%) at the third month of follow-up , a 56% . The proThe mean age-adjusted and sex-adjusted SBP at baseline was 145.0 mm Hg (95% CI 143.4 to 146.7) among the 549 people with hypertension. Those with uncontrolled blood pressure had higher mean SBP at baseline  compared with the entire sample of patients with hypertension . A substWe observed a similar pattern of change in the control of blood glucose among diabetics. At baseline, 74% (95% CI 68% to 80%) of patients with diabetes had uncontrolled blood glucose. This proportion dropped to 43% (95% CI 32% to 54%) at the third follow-up visit and 31% (95% CI 7.5% to 54.8%) at the fifth follow-up visit. The reduction in the proportion of patients with uncontrolled blood glucose was more pronounced among people with uncontrolled conditions at baseline.The proportions of patients lost to follow-up immediately after baseline and during the entire study period were 29.1% and 36.2%, respectively . FactorsIn total, 41% of follow-up visits were carried out at the community clinics. Average daily numbers of patient visits ranged from 5 to 18 for the NCD clinic and 1–10 in each community clinic . Within In this study, we tested a digital technology-supported decentralisation of diabetes and hypertension management in rural Bangladesh. The service delivery model with shifting routine hypertension and diabetes services to lowest-level primary care facilities and non-physician health workers achieved a high treatment success rate and relatively high patient retention. The results highlighted the potential of technology-supported decentralised care to address the critical shortage of health service providers and geographical barriers to access, along with the retention of patients with hypertension and diabetes.Primary care delivery of hypertension and diabetes services in Bangladesh is hampered by a critical shortage of trained health workers and poor geographical accessibility. In Bangladesh, only about one in three patients with hypertension or diabetes is currently on treatment.38Our preliminary results showed that with the support of a digital care coordination platform and proper training, non-physician health workers based in the community or community clinics can successfully deliver screening for hypertension and diabetes, routine follow-up, counselling, drug refills and other aspects of NCD management. This approach to healthcare delivery has the potential to substantially improve access to healthcare and reduce the burden on physician-managed NCD clinics. Overall, the proportion of patient follow-up visits carried out at community clinics was 41%. Routine patient follow-up at community locations could be further enhanced by introducing telemedicine and dose titration by non-physician health workers. Through technology-supported decentralisation and the involvement of a large number of peripheral primary care facilities, hypertension and diabetes services could be rapidly expanded in Bangladesh and similar global settings.6 Following international standards of interoperability throughout the designing phase, the digital platform resources developed in the present study are open-source and can be readily used by the global health community.In addition to the lack of human resources, inconsistent adherence to treatment protocols among healthcare providers, poor patient management and irregular follow-up are important obstacles to effective hypertension and diabetes management.Treatment adherence and retention are major challenges in NCD management, particularly in LMICs.Our findings showed several factors were associated with loss to follow-up, including being newly diagnosed, having a controlled condition at enrolment and/or being diagnosed with hypertension only. In addition, the results showed a particularly high attrition rate immediately after enrolment, consistent with previous studies.As a proof-of-concept study, the project comprised an uncontrolled and unblinded evaluation of a decentralised service model, supported by a digital care coordination platform. Nevertheless, the observed rates of 61% for blood pressure control and 69% for blood glucose control at the end of the study compared favourably with existing implementation studies and clinical trials of multicomponent health system interventions in developing countries.In the proof-of-concept study, a digital technology-supported decentralised care model achieved high levels of blood pressure and blood glucose control among patients with hypertension and diabetes in Bangladesh. Our results suggest that with proper training, supportive supervision and access to digital technology, non-physician health workers at peripheral primary facilities can deliver health screening, counselling, routine drug refills and other aspects of patient management for people with hypertension and diabetes. The present study highlighted the potential to involve peripheral primary care facilities and community health workers to rapidly expand much-needed hypertension and diabetes services in Bangladesh and similar global settings. Further investigations are needed to evaluate the effectiveness of decentralised hypertension and diabetes care.


The major source of ccfNAs are the cells of hematopoietic system under healthy conditions. These ccfNAs include fragmented circulating cell free DNA (ccfDNA), coding or messenger RNA (mRNA), long non-coding RNA (lncRNA), microRNA (miRNA), and mitochondrial DNA/RNA (mtDNA and mtRNA), that serve as prospective biomarkers in assessment of various clinical conditions. For, e.g., free fetal DNA and RNA migrate into the maternal plasma, whereas circulating tumor DNA (ctDNA) has clinical relevance in diagnostic, prognostic, therapeutic targeting, and disease progression monitoring to improve precision medicine in cancer. The epigenetic modifications of ccfDNA as well as circulating cell-free RNA (ccfRNA) such as miRNA and lncRNA show disease-related variations and hold potential as epigenetic biomarkers. The messenger RNA present in the circulation or the circulating cell free mRNA (ccf-mRNA) and long non-coding RNA (ccf-lncRNA) have gradually become substantial in liquid biopsy by acting as effective biomarkers to assess various aspects of disease diagnosis and prognosis. Conversely, the simultaneous characterization of coding and non-coding RNAs in human biofluids still poses a significant hurdle. Moreover, a comprehensive assessment of ccfRNA that may reflect the tumor microenvironment is being explored. In this review, we focus on the novel approaches for exploring ccfDNA and ccfRNAs, specifically ccf-mRNA as biomarkers in clinical diagnosis and prognosis of cancer. Integrating the detection of circulating tumor DNA (ctDNA) for cancer genotyping in conjunction with ccfRNA both quantitatively and qualitatively, may potentially hold immense promise towards precision medicine. The current challenges and future directions in deciphering the complexity of cancer networks based on the dynamic state of ccfNAs will be discussed.The circulating cell-free nucleic acids (ccfNAs) consist of a heterogenous cocktail of both single (ssNA) and double-stranded (dsNA) nucleic acids. These ccfNAs are secreted into the blood circulation by both healthy and malignant cells  The discovery of cell-free nucleic acids in the body fluids by Mandel and Metais  opened aThe heterogeneity of circulating cell free RNA (ccfRNA) further expands the battery of non-invasive biomarkers in reflecting the origin and state of the disease. Unlike the messenger or coding RNA, non-coding RNAs (micro RNA and long non-coding RNA) have been studied extensively in multiple diseases but the functionality of non-coding RNAs is still not clearly validated . In contEvidence based information indicates that the source of ccfDNA lies in the hematopoietic cells present in a healthy individual . LongituThe fraction of circulating cell-free DNA derived from malignant cells is termed as circulating tumor DNA (ctDNA) that constitutes less than 1% of the total ccfDNA . These cN-RAS mutation proposed that bone marrow aspiration does not consistently contain the diverse population of malignant clones that are implicated in the disease . The ccfRNAs isolated from body fluids (plasma and serum) have been used as biomarkers for diagnosis, early detection and provide insights on the recurrence, prediction of survivability and cancer monitoring and have been widely used in cancers of breast, prostate, pancreas, colon, thyroid and skin , small nuclear RNA (snRNA) and piwi-interacting RNA (piRNA). The small non-coding RNA have been studied extensively and are known to play diverse roles in gene regulation, RNA interference, modification or formation of spliceosome . MoreoveThe long non-coding RNA (lncRNA) has also gathered attention of cancer researchers in the last decades . The lncmiRNA are the short non-coding RNA that are extensively studied as biomarkers for different diseases. These are ∼20–25 nucleotide long oligonucleotides and are vital for regulating the post-transcriptional expression of genes in association with mRNAs. Studies conducted by As opposed to long non-coding RNA (RNA molecules that are not translated into proteins), mRNAs are the protein coding regions that encompass the information produced as a result of DNA transcription . These mThe quest for exploring ccfRNA [both coding (mRNA) and non-coding ] for their value as biomarkers in monitoring cancer, cardiovascular disorder, neurodegenerative disorder, and infection is ongoing. Various RNA species including mRNA and miRNA are released either in bound form with lipoproteins or inside exosomes as a means of cell-to-cell communication . ExosomeThe exosomes contain a large number of mRNA transcripts and other RNA species such as miRNA and long non-coding RNA that are protected against degradation by RNases in blood circulation  Figure . The exoThe mRNA inside exosomes can potentially act as biomarkers as they are prevented from degradation by RNases in the circulation and can also remain stable at 4°C . The staSimilar to exosomal mRNA, miRNA can also be potentially treated as diagnostic and predictive biomarkers for diseases to reflect the internal environment of the cells along with their physiological and pathological conditions . miRNA iThe extent to which ccfRNA may reflect human physiologic and disease state still remains to be elucidated. Approximately 19,000 unique transcripts of mRNA were detected in circulation among which, 65–75 percent of these coding transcripts represent the most significant proportion of circulating RNA that remains consistent with different clinical categories . Based oRecent developments in the field of RNA sequencing have allowed better and efficient transcriptome analysis in plasma to facilitate the discovery and application of novel diagnostic, prognostic, and therapeutic biomarkers to detect and monitor the disease. Next-generation sequencing revealed that ccf-mRNA is enriched in the transcripts from bone marrow when compared with the transcripts from circulating cells . To suppThe data from fewer studies conducted on mRNA transcripts indicated their presence as typically fragmented in the blood circulation. Studies have been conducted on the transcripts of human telomerase reverse transcriptase (hTR and hTERT), the two constituents of telomerase enzyme that are found in serum samples of breast cancer patients as potential biomarkers for disease prognosis . These tThe thyroid stimulating hormone receptor (TSHR) mRNA could detect the presence of thyroid nodules and was utilized for predicting the rate of recurrence in thyroid cancer . The expSimilarly, the overexpression of EGFR mRNA in circulation  was linked to non-small cell lung cancer, pancreatic cancer and colon cancer . FurtherIncreased levels of metastatin mRNA in the serum correlated with poor survival rate and metastasis to lymph nodes in breast cancer . The chap = 0.042; p = 0.008), at diagnosis predicting suboptimal debulking surgery and indicated shorter progression-free survival  and overall survival .The first study in ovarian cancer demonstrated circulating Metastasis-associated in colon cancer 1 (MACC1) and S100 calcium-binding protein A4 (S100A4) transcripts as potential liquid biopsy markers . Both MAAMACR, GOLM1, TRPM8 and NKX3-1 genes were overexpressed and were closely associated with cancer aggressiveness, extracapsular extension and vesicular seminal invasion  was able to identify high-risk prostate cancer cases (85% of sensitivity and 58% of specificity). This ccf-mRNA signature has emerged as a superior marker with a better overall performance compared with the classical biopsy Gleason score and prostate-specific antigen. Pre-transplant measurable residual disease status of WT1 ccf-mRNA expression has been assessed for prognostic value in peripheral blood obtained from patients with Acute Myeloid Leukemia and Myelodysplastic syndrome (MDS) (The ccf-mRNA has been employed for risk assessment of prostate cancer. Transcripts of invasion . A combime (MDS) .n = 46 + 30]) and non-cancer (n = 89) participants from the Circulating Cell-free Genome Atlas (NCT02889978) was reported by Larson et al.  patients, which were enriched in AD-associated biological processes . Albeit,n et al. . Of 57,8BCL2L1, CXCL9, IDO1, IL13, MIF, MYD88 and TLR4p ≤ 0.001, versus high disease burden). In patients with therapeutic response relative to baseline assessment, a statistically significant increase in BCL2L1, CCL4, CCL5, CXCL9, GZMB and TNFSF10 genes was noted. Moreover, in stage IV melanoma patients with brain metastases, CCL18, CCR1, CCR4, CD274, CSF2, EGF, and PTGS2 genes were significantly over-expressed .Notable differences were reported in the plasma transcriptomic profile between low disease burden compared to advanced melanoma patients . SeveralKPNA2, DTL, BACE2 and DTYMK genes between melanoma patients’ and healthy donors’ plasma . Furthermore, ccf-mRNA levels correlated proportionally with increasing TMB and radiological absence of disease. The cellular sources of ccfRNA were traced by comparison with transcriptomes obtained from single cells, that identified a pan-tumour origin beyond melanoma cells . The genes with low baseline ccfRNA levels were associated with a prominently longer progression-free survival (PFS) and overall survival (OS). An increase in the ccfRNA levels of KPNA2 and DTYMK at the time of therapy predicted shorter PFS. Supportively, the levels of ccfRNA substantially increased after therapy in non-responders when correlated against the responders, independent of therapy and mutational subtypes.In another study, ccf-mRNA facilitated the minimally invasive pan-tumor monitoring of melanoma therapy that was not dependent on the mutational genotype of the patient . High diPlasma ccfRNA expression levels were quantified by a tagmentation-based library sequencing in primary NSCLCs . The com+ and CD8+ T-cells at the center of tumor and also alongside its margin (Immunoscore)  , expressA pre-clinical mouse model was utilized to temporally demonstrate the potential of profiling ccf-mRNA in plasma through drug-induced molecular alterations that are associated with inflammatory and immune responses. On one hand, lipopolysaccharide induced systemic immune responses and dysregulated signaling pathways were captured by cell-free mRNA-Seq whole transcriptome profiling. On the other hand, JAK inhibitor was shown to diminish these inflammatory pathways, including interferon and STAT signaling that was reflected in the ccf-mRNA . InteresBCL2L1, GZMB, HLA-A, IRF1, MYD88, TLR2, and TP53 genes were significantly over-expressed in glioma patients . Immune-related genes GZMB, HLA-A and BCL2L1 were significantly over-expressed in high-grade glioma patients  and a concordance between differentially expressed genes in plasma- and glioma-derived RNA was reported.A noteworthy study demonstrated the differential expression of genes involved in cancer inflammation and immunity. Among patients with different glioma grades, a positive correlation between their transcriptomic profile in plasma and tumour samples was shown . The BCLA potential usability of plasma-derived exosomal RNAs was described in non-small cell lung cancer patients for the characterization of molecular phenotypes of osimertinib resistance . The traLMNB1, TGF-β, and MCM6 from plasma samples were proposed as potential biomarkers for hepatocellular carcinoma. The MYC mRNA from serum samples of B-cell lymphoma was linked to short overall PFS and partial treatment response. However, some restrictions that limit the use of mRNA in clinical practice can be attributed to the labile nature of mRNA in circulation  in the non-small-cell-lung cancer (NSCLC). The following study proposed the applicability of both ccfRNA and ccfDNA for detection of fusion genes. A standard protocol facilitated the detection of fusion genes by ccfRNA and formulated analytical validity and functionality in 30 samples from 20 patients with fusion positive NSCLC. The conclusions drawn from ccfRNA-based assays were compared against the ccfDNA based assay (Guardant360 plasma NGS assay). The cumulative sensitivity of the ccfRNA based assays was 26.7% as opposed to 16.7%, for ccfDNA based assay. The analysis that was restricted to the samples obtained at chemo-naïve or progressive disease status and available for both assays showed that sensitivity of ccfRNA based assays was comparatively higher about 77.8% than ccfDNA based assay (33.3%). The identification and characterization of fusion-genes in ccfRNA correlates with the treatment response and these results support the usefulness of ccfRNA assay in diagnosing patients with insufficient tissues to channelize proper first-line treatment. Subsequently, the assay can also be used to monitor the progression of NSCLC that can be further considered for second-line of treatment .The cell free nucleic acids in circulation hold great potential as minimally invasive biomarkers for diagnosis and prognosis of cancer. Both plasma and serum serve as a major source for isolation of cell free nucleic acids but plasma is the preferred choice for ccfDNA. Since the ctDNA is relatively low in concentration, especially in the early stages of cancer, therefore highly sensitive advanced detection methods for successful profiling of mutation, methylation, and fragment patterns are recommended. Various conventional and ultrasensitive technology platforms for genotyping ctDNA were described by our group and others earlier . The seqFurther technological advancements have been witnessed towards better characterization of ccfDNA . Cell-frTargeted error correction (TEC-Seq) based sequencing is a recent UMI-based technique for ccfDNA detection that has been employed for detection of mutations in 58 genes involved in cancer of ovaries, breast, and lung . HoweverThe lack of optimum reproducibility across different studies occurring from lack of knowledge of standard isolation methods for ccfRNA has restrained the progress in the applications and versatility of liquid biopsies. A comparative analysis of ten methods used for isolation of ccfRNA from different biofluids have provided insights on the complexity and reliability of consequent small RNA-seq profiles . An asseSimilarly, the gene expression characterization of ccfRNA involves the use of conventional methods such as quantitative real time-polymerase chain reaction , microarrays and Next-generation sequencing . Every mModifications have been introduced in small RNA sequencing strategy to facilitate the simultaneous detection of ccfRNA. Yao J et al. performed Thermostable group II intron reverse transcriptase sequencing (TGIRT-Seq) in combination with peak calling to concomitantly profile the different biotypes of RNA in human plasma that was pooled from different disease-free individuals. From this study, the researchers proposed that the plasma contains mRNA that has undergone substantial fragmentation derived from more than 19,000 protein-coding genes, high population of full length and mature tRNA and other structured small non-coding RNA with a minor population of mature and pre-miRNA . DiffereA novel detection technique that determines small fragments of RNA called “Phospho-RNA-Seq” reveals that other standard small RNA-seq techniques are not sensitive enough to detect circulating mRNA and lncRNA in human plasma that can potentially serve as biomarkers. Therefore, this new method used treatment of RNA with T4-polynucleotide kinase in assessing these small circulating RNA fragments . A paralFurther modification was described by Newer methods for biomolecule detection through graphene quantum dots (GQDs) was described that could estimate the circulating nucleic acids like DNA, mRNA, miRNA, mtDNA and lncRNA in plasma and serum samples . These GSILVER-Seq  is a newly proposed method for sequencing integral as well as fragmented form of ccfRNA in as small as a droplet of volume 5-7 µl from liquid biopsy . This teShort-read deep RNA-Seq has been reported to identify the mRNA, lncRNAs and circRNAs (circular RNAs) in plasma as novel biomarkers for diagnosing coronary artery disease (CAD). The circulating cell-free RNA was sequenced in patients with stable CAD and controls. The fragments from 160 of 3,986 mRNA, 10 of 164 lncRNAs and 2 of 405 novel lncRNAs were observed to be altered in patients with disease. The transcripts that were differentially elevated were enriched in mRNA that is coded by mitochondrial DNA that stipulates myocardial ischaemia and oxidative stress .FRET-based intramolecular kinetic fingerprinting for direct digital counting of single molecules of nucleic acid that is also known as intramolecular single molecule recognition through equilibrium Poisson sample (iSiMREPS) is employed for a quick determination and counting of miRNA and mutant ctDNA with a very high sensitivity and specificity to detect as small as single molecule  Figure . This isThe ccfDNA has been employed in the prediction of transplant rejection, disease monitoring, predicting prognosis, prenatal diagnosis, relapse of disease, and clinical trials . RegardlCirculating cell free RNA, on the other hand that has emerged alongside ccfDNA, holds the transcriptome which can be contemplated as transcript from different tissues . The ccfThe ccfRNA contains necessary signatures in the characterization of oncological patients that have a strong potential to be developed as markers alongside ctDNA for diagnosis and monitoring . This ccIn vitro studies carried out on serum had shown that RNA present inside the apoptotic vesicles does not undergo degradation (Although, the detection and study of ccfRNA is an incredible discovery but RNA is more highly prone to degradation due to the presence of several ribonucleases in the blood. It is shown that RNA is present inside apoptotic bodies, other vesicular structures, or in bound form within DNA nucleosomes to escape the denaturing action of RNases in the blood . In vitrradation . The levradation .The mRNA is released from the cells through apoptosis and necrosis however it is also shed from tumor tissues even at a low abundance of circulating tumor cells. As shown with circulating cell-free hTERT and MUC1 mRNA that can provide a screening tool for the early detection of gastric cancer and its relapse . The ccfThe major challenge that persists in the applicability of liquid biopsies as definitive screening, diagnostic and prognostic tools is the issue of biological and technical reproducibility and the absence of any gold standard for sample storage, processing, and data analysis . Apart fAn efficient RNA biomarker discovery is also hindered by biological challenges that include presence of high interpersonal variability, that implies that the expression of some genes is higher in certain individuals than others. This stipulates the importance of invariable normalization to address this biological variability.One of the challenges encountered with biomarker identification and discovery using RNA sequencing is the interpretation and analysis of the data obtained. The bioinformatics analysis depends on data quality control, quantification of the transcript, and other downstream processes. Furthermore, the input quality also serves as a critical parameter for the quality assessment of the data. RNA sequencing is an extensively used tool for estimating gene expression, however it does not provide absolute quantification of the data like RT-qPCR methods .This limitation maybe overcome by normalization based on spike-in controls that can be done during the step of library preparation that can provide absolute quantification of transcriptome and is reproducible when working with plasma samples . NormaliThe studies conducted in the field of liquid biopsies are limited. However, the application of liquid biopsies, especially ccf-mRNA biopsies is very diverse. The use of coding RNA molecules as promising biomarkers for diagnosis, prognosis, and therapeutic use holds potential in incorporating non-invasive diagnostic tests in routine clinical practice. The growing interest in RNAs as biomarkers has not been very recent, but the shift of focus from microRNA to long-coding RNAs has only been in last 5 years that has steered the discovery of several new disease-associated RNA. Even though there is much that needs to be explored and studied to successfully bring the use of mRNAs into clinical practice, the recent studies conducted on ccf-mRNA as biomarkers for various cancers and other diseases suggest that the use of ccf-RNA may complement ctDNA in bringing a new revolution for screening, diagnosis, disease monitoring and improve patient management.


The world is undergoing a demographic transition to an older population. Preventive healthcare has reduced the burden of chronic illness at younger ages but there is limited evidence that these advances can improve health at older ages. Statins are one class of drug with the potential to prevent or delay the onset of several causes of incapacity in older age, particularly major cardiovascular disease (CVD). This paper presents the protocol for the STAtins in Reducing Events in the Elderly (STAREE) trial, a randomised double-blind placebo-controlled trial examining the effects of statins in community dwelling older people without CVD, diabetes or dementia.We will conduct a double-blind, randomised placebo-controlled trial among people aged 70 years and over, recruited through Australian general practice and with no history of clinical CVD, diabetes or dementia. Participants will be randomly assigned to oral atorvastatin (40 mg daily) or matching placebo (1:1 ratio). The co-primary endpoints are disability-free survival defined as survival-free of dementia and persistent physical disability, and major cardiovascular events . Secondary endpoints are all-cause death, dementia and other cognitive decline, persistent physical disability, fatal and non-fatal myocardial infarction, fatal and non-fatal stroke, heart failure, atrial fibrillation, fatal and non-fatal cancer, all-cause hospitalisation, need for permanent residential care and quality of life. Comparisons between assigned treatment arms will be on an intention-to-treat basis with each of the co-primary endpoints analysed separately in time-to-first-event analyses using Cox proportional hazards regression models.STAREE will address uncertainties about the preventive effects of statins on a range of clinical outcomes important to older people. Institutional ethics approval has been obtained. All research outputs will be disseminated to general practitioner co-investigators and participants, published in peer-reviewed journals and presented at national and international conferences.NCT02099123. STAtins in Reducing Events in the Elderly (STAREE) is a double-blind, placebo-controlled randomised clinical trial among a primary prevention population of people aged 70 years and over that will evaluate the effect of statin therapy on two co-primary outcomes including disability-free survival and major cardiovascular events.STAREE is a pragmatic trial run through general practice across metropolitan and regional/rural Australia with usual care provided by general practitioners.STAREE is one of the largest statin trials to carry out a comprehensive assessment of several causes of incapacity including dementia and disability.Complete vital status ascertainment will be facilitated by linkage to the National Death Index.STAREE will be examining the effects of starting statins but not withdrawing statins or de-escalating therapy in older age.The world is experiencing a rapid demographic transition towards an older population.While high levels of LDL cholesterol (LDL-C) are known to increase the risk of CVD events and mortality among young and middle-aged populations,Statins have the potential to delay the onset of several causes of incapacity in older people. Their best-recognised pharmacological action is to inhibit the mevalonate pathway and biosynthesis of cholesterol and isoprenoids by binding to HMG-CoA reductase. The reduction in circulating lipid levels, especially LDL-C, underpins their ability to reduce the incidence of acute myocardial infarction (MI) and stroke  and their potential long-term sequelae .In people with known CVD, the benefits of statin therapy are clear, with the risks of cardiovascular events reduced across all ages. In people without CVD, the benefits of taking a statin are not as clear at older ages and continue to be debated.A meta-analysis of pooled individual participant data (aged 55+ years) from 28 statin trials that included participants with and without known CVD, reported a 21% reduction in risk of major vascular events per 1 mmol/L reduction in LDL-C, attributed to reduced risks of major coronary events, major stroke events and vascular death.Yet statin treatment is not without potential risk of harm.In conclusion, the gains made in increasing lifespan may not have been accompanied by similar gains in healthy life, for the additional later years of life lived. A strong potential exists for greater use of statins for primary prevention to extend healthy life and maintain independence among the ageing population however, current evidence for starting statins among older people is insufficient to support this approach. The STAtins in Reducing Events in the Elderly (STAREE) trial is a large scale randomised controlled trial (RCT) specifically designed to address this need.To determine in people aged ≥70 years the effect of statin therapy (40 mg atorvastatin) versus placebo, on two co-primary clinical endpoints:Disability-free survival defined as survival free of dementia and persistent physical disability ; andMajor cardiovascular events .We hypothesise that treatment with a statin (atorvastatin 40 mg) will: (1) prolong overall disability-free survival, and (2) reduce the incidence of major cardiovascular events among a primary prevention population aged ≥70 years.To determine the effects of statin therapy (atorvastatin 40 mg) versus placebo on each of all-cause death, dementia and other cognitive decline, persistent physical disability, fatal and non-fatal MI, fatal and non-fatal stroke, cardiovascular death, fatal and non-fatal cancer, heart failure hospitalisation, atrial fibrillation, all-cause hospitalisation, need for permanent residential care and quality of life.To determine the effects of statin therapy (atorvastatin 40 mg) versus placebo on each of fasting glucose/glycated haemoglobin, urine albumin to creatinine ratio (ACR), estimated glomerular filtration rate (eGFR), frailty phenotype and functional independence ).STAREE is a double-blind, randomised, placebo-controlled primary prevention trial. The WHO Trial Registration Data Set is shown in 10.1136/bmjopen-2022-069915.supp1Supplementary data10.1136/bmjopen-2022-069915.supp2Supplementary data10.1136/bmjopen-2022-069915.supp3Supplementary dataRecruitment is being conducted through general practices across metropolitan and regional/rural Australia with participating general practitioners (GPs) registered as STAREE co-investigators. General practice clinical database reviews create a list of potential participants aged ≥70 years without a history of clinical CVD, diabetes, dementia or other life-limiting conditions. Each GP screens the list for suitability and approves the sending of an invitation letter through the mail. Potential participants call the study centre and undergo an initial phone screening for trial inclusion and exclusion criteria. Those who are eligible are invited to take part in two baseline screening visits, either as a face-to-face visit at their usual GP clinic or as a remote phone visit with a trained study team member.At the first baseline screening visit informed consent is obtained  and thenAt the second baseline screening visit after the run-in period, participants who achieve run-in medication compliance of >85% and have GP approval to participate in the trial, complete a cognitive battery to ascertain baseline cognitive performance see .See Individuals are deemed eligible if they; (1) are aged ≥70 years; (2) live independently in the community; (3) are willing and able to provide informed consent and accept the study requirements and (4) do not have a history of clinical CVD events, diabetes or dementia. Exclusion criteria are a Modified Mini-Mental State Examination (3MS) score <78 on screening, moderate or severe chronic kidney disease, moderate or severe liver disease or serious intercurrent illness likely to cause death within the next 5 years; total cholesterol >7.5 mmol/L; current participation in another interventional clinical trial; and an absolute contraindication to statin therapy, current use of statin therapy or other lipid-lowering therapy for primary prevention and unwilling to stop therapy or current long-term or permanent use of potent cytochrome P450 3A4 inhibitors.Participants are randomly allocated to either atorvastatin or placebo treatment through a web-based system according to a randomisation schedule generated by an independent statistician. The allocation list was generated using randomly permuted blocks (of size either 2 or 4) with randomisation stratified by state  and age group . Blocks ensure ongoing approximate balance of randomisation to statin and placebo within strata.Study medication comprises active treatment with atorvastatin tablets (20 mg) or identical placebo. Participants are asked to take one tablet of study medication (20 mg of atorvastatin or placebo) per day for 4 weeks. They are then contacted by phone to assess study medication tolerability and instructed to increase the dose to two tablets per day (2×20 mg). Dose reduction of study medication to one tablet per day (20 mg) or temporary interruption is permitted throughout trial follow-up to assist in managing tolerability and to reduce study medication discontinuation.Atorvastatin has been chosen as one of the most commonly prescribed statins in Australia and many other countries with extensive GP experience in its use.Medication is posted to participants at approximately 6 monthly intervals. Both active and placebo treatments are supplied by Ramsay Community Pharmacy and dispensed by an unblinded and independent pharmacist through a Monash University-affiliated central pharmacy dispensing centre.The trial activity schedule is summarised in . ParticiA random subset of participants will also be offered the opportunity to attend STAREE sites with facilities for processing and storing biospecimens for collection of whole blood and/or urine samples, as outlined in 10.1136/bmjopen-2022-069915.supp4Supplementary dataAdherence to medication is encouraged with multiple strategies including review of study medication use at follow-up phone and face-to-face visits and through reminders included in quarterly participant newsletters and annual face-to-face or web-based study updates.Participants are advised to seek care from their usual treating GP  for any medical condition arising during the study. Any tests or measurements performed as part of the trial trigger a notification to the participant’s GP if outside the normal range or deemed clinically significant.Any participants who develop a recognised clinical indication (such as an MI or stroke) for statin treatment can take open label statin as per usual practice by their treating clinician or clinical teams and will cease study medication while continuing in the study for observation (routine follow-up visits).As a result of government and workplace restrictions implemented during the COVID-19 pandemic, and to ensure safety of staff and participants, amendments to the trial protocol were implemented in 2020 to enable remote screening and follow-up visits by phone instead of in-person visits. After easing of COVID-19 restrictions, participants were given the option of continuing with remote screening and follow-up visits or face-to-face clinic visits depending on their risk profile, personal preference and location.All STAREE staff including the investigator team, management, administration, medical and research officers and students are blinded to treatment allocation through the randomisation procedure and study duration. All GP co-investigators and practice staff will be blinded to treatment allocation. All participants will be blinded to treatment allocation for the study duration. GP co-investigators are requested to not measure lipid levels in participants after randomisation, to minimise the risk of unmasking.In the event of a clinical emergency, participants will be assumed to be on active treatment and the recommendation will be to cease study treatment if there was a requirement for statin use. The need for unblinding of study participants will be determined on a case-by-case basis after referral to a senior STAREE medical investigator who will discuss the clinical need for unblinding with the requesting clinician and then authorise the request. The process of emergency unblinding will be overseen by the Monash University-affiliated, independent pharmacist who will have access to the randomisation code.Ascertainment of primary endpoints will be performed by Endpoint Adjudication Committees of expert clinicians. These Committees will adjudicate events related to possible MI, stroke, dementia, disability, heart failure and trajectory and mode of death. Each subcommittee will have a chair and a minimum of two other members. Two members will conduct adjudications for each event and discordant cases will be resolved via consensus of all committee members.Disability-free survival is defined as survival free of dementia or physical disability as derived from the endpoints of all-cause death, dementia and persistent physical disability. It is a composite endpoint that has been used in recent clinical trials and enables an evaluation of the net benefit of statin by capturing both beneficial and adverse effects.Dementia is defined as a significant impairment in cognitive function  trial populationDFSThe recruitment rate in Australia was impacted by the COVID-19 pandemic. In addition, STAREE was originally planned as an international endeavour with proposed funding and recruitment across multiple regions including Australia, however in late 2020 it became evident that the proposed funding for recruitment outside of Australia would not be secured. These developments were considered in July 2021 in finalisation of the trial design, reduction in the sample size from an original target of n=18 000 and corresponding power calculation to allow for feasible completion of the trial in Australia alone.The sample size requirements are summarised in Loss to follow-up will occur at a rate of 2% of study participants per annum . Hence an average of 6.0 years of follow-up time per participant is expected by the anticipated end of the trial in December 2025, at which time the required number of co-primary endpoints are expected to have accrued.An age structure of STAREE participants of 56%: 27%: 12%: 5% in the respective age bands 70–74 years: 75–79 years: 80–84 years: 85+ years. These proportions are based on recruitment of the initial approximately 7500 participants to late 2020.With treatment adherence statins will reduce the rate of major cardiovascular events by 25% and the rate of disability-free survival by 18%.Treatment crossover will occur and impact the HR that will be observed in intention-to-treat analyses. In the first year of follow-up, we assume 8% of participants randomised to statin will cease taking their study medication. Thereafter we assume 3% of participants per annum in the statin group will cease taking study medication. In the first year of follow-up, we assume 1% of participants in the placebo group will commence open-label statin. Thereafter, we assume 3% of participants per annum in the placebo group will commence statin therapy. Further, we assume that there will be no subsequent treatment ‘re-crossovers’. It is assumed that the effects of statin use and non-use outside use of study medication is the same as the effects of randomised statin and placebo, respectively.Rates of the co-primary endpoint death, dementia or persistent physical disability in the placebo group of 18.3 per 1000 person-years (70–74 years), 33.9 per 1000 person-years (75–79 years), 65.7 per 1000 person-years (80–84 years) and 115.4 per 1000 person-years (85+ years) see . To alloMajor cardiovascular event rates in the placebo group, according to age at randomisation, of 9.5 per 1000 person-years (70–74 years), 11.5 per 1000 person-years (75–79 years), 18.5 per 1000 person-years (80–84 years) and 24.5 per 1000 person-years (85+ years).10.1136/bmjopen-2022-069915.supp5Supplementary dataBaseline characteristics of the two treatment groups will be tabulated and imbalance defined as a 0.25 SD difference in means (quantitative characteristics) or an OR of 1.5 (binary characteristics). Given the large sample size, randomisation is anticipated to adequately balance baseline characteristics of participants in the two treatment groups. Hence unadjusted analyses will be considered primary. All primary comparisons between treatment arms will be on an intention-to-treat basis, that is, according to the group to which participants were randomised and without reference to their actual compliance with assigned treatment. Each of the co-primary endpoints will be analysed separately in time-to-event analyses. Event rates (time to first event within each endpoint definition) will be compared between groups using an HR and 95% CI from a Cox proportional hazards regression model fitted to the endpoint, with censoring for individuals not experiencing an endpoint event at their most recent study visit, and a single covariate being an indicator of the group to which the individual was randomised, statin or placebo. The proportional hazards assumption will be tested for each model. Loss to follow-up will be considered a censoring event. This equates to an assumption that data is missing at random given the participant’s treatment group and the timing of their loss to follow-up. The adequacy of this assumption will be checked in sensitivity analyses that will include both imputation approaches and adjustment for baseline covariates predictive of propensity for dropout.A closed testing procedure will be used to allow for the multiple testing arising from two co-primary endpoints. This approach is based on the expectation that cardiovascular benefit will be the main contributor to improved disability-free survival and that a substantial effect of statins on the latter is unlikely in the absence of an effect on the former. First, major cardiovascular events will be tested at alpha=0.05 and, if the major cardiovascular events p value is <0.05 then second, disability-free survival will be tested at alpha=0.05. If the major cardiovascular events p value is not <0.05 than a p value for disability-free survival will not be presented.Secondary analyses of the co-primary endpoints will be performed using extended Cox proportional hazards regression models based on time(s) to any event within the endpoint definition (for relevant endpoints that can occur more than once). No statistical adjustment will be made for the multiple secondary endpoints and no p values will be presented for the statin versus placebo comparison for these endpoints. The reporting of all secondary endpoint analyses will make clear whether either of the co-primary endpoints were statistically significant.2), smoking status , 3MS score , eGFR  and LDL-C and HDL-C levels (terciles).Subgroup analyses will be undertaken by sex , age , systolic and diastolic blood pressure levels (terciles), body mass index , following emerging guidance on analytical approaches and estimand definitions.An independent committee comprising experts in clinical trials, cardiology and statistics are overseeing data accrual and safety. As outlined in the Data and Safety Monitoring Board (DSMB) Charter , the DSM10.1136/bmjopen-2022-069915.supp6Supplementary dataSTAREE has embedded mechanisms for ongoing participant and public involvement. These include a participant advisory committee, which reviewed study processes and documents, and a formal study that assessed participant preferences about the delivery of informed consent information.During the trial, participants may provide feedback via access to a free phone line. Participants are also invited to annual trial updates and ‘question and answer’ sessions. At the completion of the trial, participants will receive a summary of the study results and information about which treatment arm they were allocated to.STAREE is being conducted in accordance with the Declaration of Helsinki , the NHMRC Guidelines on Human Experimentation, the federal patient privacy (HIPAA) law and ICH-GCP guidelines and the International Conference of Harmonisation Guidelines for Good Clinical Practice. Institutional ethics approval has been obtained from the following:Monash University Human Research Ethics Committee (Project ID 2787 and 21528).Curtin University Human Research Ethics Committee (HR113/2015).RACGP National Research and Evaluation Ethics Committee (14 - 017).Tasmanian Health and Medical Research Ethics Committee (H0014918).University of Newcastle Human Research Ethics Committee (H-2016–0266).The trial is managed and co-ordinated by the STAREE steering committee  which is10.1136/bmjopen-2022-069915.supp7Supplementary dataAll research outputs will be disseminated to GP co-investigators and participants, published in peer-reviewed journals and presented at national and international conferences. Authorship of papers will be according to the criteria of the International Committee of Medical Journal Editors and all STAREE publications will list the lead authors and writing group, and the STAREE Investigator group.On completion of the trial, and after publication of the primary and secondary outcomes of the study, requests for access to de-identified data (to be provided through a secure online environment) may be submitted to the researchers located at the School of Public Health and Preventive Medicine, Monash University, Melbourne, Australia.Recruitment commenced in November 2015 and was expected to be completed in 2021. However, COVID-19 public health directives and work restrictions resulted in reduced access to general practices across Australia such that recruitment had to be extended for further 18 months.There is continuing debate on the utility of statins for primary prevention in older people primarily due to the lack of RCT evidence in this population.STAREE is a large primary prevention trial that is assessing whether starting statins in adults 70 years and older will extend independent and healthy lifespan. While STAREE captures the CVD endpoints that are the traditional primary outcomes of statin trials, we consider that a wider range of outcomes beyond major CVD events is important to evaluate the overall benefit of statins in an older population. To this end, our disability-free survival outcome including detailed ascertainment of dementia using well validated measures of cognition, such as those used in the ASPREE trial


Adequate skeletal loads during the adolescent growth spurt are elemental in building a robust skeleton. Adolescents with poorer motor competence are less active and develop a less robust skeleton compared to their peers with better develop motor competence. However, few longitudinal examinations of motor competence and skeletal loading through adolescence have been reported. Therefore, the purpose of the present study was to explore the association between motor competence and free-living bone-generating (osteogenic) skeletal load trajectories among adolescents.th graders (11 to 13 years-of-age) were initially included in the sample and were followed up annually for four years from 2017 to 2020 (N = 239 in 2020). The children were asked to wear a waist-worn accelerometer for 7 days and motor competence was assessed with 5-leap and side-to-side jumping tests once a year. Latent profiles were identified based on motor competence, and skeletal loading was estimated using an osteogenic index, which combines the number of loading peaks in particular intensity categories akin to counting steps but with accounting for the magnitude of the impact.A total of N = 562 5Three motor competence groups arose from the data for both boys and girls and all groups exhibited persistent low, mid or high motor competence throughout the follow-up. The latent group with the low motor competence consistently exhibited the lowest skeletal loads (10 to 20% below the highest motor competence), and the highest motor competence group the highest with the mid-group in between. All latent groups declined in skeletal loads over the follow-up (from osteogenic index 420 SD[80] to 300[90]) with no difference in the steepness of the decline among boys. However, the lowest motor competence group among girls had a slightly steeper decline in skeletal loads than the other groups.the finding that motor competence is associated with skeletal loading among adolescents was confirmed in this longitudinal study. Notably, girls with the lowest motor competence had the steepest decline in skeletal loading over the follow-up. Policies aiming to improve fundamental movement skills could be expected to improve skeletal development particularly among less motor competent girls.


Focal Hand Dystonia (FHD) is a disabling movement disorder characterized by involuntary movements, cramps and spasms. It is associated with pathological neural microcircuits in the cortical somatosensory system. While invasive preclinical modalities allow researchers to probe specific neural microcircuits of cortical layers and columns, conventional functional magnetic resonance imaging (fMRI) cannot resolve such small neural computational units. In this study, we take advantage of recent developments in ultra-high-field MRI hardware and MR-sequences to capture altered digit representations and laminar processing in FHD patients. We aim to characterize the capability and challenges of layer-specific imaging and analysis tools in resolving laminar and columnar structures in clinical research setups. We scanned N = 4 affected and N = 5 unaffected hemispheres at 7T and found consistent results of altered neural microcircuitry in FHD patients: 1) In affected hemispheres of FHD patients, we found a breakdown of ordered finger representation in the primary somatosensory cortex, as suggested from previous low-resolution fMRI. 2) In affected primary motor cortices of FHD patients, we furthermore found increased fMRI activity in superficial cortico-cortical neural input layers (II/III), compared to relatively weaker activity in the cortico-spinal output layers (Vb/VI). Overall, we show that layer-fMRI acquisition and analysis tools have the potential to address clinically-driven neuroscience research questions about altered computational mechanisms at the spatial scales that were previously only accessible in animal models. We believe that this study paves the way for easier translation of preclinical work into clinical research in focal hand dystonia and beyond. Focal Hand Dystonia (FHD) is a disabling movement disorder characterized by involuntary movements, cramps and spasms. Common sub-forms are known as writer’s cramp and musician’s cramp Karp 2017) with epidemiological prevalence up to 1 in 2,500 people 7 with ep, conventScan duration: Previous studies could collect up to 18 h of fMRI data per participant. This was achieved by inviting the participants for up to 10 two-hour scan sessions . As exemplified in Noise in task compliance: FHD patients can have a hard time following the task instructions. For example, when instructed to move the ring finger only, the middle finger and fifth finger are often moved too. This reduces the finger functional contrast-to-noise ratio.EPI Artifact level: Sub-millimeter fMRI is limited by low bandwidths and small coverages. In previous applications in healthy volunteers, this had been accounted for with iterative online fine tuning of acquisition parameters during the data acquisition phase . Within the limited scan time of patients, such fine tuning is not feasible. Thus, the raw functional images can be challenged by EPI phase-inconsistency artifacts as exemplified in Due to these challenges, the applicability of laminar and columnar functional imaging with fMRI in patient populations remains unclear. The aim of this study is to characterize and mitigate these challenges. We used the altered digit representation in FHD patients as a testbed to explore the capability of applying layer-fMRI VASO to investigate mesoscopic neural representations in patients. Specifically, we sought to investigate the quantifiability of pathological alterations of the mesoscopic finger representations in FHD patients across cortical depth as well as across topographical arrangements along the cortical sheet.ClinicalTrials.gov identifier: NCT03223623). Four FHD patients  receiving regular botulinum toxin injections were scanned at least 3 months after their last injection. Additionally, data from a prior study  using the vendor-provided IDEA environment (VB17A-UHF). For RF transmission and reception, a single-channel-transmit and 32-channel receive head coil  was used. The scanner was equipped with a SC72 body gradient coil .https://layerfmri.com/ss-si-vaso-sequence-manual/. The optimal sequence parameters were tested and optimized in previous studies .Slice-selective slab-inversion (SS-SI) VASO sequence (Huber 2014) was used in combination with a 3D-EPI readout, as previously implemented . VASO is studies . In shorThe VASO imaging slab was positioned to be approximately perpendicularly oriented to the main surface of the central sulcus of one hemisphere. This was achieved by tilting it in two directions. Scanning was conducted using preset parameters for layer-fMRI VASO experiments, without individualized settings to optimize the time of the patients in the scanner. This scanning setup can be used to exemplify the usability and scalability of the experimental procedures beyond specialized MRI-development research groups. No specific custom hardware was necessary during the scanning, beyond the common 7T MRI configuration that is available at >100 centers worldwide. We scanned N = 4 patients and N = 2 healthy volunteers. Together, we collected data from N = 5 healthy hemispheres and N = 4 affected hemispheres.via Psychopy 2 with publicly available scripts . The same task was repeated for each hand. Each run lasted approximately 33 min.For the sake of consistency and comparability with previous studies, we used the same tapping tasks as previously used (Huber 2020). Briefly, before each run, subjects were instructed on what hand to tap with. Participants were instructed to tap one finger by extension-flexion at the metacarpophalangeal joint. The same tapping task was repeated for each finger. The tapping frequency was self-paced at a frequency of approximately every 1–2 s. During the scan, a video prompted which finger to move and when to rest. The tapping timing was locked to scanner triggers in units of 16 TRs (2.2 s each) and contains visual cues when to tap which finger and for how long. The task was controlled T1-contrast (in the Mz-direction) is completely orthogonal to the BOLD Mxy-direction). Block design activation z-scores and beta estimates were extracted with FSL-FEAT . Motion-corrected time series were corrected for BOLD contaminations, by means of dynamically dividing blood-nulled signals with not-nulled BOLD signals using LayNii’s v2.2.1  LN_BOCO.FSL-FEAT . Layerifhttps://layerfmri.com/analysispipeline/.Hands-on tutorials (with maintained QnA sections), analysis manuals, and “click-along” video instruction of the entire preprocessing and layerification analysis that is performed here can be found at https://thingsonthings.org/ln2_multilaterate/ and here https://youtube.com/playlist?list=PLs_umVHtShfadNm8brOweXHUSmqVDTk4q, respectively. Layer-extraction was manually constrained to the Brodmann area BA4a. This is the evolutionary older part of the primary motor cortex (as opposed to BA4p). In order to pinpoint it, we followed previously described landmarks (https://layerfmri.com/finding-roi-of-the-double-layers-in-m1/. The specific ROIs of all patients and hemispheres as used in this study are shown in Cortical patch flattening was performed with the LN2_MULTILATERATE and LN2_PATCH_FLATTEN programs in LayNii . The algandmarks . In shorWe collected short video recordings of the FHD patients while they were performing the tapping task (example screenshots in Across all data sets, we find tapping induced activation both in M1 and in S1. This means that five out of five patient hemispheres show clear CBV changes in M1 and also four out of four patient hemispheres show clear CBV changes in S1. This is a success rate of 100% for both brain areas. Activation maps of all hemispheres in In order to explore the spatial structure of finger representation in the primary somatosensory cortex on the posterior bank of the central sulcus, we imposed a local coordinate system within the GM cortical sheet. Exemplary data of one representative participant show exemplary how the finger representation follows the somatotopic alignment for the healthy hemisphere . The spaAside from topographical finger-representations in the sensory cortex, we also explored the laminar blood volume responses in the primary motor cortex. Video material of the FHD patients performing the task inside the scanner bore suggested a qualitative trend that FHD patients might have performed the tapping at a slower pace compared to typical tapping frequencies in healthy participants. To quantify the effect of tapping frequency on the interpretation of the layer profiles shown in In this study, we explored the capabilities and challenges of high-resolution layer-fMRI to inform research questions of affected cortical microcircuitry in patients. We scanned N = 4 focal hand dystonia patients and found consistently altered somatotopic and laminar activation patterns in affected and unaffected hemispheres. The results presented here suggest that the high-resolution acquisition and analysis protocols developed here allow clinical neuroscientists to investigate pathological laminar pathologies in patients. Specifically, we found that cortical hemispheres that were affected by FHD appear to have a less structured somatotopic alignment of finger representation in the primary somatosensory homunculus of the postcentral gyrus. These results are consistent with the parallel ongoing imaging efforts of high-resolution functional mapping of finger representations at 7T . This stLooking beyond FHD, the tools developed and tested in this study provide a starting point for mapping layer-specific connections in patient populations in the general context of clinical neuroscience. Many influential theories of brain function of disorders posit hypotheses of neural deficits in distinct cortical layers and their role of feedforward and feedback processing. For example: mental disorders, such as autism and schizophrenia, and neurodegenerative diseases such as Parkinson’s or Huntington’s disease. Specifically.Layer-fMRI VASO has been discussed to be the key future technology to probe predictions of axonal loss and microcircuit dysfunction to provide insights for neurodegenerative motor disease , 27. TheLayer-fMRI has been discussed to be a key future technology to act as Occam’s razor for multiple competing hypotheses of hierarchical microcircuit disruptions in psychosis , 29. NamThese examples show that layer-fMRI applications in patients open the door to investigating computational mechanisms at the spatial scales that were previously only accessible in animal models. We believe that this study paves the way for easier translation of preclinical work into clinical research in focal hand dystonia and beyond.While there are plentiful review articles about the value of layer-fMRI for clinical neuroscience research –29 exper


RNA velocity analysis of single cells offers the potential to predict temporal dynamics from gene expression. In many systems, RNA velocity has been observed to produce a vector field that qualitatively reflects known features of the system. However, the limitations of RNA velocity estimates are still not well understood.We analyze the impact of different steps in the RNA velocity workflow on direction and speed. We consider both high-dimensional velocity estimates and low-dimensional velocity vector fields mapped onto an embedding. We conclude the transition probability method for mapping velocity estimates onto an embedding is effectively interpolating in the embedding space. Our findings reveal a significant dependence of the RNA velocity workflow on smoothing via the k-nearest-neighbors (k-NN) graph of the observed data. This reliance results in considerable estimation errors for both direction and speed in both high- and low-dimensional settings when the k-NN graph fails to accurately represent the true data structure; this is an unknown feature of real data. RNA velocity performs poorly at estimating speed in both low- and high-dimensional spaces, except in very low noise settings. We introduce a novel quality measure that can identify when RNA velocity should not be used.Our findings emphasize the importance of choices in the RNA velocity workflow and highlight critical limitations of data analysis. We advise against over-interpreting expression dynamics using RNA velocity, particularly in terms of speed. Finally, we emphasize that the use of RNA velocity in assessing the correctness of a low-dimensional embedding is circular.The online version contains supplementary material available at 10.1186/s13059-023-03065-x. Cell cycle positions are estimated using the tricycle Bioconductor package . For thek nearest neighbors in Euclidean distance of the top 30 PCs (default setting) and assigns weights to each edge  and Here, For the velocity latent time, we cannot determine whether the latent time spans a complete period. Therefore, we fit a standard loess model instead of the periodic model described above for the velocity latent time.In the simulations, we know the true values, such as the true RNA velocity at the gene level. We use both PCC and NRMSE to quantify how good the estimations are. The NRMSE is calculated asn is the number of observations. The standard deviation of the true values Here, Additional file 1: Contains Figs. S1-S20 and supplementary notes on “The differences between implementations of RNA velocity analysis” and “Visualization of 2D vector fields”.Additional file 2. Contains the review history.