This cohort study examines the association of suicide and other mortality risk within 1 year of emergency department presentation among patients presenting with deliberate self-harm, suicidal ideation, or any other chief concern. Is emergency department presentation associated with 1-year incidence of suicide and other mortality, and does the suicide rate vary by patient clinical and sociodemographic characteristics?In this cohort study including 648 646 patients who presented to California emergency departments, compared with the demographically matched general population, suicide mortality was 56.8-fold higher among patients presenting with deliberate self-harm, 31.4-fold higher among patients presenting with suicidal ideation, and 1.9-fold higher among patients presenting with any other chief concern; risk of other mortality was also increased. Sociodemographic and clinical factors associated with suicide risk varied by patient group.These findings suggest that broad implementation of suicide risk screening and intervention is needed in emergency department settings, and the scope of interventions should also consider suicidal individuals’ risk for unintentional injury and other premature mortality. Emergency departments (EDs) have the potential to play a pivotal role in suicide risk detection and prevention, yet little is known about the profile of risk of suicide after ED visits in the United States.To examine 1-year incidence of suicide and other mortality among ED patients who presented with nonfatal deliberate self-harm, suicidal ideation, or any other chief concern, and to examine sociodemographic and clinical factors associated with suicide mortality risk.This retrospective cohort study included statewide, all-payer, longitudinally linked ED patient records and mortality data from all California residents who presented to a California-licensed ED at least 1 time from January 1, 2009, to December 31, 2011, with deliberate self-harm, suicidal ideation but not self-harm, or neither (a 5% random sample). Age-, sex-, and race/ethnicity-adjusted standardized mortality ratios (SMRs) for suicide and other manners or causes of death were determined for each patient group using statewide mortality data. Data were analyzed from January 10 to July 18, 2019.International Statistical Classification of Diseases and Related Health Problems, Tenth Revision codes. Suicide rate and all mortality rates were measured per 100 000 person-years.Suicide and other manners or causes of death were ascertained using Among 648 646 individuals  who visited an ED in California from 2009 to 2011, the rates of suicide deaths per 100 000 person-years in the year after index ED presentation were 693.4 deaths among 83 507 individuals presenting with deliberate self-harm , 384.5 deaths among 67 379 individuals presenting with suicidal ideation but not self-harm , and 23.4 deaths among 497 760 reference patients . Compared with the demographically matched general population, the rates of nonsuicide external-cause mortality were also increased among patients with self-harm , patients with suicidal ideation , and reference patients . In all 3 groups, the rates of suicide mortality per 100 000 person-years were higher among men , people 65 years or older , and non-Hispanic white patients  than among their respective referent groups. Other sociodemographic factors and clinical diagnoses were associated with striking differences in suicide rates, but these patterns were heterogeneous across patient groups.These findings suggest that ED patients with deliberate self-harm or suicidal ideation are associated with substantially increased risk of suicide and other mortality during the year after ED presentation. The process of planning for ED discharge may present opportunities to help ensure safe transitions to continuing outpatient mental health care and to consider broader risk for unintentional injury and other causes of premature mortality. Practice guidelines for management of deliberate self-harm recommend clinical assessment of ongoing risk of self-harm and suicide.4 Although effective ED-based interventions for patients at high risk of suicide have been developed, they are not yet widely used in community practice.6Each year in the United States, more than 500 000 people present to emergency departments (EDs) with deliberate self-harm, and many more people present with suicidal ideation.7 and mortality-linkage studies using samples of US military patients, outpatients, and psychiatric inpatients differ considerably from ED patients in demographic characteristics, clinical severity, and treatment protocols.9 Other studies have examined mortality among single-payer  patient pools, but findings from such specialized subpopulations are not generalizable.10 Although a few studies have examined suicide among US-based general-population ED patients, they all had limited statistical power, particularly to examine variability in patient suicide rates according to demographic and clinical characteristics, and none examined nonsuicide mortality, to our knowledge.12Little is known about the profile of risk of suicide after ED visits for self-harm or suicidal ideation in the United States. Past studies using large ED databases, such as the Healthcare Cost and Utilization Project data, cannot be linked to mortality records,To help address this gap in knowledge, this study examines longitudinal patterns of mortality among residents of California who visited an ED. We focus on 3 patient groups: those presenting with deliberate self-harm , those presenting with suicidal ideation (without deliberate self-harm), and those presenting without deliberate self-harm or suicidal ideation, representing a random 5% sample, as a reference population. To our knowledge, this analysis represents the first US population-based study of suicide and other mortality after ED presentation with deliberate self-harm or suicidal ideation. To inform clinical practice, the study also advances prior work by examining how clinical correlates and sociodemographic variables of interest within each patient group are associated with suicide risk. Prior to performing these analyses, we hypothesized that both in the presence and absence of diagnosed mental disorders, a gradient in suicide risk would be observed, with patients presenting with deliberate self-harm having greater risk than those with suicidal ideation, who in turn would have greater risk than patients with neither self-harm nor suicidal ideation.STROBE) reporting guideline.This study was approved by the institutional review boards of the California Health and Human Services Agency and the University of California, Merced. Because the study comprised analyses of secondary data, a waiver of informed consent was granted by the California Health and Human Services Agency institutional review board. This study was prepared in accordance with the standards set forth by the Strengthening the Reporting of Observational Studies in Epidemiology  were not eligible for linkage and were excluded from analysis.International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM)13 external cause-of-injury code (E-code) of E950.0-958, in any diagnostic position; (2) patients with suicidal ideation, defined as those with at least 1 ED visit during the period that included an ICD-9-CM diagnosis of V62.84 in any diagnostic position but not with a diagnosis for deliberate self-harm; and (3) a 5% random sample of all other patients . California has mandated 100% reporting of E-codes since 1990. For patients with self-harm or suicidal ideation, their first qualifying ED visit during the study period was defined as their index visit. For reference patients, their first ED visit during the period was defined as the index visit. The analyses were limited to index visits.The cohort was partitioned into 3 nonoverlapping groups: (1) patients with deliberate self-harm, defined as those with at least 1 ED visit during the study period that included an For visits resulting in patient discharge or transfer to another facility, the index date was the date of ED presentation. For visits that resulted in a same-hospital admission, the index date was the date of hospital discharge from the associated hospital admission. Patients who died on the date of the index visit or whose index visit disposition was coded as death were excluded from follow-up analyses.14 and Wide-ranging Online Data for Epidemiologic Research15 systems.We obtained data on death by cause for California overall in 2009 to 2012 from the Centers for Disease Control and Prevention’s Web-based Injury Statistics Query and Reporting SystemInternational Statistical Classification of Diseases and Related Health Problems, Tenth Revision (ICD-10)16 codes X60-X84, Y87.0, or U03 as the immediate cause of death. Secondary outcomes included deaths by unintentional injury and other causes, based on corresponding ICD-10 codes.16Our primary outcome was suicide within 1 year of the index date. Suicide was defined as any death with 17Patient demographic factors of interest included sex , age group , race/ethnicity , and insurance status. Insurance was classified as private , Medi-Cal , Medicare , or self-pay or other. Patients’ urbanicity was defined via zip code of residence using the US Department of Agriculture’s Rural-Urban Commuting Areas 2010 geographic taxonomy, version 3.10 .ICD-9-CM codes and Clinical Classification Software (CCS) groupings, which aggregate ICD-9-CM diagnoses into discrete, clinically meaningful categories. We focused on psychiatric diagnoses known to be associated with suicide risk: depression , bipolar disorder (ICD-9 codes 296.0-296.1 and 296.4-296.8),13 anxiety disorder (CCS code 651), psychotic disorder (CCS code 659), alcohol-related disorder (CCS code 660), and drug-related disorder (CCS code 661); we also included suicidal ideation for patients with deliberate self-harm. We also calculated the Elixhauser Comorbidity Index, a claims-based scale that counts up to 31 categories of comorbid physical and psychological conditions and has good predictive validity for short-term mortality.18Patient clinical factors of interest included comorbid clinical diagnoses recorded at the index ED visit and, for patients with deliberate self-harm, method of index self-harm injury. Comorbid diagnoses were ascertained using 19 The ICD-9-CM codes13 used to define this variable are shown in the eTable in the Injury method among patients with deliberate self-harm was categorized according to the most common methods observed among California ED patients: poisoning, cutting or piercing, hanging, jumping, firearm, and all others.We calculated crude mortality rates per 100 000 person-years of follow-up for suicide and other manners or causes of death in the year after the index ED visit for self-harm, suicidal ideation, and reference groups. We additionally calculated crude suicide mortality rates per 100 000 person-years of follow-up within each group for the demographic and clinical categories of interest. Individuals whose information was not linked to California mortality records from 365 days after the index date were presumed alive for this period. Decedents were treated as censored on their death date; patients who died more than 365 days after their index visit were assumed alive at the 1-year point.For our second aim, we restricted analyses to each patient group and modeled incidence rate ratios estimating associations between each variable and rate of suicide by end of follow-up. We opted to estimate risk ratios (RRs) rather than hazard ratios because several variables did not meet proportional-hazards assumptions required for Cox regression. All models included an offset term consisting of person-time observed for each patient during follow-up. We first estimated bivariate unadjusted RRs. We then fit adjusted models that controlled for patient age, sex, and race/ethnicity, as these factors are important independent factors associated with suicide risk.14 and Wide-ranging Online Data for Epidemiologic Research15 mortality databases, standardized to the distribution of sex, age category, and race/ethnicity category of the respective groups in our ED cohorts. For each respective ED cohort, standardization was implemented by multiplying the proportion of patients in that cohort’s age-, sex-, and race-specific strata by the age-, sex-, and race-specific mortality rates observed statewide.As a frame of reference, we calculated annualized standardized mortality ratios (SMRs), defined as the ratio of the observed numbers of deaths in our ED cohorts to expected deaths. The rates of expected deaths in California for 2009 to 2012 for suicide and other manners or causes of death were calculated using the Centers for Disease Control and Prevention Web-based Injury Statistics Query and Reporting SystemP values were 2-tailed, and statistical significance was set at an α of .05. Data were analyzed from January 10 to July 18, 2019.Statistical analyses were conducted using Stata version 14.0 (StataCorp). The cohort included 648 646 individuals  who visited an ED in California from 2009 to 2011. There were 83 507 patients with deliberate self-harm  , 67 379 An additional 4040 individuals died on the date of the index event and were excluded from analysis, including 820 patients with deliberate self-harm (73.9% listed as dying by suicide), 104 patients with suicidal ideation (3.9% listed as dying by suicide), and 3116 reference patients (0.3% listed as dying by suicide). Of 15 patients with deliberate self-harm who were discharged alive but died on their index date, 13 died by suicide. No patients in the other groups who were discharged alive died by suicide on their index date.Among all patients, the cumulative probability of suicide in the first year after discharge was highest among patients with self-harm, then patients with suicidal ideation, then reference patients  and patients 65 years and older (1919.5 deaths per 100 000 person-years) had markedly higher rates of suicide compared with women (473.5 deaths per 100 000 person-years) and patients aged 10 to 24 years (221.3 deaths per 100 000 person-years). Patients who were non-Hispanic black (226.5 deaths per 100 000 person-years) had substantially lower rates compared with patients who were non-Hispanic white (914.1 deaths per 100 000 person-years). Suicide rates among patients with Medicaid coverage (490.4 deaths per 100 000 person-years) or self-pay or other health care payer (574.0 deaths per 100 000 person-years) were also lower than rates among privately insured patients (755.3 deaths per 100 000 person-years). Rurality of residence was not significantly associated with suicide risk.In patients whose index ED visit involved deliberate self-harm , the suiAmong patients with self-harm, several comorbid clinical diagnoses  were associated with elevated suicide risk in unadjusted models, but some associations were reduced to nonsignificance in the adjusted models. Patients with deliberate self-harm with a comorbid diagnosis of bipolar disorder , anxiety disorder , or psychotic disorder  remained statistically significantly more likely to die by suicide than patients without those diagnoses. Among patients with self-harm, higher Elixhauser Comorbidity Index scores were associated with increased suicide risk in a dose-response manner . Firearm injury at index visit was also associated with increased risk of suicide death . Patients whose nonfatal index self-harm event involved a firearm had a suicide incidence rate of 4444.1 deaths per 100 000 person-years, far higher than any other patient group.In the suicidal ideation group , the suiAmong reference patients , the suiThe Beyond our primary outcome of suicide within 1 year of ED presentation, we also examined rates of mortality by other causes . In the 11 These findings reinforce the rationale for providing ED-based within-encounter and postdischarge interventions, including safety planning, follow-up communications, mobile crisis team linkage, and text reminders, that have evidence for effectiveness but are not yet widely used.20This cohort study is the first US population-based study to examine 12-month suicide rates after an index ED visit, to our knowledge. In the state of California from 2009 to 2012, we found substantially increased and enduring risk of suicide death among patients who presented to an ED with deliberate self-harm or suicidal ideation. The suicide rate of the self-harm sample was 2-fold that reported from a self-harm sample at a university-affiliated ED.21 but which is also not yet widespread.Even ED patients with no self-harm or suicidal ideation experienced postindex suicide rates almost 2-fold that of the corresponding California population. Together, these findings reinforce the potential value of routinely screening all ED patients for suicide risk, an approach that has been found to increase the number of ED patients identified as warranting treatment for suicide risk by approximately 2-fold,22 This may reflect limitations in determining intent of death, particularly for deaths via unintentional overdoses.23 In addition, there may be common etiologic factors, such as mental states, including impulsivity, inattentiveness, and hopelessness, across these causes of death. These patterns suggest that the scope of interventions for individuals with self-harm or suicidal behavior in ED settings should consider their broader risk for unintentional injury and other premature mortality.22Patients in both the self-harm and ideation groups also had markedly increased risk for other external cause–mortality, particularly via unintentional injury. Such overlap between suicide and unintentional injury mortality risk has been observed in other settings.24 As the We found striking differences in suicide rates according to patients’ clinical diagnoses recorded at the index ED visit. These patterns were heterogeneous across patient subgroup, an observation that has not been previously reported, to our knowledge. For example, a clinical diagnosis of psychosis was associated with increased suicide risk among patients with deliberate self-harm but not among patients with suicidal ideation or reference patients. By contrast, alcohol use disorder in the absence of suicidal ideation or self-harm was associated with increased suicide risk among reference patients. In interpreting between-group variability, it is important to recognize that mental disorder diagnoses may be underrecorded in ED settings, and the extent of the underrecording likely varies with the presenting chief concern.25 patients with self-harm whose index ED visit was firearm related had the highest suicide rate of any subgroup studied. Along with a need for improved practices regarding limiting access to lethal means for individuals with suicide risk, including lethal means counseling, ED physicians should be aware that patients with self-harm who use high-lethality methods at a nonfatal event remain at highly increased risk for future death of suicide.Consistent with a 2017 study,27 Insurance status was also associated with suicide risk, albeit inconsistently. Notably, the suicide rates in this study for patients with self-harm who had Medicaid coverage closely resembled rates reported in a national study of patients with self-harm and Medicaid coverage,25 suggesting that this population experiences lower rates of suicide. This difference may be owing to better access to mental health care among patients with Medicaid coverage compared with individuals who are privately insured or differences in their sociocultural composition.28Within each patient group, sex, age, and race/ethnicity were associated with variability in suicide risk. These differences, which recreate in ED samples patterns in the general population, might be partially explained by variable access to and probability of using firearms, moral beliefs about suicide, and access to psychosocial resources and psychological treatment.29 and because California has a relatively low state suicide rate,30 the results may not be safely generalized to the entire United States. Mortality data for members of our ED cohort who died outside of California were not available for this study; we expect this artifact has a slight downward bias on the absolute mortality rates in our ED groups and on the comparison among the ED groups and California overall. Some suicide mortality occurring after the index date may have been due to the index self-harm event and therefore would be outside the reach of intervention initiated at the index ED visit. Additionally, ICD-9 E-codes13 do not distinguish between deliberate self-harm with and without intent to die. Furthermore, all diagnostic codes used in the study were based on clinical judgment and were not subject to expert independent validation. External cause-of-injury coding in California is virtually complete (>95% of injury events are E-coded); nevertheless, 4.7% of injury or poisoning events did not have an associated E-code and could have been placed in the reference group. We did not attempt to validate the determined cause of death as recorded in our death certificate data. Furthermore, we lacked information about many potentially relevant patient risk factors, such as access to lethal means and engagement with outpatient care after ED discharge.This study had several limitations. There are alternative ways to partition the ED cohort into subgroups; moreover, calculating person-level mortality rates likely underestimates visit-level mortality risk, as individuals who make multiple ED visits would presumably experience higher risk. This may be particularly true for individuals presenting with suicidal ideation, many of whom were classified into the self-harm group in this study. However, we believe alternative analytic strategies would be unlikely to change our main conclusions. Because the sociodemographic characteristics of California differ from those of other statesOur study also had important strengths. These included a census of the residents of California who visited EDs during a 3-year period, comprehensive coding of external cause of injury, which is not available in all states, and statewide ED discharge data linked to mortality records, which also may not be available in all states.31 Some promising interventions include universal screening,21 safety planning,20 active management of service transitions,32 and telephone follow-up and support.5 Moreover, conducting ongoing public health surveillance of the patterns and associations of suicide and other mortality among ED patients could inform health care quality improvement,33 help monitor progress toward national suicide prevention goals, and address issues of parity between mental and physical health care.The findings in this cohort study regarding the high risk of suicide after ED visits among patients with nonfatal deliberate harm or suicidal ideation underscores the clinical rationale for ensuring that these patients receive timely follow-up mental health care. This rationale is strengthened by evidence of low rates of outpatient mental health follow-up among patients with self-harm who are publicly or privately insured.


Background: Resistance training (RT) is recommended as part of our national physical activity guidelines which includes working all major muscle groups on two or more days a week.Older adults can gain many health benefits from RT, such as increased muscle strength,increased muscle mass, and maintenance of bone density. Additionally, certain dimensions of health-related quality of life (HRQOL) have been shown to improve in older adults due to RT intervention. The purpose of this study was to use systematic review and meta-analytic techniques to examine the effect of RT on HRQOL in older adults.Methods: A systematic review of current studies (2008 thru 2017) was conducted using PubMed. Studies were included if they used a randomized controlled design, had RT as an intervention, measured HRQOL using the SF-36/12 assessment, and included adults 50+ years of age. Eight dimension scores  and two summary scores  were extracted. Ten meta-analyses were performed using standardized mean effect sizes and random effects models. Study quality,moderator and sensitivity analyses were conducted.Results:  A total of 16 studies were included in the analyses with a mean Physiotherapy Evidence Database (PEDro) score of 4.9 (SD=1.0). Among the mental health measures, RT had the greatest effect on mental health . Among the physical health measures, RT had the largest effect on body pain .Initially, RT did not significantly affect measures of emotional role function, social function or physical role function. However, after removing a single study, RT significantly increased all HRQOL measures.Conclusion: The meta-analytic evidence presented in this research clearly supports the promotion of RT in improving HRQOL in older adults. For the aged adult, the same muscle strengthening guidelines apply, as RT may hold even greater benefit for this population. Several health problems affecting older adults can be countered by adopting a regular RT program. For example, older adults are at greater risk of premature death due to falls, which is associated with age-related declines in muscular fitness and balance.5 A recent report from the Centers for Disease Control and Prevention (CDC) states that approximately one in four older (65+ years of age) US adults fall each year and deaths from falls have increased an average of 3% annually from 2007 to 2016.6Resistance training (RT) is recommended as part of the 2008 physical activity guidelines for Americans.7 Studies have shown that RT can benefit bone mineral density,9 lipoprotein profiles,10 glycemic control,11 body composition,12 symptoms of frailty,13 metabolic syndrome risk factors,14 and cardiovascular disease markers.15 Studies have further shown that RT can decrease the risk of all-cause mortality both in observational17 and experimental18 designs. Furthermore, RT intervention has shown to effectively improve psychosocial health outcomes such as sense of coherence,19 perceived stress,20 depression,21 anxiety,22 and fatigue.23Older adults can gain other health benefits from RT, besides increased muscle mass and strength.24 HRQOL is a multidimensional construct and considers the relationship between an individual’s health status and their quality of life.25 As a recent addition to the Healthy People goals for year 2020, two objectives were issued.26 Specifically, these objectives are to increase the proportion of adults who report at least good health, with one objective specifying physical health and the other specifying mental health.Health-related quality of life (HRQOL) is another psychosocial outcome of increasing interest in health sciences research.27 Therefore, selecting an appropriate assessment is paramount to research soundness. Many different HRQOL assessments have been used in physical activity-related research, however, the Medical Outcomes Study 36-Item Short Form Survey (SF-36) and its variant (SF-12) have served as a gold-standard.28 One attractive characteristic of the SF-36 and SF-12 assessments (SF-36/12), is the many different outcome scores resulting from its administration. Specifically, ten different scores can be computed from the SF-36/12: eight dimension scores  and two summary scores .29As with any health outcome measure used in practice or in research, the use of a reliable HRQOL measure is important to the internal validity of study findings.30 this research delimited its examination to only studies using the SF-36/12 to measure HRQOL. Moreover, studies support the positive effect that RT has on HRQOL.31 However, a collective summary of the effect that RT has on a gold-standard HRQOL assessment is necessary. A collective summary through systematic review can ensure that the promotion and adoption of RT among older adults will contribute to the effort to meet our national HRQOL objectives. Therefore, the purpose of this study was to use systematic review and meta-analytic techniques to examine the effect of RT on HRQOL, assessed only using the SF-36/12, among older adults.Due to its widespread use and above standard psychometric properties,Two researchers independently engaged in all search strategy procedures. During review of the results at each stage, if discrepancies were found between the researchers, they were reviewed and discussed until an agreement was made. The search strategy steps consisted of: (1) initial search of the PubMed database using keyword search terms and review of all initial abstracts, (2) retrieval and review of all full-text articles estimated to be appropriate from the initial abstract review, and (3) agreement on the final set of full-text articles included in the study. The following terms were used in the initial PUBMED search: “(elderly OR older OR aging) (“strength training” OR “resistance training” OR “resistance exercise” OR “muscle strengthening” OR “weight training”) ”.During the search procedures stated above, inclusion criteria were used to flag abstracts and full-text articles as appropriate for the study. During the last stage of the search, included studies were excluded only if the data reported were not conducive to a standardized mean difference meta-analysis . The following inclusion criteria were used during each step of the search:Date criteria: The full-text articles must have been published within a 10-year period, beginning January 2008 and ending December 2017.Article criteria: The full-text articles had to represent first-hand research.Study design criteria: The full-text articles must have examined research outcomes from a randomized controlled pretest-posttest design. Single group studies and studies with between-group posttest only results were not included.Age criteria: The full-text articles must have examined the effect of RT of HRQOL using adults at least 50 years of age. If studies included younger and older subjects but did not stratify the analyses by age groups to focus on the 50+ age group, they were not included.Intervention criteria: The full-text articles must have examined research that used RT as a primary treatment/exposure/intervention component. RT was defined as any regimented program that worked large muscle groups by using either concentric, eccentric, or isometric muscle actions.HRQOL criteria: The research studies must have measured HRQOL using the SF-36/12 assessment.Posttest criteria: The research studies had to have measured HRQOL after the RT program. Studies that only examined long-term effects of RT on HRQOL were not included.P values, and test statistics. All data were entered into spreadsheets with the same formatting and a comparison of results was performed using the SAS PROC COMPARE procedure.32 Any discrepancies in data extraction were discussed until an agreement was made.Data were extracted from each study independently by the same two researchers. A preformatted spreadsheet was created for both researchers and included the following columns: study number, first author last name, year of publication, HRQOL form (SF-36 or SF-12), mean age of participants, minimum age of participants, gender of participants , disease status  (yes or no), length of intervention (in weeks), and whether the intervention included other components  (RT only or RT plus). Additionally, data for effect size calculations were extracted and included any number of columns such as pretest mean value and standard deviation (SD), posttest mean value and SD, mean gain score value and SD, between group mean difference in gains and SD, confidence interval limits, standard errors (SEs), 33 The effect size in this study represents the effect that RT has on HRQOL, as compared to a control. In 15 of the 16 studies, the reported pretest and posttest means and standard deviations were used to compute each effect size. The numerator of each effect size was simply the difference between the treatment mean difference and the control mean difference. The denominator of each effect size was a pooled standard deviation of the two group’s standard deviation of changes. When these standard deviations of changes were not reported, we estimated them using conventional methods.34 When pretest and posttest group standard deviations were not reported directly, we computed them from reported confidence intervals. The effect size standard errors were also calculated using the computed effect size and group sample sizes. The one unique study reported all change statistics for each group. In this case, the effect size numerator was simply the subtraction between the two reported mean differences. In this case, the denominator was the conventional pooled standard deviation of the two reported standard deviations of changes.Each of the ten HRQOL measures from the SF-36/12 assessment was considered distinct measures of HRQOL and so ten different meta-analyses were performed. Each meta-analysis was conducted using the computed standardized mean effect size (ES) and its SE.35 To describe individual study-level effect sizes and each pooled effect size, Forest plots were constructed with 95% confidence intervals (CIs).36 To further describe variability in effect sizes, the Q statistic for heterogeneity, tau-squared (τ2) representing the variance component, and I2 describing percent of heterogeneity were computed.37 Additionally, moderator analyses using random effects models were performed for four categorical factors and three continuous factors.35For this meta-analysis research, it was assumed that different populations indeed exist within the older adult population  and therefore RT would have varying effect on HRQOL across these different populations. With this assumption in mind, random effects models were pre-planned and performed on all meta-analyses.38 Second, a trim-and-fill procedure was performed to estimate the number of effect sizes needed to reproduce a symmetric funnel plot.39 An estimated mean effect size was produced as part of the trim-and-fill analysis and represents the change in pooled effect size with imputed study effect sizes required to balance each funnel plot. Third, a leave-one-out analysis was performed which estimates new pooled effect size estimates with each study deleted.40 Finally, the experimental design quality of each study included in this research was evaluated using the Physiotherapy Evidence Database (PEDro) scale.41 SAS version 9.4 ,32 R version 3.5 ,42 and STATA version 14  43 were used for all analyses. Significance was set to P < 0.05. Due to a relatively small sample size for some meta-analyses, suggestive evidence was set at P < 0.10 for the moderator analysis. Strength criteria for the standardized mean difference effect sizes were set as follows: 0.20 , 0.50 (medium), 0.80 (large).44Three procedures were employed as part of a sensitivity analysis. First, Egger’s regression was performed to test funnel plot asymmetry.60 A total of 77 effect sizes were computed from all studies with specific HRQOL measures ranging from 6 (ERF and PRF) to 12 (PF) effect sizes. Figures Q statistics were significant (P < 0.01), indicating heterogeneity in effect sizes. Additionally, I2 values were large for all ten meta-analyses, with the smallest I2showing approximately 63% variance (inconsistency) in effect sizes due to factors other than sampling error (chance).P = 0.026) and interventions containing females only showing greater RT effect on SF . Also, noteworthy, intervention length showed a significant and positive relationship with RT effect on ERF . P=0.008) and GH . Additionally, age categorized into three different groups significantly changed effect size measures for PCS and GH, with those in the 65+ years group seeing lower RT effect on PCS  and GH .61 Furthermore, the control group in Tomas-Carus (2016) was a true control group in that they were only instructed to behave in their usual manner. With this in mind, it is unlikely to see a control group experience this amount of ERF improvement and it is more likely that some subjects were measured as having abnormally lower ERF than typical. It is also likely, that this same reasoning  explains why the treatment group in Tomas-Carus (2016) appeared to suffer such a drop in SF from pretest to posttest.The purpose of this study was to use systematic review and meta-analytic techniques to examine the effect of RT on measures of HRQOL in older adults. Additionally, this research sought to use only HRQOL measures assessed by the gold-standard SF-36/12 assessment which consisted of MCS, ERF, MH, VT, SF, PCS, BP, GH, PF, and PRF. Results from this study support RT intervention as an effective means for improving HRQOL in older adults. These results, however, are not without caveats and, therefore, should be discussed. For instance, three of the ten meta-analyses  did not significantly support RT as an efficacious means for increasing HRQOL, at the initial stages of analysis. However, results from the sensitivity analysis revealed a single study was influencing the non-significant pooled mean effects. Specifically, if Tomas-Carus (2016) is removed from the meta-analyses, it is seen that RT has a significant effect on both ERF and SF. This inconsistency is clarified after further inspection into the Tomas-Carus (2016) data. Specifically, Tomas-Carus (2016) reported an unusually low pretest ERF mean value for the control group of 79.5, whereas the treatment group pretest ERF mean was 93.8. Posttest ERF means for control and treatment groups were 92.3 and 92.5, respectively. Conversely, Tomas-Carus (2016) reported an unusually high pretest SF mean value for the treatment group of 89.1, whereas the control group pretest mean was 82.7 for SF. Posttest SF means for treatment and control groups were 82.0 and 82.7, respectively. When examining the Tomas-Carus (2016) data this way, it becomes clearer that a case of statistical regression toward the mean possibly influenced these study findings.τ2).62 Tau-squared is a measure of between-study variance and adding its component to the inverse variance weights of the standard error computation is the driving mechanism behind random effects models.64 Therefore, the value of tau-squared was considerably large with Teixeira (2010) in the PRF meta-analysis. More specifically, the Teixeira (2010) effect size contributing to the PRF meta-analysis was 2.28 (CI: 1.74-2.83), which yielded a tau-squared of 0.65  is removed from the meta-analyses, it is seen that RT has a significant effect on PRF. At first glance, the pooled mean effect becoming significant after removing the Teixeira (2010) seems counterintuitive see . It woul0.65 see . With su65 This meta-analysis included studies that used a different HRQOL assessment (Minnesota Living with Heart Failure Questionnaire) with a lower bound mean age of 48 years. The findings from this meta-analysis supported RT as a strong positive factor in increasing HRQOL. Another meta-analysis examined the effect of RT on HRQOL among participants with chronic kidney disease (CKD).66 In this research, measures of HRQOL were extracted from studies that used the PF and PCS of the SF-36 assessment with a lower bound mean age of 43 years. Results from this meta-analysis also supported RT as an effective intervention in improving HRQOL in participants. A final study worth noting is a meta-analysis that examined the effect of RT on HRQOL in cancer patients.67 This meta-analysis included studies that used two different disease-specific HRQOL assessments, the Functional Assessment of Cancer Therapy (FACT) self-report questionnaire and the Cancer Rehabilitation Evaluation System Short Form (CARES-SF). Only six studies were included in this meta-analysis, with a lower bound mean age of 49 years. However, a small RT effect (ES=-0.17 in favor of intervention) on HRQOL was still seen. Given the results from these supporting studies and results from the current meta-analyses, RT clearly is an effective intervention for increasing HRQOL in older adults.With the above caveats explained, it can then be concluded that RT has a robust effect on HRQOL in older adults. This conclusion is supported by findings from similar meta-analyses. A recent meta-analysis examined the effect of RT on HRQOL among participants with chronic heart failure (CHF).30 Two, the SF-36/12 assessment has a unique attribute in that it allows for ten different HRQOL scores.29 This attribute of the SF-36/12 assessment permits a greater and more valid coverage of the various health-related dimensions that ultimately affect the quality of life of older adults.The major strength of this study was its use of the SF-36/12 assessment as inclusion criteria during the systematic review. This inclusion criteria gave strength to this research for two reasons. One, the SF-36/12 assessment, as previously mentioned, is a gold standard HRQOL assessment in physical activity research, providing both valid and reliable measures.68 Publication bias exists in a meta-analysis if studies with negative (null) findings have been systematically omitted from the data extraction process. However, this phenomenon is more likely to occur in industries such as pharmaceutical manufacturing, where organizations have a stake in the research results.69 In physical activity research, a null finding is more likely considered a valuable addition to the literature. For example, of the 77 effects extracted from the 16 studies in this research, 49 were non-significant – which arguably is evidence against publication bias. Additionally, bias was addressed in this research during the sensitivity analysis, where little bias was found. Second, this study is possibly limited due to search bias. That is, a bias introduced using a limited search strategy. Although this limitation is important to consider, this study took measures to prevent search bias. Specifically, the systematic review procedures included a search of the PubMed database as well as included a large set of keyword terms to ensure a sensitive search. Third, this study is possibly limited due to selection bias, which is related to bias in the way flagged abstracts and articles were included into the meta-analysis. This limitation is important to consider. However, this study utilized two independent researchers on all stages of the systematic review and data extraction procedures, to limit this potential bias. Finally, the use of a single database  may have limited this research and decreased the quality of the search strategy by systematically missing relevant research articles. However, PubMed, a web-based portal of MEDLINE developed by the United States Department of Health and Human Services, has been shown to be more effective with comprehensive medical-related reviews than other similar databases.70This study does have limitations worth noting. First, this study is possibly limited due to the phenomenon of publication bias.The meta-analytic evidence presented in this research clearly supports RT as an effective means for improving HRQOL in older adults. The array of specific HRQOL dimensions that RT may improve span both mental  and physical  HRQOL domains. RT may, however, be particularly effective at improving MH and BP in older adults. RT should be a priority intervention for improving HRQOL in older adults and helping to meet our national HRQOL goals.This study used already published data from journal articles. Therefore, institutional review board approval was not required.The authors declare that they have no competing interests.PDH and DJB designed, managed, and analyzed this study as well as wrote this paper.No funding was provided for this research.The authors would like to thank Dillon Barnes for helping with the beginning stages of this research.


Since their discovery in 1993, numerous microRNAs (miRNAs) have been identified in humans and other eukaryotic organisms, and their role as key regulators of gene expression is still being elucidated. It is now known that miRNAs not only play a central role in the processes that ensure normal development and physiology, but they are often dysregulated in various diseases. In this review, we present an overview of the role of miRNAs in normal renal development and physiology, in maladaptive renal repair after injury, and in the pathogenesis of renal parenchymal diseases. In addition, we describe methods used for their detection and their potential as therapeutic targets. Continued research on renal miRNAs will undoubtedly improve our understanding of diseases affecting the kidneys and may also lead to new therapeutic agents. Ribonucleic Acids (RNAs) are a ubiquitous class of unbranched polymeric molecules that serve as intermediates responsible for decoding genetic information from DNA to ribosomes in the form of messenger RNA (mRNAs), transfer of amino acids to ribosomes by transfer RNA (tRNA), and in protein synthesis itself in the form of ribosomal RNA (rRNA). In 1993, a new class of non-coding RNA molecule, microRNAs (miRNAs), was identified. These small molecules play pivotal roles in cell-growth cycle regulation, differentiation, and survival by modulating mRNA stability and translational efficiency. Over the last two decades, the role of miRNAs in various diseases, as well as their role in maladaptive repair, has been elucidated. In addition, miRNAs have been studied for their potential use in disease diagnosis and prognostication, and as therapeutic targets.In this review, we describe the role of miRNAs in renal physiology and pathology and their putative roles in various renal parenchymal diseases. We also discuss methods of their measurement as well as various strategies for using miRNAs as therapeutic agents.To identify pertinent studies for this review, we searched Medline for phrases “MicroRNAs” [Mesh] OR “MIRN632 microRNA, human” [Supplementary Concept] AND “Kidney” [Mesh]. We limited our search to papers published in the English language journals from January 2007 to June 2018. We also identified relevant studies from the previously published review papers in this field.Before 1993, it was thought that mRNAs, transcribed from the coding regions of DNA, are translated by ribosomes into proteins with no other non-coding regions of DNA playing a significant role in the processes of protein synthesis or regulation of gene expression. The processes that lead to miRNA biogenesis have been extensively described and we refer readers to comprehensive reviews for details ,3,4. At In mammals, renal development is heralded by aggregation of cells in the mesoderm called the urogenital ridge—a portion of which forms the nephrogenic cord. The nephrogenic cord undergoes changes to form the pronephros which regresses, mesonephros which forms the mesonephric duct, and the metanephros which develops as an outgrowth of cells from the mesonephric duct called the ureteric bud. Nephron progenitor cells surround the ureteric bud and form nephrons. The ureteric bud forms the collecting system of the kidneys. These processes involve a series of well-coordinated steps which require expression and suppression of various proteins that have been shown to be regulated by miRNAs.To better understand the role played by miRNAs in the developing kidney, researchers conditionally deleted Dicer in specific cell populations in the developing kidney. A mouse conditional deletion of Dicer in the nephron progenitor cells resulted in increased apoptosis with a marked reduction of cells and increased expression of pro-apoptotic Bcl-2-Like Protein 11 (BIM) protein in the embryonic kidney . NephronAlthough these observations highlight the relevance of miRNA regulation in renal development, the identity of the miRNAs and cell populations involved remains unclear. The first question was addressed by targeted deletion of the mmu-miR-17~92 cluster, which has essential roles in development and has been implicated in cancer. Deletion of this family of miRNAs resulted in the preservation of the nephron progenitor population but impaired their proliferation and thus reduced nephron number. Mice lacking the miR-17~92 cluster developed albuminuria by 6 weeks, and focal podocyte effacement and glomerulosclerosis by 3 months . In the Ren1 and Ren2 genes, leading to decreased renin concentration, hypotension, abnormal renal function, renal vascular abnormalities and strip fibrosis [+/K+ ATPase β-1 subunit [miRNAs play a diverse role in normal renal function, as demonstrated by the elimination of specific miRNAs and/or miRNA-processing enzymes in mouse models. For example, conditional deletion of Dicer in renin-expressing cells in mouse kidneys resulted in a reduced juxtaglomerular cell population, and decreased expression of fibrosis . Conditifibrosis . A major subunit . These a subunit . This faThis system is of emerging importance for understanding the development of systemic, volume-sensitive hypertension. Control of the system of miRNAs exemplifies the integration between FF kinase and epigenetic regulatory loops and thus will be examined at some length here . In the Renal fibrosis is the final common pathway of various forms of progressive renal disease. TGF-β signaling plays a central role in renal fibrosis. Renal parenchymal cells synthesize TGF-β1 and its isoforms (β2 and β3). Experimental models and human studies have shown that TGF-β1 is upregulated in diseased and fibrotic kidneys . VariousmiRNAs regulate TGF-β activity by modulating expression of various components of the TGF-β signaling pathway. In particular, hsa-miR-744-5p has been shown to post-transcriptionally inhibit expression of the TGF-β1 ligand . SimilarConversely, TGF-β signaling also influences miRNA expression. TGF-β administration increases expression of miR-192-5p in human, mouse, and rat tubular epithelial, mouse mesangial, and rat proximal tubular epithelial cells, respectively ,28,29,30The final common pathway of TGF-β signaling is the production of extracellular matrix (ECM) proteins and their deposition into the interstitium. Several lines of evidence demonstrate that this process is under miRNA control. In systemic sclerosis—a disease characterized by widespread fibrosis—expression of the mmu-miR-29 family is decreased . mmu-miRmiRNA expression profiles have been studied in many renal parenchymal diseases. Specific miRNA expression signatures have been identified for some diseases in both animal models and human studies. We will briefly review some of these associations since they provide the basis for detecting miRNAs as disease-specific biomarkers and potential therapeutic targets.miRNAs have been directly implicated in the pathogenesis of diabetic nephropathy. mmu-miR-29c-5p expression—which is associated with podocyte apoptosis—is increased in both the glomeruli and microvascular endothelial cells in a mouse diabetic model . In addiThis association may be causal since the introduction of TGF-β to proximal convoluted tubule cells exposed to high glucose conditions leads to decreased hsa-miR-192-5p expression . ConversSeveral miRNAs may be involved in the expression of the fibrotic renal phenotype. TGF-β increased mmu-miR-216a-5p and collagen type I α1 expression in mouse mesangial models of diabetes . A miRNAHypertension is a major risk factor for developing coronary artery disease, congestive heart failure, sudden death , left veOxidative stress due to inhibition of nitric oxide (NO) production and generation of reactive oxygen species could be the final common pathway for hypertension development . ProductmiRNAs may also play a role in the development of hypertension, by their effects on vascular smooth muscle cells (VSMCs). Aberrant division of VSMCs leads to vascular luminal hypertrophy and luminal narrowing which causes and propagates hypertension. hsa-miR-143-5p and hsa-miR-145-5p ensure proper development and regulation of VSMCs . VSMCs dActivation of renin-angiotensin-aldosterone systems (RAS) plays a cardinal role in pathophysiology and maintenance of different forms of hypertension. Activation of the angiotensin 1 receptor (AT1R) by Angiotensin II (Ang II) increases blood pressure by vascular smooth muscle cell proliferation, vascular constriction, cardiac remodeling, aldosterone production, and sodium retention, which plays a central role in the pathogenesis of hypertension . These aRen1 (Renin) mRNA and lower miR-181a indicating SNS mediated release of renin [Ren1 mRNA. The authors postulated that mmu-miR-181a-5p suppression potentiates sympathetic nervous system-mediated increase in renin production in BPH/2J mice during the active periods [Ren1 mRNA [Sympathetic nervous system overactivity is one of the mechanisms for development and maintenance of hypertension. The role of the sympathetic nervous system (SNS) and RAS in the maintenance of hypertension was studied in mice which were genetically prone to develop hypertension (BPH/2J mice) . Ganglioof renin . These f periods . These fen1 mRNA .BN rats are genetically modified Dahl-SS rats that have less pronounced blood pressure rise and medullary interstitial fibrosis when exposed to a high salt diet [BN rats but not in Dahl-SS rats [BN rats—a pattern opposite of miR-29b expression. Furthermore, a miR-29b knockdown Consomic SS 13BN rat model had upregulation of various collagen genes, suggesting that miR-29b expression protects rats from hypertension-associated renal injury [Various animal models have been developed to study the effects of hypertension on kidneys. Dahl salt-sensitive (Dahl-SS) rats develop hypertension with medullary interstitial fibrosis when exposed to a high salt diet. Consomic SS-13alt diet . Liu et -SS rats . Variousl injury .Kidney biopsies in patients with hypertension reveal glomerulosclerosis, tubular atrophy, interstitial fibrosis, and vascular smooth muscle cell hypertrophy. In a recent study, intrarenal miRNA expression profiles of 30 patients with hypertensive nephrosclerosis were compared to 20 normal controls. hsa-miR-200a-5p, hsa-miR-200b-5p, hsa-miR-141-5p, hsa-miR-429-5p, hsa-miR-205-5p, and hsa-miR-192-5p were significantly increased in patients with hypertensive nephrosclerosis . Table 5Glomerulonephritides (inflammation of glomeruli) are a group of diverse disorders that may present as proteinuria and or/hematuria with renal dysfunction. Kidney biopsy findings include podocyte injury, mesangial and endocapillary proliferation, and disruption of basement membranes leading to focal and segmental glomerulosclerosis, tubular atrophy, and interstitial fibrosis. We will now discuss the role of miRNAs in some of the conditions that can cause glomerulonephritis.Focal segmental glomerulosclerosis (FSGS) is a pattern seen on kidney biopsy characterized by involvement of some of the glomeruli, with part of the involved glomerulus showing obliteration of the capillary lumen and increase in mesangial matrix. FSGS is usually caused by infections, medications, and conditions that cause chronic renal injury. At times, no cause of FSGS is found, and this is labeled as primary FSGS. A molecule that increases the permeability of glomerular basement membranes has been postulated in the pathogenesis of primary FSGS ,65 but tIn a puromycin-induced FSGS rat model, researchers found diminished rno-miR-30s-5p . ReplaceRecently, the role of Brain Derived Neurotrophic Factor (BDNF) in maintaining the integrity of podocyte layer was studied . Mice pomiRNAs have also been used as biomarkers—both in serum and urine—to assess FSGS disease activity. In one study, researchers found elevated plasma hsa-miR-125b-5p, hsa-miR-186-50 and hsa-miR-193a-3p in patients with FSGS with area under curve (AUC) of 0.88, 0.78, and 0.91, respectively . PatientIgA nephropathy is the most prevalent primary glomerulonephritis in the world. Abnormal O-galactosylation of IgA causes the formation of IgA complexes against which IgG and IgA are formed with deposition in the kidneys and activation of the complement system leading to kidney injury.Genome-wide analysis has revealed various miRNAs which might play a role in IgA nephropathy. miRNA expression of 6 patients, each with biopsy-proven IgA nephropathy, compared to those with renal cell carcinoma, revealed upregulation of 11 miRNAs and downregulation of 74 miRNAs in the IgA nephropathy group . MembersN-acetylgalactosaminyltransferase 2 (GALNT2) and 1 β 1,3 galactosyltransferase 1 (C1GALT1), respectively—these enzymes play a central role in aberrant IgA galactosylation. It has been shown that these enzymes are overexpressed in the peripheral blood mononuclear cells of IgA nephropathy patients [miRNA hsa-let-7a-5p and hsa-miR-148b-5p control patients ,73.miRNA expression profiles in paraffin embedded kidney biopsy specimens and urine from patients with IgA nephropathy was studied . Both thSystemic lupus erythematosus (SLE) is a systemic disease due to dysregulated immune system activity. Kidney involvement in SLE often leads to chronic kidney disease and eventually kidney failure if left untreated, and it is the major cause of morbidity and mortality. Genetic factors have been implicated in SLE pathogenesis, but the underlying control mechanisms remain poorly defined.Various lines of evidence point towards the role of miRNAs in SLE. miRNAs regulate 72 genes labeled as “autoimmune genes” that control various aspects of the immune system . hsa-miRThe role of miRNAs in the pathogenesis of proliferative lupus nephritis has been a subject of intense research. A high-throughput analysis of paraffin embedded kidney biopsies of pediatric patients showed differential expression of various miRNAs . hsa-miRANCA vasculitis is a small vessel vasculitis involving the kidneys as well as other organs and is characterized by the presence of either anti-Proteinase 3 (PR3) or anti-Myeloperoxidase (MPO) (components of neutrophils) antibodies. Currently, it is not known whether these antibodies are pathogenic and what are the inciting factors for production of these antibodies.Pooled plasma samples from 40 patients who had active ANCA vasculitis or were in remission showed up-regulation of hsa-let-7f-5p and hsa-miR-424-5p, and downregulation of hsa-miR-106b, hsa-miR-9-5p, hsa-miR-125a-50, and hsa-miR-15b-5p . These mhsa-miR-155-5p is upregulated in patients with ANCA-associated crescentic GN . NephrotSystemic sclerosis is a condition associated with multiple organ fibrosis. It affects the kidney by causing thickening of the blood vessels, leading to hypertension, endothelial injury, and thrombotic microangiopathy. It has been shown that TGF-β  and miR-ADPKD is a disease characterized by impaired ciliary function leading to kidney and liver cyst formation and kidney failure in the vast majority of patients.In the Sprague-Dawley rat model of ADPKD, 29 miRNAs were downregulated and only 1 miRNA (miR-21) was upregulated. Most of the dysregulated miRNAs control cell-to-cell interaction and crosstalk . Global −/− mice, mmu-miR-21-5p is expressed equally in both compartments [−/− mice resulted in the preservation of renal function, reduction in albuminuria, improved survival, reduced glomerulosclerosis, crescent formation, and tubular injury [Alport syndrome is due to abnormalities in genes encoding α3, α4 or α5 chains of collagen Type IV, resulting in abnormal basement membranes in the kidney, eyes, and inner ear. These changes in the kidney lead to abnormalities of glomerular basement membrane and progressive renal disease. mmu-miR-21-5p is preferentially expressed in the tubulointerstitium instead of glomeruli in normal mice; however, in the Col4 α3artments . As descr injury . Currentr injury .The preceding section highlights the role of specific miRNAs in normal renal development and physiology, but also the initiation and the progression of the interstitial fibrosis that underlies progressive forms of chronic kidney disease. It follows, that miRNAs detected in either plasma or urine, the two fluidic compartments directly affected by renal processing, may be mechanistically plausible, rational biomarkers for diverse forms of kidney diseases. In fact, miRNA associations found in observational human studies may offer a unique opportunity to “reverse translate” such findings into animal studies, which provide mechanistic insights into novel therapeutics that are tested in rigorous interventional clinical trials in humans. .Nevertheless, detection of miRNAs poses unique challenges because of their short size and the similarity of many sequences to one another. These biochemical features of miRNAs may directly impact the performance of the three methods most commonly used for miRNA detection: quantitative real-time PCR (qPCR), microarrays, and next-generation sequencing (NGS). Each of these approaches comes with its distinct advantages, but also limitations when used as the basis for the development of miRNA biomarker assays.Of the methods listed above, qPCR has the highest sensitivity, with a theoretical limit of detection of just a few copies per sample . SeveralMicroarray-based methods allow for the simultaneous measurement of many miRNAs, making them a better choice than qPCR for profiling a large set of targets. Commercial products are available covering all the mature miRNA sequences in miRBase on a single array. However, the amount of starting material required for microarray analysis is relatively high (~100ng per sample) and it remains difficult to design probes and hybridization conditions that can distinguish between closely related miRNA sequences. In addition, the dynamic range of microarrays is typically lower than either qPCR or NGS.Unlike qPCR and microarrays, NGS requires no prior knowledge of the target sequences in the sample, and so is ideal for discovery studies. In addition, NGS is not affected by the complications of designing primers or probes with the specificity needed to distinguish between short sequences with high sequence identity. Consequently, as costs for NGS library preparation and sequencing have dropped, small RNA sequencing (sRNA-seq) has become widely used for miRNA profiling because of its ability to comprehensively interrogate the miRNAs  in a sample. As opposed to qPCR and microarrays, sRNA-seq allows the analysis of miRNAs with single nucleotide resolution, so not only can the canonical sequences be studied, but also variants arising from RNA editing or imprecise miRNA processing (isomiRs). sRNA-seq is not without drawbacks, however. Compared to qPCR and even microarray analysis, NGS is more expensive and more time intensive, both in sample preparation and data analysis. Most methods for preparing sRNA-seq libraries involve sequential ligation of adapters to the 3′ and 5′ ends of the miRNAs, followed by reverse transcription and PCR amplification to add indexes and other sequences needed for attachment to the flow cell and sequencing. The adapter ligation steps have previously been shown to introduce bias into small RNA libraries, which leads to decreased library diversity and prevents making quantitative comparisons of the expression level of different miRNAs in a sample ,103,104.Several important issues affect all methods commonly used for measuring miRNAs. For example, whereas several housekeeping genes have been widely used for normalization in mRNA expression profiling studies, there is less consensus on similar invariant transcripts suitable for miRNA expression analysis. Other options include normalization based on sample input  or based on the addition of spike-in oligonucleotides added during sample processing. Normalization based on relative read counts  is also frequently used to report sRNA-seq data, but this method can yield inaccurate results if samples have different proportions of small RNA species, either because of biological differences or technical differences such as variations in the size selection step of sRNA-seq library preparation.Another issue affecting miRNA measurement is the lack of correspondence between data generated by different methods. It is not an uncommon practice to identify changes in miRNA expression using high-throughput methods such as microarrays or NGS and subsequently validate those changes by qPCR. However, it has been known for some time that changes in miRNA expression detected by one method are often difficult to corroborate across methods . High amMeasurement of extracellular miRNAs is further complicated by low concentration and inhibition by other macromolecules in the sample. Although there is great interest in profiling RNAs present in biofluids such as blood and urine to identify disease biomarkers, obtaining accurate and reproducible results from these sources remains challenging. The primary reason that measuring miRNA in biofluids is difficult is that the RNA concentration is much lower than in cells or tissues. Typical RNA isolation methods recover only about 10–50 ng of total RNA per milliliter of cell-free plasma, with even lower yields from fluids such as urine and saliva. In addition, the presence of inhibitors derived either from the sample itself or during the collection procedure and co-purified with the RNA, can be problematic for the enzymatic steps in library preparation. Salts present in urine samples and heparin used as an anticoagulant during plasma collection are two examples of inhibitors commonly encountered with biofluid samples. Furthermore, the handling and storage methods of the samples before RNA extraction can have a significant impact on the results. Lysis of cells during collection  or incomplete removal of intact cells before RNA isolation can also significantly distort the miRNA profile from biofluids since cellular RNA content is much higher than that of the cell-free fluid. Thus, sample collection, RNA isolation, and library protocols are all critical for accurate profiling.miRNAs have a unique role among biological regulatory molecules, participating in feed-forward regulatory loops ,114, whoIn the previous sections, we discussed the roles of miRNAs in normal kidney function as well as kidney diseases associated with miRNA dysregulation. There is significant interest in the use of miRNAs as therapeutic agents since they modulate the activity of numerous genes. miRNA-based therapeutics are based on either inhibiting a deleterious miRNA or replacing a deficient beneficial miRNA. miRNA antagonists—also called antagomirs or antimiRs—are single-stranded molecules that are designed to bind directly to a mature miRNA and to block its action. Deficient miRNAs can be replaced by either making small interfering miRNAs (siRNAs), which are small double-stranded RNA molecules encapsulated in nanoparticles and delivered to the target site  or by viDesigning an effective miRNA mimic and delivering it to its intended target organ without degradation or causing unintended effects has been the subject of intense research. Designing miRNA mimics that effectively block their targets without affecting any unintended transcripts has proven problematic . TherefoThe process of delivering a miRNA molecule to its intended target is fraught with difficulties. Naked miRNAs are unstable in the blood and are rapidly degraded by the mononuclear macrophage system and removed from circulation by the kidneys and liver. Therefore, effective delivery methods that prevent miRNA degradation must be devised. These delivery vehicles should be non-toxic, have low immunogenicity, and should be able to deliver a large proportion of the miRNAs to their intended target . Some ofVarious phase I and phase II trials are underway or have been completed for miRNA-based therapeutic agents for the management of chronic hepatitis C, diabetes mellitus type 2 with fatty liver and cancers . miRNAs Regulus Therapeutics in collaboration with Genzyme has developed a single-stranded molecule RG-012 that inhibits miR-21. In a rat model of Alport syndrome, miR-21 inhibition by this molecule led to milder kidney disease and improved survival than control mice. There was less glomerulosclerosis and tubulointerstitial fibrosis in the treated mice with no adverse events . A phaseIt has been shown in practice over the past decade that extracellular miRNAs can provide informative biomarkers for multiple biological effects and pathologies. The value of understanding miRNA function, however, is much broader. In concert with the multiple factors regulating transcription, miRNAs provide an additional level of control of gene expression, largely at the post-transcriptional level. Their influence on various biological pathways is both widespread and complex and is often subtle. In the last 20 years, tremendous progress has been made in understanding their roles in renal physiology and pathology, and this is beginning to open several new lines of investigation. Research is currently underway to study and modulate miRNAs specifically to control maladaptive repair that leads to fibrosis in various renal diseases. miRNAs also provide us a novel opportunity to develop new ways of studying disease activity and to assess the efficacy of therapeutic agents. Since miRNAs can be targeted directly, although this is sometimes difficult in practice, they provide the opportunity to develop a new class of therapeutic agents. miRNA-based diagnostics and therapeutics, therefore, have the potential to lead medicine into a new era of effectiveness.


RV144 is to date the only HIV vaccine trial to demonstrate efficacy, albeit rapidly waning over time. The HVTN 702 trial is currently evaluating in South Africa a similar vaccine formulation to that of RV144 for subtype C HIV with additional boosters (pox-protein regimen). Using a detailed stochastic individual-based network model of disease transmission calibrated to the HIV epidemic, we investigate population-level impact and maximum cost of an HIV vaccine to remain cost-effective.Consistent with the original pox-protein regimen, we model a primary series of five vaccinations meeting the goal of 50% cumulative efficacy 24 months after the first dose and include two-yearly boosters that maintain durable efficacy over 10 years. We simulate vaccination programs in South Africa starting in 2027 under various vaccine targeting and HIV treatment and prevention assumptions.Our analysis shows that this partially effective vaccine could prevent, at catch-up vaccination with 60% coverage, up to 941,000 (15.6%) new infections between 2027 and 2047 assuming current trends of antiretroviral treatment. An impact of up to 697,000 (11.5%) infections prevented could be achieved by targeting age cohorts of highest incidence. Economic evaluation indicates that, if treatment scale-up was achieved, vaccination could be cost-effective at a total cost of less than $385 and $62 per 10-year series .While a partially effective, rapidly waning vaccine could help to prevent HIV infections, it will not eliminate HIV as a public health priority in sub-Saharan Africa. Vaccination is expected to be most effective under targeted delivery to age groups of highest HIV incidence. Awaiting results of trial, the introduction of vaccination should go in parallel with continued innovation in HIV prevention, including studies to determine the costs of delivery and feasibility and further research into products with greater efficacy and durability. HVTN 702 is a phase 2b/3 trial , supportThe ongoing HVTN 702 study is a multi-site, randomized, double-blinded, placebo-controlled clinical trial designed to test a regimen adapted to HIV Clade C exposed populations in South Africa. In addition to the six-month series tested in RV144, its original protocol comprises a booster dose at month 12, as well as a change in adjuvant  to the gp120 protein component of the vaccine, and a recently amended 18-month booster dose. Preliminary results of HVTN 100 [If this or an improved pox-protein HIV vaccine regimen is proven to be sufficiently efficacious and licensable, timely and efficient scale-up will require an evidence-based vaccine access plan that defines expectations and outlines commitments essential to making a licensed vaccine available to priority populations in South Africa and potentially beyond. To inform the development of an access plan, the P5 Global Access Committee , Sanofi Pasteur, GlaxoSmithKline and the South African Medical Research Council) has engaged in a variety of preparatory analyses -including commissioning the modelling work detailed in this article- to identify the populations that would benefit most from the vaccine. Here, target populations for vaccination were solely defined based on age and sex, although other key populations  could have similar benefits from vaccination should the product prove efficacious.In South Africa, HIV incidence varies considerably by age and sex. The highest HIV incidence rates are observed in young women aged 15–24. In 2012, the estimated incidence in this age group was 2.54% per year (95% 2.04–3.04%), which is five times the rate of HIV incidence in young men aged 15–24 ,12. ReceHowever, determining the optimal use of an HIV vaccine based on age patterns of incidence has several complications. First, an optimal vaccination schedule should account for any waning efficacy, recommendations for booster dose frequency, and rate of attrition from the recommended series of HIV vaccine doses. Second, a licensed vaccine may take up to a decade before being approved for widespread use in South Africa. In the interim, nationally representative HIV epidemiologic patterns are likely to change from current data, which dates back to 2012. Forecasting changes in incidence patterns is needed to predict the likely impact of implementation scenarios under consideration and design a relevant and effective scale-up strategy. Finally, the patterns of HIV transmission must be accounted for in order to fully capture the potential population-level impact of a vaccine. Effective vaccination will prevent HIV infections not only in the direct recipients, but also in their sexual partners within the contact network.Several modeling studies ,15–24 haResults from the present modelling analysis will help to inform ongoing vaccine access planning elements, including priority populations for whom the pox-protein HIV vaccine would be expected to have the greatest and/or most efficient public health impact.2.We developed an agent-based model of the South African population to forecast HIV infections, disability-adjusted life-years (DALYs), and healthcare costs from a government payer perspective over a 30-year time horizon, from year 2018 to 2047. As compared to a reference case with no HIV vaccine, we evaluate implementation of strategies for initiation of HIV vaccination.We modified EMOD-HIV v2.5, an age-stratified and individual-based network model of HIV of South Africa, to incorporate HIV vaccination according to pox-protein HIV vaccine regimens (such as the regimen currently being tested in HVTN 702). Because EMOD is an individual-based model, interventions such as a time-varying course of vaccine efficacy can be applied to each individual according to his or her own timing of vaccination and adherence to the booster series. This renders the model well suited for a nuanced analysis of the anticipated time-dependent efficacy of the pox-protein HIV vaccine regimen.To ensure that our analysis reflected the realities of the HIV epidemic and the health system in South Africa, we iteratively engaged South African government, academic and community stakeholders through one-on-one interviews, a vaccine access planning summit and a public health impact modelling workshop during which preliminary results of this work were presented and discussed. This stakeholder engagement process helped us understand perspectives on the future HIV prevention landscape, benefits of and challenges to reaching specific target populations and the economic factors that will influence vaccine access, all of which were incorporated into our analysis and considerations.http://idmod.org/software.The parameters, model input values, sources, projections, and sensitivities of the epidemic projection without vaccine, used as the reference group for comparison, have been described previously –27. A deFor detailed information on the model set-up, calibration and baseline assumptions on HIV treatment and prevention other than vaccine we refer to the ai is the efficacy increase of immunization with ALVAC-HIVC, bi is efficacy increase after ALVAC-HIV-C+gp120 immunization, ω is the efficacy decay rate per month and d is the delay between immunization and initiation of protective effect in months.We incorporated a parametric model of time-dependent vaccine efficacy that was hypothesized for the pox-protein regimen based on results from RV144. We included the time series of efficacy associated with each dose administered during the study and possible booster doses beyond the 24-month duration of the first stage of the study. The original pox-protein dosing schedule administered a series of five immunizations over 12 months (the 18-month dose recently amended to the protocol was not modeled here). ALVAC-HIV-C was administered at months 0 and 1, followed by ALVAC-HIV-C+gp120 at months 3 and 6, and ALVAC-HIV-C+gp120 dose was administered at month 12 (supplementary to RV144 schedule). Time-dependent vaccine efficacy was interpreted as a per exposure reduction in the probability of acquisition parameterized by an impulse and exponential decay model.ai = 0:08; bi = 0:34; ω = 0:065 and d = 0:1.Assuming uniformly distributed exposure over a given time span in the trial, we calculated the cumulative vaccine efficacy  as the area under the curve of the instantaneous vaccine efficacy rescaled by the length of the time span.l models  for RV14For the purpose of model projections beyond the primary trial endpoint, we also implemented up to four two-yearly boosters starting at month 36 with fixed attrition rates of 0, 20 or 50% per booster to cover a total of 10 years of vaccine efficacy. We assumed booster efficacy to follow the same parameterization as ALVAC-HIV-C + gp120 doses from the primary immunization series during the first 12 months. Booster eligibility depended on having received the primary immunization series or the booster previously. Missing a booster resulted in loss of eligibility for subsequent boosters. Individuals who tested HIV positive were not eligible for future boosters, and we did not add HIV testing to the cost. We assumed that four booster doses after the primary first-year series were necessary to confer one decade of protection. For catch-up vaccination scenarios, booster eligibility was limited by the age range of the vaccinee.Based on consultations with the P5 GAC, we evaluated 30 implementation strategies for HIV vaccine targeting and implementation. Strategies included a wide range of age- and gender-specific targeting scenarios at low (30%), medium (50%) and high (80%) coverage using two distinct roll-out approaches starting in 2027, the earliest realistic date of introduction: Cohort vaccination, aiming for immunization and follow-up boosting of a pre-specified proportion (coverage) of individuals at a particular target age every year over the period between 2027 and 2047 , and catTo account for uncertainty in predicting the next decade of the HIV epidemic, we varied the scale-up of treatment in terms of ART and oral PrEP coverage and considered three scenarios. In the most pessimistic scenario (Status Quo without PrEP), we stipulated that guideline changes have no impact on ART initiation. We excluded any use of oral PrEP. These assumptions were consistent with the 60% ART coverage under current guidelines assumed in the HIV/TB Investment Case Report for South Africa  and reflEpidemic impact was estimated in terms of the number of HIV infections prevented over the period between 2027 and 2047, calculated as differences between the total number of infections given a vaccination strategy and the reference with no HIV vaccine, over the 20-year time period. The fraction of cumulative infections prevented was calculated as the ratio of new infections averted to the number of new infections accumulated between 2027 and 2047 in the reference case (covering thus the period of vaccination). For each implementation policy strategy evaluated, we sampled with replacement from 50 simulation results  per infection prevented, defined as the ratio of the average number of vaccine regimens distributed to the average number of new infections prevented. The number of vaccine regimens distributed was defined as the number of primary series completed. Health impact was summarized in disability-adjusted life years (DALYs), calculated as the present discounted value of years of healthy life lost to disability and years of life lost to premature death over a 20-year horizon of vaccination . DisabilThe launch price and implementation cost of an HIV vaccine in South Africa are unknown. Following a value-based pricing framework for medicines –40, a thBased on strategy-dependent changes in total discounted health-care costs and DALYs, as compared to the reference with no vaccine, we estimated the maximum vaccination cost level for a vaccination strategy to be cost-effective. This cost level included both the ALVAC and the protein component, as well as implementation cost . Since we assumed complete primary series, wasted doses could potentially originate only from missed two-yearly boosters. South Africa does not have an established cost-effectiveness threshold per DALY-averted to determine value for money, although some recent estimates are now available ,41,42. TFor a given nominal vaccinecost and vaccination strategy, we calculated the net DALY burden relative to the no vaccine reference caseat a given cost-effectiveness threshold (CET):3.3.1.Varying assumptions about linkage to and drop-out from ART and uptake of oral PrEP between 2016 and the presumed start of vaccination in 2027 results in distinct future ART coverage and incidence estimates. For the scenarios ‘Status Quo without PrEP’, ‘Status Quo with PrEP’ and ‘Fast Track with PrEP’, the average percentage of HIV-diagnosed individuals on ART would be 56, 55 and 85% respectively in 2027, compared to 54% in 2016 . LikewisWithout the vaccine, model projections predict an average of 0.84%, 0.76% and 0.48% annual HIV incidence in a general population aged 15–49 by 2047 for ‘Status Quo without PrEP’, ‘Status Quo with PrEP’ and ‘Fast Track with PrEP’ respectively. This indicates that introduction of oral PrEP for high-risk groups starting in 2016 will have a smaller impact on overall HIV incidence as compared to ART scale-up.3.2.Fitting the impulse and exponential decay model to known time-dependent vaccine efficacy estimates  for the 3.3.To determine vaccination age-targeting of highest impact, we compare cohort vaccination at age 18 to age 15, with or without a 5-year age off-set between men and women  at a relA difference in vaccination age between men and women of 5 years provides additional benefit with up to 402; 000  and 642; 000  of new infections prevented for the two scale-up scenarios respectively. Missing 50% of booster would decrease the impact to 256; 000  and 434; 000  for vaccination assuming ‘Fast Track with PrEP’ and ‘Status Quo without PrEP’ respectively.Further impact can be achieved by targeting 18-year-olds resulting in 426; 000  and 604; 000  infections prevented respectively at 0% booster attrition. Echoing the age difference in the peak of HIV incidence, vaccinating 18-year-old women and 23-year-old men would increase the impact to up to 501; 000  and 697; 000  infections prevented respectively, given full booster retention.The impact of age off-setting and vaccination age is also highlighted by the number needed to vaccinate (NNV) to prevent a new infection. When vaccinating at age 15 without versus with age off-set the NNV decreases from 55:7 to 43:9 and from 34:6 to 27:0 respectively. Likewise, vaccinating 18-year-olds requires 41:0 and 28:5 NNV respectively, a five-year age off-setting would decrease these numbers to 34:2 and 24:3 NNV. To illustrate the importance of continued boosting for long-term impact, simulating the latter scenario under 50% (instead of 0%) attrition after each booster dose results in a rise to 45:4 and 32:7 NNV.Catch-up vaccination appears to be particularly impactful, i.e. by targeting age ranges between 15and 32 at the beginning of the roll-out at 60% coverage and maintaining cohort coverage of 80% thereafter, the number of new infections prevented increases from 321; 000 to 689; 000  (or 18:1% (95%CI: 17.9–18.3%)) and from 504; 000 to 941; 000  (or 15:6% (95%CI: 15.5–15.7%)) for ‘Fast Track with PrEP’ and ‘Status Quo without PrEP’ scenarios respectively . ShiftinCohort vaccination at 80% or 50% coverage would require approximately 8:4 million or 5:5 million vaccine regimens over 20 years . Catch-u3.4.For economic evaluation of vaccine cost per regimen (including delivery and implementation costs), we calculated the maximum cost at which vaccination would remain cost-effective, referred to as maximum vaccine cost in what follows. Cost-effectiveness thresholds are relative to the 2015 gross domestic product (GDP) of South Africa of 5,691 US$ per capita . Since fAmong the simulated vaccination strategies, catch-up vaccination age 18–35 is the most efficient when compared to cohort vaccination, since ART cost savings as well as averted DALYs are high, despite very similar maximum vaccine cost. Sensitivity analyses shows that maximum vaccine cost is proportional to the CET, e.g. at a threshold of approximately 750$ per DALY averted see .4.The results of this study highlight the need to exploit different vaccination targeting and roll-out scenarios to maximize the population-level impact of a partially effective HIV vaccine in South Africa. Even under high attrition rates, providing additional booster doses for up to one decade after the primary series can increase public health impact, as long as gender-specific age ranges of highest incidence are covered by vaccination. Although adolescent HIV vaccination has the potential of reaching high coverage in South Africa through school-based programs (such as Human Papilloma Virus routine vaccination ), our moThe time-dependent course of vaccine efficacy necessitates aligning vaccination such that the times of highest efficacy are within the ages of highest HIV incidence in 2027 – projected to be 20–25 years of age for women, and five to ten years older for men . SpecifiLong-term economic evaluation of vaccination suffers, in part, from the uncertainty of whether HIV treatment and prevention scale-up could be achieved between now and 2027. We derived estimates for the maximum vaccine cost  to remain cost-effective averaging-for two different treatment and prevention scale-up projections. Our analysis suggested an estimated maximum cost of 105 US$–787 US$ at which vaccination with a full 10-year regimen would be cost-effective based on a 1×GDP per capita threshold . MeasuriThere are several major limitations to this analysis. The time-dependent vaccine efficacy curves are based on limited data from RV144 and goals for the pox-protein vaccine regimen, and we did not consider any primary series attrition. Additional sources of uncertainty include differences in regimens and study populations between RV144 and the ongoing trial, statistical uncertainty in the RV144 results, and translation of cohort efficacy data to an individual-based model in the absence of HIV exposure information for the cohort under study. We did not model the impact of possible changes in voluntary medical male circumcision scale-up on vaccination, nor did we model the recently added 18-month dose in the amended trial protocol. Since available data on the distribution of earnings, non-healthcare consumption costs, and patient time costs in South Africa were insufficient to be included in this analysis, we adopted a government payer instead of a societal perspective. The economic evaluation of the vaccine does not account for indirect economic implications (e.g. productivity effects). The threshold analysis to determine the maximum cost-effective cost per vaccine regimen included product, delivery and implementation cost, which may vary across the simulated strategies, making cost comparisons difficult. Although the estimated maximum vaccine cost accounts for possible resource displacement (e.g. ART cost saving), further additional investment might be necessary to maintain treatment and prevention programs with concurrent vaccine roll-out, such that vaccine cost is likely underestimated. The lack of bounds on the vaccine price and insufficient data on other cost-drivers such as staff training, information campaigns and monitoring for such a complex regimen made it challenging to carry out a well-informed cost breakdown analysis. Furthermore, different target populations may be more or less challenging to reach. This is difficult to know a priori and was not included in the analysis, and therefore remains an area important to explore in close collaboration with implementers of HIV prevention programs. We did not model risk compensation ,51 i.e. 5.Taken together, our model suggests that, averaged across different treatment scale-up settings, vaccinating population cohorts aligned with the ages of highest HIV incidence (i.e. 18-year-old women and 23-year-old men) including continued boosting for up to 10 years could avert up to 13:1% and 11:5% of HIV infections in the coming decades (for ‘Fast Track with PrEP’ and ‘Status Quo without PrEP’ respectively). If durability of vaccine efficacy proves to be better than observed in the RV144 trial, the benefit could be appreciably larger. Adult catch-up vaccination and efforts to ensure continued boosting, could further increase the impact of the vaccination to possibly just over 18:3%  and 15:5%  of new infections prevented (for ‘Fast Track with PrEP’ and ‘Status Quo without PrEP’ respectively). However, we recognize that identifying optimal vaccine implementation platforms and deployment channels to deliver this complex vaccine regimen at high coverage will be a significant challenge–especially when vaccinating 18 year-old women instead of adolescent girls who may be more easily reached through a school-based program. Therefore, improving deployability of HIV vaccine regimen by optimizing vaccine performance characteristics  should be ultimate goal of future vaccine research. Hopefully, such optimization would be supported by the identification of immune correlates of protection in HVTN 702. Although the maximal impact of 18:3% of new infection prevented is appreciable, the roll-out of a partially effective, rapidly waning vaccine alone will not eliminate HIV as a public health priority in South Africa. Therefore, vaccination should be performed in parallel with continued innovation in HIV prevention technologies.Suppl Materials