State policy approaches designed to provide opioid treatment options have received significant attention in addressing the opioid epidemic in the United States. In particular, expanded availability of naloxone to reverse overdose, Good Samaritan laws intended to protect individuals who attempt to provide or obtain emergency services for someone experiencing an opioid overdose, and expanded coverage of medication-assisted treatment (MAT) for individuals with opioid abuse or dependence may help curtail hospital readmissions from opioids. The objective of this retrospective cohort study was to evaluate the association between the presence of state opioid treatment policies—naloxone standing orders, Good Samaritan laws, and Medicaid medication-assisted treatment (MAT) coverage—and opioid-related hospital readmissions.We used 2013–2015 hospital inpatient discharge data from 13 states from the Agency for Healthcare Research and Quality Healthcare Cost and Utilization Project. We examined the relationship between state opioid treatment policies and 90-day opioid-related readmissions after a stay involving an opioid diagnosis.Our sample included 383,334 opioid-related index hospitalizations. Patients treated in states with naloxone standing-order policies at the time of the index stay had higher adjusted odds of an opioid-related readmission than did those treated in states without such policies; however, this relationship was not present in states with Good Samaritan laws. Medicaid methadone coverage was associated with higher odds of readmission among all insurance groups except Medicaid. Medicaid MAT coverage generosity was associated with higher odds of readmission among the Medicaid group but lower odds of readmission among the Medicare and privately insured groups. More comprehensive Medicaid coverage of substance use disorder treatment and a greater number of opioid treatment programs were associated with lower odds of readmission.Differences in index hospitalization rates suggest that states with opioid treatment policies had a higher level of need for opioid-related intervention, which also may account for higher rates of readmission. More research is needed to understand how these policies can be most effective in influencing acute care use. The opioid epidemic in the United States has escalated in recent years, subsequently affecting the U.S. health care system. From 2002 to 2015, there was a 2.8-fold increase in the number of opioid-related deaths [standing orders that make naloxone directly available without an individualized provider prescription from sites such as community agencies or pharmacies [Naloxone can rapidly reverse the potentially life-threatening effects of opioids. It has low rates of adverse events and can be administered by laypersons . Some starmacies –6. As ofarmacies .Good Samaritan opioid-related legislation provides individuals who call 911 for an opioid overdose with immunity from arrest, charge, or prosecution for certain drug-related violations . As of JThe Food and Drug Administration (FDA) has approved three principal drugs as part of MAT to help individuals reduce the use of opioids—methadone, buprenorphine , and naltrexone (injectable and pill form). The American Society of Addiction Medicine (ASAM) guidelines recommend coverage of all three drugs , 9. MediNaloxone standing orders, Good Samaritan laws, and expanded MAT coverage may affect hospitalizations related to opioid-related adverse events, overdoses, and deaths, while also encouraging individuals with opioid use disorders to seek treatment , 10. HowThe objective of this retrospective cohort study was to evaluate the relationship between these three opioid treatment policies and the risk of opioid-related readmission. We used 2013–2015 hospital discharge data from 13 states with differing treatment policies to estimate the odds of a subsequent opioid-related readmission within 90 days following discharge. We hypothesized that naloxone standing orders would be associated with lower odds of readmission because they would allow individuals with an opioid overdose to obtain treatment outside of the acute care setting, thereby reducing the need for hospitalization . ConversWe used inpatient discharge data from nonfederal community hospitals in Arkansas, California, Florida, Georgia, Iowa, Maryland, Massachusetts, Nebraska, Nevada, New York, Tennessee, Vermont, and Wisconsin from the Agency for Healthcare Research and Quality (AHRQ) Healthcare Cost and Utilization Project (HCUP) . We inclWe obtained state-level data about the status and specific implementation dates of naloxone standing orders and Good Samaritan laws from The Policy Surveillance Program: A Law Atlas Project , 7. For We gathered information about presence of hospital detoxification and psychiatry units from the American Hospital Association . For eacInternational Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM) diagnosis codes that were present on admission for opioid abuse or dependence alone or in combination with other drugs  or for poisoning by opium, methadone, heroin, opiates and related narcotics, or opiate antagonists . We also included external cause of injury codes (E codes) for accidental poisoning by opium, methadone, heroin, and opiates and related narcotics (E850.0–E850.2) and adverse effects of heroin, methadone, opiates and other narcotics, and opiate antagonists . We included any-listed opioid diagnoses for our index stays to capture the potential population of individuals who could be affected by naloxone, Good Samaritan, and MAT coverage policies. We excluded index hospitalizations in which the patient died or was transferred into or out of the hospital.The study population comprised a retrospective longitudinal sample of patients aged 18 years and older with an opioid-related index hospitalization between April 2013 and June 2015 and no preceding opioid-related hospitalization within 90 days.The outcome variable was a readmission within a 90-day period with an opioid-related principal diagnosis or an opioid-related accidental poisoning or adverse effect diagnosis (E code). Consistent with other studies, we selected 90 days as the follow-up period because it would be sufficient time for patients discharged from the hospital to access potential outpatient rehabilitation services , 38. ThiThe key independent variables focused on the three state policies: (1) naloxone standing orders, (2) Good Samaritan laws, and (3) Medicaid MAT coverage and generosity.The first key independent variable indicated whether a state had a naloxone standing order that allowed pharmacies to dispense naloxone without an individual provider prescription. The second indicated whether a state had a Good Samaritan law granting immunity to users from arrest, charge, or prosecution for possession of drugs or drug paraphernalia. For these first two independent variables, we classified an index stay as having a naloxone standing order or Good Samaritan law if the date of implementation was before or on the date of the index stay , 7.The final two key independent variables represented Medicaid MAT coverage and generosity. Because the states in our sample had little variation in naltrexone and buprenorphine/naloxone coverage, we focused on two components of MAT coverage: whether a state had any coverage of methadone for Medicaid enrollees and whether a state had more or less generous coverage of buprenorphine/naloxone or naltrexone for Medicaid enrollees. Generosity of coverage of buprenorphine/naloxone or naltrexone was a composite variable based on the following four measures: (1) prior authorization requirement for buprenorphine/naloxone, (2) prior authorization requirement for injectable naltrexone, (3) buprenorphine/naloxone dosing limits , and (4) requirement for SUD counseling prior to treatment with buprenorphine/naloxone or naltrexone. If a state lacked restrictions on at least two of these four measures, it was categorized as more (vs. less) generous. As noted above, the Medicaid methadone coverage and MAT coverage generosity measures were based on data collected from a combination of sources. Because these data sources did not provide exact dates of implementation, we counted a state as having coverage in place during the years in which our sources collected the data (2013–2014) , 15–27.The analysis included covariates for patient-level factors, hospital stay and hospital characteristics, and state-level factors—all measured at the time of the index stay—that could have influenced the outcome of 90-day readmission. Patient-level factors included sociodemographic characteristics: age (continuous variable), sex, race/ethnicity , expected primary payer , community-level income based on the state-defined quartile for median household income of the ZIP Code of the patient’s residence, and urban/rural residency. To examine severity of illness, we identified whether the primary reason for the admission  was an opioid-related diagnosis or a non-opioid-related diagnosis, whether the patient was admitted with an opioid use disorder diagnosis  or a poisoning/adverse effect diagnosis , and whether the patient had a continuous opioid use disorder . We used the HCUP Clinical Classifications Software (CCS) diagnosis categories  to definHospital stay characteristics included whether patients received any treatment for drug rehabilitation or detoxification during the index stay (ICD-9-CM procedure codes 94.64–94.69) and the length of the index stay. Because SUD treatment varies across hospitals, we included covariates for characteristics related to the hospital in which the index admission occurred: percentage of hospital discharges among patients with opioid-related conditions and whether the hospital had a SUD detoxification or psychiatric acute care unit.State-level factors included the following measures of capacity for opioid treatment that were based on the year of the index stay: Medicaid coverage of all four levels of ASAM-recommended treatment services  during the 2013–2014 period ; newly cWe first conducted bivariate analysis to examine characteristics of our sample as well as the association between our key independent (policy) variables and the outcome variable, opioid-related readmission within 90 days after discharge. Next, we conducted multivariate logistic regression analysis to estimate the association between our key independent variables and an opioid-related readmission, taking into account the patient-, hospital- and state-level factors described above.All data were analyzed using SAS version 9.4. The HCUP databases are consistent with the definition of limited data sets under the Health Insurance Portability and Accountability Act Privacy Rule and contain no direct patient identifiers. The AHRQ Human Research Protections Program has determined that research using HCUP data has exempt status.During the study period, there were 383,334 index hospitalizations involving opioid-related diagnoses across the 13 sample states. Table Bivariate analysis showed that states with naloxone standing orders, Good Samaritan laws, Medicaid coverage of methadone, and more generous Medicaid coverage of MAT had a higher percentage of opioid-related readmissions within 90 days compared with states without these policies Table .Table 2BTable By insurance group, compared with states without Medicaid methadone coverage, the odds of readmission in states with Medicaid methadone coverage were higher among Medicare patients , privately insured patients , and uninsured patients . Medicaid methadone coverage was not associated with readmission among Medicaid patients.The odds of readmission in states with more generous Medicaid MAT coverage were lower among Medicare and privately insured patients  but higher among Medicaid patients  compared with these same groups of patients in states with less generous Medicaid MAT coverage. Medicaid MAT coverage generosity was not associated with readmissions among uninsured patients.Among the covariates in our model, patients treated in states where Medicaid covered all four ASAM-recommended treatment levels and states with more OTPs had lower odds of an opioid-related readmission  than did patients in states without these capacities. By contrast, patients in states with more substance use facility beds  and a higher opioid death rate had higher odds of readmission .Other factors associated with higher odds of an opioid-related readmission included the following at index hospitalization: a continuous opioid-related diagnosis, an opioid principal diagnosis, more physical codiagnoses, a mental health codiagnosis, and receipt of inpatient detoxification or rehabilitation treatment. In addition, patients with index stays in hospitals with more opioid-related discharges or with dedicated SUD treatment units had higher odds of readmission. By contrast, patients who were female, from the lowest income quartile or rural areas, with an opioid use disorder diagnosis (vs. a poisoning or adverse event diagnosis), or with a longer index stay had lower odds of readmission.In this study, patients with an opioid-related hospitalization in states with naloxone standing orders, more generous Medicaid methadone coverage , and more generous Medicaid MAT coverage (for the Medicaid group) had higher odds of an opioid-related readmission within 90 days. It is likely that policies that address opioid use disorders, such as the ones we explored, were implemented in states that had high need for opioid-related intervention. For example, states with more generous Medicaid MAT coverage had higher opioid death rates. Patients in states with these policies were more likely to be admitted for continuous opioid use and more likely to be covered by Medicaid than patients from states without these policies. Another possible explanation for our findings is that states that implemented these policies had more aggressive lobbying efforts to promote passage of such laws coupled with educational campaigns that resulted in an increase in patient awareness about the importance of acute treatment for opioids .Contrary to our hypothesis, patients in states with naloxone standing orders were more likely to be readmitted for opioid use than were patients in states without these policies. Prior research has shown that naloxone may reduce overdose mortality . For exaWe did not find any relationship between Good Samaritan laws and opioid-related readmissions. We may not have had sufficient time to see the effects of these laws, many of which were implemented recently . DespiteThis analysis showed associations of varying magnitude and significance, depending on insurance coverage, between opioid-related readmission and Medicaid coverage of methadone and Medicaid MAT coverage generosity. Methadone coverage did not have a statistically significant association with readmission in the Medicaid population. However, in all other payer populations, individuals treated in states with Medicaid methadone coverage had higher odds of readmission. In prior studies among the Medicaid population, methadone use has been associated with an increased likelihood of acute care use for overdoses from opioids –46. WhenMedicaid MAT coverage generosity was associated with lower odds of an opioid-related readmission among individuals covered by Medicare or private insurance but higher odds of readmission for those covered by Medicaid. The reason is not entirely clear. The Medicaid population historically has had high rates of readmission, particularly for SUDs . Our finThe findings also demonstrated that outpatient treatment capacity, along with coverage, is an important factor associated with readmission, particularly among the Medicaid population. Many states face barriers to distribution of MAT that extend beyond coverage, including lack of available providers, which tends to be most severe for publicly funded facilities . In thisOur study had some limitations. We did not have information in our sample on individual patient treatment or on whether individuals received treatment outside of the acute care medical or surgical hospital setting. We also did not have information on events that occurred outside of the acute care setting, such as post-discharge deaths, that may have affected our outcomes. Although we had the exact dates of implementation for naloxone standing orders and Good Samaritan laws, we did not have the exact dates of implementation for Medicaid methadone or MAT coverage policies. For the latter, we relied on general implementation information for the range of years during our study’s data collection period. Another limitation is that the association between our outcome and our policy variables may have been related to unmeasured variables; in addition, there may have been unmeasured endogenous terms that may have similarly impacted both the outcome and predictor variables. In addition, the available policy information did not distinguish between Medicaid fee-for-service and managed care for most states in our sample. Medicaid managed care plans may have separate formularies from fee-for-service formularies that may involve medication restrictions and prior authorization and utilization review requirements. Our sources indicated, however, that states in our sample had similar SUD coverage requirements under both types of plans. Finally, since our analysis was limited to 13 states, our results may not be generalizable to other regions or the nation as a whole.Policies to address opioid use disorders such as naloxone standing orders, Good Samaritan laws, and MAT coverage are still in their nascent stages but have the potential to affect acute care utilization, including readmissions. The implementation of such policies in the states included in this study may have been motivated by higher rates of continuous opioid abuse and dependence in the population. Although some policies were associated with increased rates of readmissions, others were associated with a decrease or no change in readmission rates. We propose some explanations for our findings; however, more research is needed to understand how these policies can be most effective in influencing acute care utilization, including educating the public about the existence of these benefits and ensuring that states have adequate capacity to optimize their effectiveness.


The axon is responsible for integrating synaptic signals, generating action potentials (APs), propagating those APs to downstream synapses and converting them into patterns of neurotransmitter vesicle release. This process is mediated by a rich assortment of voltage-gated ion channels whose function can be affected on short and long time scales by activity. Moreover, neuromodulators control the activity of these proteins through G-protein coupled receptor signaling cascades. Here, we review cellular mechanisms and signaling pathways involved in axonal ion channel modulation and examine how changes to ion channel function affect AP initiation, AP propagation, and the release of neurotransmitter. We then examine how these mechanisms could modulate synaptic function by focusing on three key features of synaptic information transmission: synaptic strength, synaptic variability, and short-term plasticity. Viewing these cellular mechanisms of neuromodulation from a functional perspective may assist in extending these findings to theories of neural circuit function and its neuromodulation. Neuromodulators exert powerful control over both neuronal circuit activity and animal behavior throughout the brain . NeuromoAxonal ion channels are important for many aspects of neuronal function, from the initiation and propagation of APs to the release of neurotransmitter . APs areV), potassium (KV), and calcium (CaV) channels. In particular, modulation of CaVs can strongly impact the release of neurotransmitters because vesicle release is a direct (but probabilistic and non-linear) function of calcium concentration. Intracellular calcium also influences how the release of neurotransmitter changes with different patterns of presynaptic activity, a critical component of short-term plasticity (STP). As a result, subtle changes to ion channel function at the axonal bouton can result in large effects on synaptic strength, STP, and synaptic information transmission.Neuromodulation of ion channels further down the axon also significantly affects how information is propagated to downstream synapses. Like in the AIS, modulation of ion channels at axonal boutons, the site of neurotransmitter release, also occurs through activation of GPCRs . Known tB receptor systems in which significant mechanistic detail has been elucidated. For a recent review of membrane excitability across axonal compartments  cation channels . This AP propagation itself is not perfectly reliable. For example, APs may fail at axonal branch points  in the axon , CaV2.2 (N-type), and CaV2.3 (R-type). Axonal expression of each of these isoforms varies by cell type, and, at times, postsynaptic target  calcium channels in dissociated striatal neurons via PKA-dependent signaling . This form of presynaptic regulation without marked changes in STP has also been observed for noradrenergic  synaptic strength, (2) synaptic variability, and (3) STP. By regulating presynaptic calcium influx and vesicle release, axonal neuromodulation can control the strength of specific subsets of synaptic inputs relative to others. But because transmitter release is probabilistic, it also regulates the extent to which individual presynaptic APs are reliably transmitted to the postsynaptic cell. Finally, regulation of STP controls a release site’s strength as a dynamic function of its past activity, which can create a frequency-sensitive postsynaptic signal from a sequence of all-or-none presynaptic APs. Considering synaptic neuromodulation from this functional lens can lead to insight into how cellular mechanisms in the axon might be employed in neural information processing Pr. In order to focus on broader functional impacts, we will define Pr at the level of the synapse, i.e., the probability that the active zone successfully releases a vesicle of neurotransmitter in response to an AP. This quantity is the product of many complex underlying variables, including the number of vesicles available for release, their distance to sources of calcium influx, and the probability of release of that each of these vesicles as a function of intracellular calcium. Within this framework, synaptic strength is therefore defined as the product of Pr, the number of release sites N, and the quantal size q .Neuromodulatory GPCRs can exert fast and reversible control of synaptic strength by altering the probability of neurotransmitter release, Pr and postsynaptic sensitivity to neurotransmitter q will change synaptic strength linearly. Importantly, however, this is only true on average, because presynaptic neuromodulation also changes the variability of the synapse, whereas postsynaptic modifications of q do not  can lead to frequency-dependent filtering of synaptic strength . This trPr will also affect the extent of depletion-mediated short-term depression. In fact, these effects can both occur simultaneously to conceal each other. For example, at high-Pr synapses short-term facilitation can be limited by vesicle depletion. At such a synapse, reductions in calcium influx can both reduce Pr and unveil underlying facilitation  are so common that they are considered a hallmark of presynaptic neuromodulation of release  will be required to attribute behavioral effects to these transformations of synaptic function. Furthermore, tools to precisely separate the two presynaptic effects of GABAB (reducing Pr and increasing facilitation) would allow us to better understand the function of other presynaptic neuromodulators that also employ Gβγ-dependent signaling pathways, such as the CB1 receptor. Alternatively, experiments that compare activation of presynaptic neuromodulators with different effects on synaptic transmission  could more clearly tie these effects on STP to circuit activity and behavior.A common example of synaptic suppression with changes in STP is activation of presynaptic GABAignaling . Alongsiignaling , this leignaling . This coignaling . The beh in mice , these mβγ-dependent signaling canonically regulates both synaptic strength and STP, which we will term temporal modulation, some presynaptic neuromodulators appear to regulate Pr in isolation, which we will term gain modulation. These latter cases violate the dogma that presynaptic modulation also regulates PPR and demonstrate that temporal and gain modulation do not map cleanly onto presynaptic and postsynaptic mechanisms, respectively. Indeed, any form of modulation that essentially silences synapses  can functionally be characterized as gain modulation. This includes both presynaptic mechanisms  as well as postsynaptic mechanisms . Similarly, while presynaptic mechanisms to modulate facilitation and depression have been extensively documented, some postsynaptic mechanisms have also been implicated  should be couched aside experiments designed to assess whether the same synapses utilize nano- or microdomain release mechanisms.Why do some neuromodulators that regulate ve zones . The preurations . Thus, aVs and vesicles. The aforementioned experiments examining D1 receptor modulation in PFC suggested a critical role of CaV-vesicle functional coupling in its effects on synaptic transmission (V recruitment per AP affects STP (V channel open probability at the presynaptic axon, leading to gain modulation of synaptic transmission, whereas activation of the GABAB receptor reduced CaV current per AP and led to temporal modulation. Taken together, these data led to the hypothesis that D1 activation would modulate postsynaptic responses to all input frequencies similarly, whereas GABAB activation would preferentially suppress responses to low-frequency inputs. Experiments quantifying postsynaptic AP generation confirmed this hypothesis. Whether this functional distinction between gain and temporal modulation extends to naturalistic patterns of activity seen in vivo, and what functional role it may play in circuit neuromodulation, has yet to be investigated.The neuromodulation of synaptic strength has been understudied in the context of functional coupling between Casmission . These sects STP . At thesin vivo. Similarly, many effects of neuromodulation observed in ex vivo acute slice preparations have yet to be linked to specific behavioral outcomes in vivo. Recent work has begun to investigate how different synaptic mechanisms contribute to neuronal computations in vivo (in vivo continues to improve, it is becoming increasingly possible to bridge the gap between mechanistic studies in vitro and functional studies in vivo and better incorporate synaptic neuromodulation into theories of neuronal circuit function. The highly non-linear behavior of synapses, including their variability, short-term dynamics and complex neuromodulation, are likely to be a critical component of future research into how cellular processes impact neural circuits and behavior. It will be critical to move from mere identification of synaptic connectivity to the identification of the neural codes employed by these connections, as well as identification of when, where, and how neuromodulators are engaged in vivo. To achieve this, new tools that can provide real-time measurements of neuromodulatory activity, including sensors for neurotransmitters (B activation in changing postsynaptic conductance versus presynaptic Pr and STP (While the use of fixed frequency synaptic stimulation is both common and important in investigations of cellular mechanisms of synaptic transmission, it also likely obscures how STP and its neuromodulation affect the noisy and irregular patterns of active neurons  in vivo . As the smitters  and intrsmitters , will besmitters , includismitters . Togethe and STP . The abiAll authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Understanding how crop plants evolved from their wild relatives and spread around the world can inform about the origins of agriculture. Here, we review how the rapid development of genomic resources and tools has made it possible to conduct genetic mapping and population genetic studies to unravel the molecular underpinnings of domestication and crop evolution in diverse crop species. We propose three future avenues for the study of crop evolution: establishment of high-quality reference genomes for crops and their wild relatives; genomic characterization of germplasm collections; and the adoption of novel methodologies such as archaeogenetics, epigenomics, and genome editing. Since the Neolithic, humans have domesticated a large number of different plant species to create a reliable source of nutrition for themselves and their domestic animals. Crop plants comprise a large variety of species from diverse taxa that differ in habitat, growth habit, and life cycle, such as annual grasses, perennial trees, and medicinal herbs Table , Fig. 1.Crop domestication has been studied for more than a century and benefited recently from technological innovations in genomics. Comparative analysis of population genomic data of large samples of current and past varieties of crops together with their wild progenitors provides insight into the domestication history of species , 4, for Much has been written on plant domestication. Recent review articles have focused on convergent phenotypic evolution , causatiExtant crop wild relatives are excellent tools to understand crop evolution and as sources of novel allelic diversity for future crop improvement , 25. TheGenome sequencing and assembly have been applied to many different species for decades. In the context of plant domestication research, genomic resources such as high-quality reference genome sequences of crops and their wild relatives, but also dense genetic and physical maps, have provided the infrastructure for the genetic mapping of loci underlying key domestication traits , 28 and Oryza, i.e., wild and domesticated rice, and an outgroup species (Leersia perrieri) provided a comprehensive overview of structural genome evolution, thus contributing to the overarching aim of the International Oryza Map Alignment Project to establish genus-wide comparative genomics to discover genes for crop improvement [The traditional approach of laborious and time-consuming Sanger sequencing along a minimum tiling path of bacterial artificial chromosomes (BACs)  has beenrovement .Fig. 2GeIn the large-genome cereals wheat, barley, and rye, short-read sequencing has been widely adopted to compile complementary datasets for higher-order scaffolding such as linkage maps , 52, phyIn an eye-opening study, Avni et al.  construcSolanum pennellii (genome size ~ 1 Gb). The resultant sequence scaffolds were highly contiguous (N50 2.5 Mb) but required correction with supplementary Illumina reads to increase accuracy at the single nucleotide level. Similarly, Zimin et al. [Aegilops tauschii [Long reads (> 10 kb) from the PacBio or Oxford Nanopore platforms are two orders of magnitude longer than Illumina reads (100–300 bp) but have reduced sequence accuracy . Recentln et al.  used a ctauschii . The N50tauschii ).Until recently, there was a ‘contiguity gap’ in the assembly of complex plant genomes. The contiguity of sequence assemblies was limited to kilobase-sized contigs, but conventional methods for higher-order scaffolding and assembly validation such as genetic mapping , 68, cytCapsicum annum) and achieved a scaffold N50 of 3.7 Mb.One of the limitations of the current Bionano platform is the need for input assemblies of high contiguity so that sequence scaffolds comprise a sufficient number of label sites to confidently align sequence assembly and optical map. Technologies based on Illumina short-read sequencing can better control data density by increasing sequencing depth. One of these, the Chromium 10X platform, employs a microfluidic device to create so-called ‘linked reads’, which incorporate short barcode sequences that are shared by reads originating from the same high-molecular weight DNA fragment . ChromiuArguably, the most disruptive technology in recent assembly for genome mapping has been chromosome conformation capture sequencing (Hi-C). Hi-C was originally developed as a method to assay genome-wide chromatin contact probabilities , but it We believe it is possible to obtain for any plant taxon—wild or domesticated—a high-quality reference genome sequence assembly within a year’s time frame without prior resources. But, which is the most cost-effective combination of sequencing methods and genome mapping approaches , 89? A cThe wild progenitors of most crop plants remain extant  and can The most straightforward method for assessing genetic diversity in species with a reference genome is shotgun sequencing of short-insert libraries on the Illumina platform and alignment of the sequence reads to the reference assembly . Whole-gAegilops tauschii, a wild diploid progenitor of bread wheat. Witek et al. [Solanum americanum, a diploid, non-tuber bearing wild relative of potato.An important drawback of whole-genome sequencing is the financial burden associated with amassing sufficient sequence data for large diversity panels in minor crops or those with large genomes. Several techniques have been developed to reduce genome complexity prior to sequencing so as to increase read depth in certain genomic regions. Sequence capture with oligonucleotide baits can be applied at the scale of whole exome  or gene k et al.  combinedSolanum species to detect footprints of selection based on genetic variation at the DNA sequence level, and also found selection pressure on gene expression level in the domesticate. Lemmon et al. [1 crosses between maize and teosinte to understand the changes in the regulatory architecture of gene expression as a consequence of domestication.High-throughput RNA sequencing (RNA-seq)  is an aln et al.  analyzedReduced representation sequencing  is knownPhaseolus vulgaris, the progenitor of common bean [GBS is a versatile and inexpensive method for natural and experimental populations of crops and their wild relatives. Linkage maps for wild relatives of Triticeae crops –132 and mon bean .1 hybrids between elite breeding material and diverse landraces sampled from the collection of the International Maize and Wheat Improvement Center (CIMMYT). Genome-wide association scans revealed a co-association of genes to both flowering time and altitude. Similar approaches are underway in other cereal crops such as wheat [Moreover, GBS is a cost-effective method to screen large germplasm collections. For example, Romay et al.  used GBSas wheat , barley as wheat , common as wheat , Solanacas wheat , and ricas wheat . As moreas wheat .High-throughput sequencing has also been used to obtain measurements on aspects of the genome other than nucleotide sequence variation. Here, we describe future directions in domestication research that will benefit from these technological innovations, such as epigenomics, archaeogenetics, genome editing, domestication of novel crops, and new computational methods for analysis of population genetic data Fig. .Fig. 3FuEpigenomics refers to the study of the entirety of heritable changes other than changes in DNA sequence, such as DNA methylation or histone modifications. The interaction between euchromatin and heterochromatin likely plays a role in silencing of transposable elements and influences gene expression . CompareArchaeogenetics—the extraction, sequencing, and analysis of ancient DNA fragments—has transformed our concepts of the history of human and animal species –163. UntBrassica napus) to knockout simultaneously both homeologs of ALCATRAZ, a known regulator of seed shattering in Arabidopsis [Genome editing with CRISPR-Cas9 technology  has enribidopsis . SiliqueThinopyrum intermedium; Fig. Azolla [Miscanthus species [The domestication of new plant species has been proposed as an important future contribution to sustainable agriculture. For instance, the development of perennial grain crops has received considerable attention , 175. Pr. Azolla , Miscant species , and the species .As large population genomic datasets accrue in more species, analysis methods need to keep pace with the growing amount of input data. Efficient data structures have been devised to structure and handle large marker matrices , 181. ImNew algorithms have been developed to speed up traditional analysis methods such as principal component analysis , 189 andIn summary, progress in sequencing technology and analysis methods will make it possible to study the genetics and genomics of domestication in a wider range of crop species. In the coming years, chromosome-scale reference sequence assemblies and resequencing studies of large diversity panels will contribute to understanding the past and present diversity of domesticated plants and their wild relatives.


Peritoneal dissemination is a distinct form of metastasis in ovarian cancer that precedes hematogenic or lymphatic metastasis. Exosomes are extracellular vesicles of 30–150 nm in diameter secreted by different cell types and internalized by target cells. There is emerging evidence that exosomes facilitate the peritoneal dissemination of ovarian cancer by mediating intercellular communication between cancer cells and the tumor microenvironment through the transfer of nucleic acids, proteins, and lipids. Furthermore, therapeutic applications of exosomes as drug cargo delivery are attracting research interest because exosomes are stabilized in circulation. This review highlights the functions of exosomes in each process of the peritoneal dissemination of ovarian cancer and discusses their potential for cancer therapeutics. Ovarian cancer is the most lethal human gynecological malignancy ,2. When Extracellular vesicles (EVs) have become the focus of increase research interest due to their diverse functions in physiology and pathology . EVs areDuring the process of peritoneal dissemination, ovarian cancer cells detach from the primary site of origin . Subsequently, these cancer cells spread into the peritoneal cavity and attach to the surface of peritoneal organs, most notably the omentum, which is the predominant site of ovarian cancer metastasis . All of Several studies have shown that cancer cell-derived exosomes reprogram or educate other cells to support tumor survival and promote metastasis ,18. HoweThe initial step of peritoneal dissemination is the shedding of cancer cells from their primary site throughout the peritoneal cavity . The preOnce cancer cells shed into the peritoneal cavity, they face several challenges for survival, including anoikis and immune surveillance . In thisDuring peritoneal metastases, cancer cells detach from the primary site, float into the peritoneal cavity without access to vascular supply, and are therefore exposed to hypoxic conditions. Hypoxia causes the cells to acquire a more aggressive malignant phenotype that allows them to metastasize . Recent Malignant ascites occurs in most patients with high-grade serous ovarian cancer . StudiesAll of the organs within the peritoneal cavity are lined with a single layer of peritoneal mesothelial cells (PMCs) that cover an underlying stroma composed of ECMs and stromal cells. During ovarian cancer dissemination, once ovarian cancer cells attach to this layer, they invade through the barrier of PMCs into the peritoneum, omentum, and bowel serosa ,58. UndeOnce cancer cells attach to PMCs that line the surface of the peritoneum, productive reciprocal interactions between the cancer cells and their new microenvironment can occur, which leads to the successful establishment of metastatic tumors . During MALAT1), in ovarian cancer-derived exosomes can be transferred to HUVECs [Angiogenesis is required for ovarian cancer progression and is an established therapeutic target for advanced diseases. Several large phase III clinical trials have shown the efficacy of vascular endothelial growth factor (VEGF) targeting therapy in ovarian cancer ,65. The o HUVECs . MALAT1-Fibroblasts constitute one of the most abundant cell types in the stroma. They are essential elements for normal tissue homeostasis and function and are generally anti-tumorigenic . A numbeIf metastasis is a random event, all organs in contact with peritoneal fluid would have an equal distribution of metastases. However, ovarian cancer preferentially metastasizes to adipose tissue and omentum . Indeed,In general, circulating and disseminated tumor cells acquire a variety of immune-escape mechanisms, including alterations in the expression of major histocompatibility complex (MHC) molecules, Natural killer (NK)-cell ligands, Fas, Fas ligand (FasL), and immune-checkpoint molecules including CD47 and programmed cell death 1 ligand 1 (PD-L1) . There iAccumulating evidence has demonstrated that exosomes can serve as a therapeutic modality as well as a therapeutic target for cancer treatment ,88. In tG12D. Compared to liposomes, engineered exosomes target oncogenic KRAS with an enhanced efficacy that is CD47-dependent and allows circulating monocytes to evade phagocytosis, increasing the half-life in the circulation [Because exosomes are stable in the circulation and do not induce immune rejection, their therapeutic applications as drug delivery systems have grown into an attractive research area ,89. Advaculation ,93. In oculation . These rImmunotherapy is an emerging field in cancer treatment, and exosomes can be developed for cancer immunotherapy because of their immunomodulatory potential . In geneGiven that exosomes play pivotal roles in cancer progression, targeting exosome biogenesis and secretion has potential clinical implications for future cancer therapies. However, there are currently no clinically available drugs that efficiently eliminate deleterious exosomes in cancer patients. Through high-throughput drug screening from a total of 4580 compounds, five potent exosome biogenesis and secretion inhibitors were identified: tipifarnib, neticonazole, climbazole, ketoconazole, and triadimenol . FurtherOvercoming peritoneal dissemination remains a primary challenge in treating ovarian cancer. Therefore, it is crucial to elucidate the underlying mechanism of this distinct pattern of metastasis. Emerging evidence has shown that exosomes facilitate the peritoneal dissemination of ovarian cancer in several developmental steps by mediating intercellular communication between cancer cells and the tumor microenvironment. Furthermore, recent studies have implicated the potential of both exosome-targeting and exosome-based therapies. Although there are many challenges to be addressed in the clinical use of exosome therapy, the development of the treatment of the peritoneal dissemination of ovarian cancer is still an attractive research area.


Tissue engineering is based on the interaction between stem cells, biomaterials and factors delivered in biological niches. Oral tissues have been found to be rich in stem cells from different sources: Stem cells from oral cavity are easily harvestable and have shown a great plasticity towards the main lineages, specifically towards bone tissues. Dental pulp stem cells (DPSCs) are the most investigated mesenchymal stem cells (MSCs) from dental tissues, however, the oral cavity hosts several other stem cell lineages that have also been reported to be a good alternative in bone tissue engineering. In particular, the newly discovered population of mesenchymal stem cells derived from human periapical inflamed cysts (hPCy-MSCs) have showed very promising properties, including high plasticity toward bone, vascular and neural phenotypes. In this topical review, the authors described the main oral-derived stem cell populations, their most interesting characteristics and their ability towards osteogenic lineage. This review has also investigated the main clinical procedures, reported in the recent literature, involving oral derived-MSCs and biomaterials to get better bone regeneration in dental procedures. The numerous populations of mesenchymal stem cells isolated from oral tissues  retain proliferation ability and multipotency; these features are exploited for clinical purposes, including regeneration of injured tissues and local immunomodulation; we reported on the last studies on the proper use of such MSCs within a biological niche and the proper way to storage them for future clinical use. Stem cells have attracted a growing interest in the scientific community mainly for their ability to regenerate damaged tissues and also for their potential in modulating inflammatory and immune responses.ClinicalTrials.gov website.These skills have caused stem cells to be considered as a strategic tool for a large number of clinical applications; this is demonstrated by the increasing number of clinical trials focused on stem cell therapy that nowadays amounts to about 7000 studies found for “stem cells” as searching criteria on The elevated proliferation potential and the wide differentiation ability of mesenchymal stem cells (MSCs) strongly encourages scientists to search for new unexplored sources of these precious cells, however, their extreme plasticity and their ability to modify different biological niches has caused some scientists to still have doubts about their own existence, carrying out alternative hypotheses on MSCs real nature . Dental tissues are rich in cell populations expressing mesenchymal stem cell like-features, with the advantage of having easier access to the oral cavity than other anatomical sites, and the fact that in vitro isolation of cells is very efficient; thus, oral-derived MSCs are highly attractive for research and clinical purposes . SeveralThe immunomodulatory activity of oral-derived MSCs was specifically tested in experimental treatments of autoimmune diseases; for example, stem cells from exfoliated deciduous teeth were capable of promoting the recovery of the ratio between T-reg and T-helper cells with the result to efficiently reduce systemic lupus erythematosus-associated disorders . In vitro investigations demonstrated that dental pulp stem cells can stimulate angiogenesis/vasculogenesis, thus finding applications for the cell-based treatments of ischemic diseases . Oral stem cells have embryological development from neural crests, and these cells likely maintain a kind of “neural genetic memory”; this hypothesis is supported by the presence of basal gene-expression of neurogenic markers of these cells. The neurogenic potential of oral-derived stem cells has been more consistently demonstrated in the last discovered human periapical-cyst Mesenchymal Stem Cells (hPCy-MSCs) isolated from the inner layer of an inflamed periapical cyst . Literature have consistently confirmed that dental stem cells can be easily stimulated to differentiate into osteoblasts and into odontoblasts; this ability make them have a central role in regenerative dentistry . The aim of this narrative review is to assess the state-of-the-art and main future insights of oral-derived MSCs; we strongly suggest to use specific oral-derived MSCs as a strategic tool for dental and maxillofacial applications in future MSCs-based therapies.A literature search was performed through Medline, via PubMed and Google Scholar, to identify articles published from 2000 until 2018. The following keywords have been used for the literature search: “MSCs AND Dental”, “Regeneration AND Dental”, “Bone AND Dental”. The authors have carefully revised both the title and the abstract for each of the selected papers; only papers related to our aim were included in the current narrative review. After having excluded the duplicate articles and other types of publication, a total of 571 articles were selected for the current review. Three-hundred-and-sixty-eight articles were considered unsuitable because titles were not fitting the objectives of our review. After an initial screening, a total of 91 papers were excluded. Forty papers were also excluded, since their conclusions were considered not relevant to our aims. Five further papers were excluded, mainly because they revealed methodological bias and a poor reliability of the research. Thirty-five articles met the final inclusion criteria of this narrative review: They were included in this review and we based our main assessment on the base of their scientific messages. Stem cell therapy plays a central role in regenerative medicine, with many examples of progression from the preclinical to the early-clinical levels, for a wide variety of diseases.Friedenstein first described a population of “clonal, plastic-adherent cells” residing in the blood tissue [The first adult stem cells discovered in bone marrow were hematopoietic progenitors able to reconstitute the entire blood system of an immunocompromised host  just a fd tissue . These cd tissue .Bone marrow was initially considered the main source of MSCs. Subsequently, it will be discovered that these cells reside not only in bone marrow but also in many other anatomical sites, such as: blood, cord blood, fat, lung, heart, brain, skin, muscle, bowel, liver, gonads, and teeth ,12.Furthermore, a series of studies demonstrated that these cells could choose among several further differentiating lineages including skeletal muscle, tendon and neural commitment; these data demonstrated that MSCs hold high plasticity ,14. The Collection of MSCs from human bone marrow (hBM) does not imply a quite simple procedure, indeed, the donor must undergo an invasive intervention to allow aspiration of BM from the iliac crests. Moreover, isolated cells are not abundant because of the low frequency of MSCs in BM estimated to be nearly of one MSC per 34,000 nucleated cells . These iThe discovery of ubiquitary cells carrying the typical features of MSCs in the oral cavity, strongly shifted the scientific attention on dental tissues. Oral stem cells can be easily isolated from teeth extracted for orthodontic or irreversible periodontitis reasons. The easy accessibility to the collection site and the abundance of highly immature cells are certainly attracting hallmarks for stem cell therapy studies. Over time, different investigations have shown the existence of a growing number of stem cell populations, with the typical features of MSCs, in oral tissues. In 2000, Gronthos first showed the existence of odontogenic progenitors in adult human dental pulp, which were capable of self-renewing with a high proliferative rate and were able to form colonies in in vitro experiments. Moreover, this population was able to reproduce dentin/pulp-like structures after transplantation into immunocompromised mice. These odontogenic precursors shared the same immunophenotype of bone marrow stromal cells (BMSCs) and were finally named dental pulp stem cells (DPSCs) . Thus, DThree years after the discovery of DPSCs, Miura and colleagues isolated multipotent, clonogenic and highly proliferating progenitors from the dental pulp of human exfoliated deciduous teeth (SHEDs); these cells have shown the ability to in vitro differentiate into various cell types such as odontoblasts, adipocytes and neural cells and to promote dentin formation and bone regeneration in vivo; in addition, their neural marker’s expression allows them to survive in mouse brains after transplantation .A population expressing mesenchymal stem cell-like markers was found to be located in the connective tissue that bonds cementum to alveolar bone, periodontal ligament (PDL). Periodontal ligament stem cells (PDLSCs) can differentiate into adipocytes, collagen-forming cells, and cementoblast-like cells in culture and regenerate cementum and PDL in vivo .The connective sac that surrounds the developing tooth, defined as dental follicle, contains MSCs called dental follicle progenitors (DFPCs); these cells isolated for the first time by Morsczeck in 2005 were able to begin neural differentiation and to regenerate periodontal and bone tissues .Root apical papilla of human teeth holds stem cells (SCAP) able to differentiate into osteogenic and odontogenic progenitors, and also into adipose and neural cells ,25.Also, gingival tissues have been shown to be colonized by a multipotent clonogenic stem and progenitor cells. MSCs derived from gingiva, removed for hyperplastic or aesthetic reasons, displayed regenerative potential, for cell therapy, even superior to BMSCs .Progenitors able to differentiate into chondrocytes and osteoblasts are located in the jaw periosteum . Human pThe oral cavity is certainly a complex environment where many biological niches hosting MSCs have been found in both hard tissues and soft tissues. Scientists commonly recognize Gronthos as the first isolated MSCs population from dental tissues; in fact, he first reported about MSCs from dental pulp (DPSCs). DPSCs have been considered in the last few decades as a good substitute of bone marrow stem cells (BMSCs) for dental applications, because of their ability to induce dentinogenesis in immunocompromised mice. In 2013, a new population of mesenchymal stem cells was isolated from human periapical cysts. Tatullo and his team demonstrated for the first time the existence in periapical inflamed tissues of residing cells showing the representative features of MSCs that have been termed: human Periapical Cyst-Mesenchymal Stem Cells (hPCy-MSCs) . This nehPCy-MSCs in their basal state express high levels of transcripts for neuronal markers and for neural-related transcription factors with respect to dental pulp stem cells. Moreover, this predisposition toward the neural phenotype significantly improves under neurogenic stimulating conditions, as demonstrated by the up-regulation of a broad set of proteins and genes that define neuronal cells .Preliminary data evidence shows the ability of hPCy-MSCs to differentiate into functional dopaminergic neurons, with important potential applications in the treatment of Parkinson’s disorders (Tatullo unpublished). In a comparative study, the potential to differentiate into osteoblasts and into odontoblast was investigated between DPSCs and hPCy-MSCs; these populations were cultured in standard conditions and also in osteogenic inducing medium. The obtained results elucidate the more pronounced tendency of DPSCs to undertake the odontoblastic differentiation lineage instead of hPCy-MSCs with a major propensity toward osteogenesis. These data sustain the important value of these cells for dental and maxillofacial regenerative applications . Finally, the presence of untreated pulp-periodontal infection does not preclude the possibility of finding resident DPSCs: This could indicate that hPCy-MSCs acts as a co-factor in the process of local bone regeneration. Instead, DPSCs have commonly showed a greater ability to shift towards the odontogenic phenotype. hPCy-MSCs exert extensive proliferative ability and have shown the potential to differentiate into various cell types such as adipocytes, osteoblasts and neurons. The great advantage in the use of this newly discovered oral stem cell population consists in its easy collection from the surgically-removed periapical cysts that imply the reuse of biological waste that became a very smart source of stem cells without any impact on the surrounding healthy tissues . Thus, hMesenchymal stem cells retain a central role in regenerative medicine because of their intrinsic self-renewal ability and their multipotency that allows them to differentiate into both mesenchymal and non-mesenchymal lineages . MSCs have demonstrated to be obtainable from stem cell niches that are widely represented in the human body, as well as from somatic cells induced to show MSC-like behavior through genetic manipulations: the iPS (induced pluripotent stem cells) have been widely studied in several therapeutic applications, thanks to their easiness to be obtained from any tissue. Recently, reprogrammed iPS were transplanted into the brain of Parkinson’s disease patients, showing how the reprogramming of cells towards an embryonic-like state may be an effective strategy to strengthen the clinical use of MSCs–based therapies .MSCs residing in the oral cavity tissues receive clinical interest in oral and maxillofacial surgery as a cell source that is easy to obtain, easy to manage in vitro and has an excellent behavior after they undergo several differentiating media, even if they have clearly shown a better commitment towards neurogenic, bone and dental tissues such as cementum, dentin and periodontal ligament . Furthermore, recent papers suggest the isolation of cellular-secreted nanoparticles that include numerous patterns of functional molecules physiologically used for cell-to-cell cross talking. The possibility to isolate and to subsequently cryo-store these nanovesicles represents a further advantage for the use of the product of stem cells avoiding some existing concerns about the clinical use of stem cells .The presence of a scaffold constituted by biocompatible materials, alone or associated with stem cells, represents a fundamental element in tissue engineering applications. Recent studies elucidate the important role of scaffolds, considered not only as a merely inert support for seeded cells, but a determinant substrate able to affect cell behavior. This concept is applicable to DPSCs, in fact, in their physiological niche, besides biochemical factors, these cells are influenced by biophysical and biomechanical stimuli, such as mechanical stretch, tensile strain and micro- and nano-scale surface topography interaction. DPSCs are sensitive to mechanical forces; these signals are converted into functional cellular responses including lineage specification .This narrative review has investigated about the state-of-the-art of oral-derived MSCs and on their commitment towards the most useful lineages in dental and maxillofacial applications, with special attention to bone regeneration. MSCs in regenerative medicine are still an open challenge: their use must become applicable in the daily reconstructive surgery. In this review, with particular focus on osteogenic commitment, we found that all MSCs from oral tissues were highly committed to differentiate toward osteoblasts and precursors of bone tissue. Interestingly, hPCy-MSCs showed a better suitability towards osteogenic commitment, whereas DPSCs appeared to be more direct towards dentinogenesis, even if both these cell lineages were able to achieve good results in experimental sessions related to differentiation in the above cited lineages. The authors feel that hPCy-MSCs could be a good alternative to MSCs from other sources, given the reduced ethical and biological issues affecting this specific novel MSC lineage. Different trials are ongoing in a clinical setting, however, future investigations are needed to better understand the biology of oral-derived MSCs, including further undiscovered differentiation fates, and to explore more in depth the potentialities of the secretome of these cells, aiming to reach the final goal of shifting the results from the research laboratory to patients.


Gluteal augmentation may be performed using a variety of techniques, including implant-based, autologous fat grafting, local flaps, impermanent filler injection, or, as in this case, by way of permanent filler injection with free-silicone. Of these, free-silicone injections carry one of the highest complication rates, specifically regarding migration of the filler material from the native injection site and induction of painful reactive soft tissue changes at the new filler location. A radiologist providing this diagnosis may assist the clinician, who often cannot obtain a history of illicit silicone injection for gluteal augmentation unless the suspicion is raised. Presented here is a case of painful filler migration to the knee with granuloma formation after free-silicone gluteal injection. Gluteal augmentation has become increasingly popular as a means of enhancing physical appearance. The most popular methods involve implant-based techniques, as well as autologous fat injection. Additional, less common techniques include local fat or muscle flaps, resorbable dermal filler injection using hyaluronic acid gel, and permanent filler injection using free liquid silicone , 2. FreeA 28-year-old woman with a history of uterine malignancy, deep vein thrombosis, and hepatitis B, who had been recently admitted as an inpatient for management of renal calculi presented with left knee pain. A magnetic resonance imaging (MRI) scan of the left knee was subsequently ordered by the patient’s clinician. Prior to prescribing an MRI of the left knee, the patient had undergone a recent computerized tomography (CT) scan of the abdomen and pelvis, which had demonstrated numerous partially calcified granulomas in the gluteal subcutaneous tissues bilaterally Figure . AdditioSilicone is a substance with a controversial history. It belongs to a family of synthetic polymers comprised of the element silicon, of which the liquid form was first used for breast enhancement in the United States in 1963 , 9. Due Although safety remains a topic of debate, silicone-related procedures continue to be performed routinely, and often illegally, in many countries for cosmetic purposes , 10. TheThe final diagnosis for this case included granuloma formation in the posterior thigh and popliteal fossa with caudal migration of free-silicone to the left knee. Findings on provided MR images, however, are not specific for the diagnosis. In conjunction with the aforementioned CT imaging features of the pelvis and chest, demonstrating probable injection sites with surrounding reactive soft tissue change, the final radiologic diagnosis may be suggested.This case demonstrates complications from an unusual form of gluteal and breast augmentation by free-silicone injection. Since patients do not typically disclose undergoing illicit procedures, the practicing radiologist may assist the clinician in proposing the diagnosis when the appropriate imaging findings are present.


MicroRNAs (miRNAs) are non-coding RNAs that regulate a wide range of biological pathways by post-transcriptionally modulating gene expression levels. Given that even a single miRNA may simultaneously control several genes enrolled in multiple biological functions, one would expect that these tiny RNAs have the ability to properly sort among distinctive cellular processes to drive protein production. To test this hypothesis, we scrutinized previously published microarray datasets and clustered protein-coding gene expression profiles according to the intensity of fold-change levels caused by the exogenous transfection of 10 miRNAs  in a human cell line. Through an in silico functional enrichment analysis, we discovered non-randomic regulatory patterns, proper of each cluster identified. We demonstrated that miRNAs are capable of equivalently modulate the expression signatures of target genes in regulatory clusters according to the biological function they are assigned to. Moreover, target prediction analysis applied to ten vertebrate species, suggest that such miRNA regulatory modus operandi is evolutionarily conserved within vertebrates. Overall, we discovered a complex regulatory cluster-module strategy driven by miRNAs, which relies on the controlled intensity of the repression over distinct targets under specific biological contexts. Our discovery helps to clarify the mechanisms underlying the functional activity of miRNAs and makes it easier to take the fastest and most accurate path in the search for the functions of miRNAs in any distinct biological process of interest. About two decades ago, the discovery of a comprehensive variety of miRNAs—small endogenous non-coding RNAs—broadened horizons and revolutionized scientific knowledge regarding the mechanisms of regulation of gene expression in the metazoan genome. The ancestral origin of miRNAs, their dramatic expansion in bilateral animals, and their role in providing robustness to cellular transcriptional programs ,2 suggesMiRNAs have been reported to regulate a myriad of distinct biological processes, from cell differentiation and proliferation  to the oRecently, advances both in experimental and computational methods have increased the availability of new genomes and transcriptomes thus providing resourceful data about sequence and expression levels of miRNAs and their respective target genes. However, while it is excessively laborious to query the wide bunch of biological functions of miRNAs and targets empirically through in vivo studies, computational strategies have afforded novel valuable insights on biological research as they paved the way for the validation of miRNA functions by yielding statistically significant hypotheses from the extensive collection of biological measurements .Offshoots of these burgeoning datasets generated have shed light on diverse aspects of miRNA biology, including the (co)evolution of miRNAs and their target genes  and the The peculiar miRNA-target pairing admits the occurrence of a broad spectrum of interactions, implying that a single miRNA can interact and regulate multiple genes, and/or a single gene can be regulated by several miRNAs. In fact, every miRNA may simultaneously control several target genes enrolled in a wide range of ongoing metabolic pathways within a single cell. This is relevant, as the unraveling of true interactions coupling miRNA to target genes to functions has demonstrated the existence of patterns or modules in post-transcriptional gene regulation mediated by miRNAs. Modules seem to evolve in a fashion that allows miRNAs to coordinately exert cooperative effects on functionally related target genes . CurrentSeveral studies have sought to describe features involved in the miRNA-target recognition, such as seed match  and siteIn this paper, we seek to test the hypothesis of whether miRNAs regulate with similar intensities the expression profiles of genes enrolled in closely related biological functions. Using an in-depth detailed analysis of a broad set of global expression data of target messenger RNAs after perturbation of the expression of 10 selected miRNAs, we characterized the differential level of regulation/modulation of targets expression profiles and its potential relationship to the different functions exerted by miRNAs. We discovered a complex and highly organized scheme of gene expression levels control, which depends on the intensity of repression of the miRNA over target genes and it is structured into interdependent regulatory cluster. Our analysis shows that regulatory clusters are not limited to miRNA canonical targets, but also rely on other types of miRNA regulation which comprises a significant part of the miRNA regulatory networks, thereby clarifying the mechanisms underlying the functional activity of different miRNAs and facilitating a more meaningful analysis of their enrichment into distinct biological processes.By analyzing gene expression alterations following the transfection of 10 miRNAs in HeLa cells , we foup-value < 0.05; After, we performed functional enrichment analysis on each regulatory cluster in order to identify putative regulatory properties of such clusters. Interestingly, we observed that for six out of the 10 analyzed miRNAs , there was no correlation between the number of enriched terms and the number of the mRNAs within each cluster  is randomly distributed or not, we performed an identical enrichment analysis on random datasets constituted of artificial clusters containing the same number of genes of each analyzed cluster, and tested their distribution through a chi-squared test . Only seAlong with the observed differential quantitative distribution of enriched terms among clusters, we proposed two possibilities for what we would expect from a distinctive qualitative distribution: (i) several enriched terms would take part in more than one cluster, or (ii) enriched terms would tend to be present only in a unique cluster. Interestingly, by comparing the functional enriched terms within each cluster, we observed almost no overlap between clusters .As shown in Regulatory clusters from distinct miRNAs can also be enrolled in correlated biological functions. For instance, miR-9 cluster (0.047:0.402), miR-124 cluster (−0.576:−0.091), miR-128a clusters (−0.413:−0.106) and (0.073:0.339) control genes related to “cell cycle process” . These rTo access how many of the miRNA-responsive genes were, in fact, interacting with the miRNAs, we performed a target prediction analysis using TargetScan 7  target pIt is widely known that target prediction tools still return a considerable number of false negative results. To address this issue, we accounted all targets predicted by TargetScan (not only the conserved ones), in order to maximize sensitivity values and obtain a better estimation regarding the proportion of genes with or without target sites among the responsive genes. However, to access if we were still missing a significant amount of data, we searched for experimentally validated targets of the 10 miRNAs to evaluate how many of them were not predicted by TargetScan. We identified 699 validated targets, of which 354 (an average of 35.4 targets per miRNA) had not been predicted as targets by TargetScan  showed a smaller proportion of predicted targets than cluster (4.252:6.957). However, this may be an artifact caused by the small number of perturbed genes (only seven) in the (−6.795:−6.473) cluster. Here, we describe canonical interactions as the silencing properties of miRNAs over their target genes.Additionally, even though most of the enriched biological pathways contain genes predicted as canonical targets of the analyzed miRNA A, some bHomo sapiens, Pan troglodytes, Macaca mullata, Mus musculus, Rattus norvegicus, Bos taurus, Canis familiaris, Monodelphis domestica, Gallus gallus, and Xenopus tropicalis) using TargetScan7 and compared the Context++Score obtained for each interaction. We found the Context++Score has shown a high correlation to the fold change of the target mRNA after miRNA transfection  and rodents (M. musculus and R. norvegicus) exhibited a closer pattern within these groups than to other species. Additionally, placental mammals  showed more similar Contex+t+Scores among each other than in relation to the external group . In the same way, Context++Scores for miR-9 and its targets are closer among mammals, slightly increasing the divergence with phylogenetic distance.We also observed that phylogenetically related groups presented more similar Context++Scores for a given miRNA and its targets compared to more distant groups. For instance, miR-1 target regulation in primates , such as PIWI-interacting RNAs (piRNAs), tRNA-Derived Fragments (tRFs), small nuclear RNAs (snRNAs) and small nucleolar RNAs (snoRNAs) would act in a similar manner or not. However, for some ncRNAs such as piRNAs, this becomes difficult to associate, because the rules governing piRNAs target recognition are yet to be fully understood. It is known that piRNAs possibly do not have recognition elements in their targets as do have miRNAs, although some useful nucleotide signatures for its identification exist (such as an Adenine in position 10 and a Uracil in position 1) . HoweverEven though most of the knowledge obtained regarding miRNA regulation comes from the study of canonical interactions, we demonstrate that they comprehend only a fraction of the miRNA regulatory universe, indicating that miRNAs may undertake a larger and more complex regulatory mechanism than simply interact with its target genes to promote mRNA/protein down-regulation. Although we have sought to reduce methodological bias by accounting all miRNA targets predicted by TargetScan (and not only the conserved ones), miRNA target prediction still lacks in sensitivity, often returning false-negative results. Our analysis demonstrated that approximately 51% of the validated targets of the analyzed miRNAs were not predicted by TargetScan. However, this number represents only a small fraction of the total predicted targets . It is important to keep in mind that the yet limited number of validated targets available in the literature  makes it very difficult to estimate the real proportion of missing interactions. The main reason for that is the possible bias when choosing candidate targets to validate. Once the target prediction tools available contain distinct parameters, they identify distinct sets of predicted targets. This creates a scenario in which target prediction tools tend to identify more or less validated targets depending on the frequency and purpose of its use . In this sense, the validation of an increasing number of new interactions as well as new updates of the target prediction tools will allow a more detailed analysis of the real proportion of canonical and non-canonical regulation done by miRNAs.As expected, we observed that the relative number of non-canonical targets was inversely related to the potency of target gene down-regulation. Clusters in which down-regulation was stronger had more interactions predicted by TargetScan than clusters with weak down-regulatory efficiency or even up-regulated genes. However, even within the clusters with stronger down-regulation, about half of the perturbed genes were not predicted by TargetScan as targets.Several studies have highlighted atypical mechanisms in which miRNAs may perform functional regulation . For exaC. elegans, can recruit other proteins to promote autoactivation and final maturation of its own precursors, respectively.The similar number of down- and up-regulated genes identified after miRNA transfection also suggest a massive role of miRNAs as gene enhancers. Previous studies have described the ability of miRNAs to up-regulate protein output post-transcriptionally ,31,32 byInterestingly, some up-regulated mRNAs were also predicted as bonafide targets of the 10 transfected miRNAs. In this case, it is likely that the miRNA promotes up-regulation of that gene (either by direct or indirect regulation as described above) and also directly modulates its protein output by fine-tuning its expression  38]..38].Altogether, such miscellaneous of silencing-independent properties of miRNAs could help explain the massive number of observed mRNA fold-changes without a direct interaction predicted by TargetScan. Future studies focusing on these unusual mechanisms, which still stand for a very limited number of reported cases, may help to better address miRNA functioning under distinct biological contexts. Currently, we still lack predictive tools and computational models to evaluate the impact of these regulations on a large scale.The realization that miRNAs are able to coordinate the regulation of highly distinct biological functions by sorting them into regulatory clusters raises the question of how miRNAs perform such clusterization. Although our analysis per se may not be sufficient to solve that question in full, the vast knowledge regarding miRNA properties allows us to draw hypothesis on how they could orchestrate such molecular mechanism.An important feature of miRNA regulatory dynamics is the occurrence of competition between all possible sites that can be targeted by a single miRNA. Usually, in a cell, the total number of available sites, accounted by the number of sites in all transcripts times the expression of each of them overpass the number of miRNAs available ,39, leadOur results disclose the underlying mechanism by which miRNAs accurately regulate cell transcriptome, opening new doors to the miRNA research field and raising several new questions that can lead to interesting forthcoming investigations.For example, one of the greatest limitations of using miRNAs as therapeutic targets is the high number of off-targets  and unde-MiRNAs shares similar regulatory properties with several non-coding RNAs (ncRNAs), such as tRFs and other siRNAs. Studies focusing on identifying if regulatory clusters also occurs on other ncRNAs regulation could bring new insights on regulatory properties of non-coding RNAs.-Target prediction analysis suggests that the regulatory clusters are a conserved phenomenon. This allows in vivo experiments to be performed in model organisms, bringing novel possibilities for studying how regulatory clusters would respond in systemic environments.-Features of target binding sites may be largely relevant to the inception of regulatory clusters. However, our data shows that non-canonical and indirect interactions are part of miRNA regulation. Prospective high-throughput studies would benefit from this knowledge while performing functional enrichment analysis to map biological functions controlled by miRNAs.Additionally, we list here some other researches that would benefit from the knowledge of miRNA regulatory clusters:We analyzed 10 previously published microarray datasets that evaluated mRNA expression alteration after the transient transfection of distinct miRNA mimics into HeLa cells: miR-7 (GSM210897), miR-9 (GSM210898), miR-128a (GSM210903), miR-132 (GSM210904), miR-133a (GSM210907), miR-142 (GSM210909), miR-148b (GSM210911), miR-181a (GSM210913), miR-1 (GSM37599), and miR-124 (GSM37601) . Since aFor each array, we clustered the mRNAs according to their fold changes  by the a  [http://revigo.irb.hr/; allowed similarity = 0.5) [We performed functional enrichment analysis on each cluster using the ToppCluster tool (mc.org/) , searchiy = 0.5)  in ordery = 0.5) . Finallyy = 0.5) .To generate the random sets we used all genes, regardless of their fold change. We randomly grouped the same number of genes identified in each cluster for each miRNA and, then, we performed the functional enrichment analysis of these random sets, using the same parameters used in item 4.3. Finally, we performed the chi-squared test to assess if the distribution of the enriched terms of the random sets  matches the distribution of the original enriched terms identified . For the chi-squared tests, we did not use REVIGO summarizing for either observed or expected values. All analyses were conducted in triplicates for each cluster.https://www.osbsoftware.com.br/produto/graphpad-prism/). For the random sets, the average number of terms of the triplicates were used.To identify the correlation between the number of enriched terms and the number of mRNAs contained in each cluster, we performed a linear regression analysis using GraphPad Prims software  evaluate the proportion of canonical and non-canonical interactions of the miRNAs analyzed, in both global context and within enriched biological pathways  dataset of the TargetsScan website  [For the evaluation of the proportion of canonical and non-canonical interactions, we obtained the complete miRNA target prediction data for the 10 miRNAs studied from the human (can.org) , irrespeHomo sapiens, Pan troglodytes, Macaca mullata, Mus musculus, Rattus norvegicus, Bos taurus, Canis familiaris, Monodelphis domestica, Gallus gallus and Xenopus tropicalis).For the evolutionary conservation analysis we obtained TargetScan data for conserved sites from 10 vertebrate species  [http://mirtarbase.mbc.nctu.edu.tw/php/index.php) [To access how many targets predicted by TargetScan were previously validated, and how many validated targets of the miRNAs analyzed were not predicted by TargetScan, we used TarBase   and miRTdex.php)  databasedex.php) ). Howevedex.php) . In thisWe used the Pathview package (v.1.22.1)  from R BP. troglodytes and G. gallus (miR-132), and C. familiaris (miR-142). Thus, such species were not integrated into the analysis of these miRNAs.We compared the TargetScan Context++Score of the miRNA-target interactions for 10 vertebrate species to evaluate if the degree of responsiveness of mRNA targets, inferred through mRNA fold changes after miRNA transfections, was conserved across vertebrates. In order to improve data visualization and comprehension, we only consider target sites that were predicted into all 10 selected species, with exception of miR-132 and miR-142 that did not have any prior predicted target for Our study reveals that miRNAs are able to simultaneously coordinate a wide range of biological functions by inducing similar fold changes in functionally related protein-coding genes, thereby aggregating them into specific regulatory clusters. Peculiarly, miRNA regulatory clusters are not limited to canonical targets, but also rely on non-canonical regulation which comprises a significant part of the miRNA regulatory universe. Our data also evidenced that this miRNA modus operandi is evolutionarily conserved implying that it must have been evolving adaptively in vertebrates. We envision that further experimental studies based on miRNA transfections in different cell lines and from several species will further help to pave the way for an even better picture of the modus operandi and evolution of miRNA regulatory clusters.


Cataract is the leading cause of blindness and low vision worldwide. Presently, cataract surgery is the only treatment for cataract and is very effective in restoring sight. In cataract surgery, the natural lens of the eye that becomes clouded is removed and replaced with an artificial intraocular lens. There are multiple techniques for removal of lens as well as many types of intraocular lenses available for implantation. For this reason, it becomes imperative to monitor the impact of different surgical techniques and different intraocular lenses on health-related quality of life (HRQoL) of the patients. This systematic review aims to evaluate HRQoL evidences on effects of different types of cataract surgeries and intraocular lenses on visual function and quality of life in age-related cataract patients.ClinicalTrials.gov, Current Controlled Trials and World Health Organization International Clinical Trials Registry Platform (WHO ICTRP) will be searched systematically. Two reviewers will independently screen studies using predefined inclusion and exclusion criteria along with the extraction of data, and assessment of methodological quality using a standard checklist.Databases like Cochrane, EMBASE, SCOPUS, NHS Economic Evaluation Database (NHS EED), Health Technology Assessment (HTA) database, MEDLINE, This systematic review will help in understanding how different types of cataract surgeries and intraocular lenses make a difference on quality of life of age-related cataract patients in terms of visual function and health-related quality of life. As the review attempts to bring together all the cataract-related HRQoL evidences pertaining to different cataract surgical techniques, different intraocular lenses and cataract-related complications, it will also identify gaps in evidence.CRD42018092377PROSPERO The online version of this article (10.1186/s13643-019-1113-6) contains supplementary material, which is available to authorized users. Health Technology Assessment (HTA) is an internationally accepted tool to inform decision making for better management of existing resources for Universal Health Coverage (UHC) –3. HealtThe first HTA topic selected for study at HTAIn secretariat was ‘Health Technology Assessment of intraocular lenses for treatment of age-related cataracts’. The request for this topic came from Rashtriya Swasthya Bima Yojana (RSBY), a Government health insurance scheme that has now been subsumed by the National Health Protection Scheme (NHPS) under Ayushman Bharat Mission , 8. UndeCataract is a condition in which the lens of the eye becomes clouded preventing clear vision . CataracPresently, cataract surgery is the only treatment for cataract, with high success rates, in restoring sight. The opaque lens of the eye is removed and replaced by an artificial intraocular lens , 18. CatThis systematic review aims to evaluate health-related quality of life evidences on effects of different cataract surgeries and intraocular lens implantation on visual function and quality of life in age-related cataract patients. This review also attempts to bring together all the cataract-related HRQoL evidences pertaining to different cataract surgical techniques, different intraocular lenses and cataract-related complications.CRD42018092377) [This protocol has been registered a priori in PROSPERO #CRD4208092377 [What are the changes in quality of life and visual function in age-related cataract patients after undergoing a particular type of cataract surgery and intraocular lens implantation?ClinicalTrials.gov, Current Controlled Trials and World Health Organization International Clinical Trials Registry Platform (WHO ICTRP) systematically. For each database, we will use words and expressions from controlled vocabulary  and free text searching. There will be no language or date restrictions for the literature search. Results will be managed using Covidence online software to facilitate automatic and manual removal of duplicate records, study screening and selection and record keeping. An example search strategy for EMBASE is given as Additional file Strategies will be designed to identify all relevant studies for HRQoL among age-related cataract patients. We will search the bibliographic electronic databases like the Cochrane Library including the Cochrane Database of Systematic Reviews (CDSR), Cochrane Central Register of Controlled Trials (CENTRAL), EMBASE, SCOPUS, the NHS Economic Evaluation Database (NHS EED), Health Technology Assessment database, MEDLINE, The studies will be included based on the following criteria:Population: adult patients with age-related cataracts without any other ocular comorbidityInterventions: phacoemulsification, small-incision cataract surgery (SICS), extracapsular cataract extraction (ECCE), intracapsular cataract extraction (ICCE), rigid lens, foldable lens, monofocal lens, multifocal lensSurgery: ICCE, ECCE, SICS, phacoemulsification.Lenses: rigid IOL, foldable IOL, monofocal IOL, multifocal IOLType of cataract: unilateral cataract, bilateral cataractComparators: The comparators for the study would be as given below:Outcomes: generic quality of life (QoL), HRQoL, vision-related quality of life (VRQoL), visual function (VF), cataract surgery-related complicationsStudy design: systematic reviews, meta-analysis, randomised controlled trials (RCTs), original observational studies, case-control studies, cohort studies, cost-effectiveness studiesIOLs for non-age-related cataracts like congenital or paediatric casesStudies reporting insufficient data for analysisStudies evaluating the association between QoL and causes of visual impairment which are unrelated to cataractStudies validating the test questionnaire or construct validation studiesOther eye disorders/diseases along with cataractNarrative review articles, study protocols, opinions, abstractsEligibility will not be restricted by year nor by language of publication.Studies will be selected for inclusion through a two-stage process. The first stage will be to screen the literature search results  identified by the search strategy to identify all citations that potentially meet the inclusion/exclusion criteria detailed above. The second stage will be a ‘preliminary’ data extraction to aid in the study selection process. Full manuscripts of selected citations that appeared potentially relevant will be obtained and preliminary data extraction will be done in order to ensure that all included studies have sufficient data pertaining to health-related generic quality of life and/or vision-related quality of life Table .Table 1PAll studies will be assessed by two reviewers and checked independently by a third reviewer before taking a final decision. At each stage, any disagreements will be resolved by discussion.Two authors will extract data using a standardised data extraction form Table  in MicroThe quality of included studies will be assessed by using a modified checklist recommended by Ara et al.  and insuStudies finally selected for inclusion in the review will be classified into different categories based on the type of cataract surgery and/or intraocular lens it involves and then each category will be classified into the subcategories based on the type of QoL instrument used in the study. Data will be separately analysed for categories and subcategories accordingly.τ2 > 0). The heterogeneity level will be investigated by using I2 index. An I2 value of more than 75% will be considered as an indication of significant heterogeneity. If there is evidence for substantial heterogeneity or inconsistency, we will not pool the results. In circumstances where pooling of studies will be deemed inappropriate, we will only provide a qualitative discussion of the findings with tables of findings and a narrative description.We will pool the data for each category by performing a meta-analysis if we get enough studies with comparable outcome measures and with similarities in terms of study design, population, instruments used for measuring the health states, value sets used for assigning utility weights and reporting results. A fixed effects meta-analysis will be applied to obtain the pooled effect size with 95% confidence interval (CI) or else a random effects meta-analysis would be performed  and type of instruments used  as there is no set pattern for reporting HRQoL. The diversity of studies is seen as some compare HRQoL between different types of intraocular lenses  where others compare it between different types of cataract surgical techniques . Still, others measure the HRQoL of cataract patients before and after surgery without mentioning much the details of type of surgical technique and IOLs used. Against this backdrop, the current study will be a broad review to systematically explore, critically appraise and provide insight to the effects of different types of cataract surgeries and intraocular lens implantations on visual function and quality of life in age-related cataract patients. If adequate evidence is available, this review may provide requisite data for decision modelling for economic evaluation studies. The review will also help in identifying gaps in availability of data that will help planning future modelling studies. As the review attempts to bring together all the cataract-related HRQoL evidences pertaining to different cataract surgical techniques, different intraocular lenses and cataract-related complications, it will also identify gaps in evidences and areas where future research is required.Additional file 1:PRISMA-P 2015 Checklist. (DOCX 30 kb)Additional file 2:An example search strategy for EMBASE. (DOCX 19 kb)