Large language models for preventing medication direction errors in online pharmacies.

Errors in pharmacy medication directions, such as incorrect instructions for dosage or frequency, can increase patient safety risk substantially by raising the chances of adverse drug events. This study explores how integrating domain knowledge with large language models (LLMs)-capable of sophisticated text interpretation and generation-can reduce these errors. We introduce MEDIC (medication direction copilot), a system that emulates the reasoning of pharmacists by prioritizing precise communication of core clinical components of a prescription, such as dosage and frequency. It fine-tunes a first-generation LLM using 1,000 expert-annotated and augmented directions from Amazon Pharmacy to extract the core components and assembles them into complete directions using pharmacy logic and safety guardrails. We compared MEDIC against two LLM-based benchmarks: one leveraging 1.5 million medication directions and the other using state-of-the-art LLMs. On 1,200 expert-reviewed prescriptions, the two benchmarks respectively recorded 1.51 (confidence interval (CI) 1.03, 2.31) and 4.38 (CI 3.13, 6.64) times more near-miss events-errors caught and corrected before reaching the patient-than MEDIC. Additionally, we tested MEDIC by deploying within the production system of an online pharmacy, and during this experimental period, it reduced near-miss events by 33% (CI 26%, 40%). This study shows that LLMs, with domain expertise and safeguards, improve the accuracy and efficiency of pharmacy operations.

Genomic regions underlying the species-specific mating songs of green lacewings.

Rapid species radiations provide insight into the process of speciation and diversification. The radiation of Chrysoperla carnea-group lacewings seems to be driven, at least in part, by their species-specific pre-mating vibrational duets. We associated genetic markers from across the genome with courtship song period in the offspring of a laboratory cross between Chrysoperla plorabunda and Chrysoperla adamsi, two species primarily differentiated by their mating songs. Two genomic regions were strongly associated with the song period phenotype. Large regions of chromosomes one and two were associated with song phenotype, as fewer recombination events occurred on these chromosomes relative to the other autosomes. Candidate genes were identified by functional annotation of proteins from the C. carnea reference genome. The majority of genes that are associated with vibrational courtship signals in other insects were found within QTL for lacewing song phenotype. Together these findings suggest that decreased recombination may be acting to keep together loci important to reproductive isolation between these species. Using wild-caught individuals from both species, we identified signals of genomic divergence across the genome. We identified several candidate genes both in song-associated regions and near divergence outliers including nonA, fruitless, paralytic, period, and doublesex. Together these findings bring us one step closer to identifying the genomic basis of a mating song trait critical to the maintenance of species boundaries in green lacewings.

A Novel Measure of Real-Time Perseverative Thought.

Perseverative thinking (PT), or repetitive negative thinking, has historically been measured using global self-report scales. New methods of assessment are needed to advance understanding of this inherently temporal process. We developed an intensive longitudinal method for assessing PT. A mixed sample of 77 individuals ranging widely in trait PT, including persons with PT-related disorders (generalized anxiety disorder, major depression) and persons without psychopathology, used a joystick to provide continuous ratings of thought valence and intensity following exposure to scenarios of differing valence. Joystick responses were robustly predicted by trait PT, clinical status, and stimulus valence. Higher trait perseverators exhibited more extreme joystick values overall, greater stability in values following threatening and ambiguous stimuli, weaker stability in values following positive stimuli, and greater inertia in values following ambiguous stimuli. The joystick method is a promising measure with the potential to shed new light on the dynamics and precipitants of perseverative thinking.

Nuclease resistance and protein recognition properties of DNA and hybrid PNA-DNA four-way junctions.

Nucleic acids possess unique biochemical features that make them ideal candidates to inhibit "difficult to target" proteins. The limited stability of nucleic acids in vivo presents a major obstacle to their development as drugs. Here, immobile four-way junctions (4WJs) are used to target the DNA-binding cytokine, High Mobility Group B1. Hybrid 4WJs composed of DNA and peptide nucleic acids (PNA) are investigated. PNA possess enhanced nuclease stability vs. DNA. 4WJs are incubated with Exonuclease III and DNase I. The nuclease assays show that 4WJs containing multiple PNAs possess significantly higher stability. Circular dichroism assays are used to probe the groove topology of 4WJs with the minor groove binder, DAPI. The CD data indicates that multi-PNA 4WJs possess altered minor groove dimensions that reduces DAPI binding affinity. Logic suggests that the minor groove of multi-PNA hybrids possess significant perturbations to the topology and local electrostatic environment that prevents proper binding/recognition by nucleases and thus enhances stability.

Detecting and Removing Sample Contamination in Phylogenomic Data: An Example and its Implications for Cicadidae Phylogeny (Insecta: Hemiptera).

Contamination of a genetic sample with DNA from one or more nontarget species is a continuing concern of molecular phylogenetic studies, both Sanger sequencing studies and next-generation sequencing studies. We developed an automated pipeline for identifying and excluding likely cross-contaminated loci based on the detection of bimodal distributions of patristic distances across gene trees. When contamination occurs between samples within a data set, a comparison between a contaminated sample and its contaminant taxon will yield bimodal distributions with one peak close to zero patristic distance. This new method does not rely on a priori knowledge of taxon relatedness nor does it determine the causes(s) of the contamination. Exclusion of putatively contaminated loci from a data set generated for the insect family Cicadidae showed that these sequences were affecting some topological patterns and branch supports, although the effects were sometimes subtle, with some contamination-influenced relationships exhibiting strong bootstrap support. Long tip branches and outlier values for one anchored phylogenomic pipeline statistic (AvgNHomologs) were correlated with the presence of contamination. While the anchored hybrid enrichment markers used here, which target hemipteroid taxa, proved effective in resolving deep and shallow level Cicadidae relationships in aggregate, individual markers contained inadequate phylogenetic signal, in part probably due to short length. The cleaned data set, consisting of 429 loci, from 90 genera representing 44 of 56 current Cicadidae tribes, supported three of the four sampled Cicadidae subfamilies in concatenated-matrix maximum likelihood (ML) and multispecies coalescent-based species tree analyses, with the fourth subfamily weakly supported in the ML trees. No well-supported patterns from previous family-level Sanger sequencing studies of Cicadidae phylogeny were contradicted. One taxon (Aragualna plenalinea) did not fall with its current subfamily in the genetic tree, and this genus and its tribe Aragualnini is reclassified to Tibicininae following morphological re-examination. Only subtle differences were observed in trees after the removal of loci for which divergent base frequencies were detected. Greater success may be achieved by increased taxon sampling and developing a probe set targeting a more recent common ancestor and longer loci. Searches for contamination are an essential step in phylogenomic analyses of all kinds and our pipeline is an effective solution. [Auchenorrhyncha; base-composition bias; Cicadidae; Cicadoidea; Hemiptera; phylogenetic conflict.].

Usability of Electronic Health Record-Generated Discharge Summaries: Heuristic Evaluation.

BACKGROUND: Obtaining accurate clinical information about recent acute care visits is extremely important for outpatient providers. However, documents used to communicate this information are often difficult to use. This puts patients at risk of adverse events. Elderly patients who are seen by more providers and have more care transitions are especially vulnerable. OBJECTIVE: This study aimed to (1) identify the information about elderly patients' recent acute care visits needed to coordinate their care, (2) use this information to assess discharge summaries, and (3) provide recommendations to help improve the quality of electronic health record (EHR)-generated discharge summaries, thereby increasing patient safety. METHODS: A literature review, clinician interviews, and a survey of outpatient providers were used to identify and categorize data needed to coordinate care for recently discharged elderly patients. Based upon those data, 2 guidelines for creating useful discharge summaries were created. The new guidelines, along with 17 previously developed medical documentation usability heuristics, were applied to assess 4 simulated elderly patient discharge summaries. RESULTS: The initial research effort yielded a list of 29 items that should always be included in elderly patient discharge summaries and a list of 7 "helpful, but not always necessary" items. Evaluation of 4 deidentified elderly patient discharge summaries revealed that none of the documents contained all 36 necessary items; between 14 and 18 were missing. The documents each had several other issues, and they differed significantly in organization, layout, and formatting. CONCLUSIONS: Variations in content and structure of discharge summaries in the United States make them unnecessarily difficult to use. Standardization would benefit both patients, by lowering the risk of care transition-related adverse events, and outpatient providers, by helping reduce frustration that can contribute to burnout. In the short term, acute care providers can help improve the quality of their discharge summaries by working with EHR vendors to follow recommendations based upon this study. Meanwhile, additional human factors work should determine the most effective way to organize and present information in discharge summaries, to facilitate effective standardization.

Conformation and protein interactions of intramolecular DNA and phosphorothioate four-way junctions.

The objectives of this study are to evaluate the structure and protein recognition features of branched DNA four-way junctions in an effort to explore the therapeutic potential of these molecules. The classic immobile DNA 4WJ, J1, is used as a matrix to design novel intramolecular junctions including natural and phosphorothioate bonds. Here we have inserted H2-type mini-hairpins into the helical termini of the arms of J1 to generate four novel intramolecular four-way junctions. Hairpins are inserted to reduce end fraying and effectively eliminate potential nuclease binding sites. We compare the structure and protein recognition features of J1 with four intramolecular four-way junctions: i-J1, i-J1(PS1), i-J1(PS2) and i-J1(PS3). Circular dichroism studies suggest that the secondary structure of each intramolecular 4WJ is composed predominantly of B-form helices. Thermal unfolding studies indicate that intramolecular four-way junctions are significantly more stable than J1. The Tm values of the hairpin four-way junctions are 25.2° to 32.2°C higher than the control, J1. With respect to protein recognition, gel shift assays reveal that the DNA-binding proteins HMGBb1 and HMGB1 bind the hairpin four-way junctions with affinity levels similar to control, J1. To evaluate nuclease resistance, four-way junctions are incubated with DNase I, exonuclease III (Exo III) and T5 exonuclease (T5 Exo). The enzymes probe nucleic acid cleavage that occurs non-specifically (DNase I) and in a 5'→3' (T5 Exo) and 3'→5' direction (Exo III). The nuclease digestion assays clearly show that the intramolecular four-way junctions possess significantly higher nuclease resistance than the control, J1.

Effect of Inpatient Medication-Assisted Therapy on Against-Medical-Advice Discharge and Readmission Rates.

BACKGROUND: Patients who present to the hospital for infectious complications of intravenous opioid use are at high risk for against-medical-advice discharge and readmissions. The role of medication-assisted treatment for inpatients is not clear. We aimed to assess outcomes prior to and after rollout of an inpatient buprenorphine-based opioid use disorder protocol, as well as to assess outcomes in general for medication-assisted therapy. METHODS: This was a retrospective observational cohort study at our community hospital in New Hampshire. The medical record was searched for inpatients with a complication of intravenous opioid use. We searched for admissions 11 months prior to and after the November 2018 buprenorphine protocol rollout. RESULTS: Rates of medication-assisted therapy usage and buprenorphine linkage increased significantly after protocol rollout. Rates of against-medical-advice discharge did not decrease after protocol rollout, nor did readmissions. However, when evaluating the entire group of patients regardless of date of presentation or protocol use, against-medical-advice discharge rates were substantially lower for patients receiving medication-assisted therapy compared with those receiving supportive care only (30.0% vs 59.6%). Readmissions rates were lower for patients who were discharged with any form of ongoing medication-assisted therapy compared with those who were not (30-day all-cause readmissions 18.8% vs 35.1%; 30-day opioid-related readmissions 10.1% vs 29.9%; 90-day all-cause readmissions 27.3% vs 42.7%; 90-day opioid-related readmissions 15.1% vs 33.3%). CONCLUSIONS: There is a strong association between medication-assisted therapy and reduced against-medical-advice discharge rates. Additionally, maintenance medication-assisted therapy at time of discharge is strongly associated with reduced readmissions rates.

A molecular phylogeny of the cicadas (Hemiptera: Cicadidae) with a review of tribe and subfamily classification.

A molecular phylogeny and a review of family-group classification are presented for 137 species (ca. 125 genera) of the insect family Cicadidae, the true cicadas, plus two species of hairy cicadas (Tettigarctidae) and two outgroup species from Cercopidae. Five genes, two of them mitochondrial, comprise the 4992 base-pair molecular dataset. Maximum-likelihood and Bayesian phylogenetic results are shown, including analyses to address potential base composition bias. Tettigarcta is confirmed as the sister-clade of the Cicadidae and support is found for three subfamilies identified in an earlier morphological cladistic analysis. A set of paraphyletic deep-level clades formed by African genera are together named as Tettigomyiinae n. stat. Taxonomic reassignments of genera and tribes are made where morphological examination confirms incorrect placements suggested by the molecular tree, and 11 new tribes are defined (Arenopsaltriini n. tribe, Durangonini n. tribe, Katoini n. tribe, Lacetasini n. tribe, Macrotristriini n. tribe, Malagasiini n. tribe, Nelcyndanini n. tribe, Pagiphorini n. tribe, Pictilini n. tribe, Psaltodini n. tribe, and Selymbriini n. tribe). Tribe Tacuini n. syn. is synonymized with Cryptotympanini, and Tryellina n. syn. is synonymized with an expanded Tribe Lamotialnini. Tribe Hyantiini n. syn. is synonymized with Fidicinini. Tribe Sinosenini is transferred to Cicadinae from Cicadettinae, Cicadatrini is moved to Cicadettinae from Cicadinae, and Ydiellini and Tettigomyiini are transferred to Tettigomyiinae n. stat from Cicadettinae. While the subfamily Cicadinae, historically defined by the presence of timbal covers, is weakly supported in the molecular tree, high taxonomic rank is not supported for several earlier clades based on unique morphology associated with sound production.

A Multi-hospital Before-After Observational Study Using a Point-Prevalence Approach with an Infusion Safety Intervention Bundle to Reduce Intravenous Medication Administration Errors.

INTRODUCTION: We previously found a high rate of errors in the administration of intravenous medications using smart infusion pumps. OBJECTIVES/DESIGN: An infusion safety intervention bundle was developed in response to the high rate of identified errors. A before-after observational study with a prospective point-prevalence approach was conducted in nine hospitals to measure the preliminary effects of the intervention. MAIN OUTCOME MEASURES: Primary outcome measures were overall errors and medication errors, with the secondary outcome defined as potentially harmful error rates. RESULTS: We assessed a total of 418 patients with 972 medication administrations in the pre-intervention period and 422 patients with 1059 medication administrations in the post-intervention period. The overall error rate fell from 146 to 123 per 100 medication administrations (p < 0.0001), and the medication error rate also decreased from 39 to 29 per 100 medication administrations (p = 0.001). However, there was no significant change in the potentially harmful error rate (from 0.5 to 0.8 per 100 medication administrations, p = 0.37). An intervention component aiming to reduce labeling-not-completed errors was effective in reducing targeted error rates, but other components of the intervention bundle did not show significant improvement in the targeted errors. CONCLUSION: Development and implementation of the intervention bundle was successful at reducing overall and medication error rates, but some errors remained and the potentially harmful error rate did not change. The error-rate reductions were not always correlated with the specific individual interventions. Further investigation is needed to identify the best strategies to reduce the remaining errors. CLINICAL TRIALS REGISTRATION: Registered at ClinicalTrials.gov, identifier: NCT02359734.

The confounding effects of hybridization on phylogenetic estimation in the New Zealand cicada genus Kikihia.

Phylogenetic studies of multiple independently inherited nuclear genes considered in combination with patterns of inheritance of organelle DNA have provided considerable insight into the history of species evolution. In particular, investigations of cicadas in the New Zealand genus Kikihia have identified interesting cases where mitochondrial DNA (mtDNA) crosses species boundaries in some species pairs but not others. Previous phylogenetic studies focusing on mtDNA largely corroborated Kikihia species groups identified by song, morphology and ecology with the exception of a unique South Island mitochondrial haplotype clade-the Westlandica group. This newly identified group consists of diverse taxa previously classified as belonging to three different sub-generic clades. We sequenced five nuclear loci from multiple individuals from every species of Kikihia to assess the nuclear gene concordance for this newly-identified mtDNA lineage. Bayes Factor analysis of the constrained phylogeny suggests some support for the mtDNA-based hypotheses, despite the fact that neither concatenation nor multiple species tree methods resolve the Westlandica group as monophyletic. The nuclear analyses suggest a geographic distinction between clearly defined monophyletic North Island clades and unresolved South Island clades. We suggest that more extreme habitat modification on South Island during the Pliocene and Pleistocene resulted in secondary contact and hybridization between species pairs and a series of mitochondrial capture events followed by subsequent lineage evolution.

Dextromethorphan/quinidine pharmacotherapy in patients with treatment resistant depression: A proof of concept clinical trial.

BACKGROUND: At least one-third of patients with major depressive disorder (MDD) have treatment-resistant depression (TRD), defined as lack of response to two or more adequate antidepressant trials. For these patients, novel antidepressant treatments are urgently needed. METHODS: The current study is a phase IIa open label clinical trial examining the efficacy and tolerability of a combination of dextromethorphan (DM) and the CYP2D6 enzyme inhibitor quinidine (Q) in patients with TRD. Dextromethorphan acts as an antagonist at the glutamate N-methyl-d-aspartate (NMDA) receptor, in addition to other pharmacodynamics properties that include activity at sigma-1 receptors. Twenty patients with unipolar TRD who completed informed consent and met all eligibility criteria we enrolled in an open-label study of DM/Q up to 45/10mg by mouth administered every 12h over the course of a 10-week period, and constitute the intention to treat (ITT) sample. Six patients discontinued prior to study completion. RESULTS: There was no treatment-emergent suicidal ideation, psychotomimetic or dissociative symptoms. Montgomery-Asberg Depression Rating Scale (MADRS) score was reduced from baseline to the 10-week primary outcome (mean change: -13.0±11.5, t19=5.0, p<0.001), as was QIDS-SR score (mean change: -5.9±6.6, t19=4.0, p<0.001). The response and remission rates in the ITT sample were 45% and 35%, respectively. LIMITATIONS: Open-label, proof-of-concept design. CONCLUSIONS: Herein we report acceptable tolerability and preliminary efficacy of DM/Q up to 45/10mg administered every 12h in patients with TRD. Future larger placebo controlled randomized trials in this population are warranted.

The frequency of intravenous medication administration errors related to smart infusion pumps: a multihospital observational study.

INTRODUCTION: Intravenous medication errors persist despite the use of smart pumps. This suggests the need for a standardised methodology for measuring errors and highlights the importance of identifying issues around smart pump medication administration in order to improve patient safety. OBJECTIVES: We conducted a multisite study to investigate the types and frequency of intravenous medication errors associated with smart pumps in the USA. METHODS: 10 hospitals of various sizes using smart pumps from a range of vendors participated. Data were collected using a prospective point prevalence approach to capture errors associated with medications administered via smart pumps and evaluate their potential for harm. RESULTS: A total of 478 patients and 1164 medication administrations were assessed. Of the observed infusions, 699 (60%) had one or more errors associated with their administration. Identified errors such as labelling errors and bypassing the smart pump and the drug library were predominantly associated with violations of hospital policy. These types of errors can result in medication errors. Errors were classified according to the National Coordinating Council for Medication Error Reporting and Prevention (NCC MERP). 1 error of category E (0.1%), 4 of category D (0.3%) and 492 of category C (excluding deviations of hospital policy) (42%) were identified. Of these, unauthorised medication, bypassing the smart pump and wrong rate were the most frequent errors. CONCLUSION: We identified a high rate of error in the administration of intravenous medications despite the use of smart pumps. However, relatively few errors were potentially harmful. The results of this study will be useful in developing interventions to eliminate errors in the intravenous medication administration process.

Complex within a Complex: Integrative Taxonomy Reveals Hidden Diversity in Cicadetta brevipennis (Hemiptera: Cicadidae) and Unexpected Relationships with a Song Divergent Relative.

Multiple sources of data in combination are essential for species delimitation and classification of difficult taxonomic groups. Here we investigate a cicada taxon with unusual cryptic diversity and we attempt to resolve seemingly contradictory data sets. Cicada songs act as species-specific premating barriers and have been used extensively to reveal hidden taxonomic diversity in morphologically similar species. The Palaearctic Cicadetta montana species complex is an excellent example where distinct song patterns have disclosed multiple recently described species. Indeed, two taxa turned out to be especially diverse in that they form a "complex within the complex": the Cicadetta cerdaniensis song group (four species studied previously) and Cicadetta brevipennis (examined in details here). Based on acoustic, morphological, molecular, ecological and spatial data sampled throughout their broad European distribution, we find that Cicadetta brevipennis s. l. comprises five lineages. The most distinct lineage is identified as Cicadetta petryi Schumacher, 1924, which we re-assign to the species level. Cicadetta brevipennis litoralis Puissant & Hertach ssp. n. and Cicadetta brevipennis hippolaidica Hertach ssp. n. are new to science. The latter hybridizes with Cicadetta brevipennis brevipennis Fieber, 1876 at a zone inferred from intermediate song patterns. The fifth lineage requires additional investigation. The C. cerdaniensis and the C. brevipennis song groups exhibit characteristic, clearly distinct basic song patterns that act as reproductive barriers. However, they remain completely intermixed in the Bayesian and maximum likelihood COI and COII mitochondrial DNA phylogenies. The closest relative of each of the four cerdaniensis group species is a brevipennis group taxon. In our favoured scenario the phylogenetic pairs originated in common Pleistocene glacial refuges where the taxa speciated and experienced sporadic inter-group hybridization leading to extensive introgression and mitochondrial capture.

Using Electroencephalography for Treatment Guidance in Major Depressive Disorder.

Given the high prevalence of treatment-resistant depression and the long delays in finding effective treatments via trial and error, valid biomarkers of treatment outcome with the ability to guide treatment selection represent one of the most important unmet needs in mood disorders. A large body of research has investigated, for this purpose, biomarkers derived from electroencephalography (EEG), using resting state EEG or evoked potentials. Most studies have focused on specific EEG features (or combinations thereof), whereas more recently machine-learning approaches have been used to define the EEG features with the best predictive abilities without a priori hypotheses. While reviewing these different approaches, we have focused on the predictor characteristics and the quality of the supporting evidence.

Isolation and characterization of microsatellite markers useful for exploring introgression among species in the diverse New Zealand cicada genus Kikihia.

The New Zealand cicada genus Kikihia Dugdale 1971 exhibits more than 20 contact zones between species pairs that vary widely in their divergence times (between 20,000 and 2 million years) in which some level of hybridization is evident. Mitochondrial phylogenies suggest some movement of genes across species boundaries. Biparentally inherited and quickly evolving molecular markers like microsatellites are useful for assessing gene flow levels. Here, we present six polymorphic microsatellite loci that amplify DNA from seven species across the genus Kikihia; Kikihia "northwestlandica," Kikihia "southwestlandica," Kikihia muta, Kikihia angusta, Kikihia "tuta," Kikihia "nelsonensis," and Kikihia "murihikua." The markers were developed using whole-genome shotgun sequencing on the 454 pyrosequencing platform. Moderate to high levels of polymorphisms were observed with 14-47 alleles for 213 individuals from 15 populations. Observed and expected heterozygosity range from 0 to 1 and 0.129 to 0.945, respectively. These new markers will be instrumental for the assessment of gene flow across multiple contact zones in Kikihia.

Commissioning reform in the NHS: will he who pays the piper ever really call the tune?

Since the purchaser/provider split was first introduced in the early 1990s, there have been successive attempts to enhance and strengthen the role of commissioners in the English NHS. Their role is to ensure that health services are planned and delivered in a way that meets the interests of patients and taxpayers rather than healthcare providers. The new coalition government has recently set out its proposals to transfer commissioning responsibilities from primary care trusts to a national NHS Commissioning Board and a set of general practice-led commissioning consortia. It is too early to say whether these reforms are likely to transform commissioning and finally place payers, rather than providers, in the driving seat of the NHS. However they unfold they are likely to have a significant impact on healthcare professionals in commissioning, primary care and specialist roles.

Cell cycle genes are the evolutionarily conserved targets of the E2F4 transcription factor.

Maintaining quiescent cells in G0 phase is achieved in part through the multiprotein subunit complex known as DREAM, and in human cell lines the transcription factor E2F4 directs this complex to its cell cycle targets. We found that E2F4 binds a highly overlapping set of human genes among three diverse primary tissues and an asynchronous cell line, which suggests that tissue-specific binding partners and chromatin structure have minimal influence on E2F4 targeting. To investigate the conservation of these transcription factor binding events, we identified the mouse genes bound by E2f4 in seven primary mouse tissues and a cell line. E2f4 bound a set of mouse genes that was common among mouse tissues, but largely distinct from the genes bound in human. The evolutionarily conserved set of E2F4 bound genes is highly enriched for functionally relevant regulatory interactions important for maintaining cellular quiescence. In contrast, we found minimal mRNA expression perturbations in this core set of E2f4 bound genes in the liver, kidney, and testes of E2f4 null mice. Thus, the regulatory mechanisms maintaining quiescence are robust even to complete loss of conserved transcription factor binding events.