Fabry disease is a monogenic disease characterized by a deficiency or loss of the α-galactosidase A (GLA). The resulting impairment in lysosomal GLA enzymatic activity leads to the pathogenic accumulation of enzymatic substrate and, consequently, the progressive appearance of clinical symptoms in target organs, including the heart, kidney, and brain. However, the mechanisms involved in Fabry disease-mediated organ damage are largely ambiguous and poorly understood, which hinders the development of therapeutic strategies for the treatment of this disorder. Although currently available clinical approaches have shown some efficiency in the treatment of Fabry disease, they all exhibit limitations that need to be overcome. In this review, we first introduce current mechanistic knowledge of Fabry disease and discuss potential therapeutic strategies for its treatment. We then systemically summarize and discuss advances in research on therapeutic approaches, including enzyme replacement therapy (ERT), gene therapy, and chaperone therapy, as well as strategies targeting subcellular compartments, such as lysosomes, the endoplasmic reticulum, and the nucleus. Finally, the future development of potential therapeutic strategies is discussed based on the results of mechanistic studies and the limitations associated with these therapeutic approaches. GLA deficiency in patients with Fabry disease results in abnormal glycosphingolipid metabolism and the progressive accumulation of Gb3 , gene therapy, and chaperone therapy . DespiteIn this review, we first analyze and discuss recent mechanistic research into Fabry disease, focusing on lysosomal function, autophagy, and lipid metabolism, as well as their connectivity. Subsequently, we systemically summarize the progress in the development of therapeutic modalities for treating Fabry disease, including ERT, gene therapy, and chaperone therapy , as wellin vitro models of Fabry disease -adenosine triphosphatases (V-ATPases), which use ATP hydrolysis to pump H+ into the lysosome, play a central role in maintaining the acidic environment of this organelle (pH = 5.2–6.1) and normal lysosomal function. Lysosomal substrate accumulation in cells leads to increased expression of V-ATPase. AMP-activated protein kinase (AMPK) is also activated in response to depleted ATP levels, and AMPK phosphorylation was observed to be increased in Fabry cardiomyocytes in an in vitro model of the disease , which is involved in a variety of signal transduction pathways, including the induction of autophagic activation and lysosomal biogenesis. Interestingly, mTOR activity has been reported to be decreased in Fabry podocytes  and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α) are the main regulators of mitochondrial biogenesis via gene transcription. When cellular autophagy is inhibited, the levels of the PPARα repressors histone deacetylase 3 (HDAC3) and nuclear receptor co-repressor 1 (NCoR1) continuously increase as they cannot be degraded in the lysosome  are upregulated in glycosphingolipid-treated fibroblasts , the major component of lipid droplets, can be reused by mitochondria or removed from cells by high-density lipoprotein (HDL). Interestingly, cholesterol metabolism has been reported to fluctuate in various Fabry disease models as well as in patients with Fabry disease . Some stroblasts . Additioroblasts . Moreoveroblasts . In a reroblasts . Patientroblasts  exert a l efflux . HoweverInflammation is a series of adaptive biological responses triggered by several internal or external stimuluses. Controlling inflammatory responses can help restoring host homeostasis and adaptations, while dysregulated inflammatory responses could induce tissue injury and even tissue fibrosis . Progresi. v. 1 mg·kg−1 every 2 weeks) and replagal  have both been approved by the United States Food and Drug Administration for the treatment of Fabry disease, and both have shown the potential to significantly reduce Gb3 deposition and improve the prognosis of patients. However, the clinical application of this approach is partly limited by poor biodistribution, a short half-life, and immunogenic responses after intravenous administration  compared with that for the commercially available enzyme. Importantly, Fabry male mice treated with PEGylated GLA display reduced anti-drug antibody production. Recently, extracellular vehicles (EVs) derived from GLA-overexpressing cells have been developed as a therapeutic vehicle for the treatment of GLA defects . Third, the treatment of central nervous system (CNS) manifestations in Fabry disease is hampered by the inability of the enzymes used for ERT to cross the blood-brain barrier; accordingly, strategies that allow these enzymes to access the brain parenchyma to exert their therapeutic effects merit further exploration. Transferrin was recently reported to be able to cross the blood-brain barrier and improve ERT effects through targeting the transferrin receptor that is highly expressed in brain endothelial cells , designed as carriers for the delivery of mRNA for nonviral gene therapy  also show promise as small-molecule therapeutic chaperones. HSPs comprise a family of endogenous chaperone proteins that play multiple roles in response to cellular stress, including facilitating protein folding. HSPs can be classified into several groups according to their molecular weight, which ranges from 12 to over 100 kDa. Importantly, HSPs are important mediators of various diseases, including cancer, myocardial infarction, and ischemic stroke . Additiot HSP 70 . These st HSP 70 . In an i. constructed a self-assembling micelle to mimic HSP 70 function by combining poly (-amino ester: hydrophobic microdomains)-block-poly (ε-caprolactone) and poly (ethylene oxide: hydrophilic chain segment)-block-poly (ε-caprolactone). The hydrophobic microdomains could selectively capture amyloid-beta (Aβ) and prevent its aggregation in a mouse model of Alzheimer’s disease  is where most protein folding occurs, and targeting the delivery of drugs to the ER has been applied in biomedicine . Shiga tNCT02489344).Substrate reduction therapy (SRT) refers to the application of molecular medications to reduce the storage of undigested macromolecules by inhibiting the synthesis of the corresponding substrates . The advThe mechanism underlying Gb3-induced organ dysfunction in Fabry disease remains largely unknown. Further studies on the fundamental mechanism involved in the pathophysiology of Fabry disease is expected to lead to improved strategies for its management. Autophagy provides new insights of how Gb3 accumulation and autophagic disorder benefit disease treatments and drug development. In this regard, the use of autophagic inhibitors may represent a promising avenue for the treatment of Fabry disease via reducing secondary lysosomal accumulation. Additional research efforts are required to reveal the therapeutic benefits of autophagic inhibitors. Previous evidence has revealed that activation of the AMPK-mTOR pathway has an important role in the modulation of autophagy. Thus, targeting processes upstream of the lysosomal signal for managing Fabry disease has shown great potential in preclinical studies. Interestingly, some studies have established a connection between Fabry disease and lipid hemostasis, which may provide new insights into disease progression and innovative therapies. Nevertheless, the therapeutic benefits of modulation of lipid hemostasis and inflammatory response have not been demonstrated, even in preclinical studies. Fundamental efforts are needed to further clarify the role of cholesterol metabolism and inflammatory activation in Fabry disease.With the progress of nanotechnology and nanoscience, the limitation of these modalities can be partly solved. Designing a lysosome-based subcellular-targeted nanoparticle may be a feasible strategy for disease intervention. This targeting strategy has resulted in excellent therapeutic outcomes in lysosomal storage disease by targeting nanoparticles to the lysosome and subsequently triggering high pH-dependent drug release. Several lysosomal targeting delivery systems have been developed in experimental studies. Nevertheless, issues associated with systemic application limit their clinical translation potential. Many medications are still premature for intracellular localization. Therefore, tissue targeting design should be considered prior to the lysosomes targeting design. Accordingly, combining subcellular targeting with active targeting may be an effective strategy for treating Fabry disease. Another constraint to achieving targeted and responsive therapy relates to the complexity of synthetic processing, which can influence the biosafety of nanocarriers. Thus, it is necessary to design simpler nanoplatforms to achieve sufficient therapeutic effects without long-term systemic toxicity.


The integrity of the genome is governed by multiple processes to ensure optimal survival and to prevent the inheritance of deleterious traits. While significant progress has been made to characterize components involved in the DNA Damage Response (DDR), little is known about the interplay between RNA processing and the maintenance of genome stability. Here, we describe the emerging picture of an intricate bidirectional coupling between RNA processing and genome integrity in an integrative manner. By employing insights from a recent large-scale RNAi screening involving the depletion of more than 170 components that direct  polyadenylation, we provide evidence of bidirectional crosstalk between co-transcriptional RNA 3′end processing and the DDR in a manner that optimizes genomic integrity. We provide instructive examples illustrating the wiring between the two processes and show how perturbations at one end are either compensated by buffering mechanisms at the other end, or even propel the initial insult and thereby become disease-eliciting as evidenced by various disorders. The exposure of cells to exogenous or endogenous stresses  can result in structural alterations of the DNA. To maintain the integrity of the genome, cells are equipped with intricate (and sometimes redundant) molecular networks that are ready to both detect and correct DNA damage. These networks take part in a protective cellular program known as DNA damage response (DDR) that, beyond merely detecting the damage, is also responsible for activating cellular checkpoints, regulating gene expression, repairing the lesions, and inducing apoptosis, in case the damage exceeds the cell-intrinsic repair capacity. The coordinated sequence of these events involves dedicated components, including DNA damage sensors  as well as effector proteins (including the checkpoint kinases Chk1 and Chk2), which in turn arrest the cell cycle until the damage is repaired. Although there is still much to be learned, the main processes underlying the DDR are understood in great detail . It is bThe present work focuses on the bidirectional connection between DDR and 3′end processing of genes, i.e. how the DDR affects 3′ end processing and how this, in turn, impinges on the DDR. Apart from illustrating a few well-known examples that connect DDR with 3′end processing, we are also showcasing insights from a recently performed large-scale screening  suggesticleavage and polyadenylation specificity factor (CPSF), cleavage factor I (CFI), cleavage factor II (CFII), and the cleavage stimulation factor (CstF)  before the mature RNA can be exported into the cytoplasm  and ref. CPA is r (CstF) . The recr (CstF)  ensures r (CstF) . 3′end pr (CstF) . Surprisr (CstF) . For exar (CstF) , both ofr (CstF)  leading r (CstF) . Moreover (CstF) . Consistr (CstF) . Similarcleavage . Thus, mcleavage . Here, tcleavage . Additiocleavage . Under DTaken together, these mechanisms involving core CPA factors during DDR evidence an important role of 3′end processing in minimizing the risk of generating inadequate polyadenylated mRNA isoforms resulting from damaging conditions.Although evidence linking 3′end processing and DDR exists, the underlying molecular connections are still poorly defined. Insights may emerge through the realization that several factors engaged in the DDR are also a part of the 3′end processing complex . This isThe efficient response to DNA damage requires the expression of multiple DDR genes for the repair process to begin. DDR expression must occur, despite the above-described global inhibition of transcription and 3′UTR processing . Due to Apart from processes acting in cis, 3′end processing of DDR genes can also be facilitated through determinants in trans. For instance, select genes that belong to the p53 pathway bypass the requirement for RNAP II phosphorylation on Ser2, which is usually required for proper 3′end processing . However, leads to rapid degradation of ARE-containing target mRNAs through recruitment of deadenylases  to AU-rich elements (ARE) typically residing in this region. These interactions can either stabilize or destabilize transcripts. For instance, the ARE-binding protein AUF1 can destabilize the growth arrest and DNA damage-inducible gene GADD45a under healthy conditions . Here, Aenylases . Conversenylases . CollectAlthough mounting evidence suggests that 3′end processing may be a key regulator taking part in genome stability and the damage response, the reciprocal role of CPA components in modulating the DDR remains elusive (see above). Evidence for a critical function of CPA for the maintenance of genome integrity, independent from a primary genotoxic insult, is provided by R-loops . R-loopsCPA regulated replication stress can however also take other forms (beyond R-loops defects) and different players come into action. For instance, CFIm depletion mediates a delayed activation of DNA damage checkpoint signaling and RNA polymerase II degradation following DNA damage  as well The role of CPA factors in safeguarding genome integrity has also been documented beyond their function in RNA processing. After ubiquitin-mediated degradation of RNAP II, chromatin must be made accessible to allow the repair machinery to access the lesion. Here, co-recruitment of CstF-50 with the ubiquitin escort factor p97 contributes to the displacement of ubiquitinated histones H2A, H2B, and nucleosome remodeling . MoreoveThese findings indicate that individual CPA components (and associated factors) can likely impair central processes that are directly involved in the suppression or the propagation of genotoxic stress. However, what is the evidence that CPA components can be more globally linked to the surveillance of genome integrity?Early reports on UV-irradiated sun-damaged fibroblasts suggested a link between the usage of different polyA signals (PAS) and genotoxic stress . The reghttp://shiny.imbei.uni-mainz.de:3838/trend-db/)  but also with other entities including cancer. This corresponds to the previously made observations indicating a central role of PCF11 for differentiation/dedifferentiation and developmental programs    . The DNAprograms .As one would expect based on the pervasive function of several CPA components for APA modulation  and the An interesting, yet to be explored, hypothesis arising from these findings is that such a coupling could serve as a buffering system conferring robustness to biological systems. This would ensure that the otherwise detrimental consequences arising from aberrant RNA processing (such as R-loop formation) result in the induction of compensatory mechanisms governing genome integrity ; Figure Collectively, CPA components that mediate RNA 3′processing and shape the transcriptome diversity are thus emerging as important pillars in the maintenance of genome integrity and vice versa. In view of the emerging opportunities to interfere with CPA (and APA) in a targeted manner  further Tools capable of elucidating polyadenylation and involved components in a transcriptome-wide manner  bear gre


JCI, Grudda et al. developed a PCR-based assay that differentiated the source of HBsAg and explored the contributing sources of HBsAg in patients on nucleos(t)ide analog antivirals. These findings provide a tool for understanding the contribution of iDNA in HBV infection and may guide therapies toward a functional HBV cure.Chronic hepatitis B virus (HBV) infection remains a major global health problem. Hepatitis B surface antigen (HBsAg) loss has been accepted as the definition of a functional HBV cure. Recent studies found that while covalently closed circular DNA (cccDNA) is the predominant source of HBsAg in hepatitis B e antigen–positive (HBeAg-positive) patients, integrated HBV DNA (iDNA) is the main source in HBeAg-negative patients. Consequently, achieving a functional HBV cure will require not only silencing of cccDNA but also iDNA. Assays that distinguish the source of HBsAg are needed to evaluate emerging therapies. In this issue of the  Hepatitis B virus (HBV) is a small DNA virus that infects the liver. Its entry into hepatocytes is facilitated by the sodium-taurocholate–cotransporting polypeptide (NTCP), a bile acid receptor, which contributes to its hepatotropism . After eS transcript expression, resulting in production of hepatitis B surface antigen (HBsAg)  genome instead of the rcDNA genome. These dslDNA forms may integrate randomly into the host genome by utilizing sites of host cell DNA breaks . Recentl (HBsAg) .Globally, an estimated 296 million persons have chronic HBV infection, resulting in approximately 820,000 deaths annually predominantly from complications of cirrhosis and hepatocellular carcinoma (HCC) . Two claS transcripts from HBeAg-negative patients as well as chimpanzees frequently lacked the 3′ ends that were present in S transcripts from HBeAg-positive patients and chimpanzees. Altering the siRNA sequence to target a different region resulted in a decline in HBsAg levels in both HBeAg-positive and HBeAg-negative patients. These findings led to the revelation that the source of HBsAg in HBeAg-positive and HBeAg-negative patients may differ, being predominantly cccDNA in the former and iDNA in the latter. This notion of differential sources of HBsAg during different phases of chronic HBV infection has since been confirmed by other investigators  patients, it had minimal effects on HBsAg levels in HBeAg-negative patients . Furthertigators , 14.The knowledge that HBsAg may be derived from both cccDNA and iDNA presents a challenge for using HBsAg loss as a definition of functional cure and an endpoint in clinical trials of new therapies, as HBsAg may remain positive despite complete silencing and even elimination of cccDNA.S transcripts are chimeric with host DNA and lack the common 3′ ends that all cccDNA-derived transcripts share  assay that can differentiate the source of HBsAg . The assts share . Thus, Hed HBsAg .S transcripts or cells that produce them, will be needed (Grudda and colleagues next used laser capture microdissection to analyze viral transcription from individual hepatocytes from paired liver biopsies of three patients. One patient with marked HBsAg decline had a corresponding reduction in infected cells with transcriptionally active cccDNA, while the two patients without HBsAg decline had a stable or increased percentage of cells producing HBsAg from iDNA. The authors suggested their findings support a role for NA in transcriptional silencing of cccDNA but not a reduction in the number of infected cells. They further suggested that in patients whose HBsAg is mainly derived from iDNA, continuation of NA therapy will not achieve additional reduction in HBsAg levels and different therapies, targeting iDNA-derived HBV e needed . These fe needed . It is pe needed .The findings from Grudda et al.  and othe


Glioblastoma multiforme (GBM) is known as the most aggressive and prevalent brain tumor with a high mortality rate. It is reported in people who are as young as 10 years old to as old as over 70 years old, exhibiting inter and intra tumor heterogeneity. There are several genomic and proteomic investigations that have been performed to find the unexplored potential targets of the drug against GBM. Therefore, certain effective targets have been taken to further validate the studies embarking on the robustness in the field of medicinal chemistry followed by testing in clinical trials. Also, The Cancer Genome Atlas (TCGA) project has identified certain overexpressed targets involved in the pathogenesis of GBM in three major pathways, i.e., tumor protein 53 (p53), retinoblastoma (RB), and receptor tyrosine kinase (RTK)/rat sarcoma virus (Ras)/phosphoinositide 3-kinase (PI3K) pathways. This review focuses on the compilation of recent developments in the fight against GBM thus, directing future research into the elucidation of pathogenesis and potential cure for GBM. Also, it highlights the potential biomarkers that have undergone extensive research and have promising prognostic and predictive values. Additionally, this manuscript analyses the advent of gene therapy and immunotherapy, unlocking the way to consider treatment approaches other than, or in addition to, conventional chemo-radiation therapies. This review study encompasses all the relevant research studies associated with the pathophysiology, occurrence, diagnostic tools, and therapeutic intervention for GBM. It highlights the evolution of various therapeutic perspectives against GBM from the most conventional form of radiotherapy to the recent advancement of gene/cell/immune therapy. Further, the review focuses on various targeted therapies for GBM including chemotherapy sensitization, radiotherapy, nanoparticles based, immunotherapy, cell therapy, and gene therapy which would offer a comprehensive account for exploring several facets related to GBM prognostics. Over the past century, therapeutic approaches for cerebral malignant tumors have remained a critical challenge. Though after several refinements in various techniques that range from neurosurgical options to the breakthrough of effective chemotherapeutic agents, improvements in radiotherapy, biotechnological advancements for targeted delivery, etc., have led to an extended and improvised survival in patients suffering from many other types of brain tumors, except glioblastoma multiforme (GBM) . It is sde novo types, as they get developed without a known precursor, or secondary ones when a low-grade tumor develops into a GBM over time. Most of the GBMs are primary, and patients with primary GBMs are older and have a worse prognosis than those with secondary forms of GBMs. Earlier, GBMs were assumed to develop from glial cells, however, the research data in the last decade and a half reveals that they can come from a variety of cell types with neural stem cell-like features  are not involved in the survival of GBM patients, instead of the fact that mut-p53 has been linked to a worse cancer prognosis. The most often disrupted biomarkers of the p53 pathway are a homozygous deletion of the cyclin dependent kinase inhibitor 2A (CDKN2A)/ARF gene, common in GBM patients. ARF acts as a tumor suppressor by promoting MDM2 degradation and preventing the loss of p53 tumor suppressor activity. Consequently, ARF loss is also associated with tectonic family member 1 (TCTN1) overexpression, involved in various biological processes, including better GBM cell proliferation. GBM tumors with substantial adipocytic-like tumor cell differentiation have also been associated with CDKN2A/ARF deletion. As a result, the phenotypic differs from that of typical GBMs and CDKN2A/ARF and p53 are co-dysregulated. Their absence exerts conflicting effects on GBM malignancy  7Li, the nuclear reaction between 10B and thermal neutrons produce high linear energy transfer (LET) and 7Li particles. Because particles and 7Li have such a short path length (9 m), high-LET irradiation of 10B-loaded tumor cells is possible while causing minimal damage to 10B-unloaded normal cells. The number of these particles and the selectivity of the boron compound in tumor cells determines the efficiency of BNCT. The minimal tumor dose of gross tumor volume (GTV) in the BNCT clinical investigation was roughly 30 Gy .Salmonella strains) have been genetically changed and utilized to carry anticancer medications to tumor sites. Earlier, bacteria were employed as an oncolytic treatment for malignant brain tumors and the use of genetically modified bacteria (GM bacteria) that selectively attack tumor cells while preserving normal brain tissue was the most potential one against GBM. Bacteria that have been genetically modified can be kept alive within the boundaries of a brain tumor, where they thrive and are unable to survive if they escape into the brain or other tissues. Once the tumor has been destroyed, microorganisms can be eliminated with a bactericidal antibiotic  have found widespread use in oncology theranostics. These IONPs are highly biocompatible and once degraded, they can be integrated into the system’s iron cycle. Furthermore, the surfaces of IONPs can be changed to (i) increase biocompatibility and aqueous colloidal stability; (ii) prolong their overall circulation duration within the blood by limiting non-specific phagocytosis even by the reticuloendothelial system (RES); (iii) offer an active surface area for drug loading. For surface modification of IONPs, several biopolymers including PEG, poly(ethylene imine) (PEI), dextran, and chitosan16 it have been used , type 2 (NK-2R) and type 3 (NK-3R). Phospholipase C is activated by NK-1R, which produces inositol triphosphate. Overexpression of NK-1R in glioma tumors has led to the development of NK-1R targeted therapies for the treatment of glioma tumors .Recombinant interleukin-2 (IL-2) is a lymphocyte-produced immunoregulatory protein with a wide range of immunological effects, which is recently approved. It is a specific biological response modifier because it has no known anticancer action and instead causes cytotoxicity by activating effector cells such as T cells, natural killer cells, and lymphokine-activated killer cells. In patients with renal cell carcinoma and melanoma, recombinant IL-2 has shown efficacy with objective response rates of around 15–20% .Owing to excellent qualities over MAbs and traditional chemotherapeutic drugs, recombinant immunotoxins (RITs) are a viable method for GBM therapy. For starters, RITs have a substantially lower molecular size than MAbs which allows them to infiltrate solid tumors more efficiently. Second, RITs have the same selectivity as MAbs, but they are much more powerful and have no known drug resistance mechanisms. Third, unlike typical chemotherapy medications, RITs are capable of killing non-dividing cells that are dormant. Finally, RITs have little drug cross-resistance and are effective in the treatment of chemo-refractory cancer. Immunotoxins have been created using MAbs or endogenous specific ligands, as well as protein toxins. Immunotoxins have progressed over time and through technological advancements and the immunotoxins currently in use are of the third generation. Most RITs use modified toxin components derived from pseudomonas exotoxin A (PE) or diphtheria toxin (DT). Human endogenous cytotoxic enzymes such as RNase, granzyme B, and death-associated protein kinase 2 have also been utilized in some RITs to reduce the immunogenicity generated by PE and DT components. Some researchers refer to these immunotoxins as fourth-generation immunotoxins. However, these human endogenous enzymes have significantly lower activity than PE and DT, resulting in poor anticancer effectiveness . The mosin vivo proliferation, long duration of the activity, and the capacity to transfer genes to battle tumor immune evasion. Multiple CAR-T cell infusions targeting the tumor-associated antigen IL-13 receptor alpha 2 (IL13Rα2) resulted in regression of GBM in patients with persistent multifocal GBM, with no adverse effects in the resected cells. After the tumor cavity was discovered, infusions were observed in the ventricular system. Intravenous injection of autologous CAR-T cells with the target of mutation in EGFRvIII in adults with chronic GBM is feasible and safe, with no evidence of off-tumor damage or cytokine release syndrome [Chimeric antigen receptor (CAR) T cells integrate a MAb’s antigen binding site with a T cell’s signaling apparatus, allowing antigen recognition to be unrestricted by major histocompatibility complex (MHC), removing one of cell therapy’s major hurdles. CAR-T technology uses retroviral or lentic  to create CARs that impose arbitrary specificity on immune effector cells like T cells. The patient is subsequently given these reprogrammed T cells. CAR-T cell targeting is comparable to MAb targeting, but with the added advantages of active passage to tumor areas, Neural stem cells (NSCs) that have been implanted inside the human body are demonstrated to relocate to GBM and diffuse inside the tumor in experimental animals, implying that they could be employed for targeted therapies as delivery systems, commonly called gene therapy. Genetically engineered NSCs have been shown to precisely target GBM cells and limit tumor growth. NSC-based gene therapy for brain malignancies is important because it uses the tumor tropism of these cells to give effective tumor-selective therapy. Human embryonic stem cells (hESCs) have been shown to reduce tumor development and prolong survival after being injected intravenously into GBM xenografts in the brain .Fas/APO-1 (CD95) is a transmembrane protein belonging to the tumor necrosis factor-α/nerve growth factor receptor family that signals apoptosis in vulnerable target cells. Anti-Fas antibodies cross-link with Fas/APO-1, causing apoptosis in human glioma cell lines, suggesting that Fas/APO-1 mediated death could be a promising immunotherapeutic treatment for malignant glioma. It was discovered that a Fas-associated death domain protein (FADD)/Mort1 preferentially interacts with the intercellular domain of Fas/APO-1 .Gemcitabine  is a nucleoside analog that must be transformed into monophosphorylated, dephosphorylated, or triphosphorylated forms to operate as a nucleoside analog and achieve full cytotoxic action. The first two stages of phosphorylation are rate-limiting in this cascade. Deoxycytidine kinase (DCK) covers gemcitabine to gemcitabine monophosphate in mammalian cells. The gemcitabine diphosphate is then produced by uridine monophosphate kinase (UMK). Antitumor action is elicited by gemcitabine derivatives that influence intracellular nucleotide synthesizing enzyme nucleotide pools or directly impede DNA synthesis. The diphosphate form inhibits the nucleotide synthesizing enzyme ribonucleotide reductase, whereas the triphosphate form binds to the elongating DNA strand and prevents DNA replication during the cell cycle .de novo protein synthesis, and they are found in several viruses [The product enzyme for the suicide gene should be missing or present at low levels in the host. It should have high catalytic properties so that tumor cells can convert this prodrug even when the substrate concentration is low. Following are the conditions that must be met for a treatment agent to be termed optimal, 1) non-toxic or minimal toxic medicine before enzymatic activation and extremely toxic afterward, 2) prodrug must penetrate and distribute itself in the tumor, 3) high affinity for the suicide gene that has been transduced but a low affinity for the cellular enzyme, and 4) long enough half-life to destroy the tumor and prevent the drug from being lost before reaching its concentration. TK is a 376-aminoacid protein produced by one of the immediate early (IE) genes. IE genes are genes that express rapidly and transiently in the absence of  viruses .EGFR promote uncontrolled proliferation and synthesis of the gene encoding normal EGFR or a shortened form called EGFRvIII. Because pharmaceutical EGFR inhibitors are in low supply, RNA interference (RNAi) might be an appropriate technique to target EGFRvIII to destroy cancer cells in the brain while sparing healthy cells. GBM cells treated with small interfering RNA (siRNA) in vitro showed reduced EGFR and catenin gene expression, as well as a significant reduction in their capacity to migrate and invade. This could be an effective treatment for human GBM, and more in vivo research is needed [Since 1968, the antisense messengers are generated and destroyed naturally during DNA replication, according to scientists. Fluorescence microscopy and flow cytometry can be used to study antisense oligonucleotides in combination with the photoluminescent amino-terminated polyamidoamine dendrimer. Antisense sequences can block messenger RNA translation, preventing the production of specific proteins. For nearly two decades, many tumor therapies focused on the antibodies injections which have been directed against certain antigens and stem cells. Because of the tumor’s non-selective affinity, the results were unsuccessful. Furthermore, intravenous administration of protein-coupled to an isotope has raised the issue of the protein distribution’s restricted specificity. After then, the researchers took a completely different strategy to the study, attempting to halt the creation of insulin-like growth factor-1 (IGF-I), which works directly on messenger RNA. The authors have effectively demonstrated that this phenomenon can occur in a variety of animals. Fortunately, the scientists were able to synthesize a large number of artificial antisense messengers. Antisense oligonucleotides coupled with the dendrimer can also be studied directly utilizing fluorescence microscopy and flow cytometry. Antisense sequences can stop messenger RNA translation, which prevents particular proteins from being made. In roughly 40–50% of GBM tumors, amplification, truncation, or mutations in the s needed . In a ges needed .The bottleneck of GBM treatment has been explored at various levels and approaches ranging from modified molecular therapies to neoteric therapies like immuno/gene/cell therapy and nanoparticles targeting too. The range of available genetic data guides research approaches, allowing scientists to pursue significant hypotheses supported by population-level genomic trends. In addition, emerging proteomic methods are significant resources that will help us better comprehend the intricacy of GBM tumors. Furthermore, genomic approaches have already identified a disease’s molecular fingerprint and pathways with a focus on GBM research. Despite the development of more detailed molecular classifications for GBM, targeted therapeutics for specific GBM subtypes need more exploration. Several unsuccessful clinical trials show that combination therapy will be the most effective option for GBM treatment and that medication design and pharmacokinetic features should be addressed. We discovered numerous genes which may play an essential role in GBM progression in this work, and these genes can be confirmed as prospective targets for GBM therapy development. It’s worth noting that the expression of genes against these targets could be a result of oncogenic stress rather than tumor growth, so target confirmation is still needed. These findings could refocus research efforts on glioma receptors and proteins that are crucial in the disease’s progression. In the future, genomic and proteomic methods will be common tools for identifying noninvasive biomarkers for diagnosis and therapy response, as well as for identifying novel therapeutic targets.


Competitive endogenous RNAs (ceRNAs) can regulate gene expression at the posttranscriptional level by competitively binding to microRNAs. In cancer, ceRNA activity is dysregulated. The imbalanced ceRNA-related regulatory network has been theorized to play an important role in cancer progression. In this review, we summarize the biological functions and clinical implications of long noncoding RNAs and circular RNAs as ceRNAs in nasopharyngeal carcinoma (NPC).Nasopharyngeal carcinoma (NPC) is a kind of head-and-neck malignant tumor, and distant metastasis treatment resistance is the leading cause of patient death. In-depth understanding of NPC progression and treatment failure remains to be explored. Long noncoding RNAs (lncRNAs) and circular RNAs (circRNAs) are noncoding RNAs that play key regulatory role in shaping tumor cell activities. Recent studies have revealed that lncRNA and circRNA function as competitive endogenous RNAs (ceRNAs) by regulating the posttranscriptional expression of genes as miRNA baits. The imbalanced ceRNA networks derived from lncRNA/circRNA-miRNA-mRNA interaction are widely found to contribute to NPC development. Herein, we summarize typical examples of lncRNA/circRNA-associated ceRNAs in recent years, which involved the potential molecular mechanisms in the regulation of proliferation, apoptosis, treatment resistance and metastasis of NPC, and discuss their potential clinical significance in the prognosis and treatment of NPC. Interpreting the involvement of ceRNAs networks will provide new insight into the pathogenesis and treatment strategies of NPC. However, ceRNA regulatory mechanism has some limitations currently. Screening the most effective ceRNA targets and the clinical application of ceRNA still has many challenges. The incectively . Epsteinectively . The occectively . Owing tectively . Currentectively . In addiectively . Cisplatectively ,12,13,14Long noncoding RNAs (lncRNAs) are noncoding RNAs (ncRNAs) longer than 200 bp. When initially discovered, lncRNAs were considered to have no biological function . HoweverAs a newly discovered type of noncoding RNA molecule, circular RNA (circRNA) forms a closed loop by backsplicing to protect it from ribonuclease R and it is stably expressed . Based oIn eukaryotes, some small RNA molecules are often used to guide gene silencing, such as small interfering RNA (siRNA) and miRNA, a mechanism known as RNA interference (RNAi) . MiRNA iThe ceRNA hypothesis was proposed by the Pier Paolo Pandolfi research group of Harvard Medical College in 2011 . HoweverWe searched several databases to identify relevant studies: Google Scholar, PubMed, and Medline. Search terms such as “ceRNA,” “NPC,” “non-coding RNA,” “lncRNA,” and “circRNA” were used in various combinations to obtain relevant information. We collected research papers on the ceRNA mechanism involved in NPC progression and integrated information to determine the content of the narrative review. Date restrictions (published ≥2017) were only used to obtain and summarize evidence of popular literature.Accumulating evidence from studies has shown that lncRNA and circRNA participate in the occurrence and development of various cancers by adsorbing miRNAs to inhibit the expression of target genes. As a novel mode of gene regulation, ceRNA reveals the potential function of ncRNAs. In this section, we review lncRNA/circRNA-miRNA-target gene networks regulating biological functions of NPC.Tumor cells have self-sufficient proliferative capacity, even without external stimulation. The ceRNA mechanism is one of the ways to maintain proliferation signal transduction in NPC cells. Almost all lncRNAs/circRNAs function as ceRNAs, which can promote the proliferation of NPC tumor cells. We focus on ceRNA networks that maintain the proliferation signal transduction ability of NPC tumor cells. Their specific molecular mechanisms are shown in For instance, tumor cells increase de novo fatty acid synthesis to promote proliferation. Oncogenic LNC02570 is upregulated in advanced stage NPC patients. High expression of LINC02570 significantly promotes NPC proliferation. As a ceRNA, LINC02570 upregulates the expression level of the key gene SREBP1 in the lipid biosynthesis pathway by adsorbing miR-4649-3p . SREBP1 The atypical cell cycle process caused by abnormal activation of the PI3K/Akt signaling pathway is considered a basic feature of many malignant tumors, including NPC. Excessive activation of this signaling pathway leads to abnormal cell proliferation. CeRNA networks at least partially promote NPC proliferation by inhibiting PTEN expression. PTEN is a tumor suppressor of NPC and a key negative regulatory gene of PI3K/Akt signaling pathway ,61.Low expression of PTEN predicts poor prognosis and shorter progression-free survival in NPC patients . OverexpTherefore, lncRNA/circRNA-associated ceRNA cross talk may be a potential therapeutic target for NPC. Understanding the specific mechanism of the ceRNA network involved in the regulation of proliferation-related genes and signaling pathways in NPC is of great significance for the development and treatment of NPC.Apoptosis is a programmed death, which plays a vital role in the development of organisms and defense against intracellular infection factors. Contrary to the effect of cell proliferation, most ceRNA-mediated abnormal expression of apoptosis-related genes inhibits apoptosis of NPC tumor cells and promotes their survival. B cell lymphoma 2 (Bcl-2) is an antiapoptotic protein. It inhibits apoptosis by regulating mitochondrial outer membrane and preventing the release of proapoptotic molecules into the cytoplasm. Bcl-2 is the main target of apoptosis molecular mechanism research, and is also the target gene of lymphoma and other cancer treatment. Some researchers have confirmed that Bcl-2 plays an important role in the antiapoptotic activity of EBV latent proteins  in the development of NPC. Xue et al. confirmed that the abnormal expression of Bcl-2 induced by lncRNA NEAT1/miR-129 was the main reason for suberoylanilide hydroxamic acid (SAHA)-induced apoptosis . InhibitImproving the radiosensitivity of NPC is a challenge clinically. CircRNA_000543 increased in radiation-resistant NPC tissue, and the overall survival of patients was poor . CircRNAThe Gene Expression Omnibus (GEO) database was used to identify lncRNAs with abnormally high PTPRG-AS1 expression. LncRNA PTPRG-AS1 promotes radiosensitivity of NPC cells as a ceRNA . LncRNA Recent studies have confirmed that lncRNA/circRNA-mediated ceRNA networks play an important role in regulating drug resistance of NPC cells . The trePaclitaxel is also one of the commonly used chemotherapy drugs for recurrent NPC. It inhibits tumor cell division by promoting tubulin polymerization and inhibiting its depolymerization. CPEB2 is an RNA-binding protein, and its role in tumors is contradictory. The identical CPEB2A of CPEB2 is a tumor suppressor and inhibits the translation of target mRNA, while the identical CPEB2 can competitively bind to the target RNA of CPEB2A, thus allowing subsequent translation. CPEB2B is a tumor promoter ,69. LncRAfter chemoradiotherapy, 20–30% of NPC patients still face metastasis . Their mEpithelial–mesenchymal transition (EMT) is a process in which epithelial cells lose their characteristics and acquire mesenchymal characteristics. In cancer, EMT is considered to be the key mechanism in metastasis. EMT is often regarded as a binary process of epithelial cells and mesenchymal cells .TGF-β is considered to be the most important signaling pathway of EMT induced by tumor cells. TGF-β-mediated EMT can occur in two ways. One is TGF-β signaling activates Smad2 and Smad3, and then forms a complex with Smad4 to transfer into the nucleus and mediates the inhibition and activation of target genes with EMT-related transcription factors (TF). Snail, Slug, and ZEB1 were identified as the main transcription activators of EMT. The loss of epithelial gene E-cadherin is the marker of EMT, and the upregulation of mesenchymal gene N-cadherin is also the key phenomenon of EMT . In addiRecent studies have shown that lncRNA/circRNA-mediated ceRNA networks induce tumor EMT formation by activating TGF-β signals, e.g., lncRNA AATBC regulates PNN by adsorbing miR-1237-3p. The interaction between PNN and ZEB1 can also promote EMT of NPC . Circ_00In addition, the reconstruction of the cytoskeleton can also trigger EMT formation. CircRNA participates in the assembly of the dynamic cytoskeleton through a ceRNA mechanism, thereby promoting metastasis in NPC. CircSETD3 competitively adsorbs miR-615-5p and miR-1538, and ultimately upregulates MAPRE1 .The upreVirus ncRNA can also be used as miRNA decoys to participate in ceRNA network regulation. This regulation pattern has been defined as “competitive viruses and host RNA” (cvhRNAs) . This noUnderstanding the ceRNA networks mediated by lncRNAs and circRNAs can provide valuable insights into the molecular mechanisms underlying the pathogenesis of a variety of human malignancies, including NPC .The TNM (tumor–lymph node–metastasis) staging system is a general guideline for cancer treatment. In clinical practice, it cannot accurately identify the risk of recurrence or distant metastasis in NPC patients. Stage II patients are generally considered at low risk. Final recurrence occurs in 15–20% of patients without adjuvant chemotherapy. Conversely, more than half of patients in stage III and IV were cured only by chemoradiation without adjuvant chemotherapy . Thus, nCircCRIM1, as a ceRNA, plays an important prognostic role in NPC metastasis and chemotherapy resistance . Hong etKnockdown of oncogenic lncRNAs/circRNAs may be an effective strategy to interfere with the progression of NPC, but how to deliver therapeutic nucleic acids accurately into cells based on the ceRNA model is a challenge. Based on the CRISPR/Cas system, the precision of lipid nanoparticles and polymer hydrogel nanoparticles to treat NPC may be a potential solution ,86.In a word, miRNAs are considered promising targets for cancer therapy. Given that miRNAs are key points in ceRNA networks, lncRNAs and circRNAs serving as ceRNAs may therefore serve as potential therapeutic targets.Since lncRNAs and circRNAs combine noncoding RNAs with protein-coding RNAs through complex ceRNA networks, they play indispensable regulatory roles in cancers like NPC. Exploring the specific molecular mechanism of lncRNAs/circRNAs as ceRNAs could provide targeted molecular therapy and clinical prevention strategies in NPC. Emerging studies have indicated that a variety of dysregulated lncRNA/circRNA-mediated ceRNAs may form networks to regulate multitudinous biological functions in NPC, including tumor cell proliferation, apoptosis, invasion, migration, metastasis, and treatment resistance.In fact, any transcript containing miRNA seed matches can function as a ceRNA. However, in the complex ceRNA networks, how to select an effective ceRNA as a target and how to specifically manipulate the target ceRNA without affecting other ceRNAs for the treatment of NPC? This is a challenging task for clinical applications of ceRNA. Moreover, the occurrence of NPC is closely related to EBV infection. Although some individual EBV-encoded circRNAs have been confirmed as ceRNAs to accelerate NPC progression, research on EBV-encoded lncRNAs/circRNAs as ceRNAs is still rare in NPC. Targeting EBV-encoded genes is also a specific therapeutic strategy for NPC. All in all, further understanding of the regulatory networks of lncRNA/circRNA-mediated ceRNAs originating from both tumor cells and EBV will provide more novel insight into the pathogenesis and therapy of NPC.


Cancer-related fatigue (CRF) is a common and debilitating consequence of cancer and its treatment. Numerous supportive care interventions have been developed to alleviate CRF; however, the diversity of outcome measures used to assess CRF limits comparability of findings. We aimed to evaluate the content and psychometric properties of measures used to assess CRF in interventions targeting fatigue, to inform the selection of suitable measures in future research.Included measures were identified from a systematic review of interventions targeting CRF. General characteristics of each measure were extracted, and item content was assessed against domains specified by the National Comprehensive Cancer Network (NCCN) definition of CRF. Psychometric properties were evaluated against COnsensus-based Standards for the selection of heath Measurement INstruments (COSMIN) criteria.Of 54 measures identified, 25 met inclusion criteria. Seventeen were fatigue-specific and eight a fatigue subscale or single item within a broader measure. Only 14 (56%) were specifically developed for cancer populations. Content coverage according to the NCCN CRF definition ranged from 0 to 75%. Evidence for fulfilment of COSMIN criteria in cancer populations ranged from 0 to 93%, with only five measures meeting > 70% of the COSMIN criteria.The Piper Fatigue Scale-Revised had good content coverage, but did not comprehensively address COSMIN criteria. The EORTC-FA12 and FACIT/FACT-F had excellent psychometric properties, with each capturing different aspects of fatigue. Ultimately, the choice of CRF measure should be guided by the research question and the CRF domains most relevant to the particular research context.The online version contains supplementary material available at 10.1007/s00520-022-07305-x. Cancer-related fatigue (CRF) is a common and debilitating consequence of cancer and its treatments, impacting over half of people with cancer . CRF is The National Comprehensive Cancer Network (NCCN) defines CRF as “a distressing, persistent, subjective sense of physical, emotional, and/or cognitive tiredness or exhaustion related to cancer and/or cancer treatment not proportional to recent activity, and interferes with usual functioning” . Thus, CTo address the significant burden of CRF on cancer patients and survivors, numerous psychosocial, lifestyle, and pharmacological interventions have been evaluated in an attempt to alleviate CRF . A recenAlthough previous reviews have identified and examined CRF measures –14, nonePatient-reported outcome measures were identified from a systematic review of systematic reviews and meta-analyses of interventions targeting CRF  content evaluation and (2) appraisal of psychometric properties.Items from included measures were assessed according to whether their content captured the eight aspects specified by the NCCN definition of CRF . SpecifiA search for psychometric validation data in cancer populations was conducted for each measure by reviewing all validation studies citing the original development paper. We also searched MEDLINE and PsychInfo databases using the following terms: *name of specific measure* + “development” or “validation” or “validity” or “reliability” or “psychometric”. The psychometric properties of included measures were assessed against international COnsensus-based Standards for the selection of health Measurement INstruments (COSMIN) , 17. TheExtraction of measure characteristics and analysis of the content and psychometric properties of each measure were conducted independently by two members of the reviewer team . Any discrepancies were resolved through team discussion until consensus was reached. The percentage of (i) NCCN CRF domains covered and (ii) COSMIN criteria addressed was calculated for each included measures. For the purpose of this review, measures were considered to have “acceptable” or “good” content coverage and psychometric properties if they captured 50% or 70% of NCCN-specified CRF domains and COSMIN criteria, respectively.Of the 54 measures identified, 25 were eligible for inclusion Fig. . CharactFatigue-specific items from the included measures were assessed against whether they captured the eight aspects specified by the NCCN definition of CRF Table . None ofTable Table This review evaluated a diverse range of patient-reported outcome measures used to assess CRF in intervention studies. Many of the reviewed measures had poor content coverage of CRF, and less than half met at least 50% of the COSMIN standards for selection of health measurement instruments. Only five (20%) measures had acceptable/good content coverage (≥ 50% of NCCN specified domains), and only 12 (48%) had acceptable/good evidence for psychometric validation (≥ 50% of COSMIN criteria) in cancer populations. This suggests that many measures used as a primary or secondary outcome measure were not fit for purpose for outcome assessment in intervention studies targeting fatigue in cancer populations. This is concerning as their inclusion may have contributed to inaccurate assessment of fatigue and poor-quality data , 19.With regard to the content of measures reviewed, the Piper Fatigue Scale-Revised most comprehensively captured the domains specified by the NCCN definition, covering 75% of domains, respectively. However, in terms of the COSMIN criteria, the Piper Fatigue Scale-Revised only had evidence for approximately one-third (29%) of the criteria. Thus, even though the Piper Fatigue Scale-Revised captured relevant content, further validation work in cancer populations is needed for this measure to be recommended for inclusion in future studies. Four other measures had acceptable content coverage, capturing 50% of NCCN specified domains . These measures had evidence for 57%, 71%, 0%, and 93% of the COSMIN criteria, respectively. Following further validation, the brevity of the PROMIS Fatigue Short Form (7 items) in particular may render it useful for inclusion in studies where minimising patient burden is a key consideration . Of note, PROMIS measures are also designed for computer adaptive testing, further minimising respondent burden . OverallThe most psychometrically robust measures were the European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire-Fatigue (EORTC QLQ-FA12) and the Functional Assessment of Chronic Illness Therapy-Fatigue (FACIT/FACT-F) questionnaire, each fulfilling over 80% of the COSMIN criteria in cancer populations. However, both measures are designed to be administered in conjunction with core quality of life measures , and their length (> 40 items) may limit their use. As such, their inclusion as an outcome measure may be especially useful for researchers wishing to examine quality of life outcomes in addition to fatigue. Further research is needed to examine the benefits and limitations of administering either of their fatigue-specific subscales alone. The NCCN domains covered also differed between these measures with the EORTC QLQ-FA12 assessing physical, emotional, and cognitive aspects of fatigue and the FACIT/FACT-F capturing only physical aspects of fatigue. Meaning, the choice of which measure to use will depend on the aspect of CRF most relevant to the particular research context.The interpretation of scores generated by CRF measures is facilitated by establishing a minimally important difference (MID), defined as the smallest change in a score considered to be clinically meaningful . The MIDOne measure did not assess any of the NCCN-specified domains  and four measures  did not have any psychometric data available in cancer populations. Clearly, these measures are not recommended for use as outcome measures until further work is done to assess their content validity and psychometric properties in cancer populations. Their use as outcome measures in intervention research highlights the need for researchers to actively follow minimum standards for selection of patient-reported outcome measures , the relevant timeframe of interest, whether a measure captures other outcomes of interest . and the existence of interpretation/scoring guidelines (i.e. cut-off scores/MIDs). When choosing between unidimensional and multidimensional CRF measures, there are advantages and disadvantages of both to consider. Unidimensional measures tend to be more limited in their assessment of CRF, whereas multidimensional measures can provide more comprehensive assessment. Unidimensional measures may be appropriate if the main outcome of interest is a single outcome , whereas multidimensional measures are more suitable for exploring intervention impacts on different fatigue domains. For example, an exercise intervention may improve physical fatigue but may not have any effect on emotional fatigue. Finally, unidimensional measures tend to be briefer and less burdensome so may be more suited for repeated longitudinal assessments and contexts where limiting questionnaire length is desirable. Although not the focus of this review, additional considerations especially important when selecting CRF measures for use in clinical practice include ease of administration, completion, and scoring. A future evaluation should focus on the clinical utility of CRF measures and their feasibility for use in clinical practice.A key strength of this review is the evaluation of a comprehensive range of patient-reported outcome measures identified from 30 review articles reporting over 300 interventions targeting CRF . AlthougThis review had some limitations. First, the COSMIN checklist was only released in 2010, and 24 96%) of the measures included in this review were developed between 1981 and 2009. It is possible that certain COSMIN criteria may have been addressed during the initial measure development phase, but these details were not reported due to the absence of reporting guidelines. To attempt to counteract this, we not only relied on primary development papers but also searched the literature for more recent development/validation papers. We also acknowledge that new data may have become available after our search for psychometric validation data in cancer populations was completed, so more recently published validation studies may have been excluded. We also limited our evaluation to key COSMIN criteria, and did not examine reports of the face validity or floor and ceiling effects of the CRF measures; future evaluation of these properties may further inform the selection of CRF measures. Moreover, this review did not evaluate the quality of available psychometric data. Consequently, some of the psychometric evidence may be based on validation studies with methodological flaws. In addition, we only assessed evidence from validation papers published in English, possibly excluding relevant validation studies published in other languages. Finally, we adopted the widely used NCCN CRF definition as our external criterion for assessing content coverage of CRF measures. However, we acknowledge there is a lack of a consensus-based definition of CRF in the literature 6% of the–26. If wIn summary, this review demonstrated the majority of measures used to assess CRF as a primary or secondary outcome measure in intervention studies were not fit for purpose. Based on the content and psychometric appraisal conducted, the EORTC-FA12 and FACT-F/FACIT-F are the most valid measures available for assessing CRF in research contexts. Although the Piper Fatigue Scale-Revised demonstrated good content coverage, further validation work in cancer populations is needed for this measure to be recommended for use. The development of a gold standard definition and conceptual framework of CRF would significantly help to inform and guide CRF measure selection in future studies.Supplementary file1 (PDF 117 KB)Supplementary file2 (DOCX 13 KB)Below is the link to the electronic supplementary material.


Point-of-care ultrasound (POCUS) training has become a standard component of Canadian emergency medicine (EM) residency programs. In resource-limited contexts, including Ethiopia, there is a critical shortage of local clinicians who can perform and teach POCUS. Our aim was to establish an introductory POCUS rotation within the EM residency program at Addis Ababa University (AAU) through The Toronto Addis Ababa Academic Collaboration in Emergency Medicine (TAAAC-EM).Through stakeholder engagement, the authors completed a quality improvement initiative and conducted a survey of AAU EM faculty and residents to understand which POCUS scans should be included in a core residency POCUS curriculum, “POCUS1”.17 residents completed the POCUS1 program and 16 residents completed the written survey. Focused assessment with sonography for trauma, inferior vena cava, and lung  were identified as core introductory topics. Seventeen residents completed the initial POCUS1 program. Three program graduates were supported to become “POCUS1 Master Instructors” to continue the program during the SARS-CoV-2 global pandemic.The authors identified the highest yield POCUS scans through a written survey, successfully introduced a sustainable core POCUS curriculum at AAU for EM residents, and graduated three master instructors for curriculum continuation. We outline the structure and materials for implementation of POCUS programs for EM trainees and staff in similar low- and middle-income countries. Point-of care ultrasound (POCUS) training has become a standard component of Canadian emergency medicine (EM) residency training programs. In resource-limited contexts, where formal imaging may be unavailable or unaffordable for patients, availability and clinical application of POCUS training is of even greater importance.In Addis Ababa, Ethiopia, there is a scarcity of radiologists serving the 10 public hospitals and population of approximately 5 million people .Compounding this need is a critical shortage of local clinicians who can perform and teach POCUS. AAU is a well-established academic institution, with multiple educational partnerships aimed at capacity-building. The Toronto Addis Ababa Academic Collaboration in Emergency Medicine (TAAAC-EM) began in 2010 and supports local faculty in providing a three-year EM residency training program. Within this context, we sought to establish an introductory POCUS rotation that could be sustainably run by local faculty .The development of EM POCUS services in low- and middle-income countries (LMICs) requires understanding the local context and balancing the need for diagnostic testing with the capacity to provide high quality training to EM residents . Other sThe authors sought to determine which EM POCUS scans are of the highest clinical benefit in the local context through stakeholder engagement and a written survey of faculty and residents and introduce a sustainable basic POCUS curriculum which could be sustained by academic faculty at the TASH ED at AAU.This was a quality improvement initiative conducted as part of the regular curriculum development process that took place at the TASH ED at AAU through six one-month teaching trips from visiting University of Toronto (UofT) faculty and residents from the Toronto Addis Ababa Academic Collaboration in Emergency Medicine (TAAAC-EM) from February 2018 to February 2020.Our curriculum design process was inspired by Kern's six step approach : 1) IdenIn order to create appropriate skills-based objectives, we conducted a written survey of AAU EM faculty and residents to determine which POCUS scans should be prioritized in a PGY (post-graduate year) 1 POCUS rotation (Appendix 1). POCUS scans addressed in the survey included: focused assessment with sonography in trauma (FAST), inferior vena cava (IVC), cardiac, lung , gallbladder, abdominal aorta, advanced abdominal , renal and bladder, first trimester pregnancy, deep venous thrombosis (DVT), ocular, musculoskeletal, endotracheal tube placement confirmation, and procedural . After the results of the survey were analyzed, three scans were selected for the POCUS1 curriculum. All participants completed a written pre-scanning quiz, performed in-person supervised scans for each selected application, and completed a supervised visual and written exam. Highly performing residents were selected through an application process to become master instructors to sustain the POCUS1 curriculum. As this was a quality improvement initiative and not research governed by the research and ethics board, REB approval was not indicated.Seven PGY1 residents, ten PGY2 residents, and seven EM faculty participated in this study. Sixteen (94%) residents and three (43%) EM faculty completed the survey. The most requested scans from residents were FAST, cardiac, lung, DVT, ocular, and nerve block scans. Faculty members' most requested scans were advanced cardiac and endotracheal tube confirmation scans.After discussions with faculty at AAU, we narrowed the focus to (a) topics with greatest relevance to the TASH ED population, and (b) topics which could be feasibly taught by local POCUS-trained faculty: FAST, IVC, and lung.The POCUS1 rotation includes a flipped classroom model reviewing materials on image generation and interpretation. To be eligible to participate in in-person scanning, residents are required to pass a pre-scanning quiz with a score of 70% (Appendix 2). Then, each resident is required to perform 25 in-person supervised scans for each application and pass a supervised visual and written exam with a score of 100%. Educational materials and scanning techniques were based on the Canadian Emergency Department Echo (EDE) program .Throughout the study period, all 17 residents completed the POCUS1 program. The next step was introduction of a POCUS1 Master Instructor (MI) certification. The SARS-CoV-2 pandemic disrupted travel for in-person teaching by UofT faculty and the need for local instructors became more urgent. Three POCUS-trained faculty were practicing at TASH, including two who were pursuing advanced ultrasound training through the Ultrasound Leadership Academy. Through an application process, three residents were selected by POCUS-trained AAU faculty to become MI candidates.MI candidates underwent a virtual orientation co-taught by AAU and UofT POCUS faculty. MI candidates supervised junior resident scanning. They were supervised in real-time and provided monitoring and feedback by AAU faculty. MI candidates were provided with a series of POCUS images and descriptive techniques on their mobile devices for use while teaching junior residents at the bedside (Appendix 3). These prompts were embedded on instructors' smart phones and did not require an internet connection to access.Junior residents logged scans electronically using the EchoLog  application. A total of ten junior residents are currently undergoing the POCUS1 training under the supervision of the MI candidates and POCUS AAU faculty. The program will be continuously refined based on continuous feedback from learners and AAU faculty.We propose a feasible and sustainable model for establishing a core POCUS rotation embedded in an emergency medicine residency training program in LMICs.Adapting the Canadian EDE curriculum to the Ethiopian emergency medicine context, we did not include first trimester obstetrics and aortic scans in the POCUS1 curriculum. Obstetric patients in their first trimester bypass the TASH ED to attend a dedicated obstetrical emergency intake area run by obstetricians. Given that some graduates will practice outside of TASH, it may be important to introduce first-trimester obstetric scans in the future. For the pragmatic purpose of establishing the POCUS1 program, it was not feasible to have trainees perform scanning shifts in both the ED and dedicated obstetrics triage area.The abdominal aortic aneurysm (AAA) scan which is included in the Canadian EDE curriculum was also excluded from the POCUS1 curriculum. The mean age of patients undergoing ED POCUS in Addis Ababa was previously estimated to be 35 years, making the prevalence of AAA pathology less relevant in the Ethiopian clinical context .Although not typically included in Canadian introductory POCUS curricula, IVC and advanced lung scanning were considered essential to the POCUS1 rotation. IVC was felt to be high yield in the TASH ED where patients present frequently with hypovolemic or cardiogenic shock. Additionally, many patients may be boarded in the ED for several days due to limited hospital capacity, resulting in Ethiopian EM physicians providing substantial amounts of ongoing critical care. Serial IVC and lung scans assessing volume status have been previously identified as amongst the most clinically relevant scans in an Ethiopian context .Through TAAAC-EM we were able to establish an introductory POCUS curriculum and a Master Instructor program to create a sustainable initiative for POCUS training within an Ethiopian EM residency training program.While the POCUS1 and MI programs have been successful, we are mindful of the challenges of maintaining a POCUS curriculum. The largest barrier to self-sustaining programming is training a critical mass of local POCUS experts who can independently coordinate rotations over the long-term. Faculty at academic institutions in Ethiopia frequently balance multiple leadership roles with limited support, which makes teaching time-intensive image generation skills an organizational challenge. In retrospect, our initial curriculum model did not explicitly have AAU faculty leading the bedside scanning shifts. In the future, we plan to have UofT faculty provide a mentorship role now that we have a critical mass of trained POCUS1 certified practitioners.The second largest barrier to self-sustaining programming is the availability of functioning hardware. During the creation of our MI program, the ultrasound machine in the ED was only intermittently available, making it challenging to schedule scanning shifts. Engagement with the decision makers for ongoing support of POCUS infrastructure in the context of emergency residency training programs remains critical to the success of any academic POCUS program in LMICs.This study should be interpreted in light of several limitations. The POCUS1 curriculum scans are based off survey results from 16 residents and 3 staff and may not represent the highest yield scans for the broader emergency medicine community in Ethiopia, or in other LMICs. As well, the authors are participating in ongoing analysis of the POCUS1 and MI programs, and the sustainability of this program is not yet understood.Our future plans include introduction of an advanced POCUS rotation, continuing medical education opportunities for faculty, and establishing AAU as a POCUS center of excellence in East Africa. We will also be conducting regular program assessments and modifying curricula with local ultrasound leads in response to local needs.


Accurate human immunodeficiency virus (HIV) risk assessment can guide optimal HIV prevention. We evaluated the performance of risk prediction models incorporating geospatial measures.We developed and validated HIV risk prediction models in a population-based cohort in South Africa. Individual-level covariates included demographic and sexual behavior measures, and geospatial covariates included community HIV prevalence and viral load estimates. We trained models on 2012–2015 data using LASSO Cox models and validated predictions in 2016–2019 data. We compared full models to simpler models restricted to only individual-level covariates or only age and geospatial covariates. We compared the spatial distribution of predicted risk to that of high incidence areas (≥ 3/100 person-years).Our analysis included 19 556 individuals contributing 44 871 person-years and 1308 seroconversions. Incidence among the highest predicted risk quintile using the full model was 6.6/100 person-years (women) and 2.8/100 person-years (men). Models using only age group and geospatial covariates had similar performance  to the full models . Geospatial models more accurately identified high incidence regions than individual-level models; 20% of the study area with the highest predicted risk accounted for 60% of the high incidence areas when using geospatial models but only 13% using models with only individual-level covariates.Geospatial models with no individual measures other than age group predicted HIV risk nearly as well as models that included detailed behavioral data. Geospatial models may help guide HIV prevention efforts to individuals and geographic areas at highest risk. HIV risk prediction models incorporating geospatial covariates identified individuals and geographic areas at high risk in South Africa. Simple models using age and local HIV prevalence had similar performance to models based on detailed demographic and seuxal behavioral measures. Despite progress in expanding access to treatment and prevention services, human immunodeficiency virus (HIV) incidence in sub-Saharan Africa remains high. An estimated 870 000 people were newly infected in 2020, and annual incidence among adults exceeds 5 per 1000 population in several countries [Accurate and efficient identification of individuals at high risk remains a key challenge for HIV prevention programs. Several model-based HIV risk prediction tools have been developed for women , men whoCommunity-level measures of HIV prevalence, viral load, and treatment coverage offer an alternative approach to identifying individuals at elevated HIV risk. These measures are associated with HIV incidence  and act Using population-based data from a large prospective HIV cohort in South Africa, we developed and evaluated the predictive performance of gender-specific HIV risk prediction models. We compared the performance of models including both geospatial and individual-level covariates (full models) with models using only individual-level covariates  and models restricted to age and geospatial covariates . We also mapped the geospatial distribution of predicted risk from each model and compared the alignment with high incidence areas, as defined by the 2015 WHO PrEP guidelines (≥3 per 100 person-years ).We used data from a large demographic surveillance system (DSS) run by the Africa Health Research Institute (AHRI) in the Hlabisa sub-district of KwaZulu-Natal, South Africa . The souEligible individuals for this analysis were aged 15–54 years with an initial HIV-negative test and at least 1 subsequent HIV test. For individuals with a subsequent positive HIV test, we randomly imputed a single seroconversion date from a uniform distribution defined between the last HIV-negative test and first HIV-positive test . Follow-We constructed our models using a suite of time-varying individual-level and geospatial covariates. Individual-level predictors included 5-year age group, gender, marital status, education, employment, migration history, prior pregnancies or children, circumcision status, contraception use, number of sexual partners, and characteristics of the most recent partner. A full list of covariates and missingness frequency is available in the Supplementary Material . We estiWe modeled time to seroconversion separately for men and women using Cox proportional hazards with least absolute shrinkage and selection operator (LASSO) penalties . We seleWe validated each of the models by predicting hazard ratios in the validation dataset, and we evaluated model discrimination using AUROC. We evaluated model sensitivity by calculating the proportion of incident infections that occur within fixed percentiles of predicted risk for each model. We also calculated HIV incidence rates across quintiles of predicted HIV risk for each model.We evaluated how well our models identified geographic areas of high HIV risk by comparing the spatial distribution of predicted risk to the areas in which observed incidence exceeded 3 per 100 person-years. We estimated the spatial distribution of incidence by separately smoothing counts of cases and total person-time at risk using Gaussian kernels and then dividing the value from the cases surface by the value of the person-time surface across the study area. Because the number of incident cases is smaller and therefore leads to noisier geospatial estimates, we generated a single incidence surface across the study area by combining data from both men and women from 2012 to 2019 and increased the kernel bandwidth to 10 kilometers. We compared this surface to the spatial distribution of predicted risk over the same time period from each of the 4 models. For each modeled surface, we calculated the percentage of the area with incidence at least 3 per 100 person-years that was contained within the 20%, 40%, or 60% of the area with the highest predicted risk. We varied the incidence kernel bandwidth in sensitivity analyses. Geospatial analyses were conducted using ArcGIS , whereas all other analyses used R version 4.0.2.All participants provided written informed consent prior to the household-based interview and collection of dried blood spots. Approval for data collection and use was obtained from the biomedical and ethics committee (BREC) of the University of KwaZulu-Natal, Durban, South Africa .The funders of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report.The development datasets contained 9623 individuals (5910 women and 3713 men) with 841 seroconversions (679 among women and 162 among men) . The valThe full models retained 38 predictors for men and 28 predictors for women . The strThe sensitivity of the models at varying predicted risk thresholds is shown in The geographic distribution of predicted risk from models incorporating geospatial covariates aligned much more closely to the geographic distribution of observed HIV incidence than predictions from models using only individual-level covariates . The 20%We developed and validated human immunodeficiency virus type 1 (HIV-1) risk prediction models using a suite of individual-level and geospatial covariates to identify men and women at very high risk for HIV acquisition in rural KwaZulu-Natal, South Africa. Men and women within the top 20% of predicted risk had incidence rates of at least 2.1 and 4.9 per 100 person-years, respectively, across all models. Simple models using only age group and geospatial covariates  predicted individual-level risk of HIV infection nearly as accurately as models that included demographic, sexual behavioral, and socioeconomic predictors. Furthermore, spatially smoothed predictions from models that included geospatial covariates aligned closely with high incidence areas while predictions from models with only individual-level covariates did not.Few HIV risk prediction models have incorporated geospatial measures, but those that have considered HIV prevalence have consistently found it to be predictive. An analysis from the communities in the vicinity of Rakai District in rural Uganda found that a 1 percentage point increase in community HIV prevalence was associated with a three percent increase in the hazard for HIV acquisition . A risk Risk scores based on detailed clinical and sexual behavior measures rely on accurate reporting of sensitive and potentially stigmatizing behaviors. Such approaches are best suited to clinical settings with sufficient resources for lengthy individual-based assessment. Our results indicate that similar predictive performance may be obtained from models using age group and smoothed HIV test results from population-based surveys. These models enable HIV prevention strategies focused more on geographic context than on individual risk behaviors. Additional investment in routine population-based HIV and viral load testing could facilitate resource prioritization to communities with the highest need.Although our model identified individuals and geographic areas at the highest risk, incidence was still elevated among women even at lower predicted risk thresholds. Women in the second lowest quintile of predicted risk using the full model experienced an incidence rate of 1.9/100 person-years between 2016 and 2019, and women in the middle quintile had an incidence rate of 3/100 person-years. Our results indicate that, in a hyperendemic South African setting, incidence was high even among women who reported few individual-level risk factors. Similar gender disparities have been demonstrated across sub-Saharan Africa , and ourOur analysis has several strengths. We used a robust internal validation strategy on a large dataset to develop our models, and we validated our predictions on data from subsequent years that were not used in model development. This validation strategy was chosen to estimate performance in prospective prediction, and our models performed well despite temporal changes in incidence and risk factor distribution. For future predictions, the models can easily be retrained using the same methods on an updated dataset. Additionally, our suite of models with different covariate restrictions allows for prediction in a variety of settings with different data availability. Models based solely on individual-level covariates could be applied in a clinical setting where geospatial covariates may not be available. In contrast, models based on age and geospatial covariates could guide geographic prioritization of interventions, such as focused community HIV testing campaigns, home-based ART delivery, or community PrEP services . ApproacOur analysis also has limitations. Some of the sexual behavior variables had high amounts of missing data, and we lacked other indicators that have been previously incorporated into risk scoring tools, such as alcohol use, whether sex partners provide financial support, and whether sex partners have other partners . TherefoIn summary, we developed and validated a suite of risk prediction models informed by individual-level and geographic covariates that identified individuals and geographic areas at high risk for HIV acquisition. These results may guide efforts to prioritize HIV prevention resources to maximize impact.Clinical Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at ciac069_suppl_Supplementary_MaterialClick here for additional data file.


IntroductionContinuous electroencephalography (EEG) is an important monitoring modality in the intensive care unit and a key skill for critical care fellows (CCFs) to learn. Our objective was to evaluate with CCFs an EEG educational curriculum on a web-based simulator.MethodsThis prospective cohort study was conducted at a major academic medical center in Florida. After Institutional Review Board approval, 13 CCFs from anesthesiology, surgery, and pulmonary medicine consented to take an EEG curriculum. A 25-item EEG assessment was completed at baseline, after 10 EEG interpretations with a neurophysiologist, and after 10 clinically relevant EEG-based simulations providing clinical EEG interpretation hints. A 50-minute tutorial podcast was viewed after the baseline assessment. Main assessment outcomes included multiple outcomes related to web-based simulator performance: percent of hints used, percent of first words on EEG interpretation correct, and percent hint-based EEG interpretation score correct, with higher scores indicating more correct answers. Participants completed a 25-item EEG assessment before (baseline) and after the web-based simulator.Results9,108 = 11.7, p < 0.001), percent of first words correct , and overall percent hint-based score . Nonconvulsive status epilepticus had the lowest percent of hints used (15%) and the highest hint-based score (87%). Overall percent hint-based score  was positively correlated with change in performance as the number of correct answers on the 25-item EEG assessment from before to after the web-based simulator activity .All 13 CCFs completed the curriculum. Between scenarios, there were differences in percent of hints used (FConclusionsA self-paced EEG interpretation curriculum involving a flipped classroom and screen-based simulation each requiring less than an hour to complete significantly improved CCF scores on the EEG assessment compared to baseline. In the intensive care unit (ICU), continuous electroencephalography (EEG) is an important monitoring modality. Accurate identification of EEG patterns that require intervention in a timely manner is a key skill for critical care fellows (CCFs) to acquire ,2. ContiThe Critical Care Continuous EEG Task Force of the American Clinical Neurophysiology Society consensus panel  noted thUsing the expertise of a neurophysiology expert (JEC), we previously developed an interdisciplinary EEG curriculum using a flipped-classroom approach that entailed an educational podcast before class, followed by interpretation of EEGs with a neurologist during class, then a screen-based simulator approach for further learning that was completed outside the classroom. The curriculum was well-received by adult CCFs from varied training backgrounds, including anesthesiology, surgery, and internal medicine  . This apThe purpose of this prospective cohort study was to further evaluate the impact of this EEG educational curriculum on performance on the screen-based simulator and assessment tools and to identify factors associated with better performance on the screen-based simulation and assessment tools. Secondary aims included evaluation of fellows’ podcast and technology experiences to better understand how previous experiences with podcasts and technology may impact learning that uses simulation as a teaching strategy.The University of Florida Institutional Review Board IRB201701046) approved this study and 13 of the CCFs who rotated through a neurocritical care unit consented to participate in the EEG educational curriculum. After enrollment, the CCFs took a survey on their previous podcast/flipped classroom, simulation, and web-based educational experiences and technology assessment tool, as previously described . They al701046 apThe actual curriculum began with participants watching a 50-minute EEG podcast, which covered the basics of EEG as described previously . It inclThe simulation EEG tracing automatically pauses at appropriate points and a question is displayed for up to 1 minute, requiring an answer entry in a free-text box. When answered correctly, the simulation scenario proceeded to the next question point or a new clinical vignette began. When answered incorrectly, one or more hints were provided, with an additional 30 seconds allowed for the learner to enter an answer after a hint. Then the simulation continued with the correct answer entry. Up to two hints were available for each question after an incorrect answer entry. If the available hints were exhausted, the answer was displayed and participants needed to type that correct answer in the free-text box to continue the simulation. The simulator design allowed the participant to suspend the session and resume where they had stopped. The main assessment outcomes for each scenario included percent of hints used (number of hints used/number of hints available), percent of correct first words , and percent hint-based score, with correct answers weighted by the number of hints used. Higher scores indicated more correct answers using fewer hints . After the 10 EEG simulation scenarios, the CCFs completed another 25-item EEG assessment. Additionally, CCFs completed a survey on the previous podcast/flipped classroom, simulation, and web-based educational experiences that have been described previously .Simulation performance metrics were summarized as mean (standard deviation), along with 95% CIs for each scenario and overall. Repeated measures ANOVA was used to assess between scenario differences in performance; a repeated measures approach was used to account for the same fellows completing each scenario. To assess the reliability of scenarios, Cronbach α was calculated by using an average percent hint-based score from each scenario. One-way ANOVA was also used to evaluate percent hint-based score differences across CCF training programs . Welch’s correction was performed for unequal variances. The overall change in the 25-item EEG assessment as the number of correct answers before (baseline) and after web-based simulator activity was evaluated using a paired t-test, with a statistically significant result supporting improvement following the simulator activity. Spearman’s correlations (rho) were calculated to assess the association between improvement in the 25-item EEG assessment and percent hint-based scores in the web-based simulator activity. They were calculated for overall hint-based score  and separately for hint-based scores of each scenario, with bootstrapped 95% CIs. For correlation analyses, change in the 25-item EEG assessment was calculated as a residual change score from before (baseline) and after web-based EEG simulator activity, which accounts for participant differences at baseline. p < 0.05 was considered statistically significant. JMP Pro 15  was used for analysis.Thirteen (n = 13) CCFs completed the web-based EEG simulator; six fellows were from an anesthesiology background, four from surgery, and three from pulmonary medicine. Twelve completed the previous podcast/flipped classroom experiences survey. The CCF baseline performance on the 25-item EEG assessment was 10.4 ± 3.0 correct interpretations.9,108 = 11.7, p < 0.001), percent of first words correct , and overall percent hint-based score  did not have a significantly different overall percent hint-based scores compared to those with more previous experience  .Figure EEG is an increasingly important diagnostic and prognostic monitor in the ICU. One example of the effect of CCCEEG monitoring is early detection of nonconvulsive status epilepticus, which enables rapid treatment of this neurological emergency. Often, treatment for this emergent condition is delayed because of its nonconvulsive presentation ,15. EEG Our study demonstrated that a brief interdisciplinary EEG curriculum involving a flipped classroom and screen-based simulation significantly improved EEG knowledge based on performance on the EEG assessment among CCFs from various backgrounds. Improvement on our EEG assessment was correlated not only with overall simulation performance but also with performance in several specific scenarios, mainly ones involving normal EEG and EEGs related to seizures and epilepsy. This provides support that our web-based simulations, especially with these specific scenarios, supported learning. However, not all scenarios were associated with improvement in the EEG assessment; furthermore, there was variability in overall performance across scenarios. This provides valuable information on specific areas of need for enhancement of the EEG training curricula. The educational approach of this hybrid EEG curriculum can be adapted to the particular needs of trainees from varying backgrounds. This curriculum was designed to provide basic and clinically relevant knowledge for managing critically ill patients monitored by CCCEEG and was not intended to be comprehensive training for EEG interpretation.Important considerations for this curriculum are how will it translate to patient care and whether there is long-term retention. We have previously reported on trainee satisfaction and impressions of our simulation curriculum . A majorThe training in this study was required as part of the education for CCFs for neurocritical care. Current medical trainees are adult learners who are familiar with electronic communications and prefer independent learning options ,18. A grThis EEG curriculum attempted to use higher levels of learning by applying principles learned to a simulated clinical situation. This curriculum used self-directed learning, as well as spaced learning between curriculum activities, which contribute to long-term retention . The intAlthough learners had different primary backgrounds, limitations include that all were from a single institution university-based program, constituting a small sample size. Thus, the sample may not be representative of all CCFs and it was underpowered to detect smaller differences in performance on simulation. Other measurements of performance including technical and non-technical skills were not evaluated, recognizing that associations may be different from other performance measurements.This pilot study provides evidence that an interdisciplinary EEG curriculum with simulation can be an effective educational method as evaluated by an EEG interpretation assessment tool to impart EEG knowledge to CCFs from different backgrounds . EEG abnormalities with lower EEG interpretation scores indicate opportunities for future education and study. Further studies are required to explore the impact of better EEG interpretation skills on patient outcomes.