Entry Efficiency Research Articles

Intranasal Delivery of BACE1 siRNA and Rapamycin by Dual Targets Modified Nanoparticles for Alzheimer's Disease Therapy.

Alzheimer's disease (AD), as a progressive and irreversible brain disorder, remains the most universal neurodegenerative disease. No effective therapeutic methods are established yet due to the hindrance of the blood-brain barrier (BBB) and the complex pathological condition of AD. Therefore, a multifunctional nanocarrier (Rapa@DAK/siRNA) for AD treatment isconstructed to achieve small interfering RNA of β-site precursor protein (APP) cleaving enzyme-1(BACE1 siRNA) and rapamycin co-delivery into the brain, based on Aleuria aurantia lectin (AAL) and β-amyploid(Aβ)-binding peptides (KLVFF) modified PEGylated dendrigraft poly-l-lysines (DGLs) via intranasal administration. Nasal administration provides an effective way to deliver drugs directly into the brain through the nose-to-brain pathway. AAL, specifically binding to L-fucose located in the olfactory epithelium, endowsRapa@DAK/siRNA with high brain entry efficiency through intranasal administration. KLVFF peptide as an Aβ targeting ligand and aggregation inhibitor enablesnanoparticles to bind with Aβ, inhibit Aβ aggregation, and reduce toxicity. Meanwhile, the release of BACE1 siRNA and rapamycin isconfirmed to reduce BACE1 expression, promote autophagy, and reduce Aβ deposition. Rapa@DAK/siRNA isverified to improve the cognition of transgenic AD mice after intranasal administration. Collectively, the multifunctional nanocarrier providesan effective and potential intranasal avenue for combination therapy of AD.

Abstract 369: Improving the nanomaterial delivery by using cell-penetrating peptides in the bystander manner

Abstract While nanoparticles (NPs) can be useful to improve the diagnosis and treatment of various diseases including cancer, a major limiting factor for their applications is the poor efficiency of cell entry. A common solution to this problem is to covalently link NPs with cell-penetrating peptides (CPPs), such as the transactivator of transcription (TAT) peptide. A few cationic CPPs were previously shown to increase the cellular uptake of co-administered, but not covalently coupled, NP cargo through an endocytic pathway, macropinocytosis. This so-called bystander uptake process is of unique advantage for NP intracellular delivery as it bypasses the requirements of additional chemical modifications. Here, we set out to determine whether other classes of CPPs (e.g. hydrophobic and amphiphilic) exhibit similar bystander activities, and what physico-chemical properties of NPs affect the bystander uptake process. First, we discovered that transportan (TP) peptide, an amphiphilic CPP, can initiate similar bystander uptake process for a variety of NPs and solute tracers. TP-induced bystander uptake relies on macropinocytosis as well, and occurs in a variety of cell types and in physiological tissues. Second, using TAT-functionalized NPs to stimulate the bystander effect, we showed that the bystander activity depends on the size of bystander NPs, with an optimal range around 50 nm in diameter. Compared to rod-like and triangular NPs, spherical NPs showed higher uptake in the bystander manner. Additionally, we investigated how CPP-NPs, once inside cells, get out of one cell and enter another, and found again that NP sizes affect the efficiency of CPP-NP intercellular transport as well. Overall, these efforts lay the foundation to further understand the bystander activity of CPP-assisted cell entry and improve the efficacy of nanomedicine in cancer and other human diseases. Citation Format: Yue-Xuan Li, Yushuang Wei, Hong-Bo Pang. Improving the nanomaterial delivery by using cell-penetrating peptides in the bystander manner [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 369.

Efficient and genotype independent maize transformation using pollen transfected by DNA-coated magnetic nanoparticles.

Current gene delivery methods for maize are limited to specific genotypes and depend on time-consuming and labor-intensive tissue culture techniques. Here, we report a new method to transfect maize that is culture-free and genotype independent. To enhance efficiency of DNA entry and maintain high pollen viability of 32%-55%, transfection was performed at cool temperature using pollen pretreated to open the germination aperture (40%-55%). Magnetic nanoparticles (MNPs) coated with DNA encoding either red fluorescent protein (RFP), β-glucuronidase gene (GUS), enhanced green fluorescent protein (EGFP) or bialaphos resistance (bar) was delivered into pollen grains, and female florets of maize inbred lines were pollinated. Red fluorescence was detected in 22% transfected pollen grains, and GUS stained 55% embryos at 18 d after pollination. Green fluorescence was detected in both silk filaments and immature kernels. The T1 generation of six inbred lines showed considerable EGFP or GUS transcripts (29%-74%) quantitated by polymerase chain reaction, and 5%-16% of the T1 seedlings showed immunologically active EGFP or GUS protein. Moreover, 1.41% of the bar transfected T1 plants were glufosinate resistant, and heritable bar gene was integrated into the maize genome effectively as verified by DNA hybridization. These results demonstrate that exogenous DNA could be delivered efficiently into elite maize inbred lines recalcitrant to tissue culture-mediated transformation and expressed normally through our genotype-independent pollen transfection system.

Cell-permeable fluorescent indicator for imaging formaldehyde activity in living systems

Formaldehyde (FA), as a reactive signaling molecule, plays an important role in living systems through a diverse array of cellular pathways. However, no systematic investigation for detection and imaging of FA by rendering cells transiently permeable has been reported yet. Specifically, we developed a new cell-permeable fluorescence probe functionality that was enhanced cellular entry efficiency and well retained intracellularly after activation for visualizing endogenous FA changes. Moreover, a smart “multi-lock system -key-and-lock” strategy，which have provoked a starting point for the use of probe and related biochemical tools to monitor FA in lysosomes. The versatile “latent” fluorophore that can undergo a subsequent self-immolative spacer for interrogating the roles and functions of FA in living systems as well as related biomedical applications.

Anti-L1 antibody-bound HPV16 pseudovirus is degraded intracellularly via TRIM21/proteasomal pathway

BackgroundPersistent HPV16 infection is the leading risk factor for developing cervical cancer. Anti-L1 antibodies against HPV16 produced in HPV16 infections play diverse roles in the clearance of virus infection and prevention of persistence. It has been implicated that the cervicovaginal squamous epithelial cells actually express TRIM21 and that some HPV16 particles could escape leaky endosomal compartment into the cytosol and that Fc receptor TRIM21 directly neutralize infection by targeting antibody-opsonized viruses for proteasomal degradation. We explored whether anti-L1 antibody opsonized HPV16 pseudovirus (PsV) entered into the cytosol could be neutralized by TRIM21-mediated activation of a proteasomal pathway to reduce the chance of persistent HPV16 infection.MethodsHPV16 PsV were generated and extracted in HEK 293FT cells co-transfected with pcDNA3.1-eGFP and p16sheLL plasmids according to the standard protocol. The HPV16 PsV with capsid protein L1 was characterized by fluorescence microscopy and western blot, and the HPV16 PsV titer and anti-L1-bound PsV entry efficiency were detected by flow cytometry. The expressions of transcription factors (TF) and cytokines elicited by the TRIM21-activated proteasomal pathway were confirmed by dual-luciferase reporter assay and RT-qPCR. The changes in HPV16 PsV load with or without inhibitors in the infected HEK 293FT cells were determinated by qPCR.ResultsSimultaneous transfection with pcDNA3.1-eGFP and p16sheLL plasmids into the HEK 293FT cells resulted in the self-assembly of HPV16 PsV with capsid protein L1. Both HPV16 PsV and anti-L1-bound HPV16 PsV could infect HEK 293FT cells. Anti-L1-bound PsV up-regulated TRIM21 mediated-activation of proteasome and increased expressions of TF and cytokines in the infected cells where HPV16 PsV load reduced by ~ 1000-fold in the presence of anti-L1 antibody, but inhibition of proteasomal activity increased HPV16 PsV load.ConclusionOur preliminary results indicate that anti-L1 antibody entered with HPV16 PsV into the cells could mediate degradation of HPV16 PsV by TRIM21-activated proteasomal pathway intracellularly, giving anti-capsid protein L1 antibody a role in host defense of persistent HPV16 infection.

Social Efficiency of Market Entry Under Tax Policy

Abstract We provide a new rationale for socially insufficient market entry. We show that if the shadow cost of public funds is sufficiently high, the number of firms under free entry can be socially insufficient if the tax policies are “time inconsistent”, so that the governments cannot commit to the tax policies before market entry of firms. Hence, strategic tax policies may provide a reason why policymakers should engage in pro-competitive policies. Lump-sum subsidies to firms may be a way to achieve that goal.

Usability-Focused Development and Usage of NeoTree-Beta, an App for Newborn Care in a Low-Resource Neonatal Unit, Malawi.

BackgroundNeonatal mortality is high in low-resource settings. NeoTree is a digital intervention for neonatal healthcare professionals (HCPs) aiming to achieve data-driven quality improvement and improved neonatal survival in low-resource hospitals. Optimising usability with end-users could help digital health interventions succeed beyond pilot stages in low-resource settings. Usability is the quality of a user's experience when interacting with an intervention, encompassing their effectiveness, efficiency, and overall satisfaction.ObjectiveTo evaluate the usability and usage of NeoTree beta-app and conduct Agile usability-focused intervention development.MethodA real-world pilot of NeoTree beta-app was conducted over 6 months at Kamuzu Central Hospital neonatal unit, Malawi. Prior to deployment, think-aloud interviews were conducted to guide nurses through the app whilst voicing their thoughts aloud (n = 6). System Usability Scale (SUS) scores were collected before the implementation of NeoTree into usual clinical care and 6 months after implementation (n = 8 and 8). During the pilot, real-world user-feedback and user-data were gathered. Feedback notes were subjected to thematic analysis within an Agile “product backlog.” For usage, number of users, user-cadre, proportion of admissions/outcomes recorded digitally, and median app-completion times were calculated.ResultsTwelve overarching usability themes generated 57 app adjustments, 39 (68%) from think aloud analysis and 18 (32%) from the real-world testing. A total of 21 usability themes/issues with corresponding app features were produced and added to the app. Six themes relating to data collection included exhaustiveness of data schema, prevention of errors, ease of progression, efficiency of data entry using shortcuts, navigation of user interface (UI), and relevancy of content. Six themes relating to the clinical care included cohesion with ward process, embedded education, locally coherent language, adaptability of user-interface to available resources, and printout design to facilitate handover. SUS scores were above average (88.1 and 89.4 at 1 and 6 months, respectively). Ninety-three different HCPs of 5 cadres, recorded 1,323 admissions and 1,197 outcomes over 6 months. NeoTree achieved 100% digital coverage of sick neonates admitted. Median completion times were 16 and 8 min for admissions and outcomes, respectively.ConclusionsThis study demonstrates optimisation of a digital health app in a low-resource setting and could inform other similar usability studies apps in similar settings.

Why Does the Omicron Variant Largely Spare Olfactory Function? Implications for the Pathogenesis of Anosmia in Coronavirus Disease 2019.

The omicron variant of severe acute respiratory syndrome coronavirus 2 causes much less olfactory dysfunction than the previous variants. There are several potential mechanisms for how omicron may change tissue tropism and spare olfactory function. The new mutations make omicron more hydrophobic and alkaline than previous variants, which may reduce penetration of the mucus layer. Overall, the new mutations minimally change receptor binding affinity, but entry efficiency into host cells is reduced in cells expressing transmembrane serine protease 2 (TMPRSS2). Because the support cells in the olfactory epithelium abundantly express TMPRSS2, these main target cells in the olfactory epithelium may become infected less by the new omicron variant.

Semi-Mechanistic Pharmacokinetic-Pharmacodynamic Model of Camostat Mesylate-Predicted Efficacy against SARS-CoV-2 in COVID-19.

ABSTRACTThe SARS-CoV-2 coronavirus, which causes COVID-19, uses a viral surface spike protein for host cell entry and the human cell-surface transmembrane serine protease, TMPRSS2, to process the spike protein. Camostat mesylate, an orally available and clinically used serine protease inhibitor, inhibits TMPRSS2, supporting clinical trials to investigate its use in COVID-19. A one-compartment pharmacokinetic (PK)/pharmacodynamic (PD) model for camostat and the active metabolite FOY-251 was developed, incorporating TMPRSS2 reversible covalent inhibition by FOY-251, and empirical equations linking TMPRSS2 inhibition of SARS-CoV-2 cell entry. The model predicts that 95% inhibition of TMPRSS2 is required for 50% inhibition of viral entry efficiency. For camostat 200 mg dosed four times daily, 90% inhibition of TMPRSS2 is predicted to occur but with only about 40% viral entry inhibition. For 3-fold higher camostat dosing, marginal improvement of viral entry rate inhibition, up to 54%, is predicted. Because respiratory tract viral load may be associated with negative outcome, even modestly reducing viral entry and respiratory tract viral load may reduce disease progression. This modeling also supports medicinal chemistry approaches to enhancing PK/PD and potency of the camostat molecule.IMPORTANCE Strategies to repurpose already-approved drugs for the treatment of COVID-19 has been attractive since the beginning of the pandemic. Camostat mesylate, a serine protease inhibitor approved in Japan for the treatment of acute exacerbations of chronic pancreatitis, inhibits TMPRSS1, a host cell surface serine protease essential for SARS-CoV-2 viral entry. In vitro experiments provided data suggesting that camostat might be effective in the treatment of COVID-19. Multiple clinical trials were planned to test the hypothesis that camostat would be beneficial for treating COVID-19 (for example, clinicaltrials.gov, NCT04353284). The present work used a one-compartment pharmacokinetic (PK)/pharmacodynamic (PD) mathematical model for camostat and the active metabolite FOY-251, incorporating TMPRSS2 reversible covalent inhibition by FOY-251, and empirical equations linking TMPRSS2 inhibition of SARS-CoV-2 cell entry. This work is valuable to guide further development of camostat mesylate and possible medicinal chemistry derivatives for the treatment of COVID-19.

Discovery of a Cyclic Cell-Penetrating Peptide with Improved Endosomal Escape and Cytosolic Delivery Efficiency.

Cyclic cell-penetrating peptide 12 (CPP12) is highly efficient for the cytosolic delivery of a variety of cargo molecules into mammalian cells in vitro and in vivo. However, its cytosolic entry efficiency is substantially reduced at lower concentrations or in the presence of serum proteins. In this study, CPP12 analogs were prepared by replacing its hydrophobic residues with amino acids of varying hydrophobicity and evaluated for cellular entry. Substitution of l-3-benzothienylalanine (Bta) for l-2-naphthylalanine (Nal) resulted in CPP12-2, which exhibits up to 3.8-fold higher cytosolic entry efficiency than CPP12, especially at low CPP concentrations; thanks to improved endosomal escape efficiency. CPP12-2 is well suited for the cytosolic delivery of highly potent cargos to achieve biological activity at low concentrations.

Functional analysis of polymorphisms at the S1/S2 site of SARS-CoV-2 spike protein.

Several SARS-CoV-2 variants emerged that harbor mutations in the surface unit of the viral spike (S) protein that enhance infectivity and transmissibility. Here, we analyzed whether ten naturally-occurring mutations found within the extended loop harboring the S1/S2 cleavage site of the S protein, a determinant of SARS-CoV-2 cell tropism and pathogenicity, impact S protein processing and function. None of the mutations increased but several decreased S protein cleavage at the S1/S2 site, including S686G and P681H, the latter of which is found in variants of concern B.1.1.7 (Alpha variant) and B.1.1.529 (Omicron variant). None of the mutations reduced ACE2 binding and cell-cell fusion although several modulated the efficiency of host cell entry. The effects of mutation S686G on viral entry were cell-type dependent and could be linked to the availability of cathepsin L for S protein activation. These results show that polymorphisms at the S1/S2 site can modulate S protein processing and host cell entry.

Social Efficiency of Entry in a Vertical Structure with Third Degree Price Discrimination

Abstract We study social efficiency of entry in the presence of downstream cost asymmetry and upstream price discrimination. We show that entry is excessive when the entrants are highly inefficient, and it is insufficient when either the entrants are efficient or their inefficiency is low. The results are in sharp contrast to the existing literature considering upstream uniform pricing (Cao, H., and L. F. S. Wang. 2020. “Social Efficiency of Entry in a Vertically Related Industry Revisited.” Economics Letters 129. Art. no. 109200), as discriminatory pricing alters the relative strengths of the business-stealing, business-creation and production-(in)efficiency effects.

Drastic decline in sera neutralization against SARS-CoV-2 Omicron variant in Wuhan COVID-19 convalescents

ABSTRACT Global concern has been raised by the emergence and rapid transmission of the heavily mutated SARS-CoV-2 Omicron variant (B.1.1.529). So far, the infection features and immune escape ability of the Omicron variant have not been extensively studied. Here, we produced the Omicron pseudovirus and compared its entry, membrane fusion, and immune escape efficiency with the original strain and the dominating Delta variant. We found the Omicron variant showed slightly higher infectivity than the Delta variant and a similar ability to compete with the Delta variant in using Angiotensin-converting enzyme 2 (ACE2) in a BHK21-ACE2 cell line. However, the Omicron showed a significantly reduced fusogenicity than the original strain and the Delta variant in both BHK21-ACE2 and Vero-E6 cells. The neutralization assay testing the Wuhan convalescents’ sera one-year post-infection showed a more dramatic reduction (10.15 fold) of neutralization against the Omicron variant than the Delta variant (1.79 fold) compared with the original strain with D614G. Notably, immune-boosting through three vaccine shots significantly improved the convalescents’ immunity against the Omicron variants. Our results reveal a reduced fusogenicity and a striking immune escape ability of the Omicron variant, highlighting the importance of booster shots against the challenge of the SARS-CoV-2 antigenic drift.

Altered TMPRSS2 usage by SARS-CoV-2 Omicron impacts infectivity and fusogenicity

The SARS-CoV-2 Omicron BA.1 variant emerged in 20211 and has multiple mutations in its spike protein2. Here we show that the spike protein of Omicron has a higher affinity for ACE2 compared with Delta, and a marked change in its antigenicity increases Omicron’s evasion of therapeutic monoclonal and vaccine-elicited polyclonal neutralizing antibodies after two doses. mRNA vaccination as a third vaccine dose rescues and broadens neutralization. Importantly, the antiviral drugs remdesivir and molnupiravir retain efficacy against Omicron BA.1. Replication was similar for Omicron and Delta virus isolates in human nasal epithelial cultures. However, in lung cells and gut cells, Omicron demonstrated lower replication. Omicron spike protein was less efficiently cleaved compared with Delta. The differences in replication were mapped to the entry efficiency of the virus on the basis of spike-pseudotyped virus assays. The defect in entry of Omicron pseudotyped virus to specific cell types effectively correlated with higher cellular RNA expression of TMPRSS2, and deletion of TMPRSS2 affected Delta entry to a greater extent than Omicron. Furthermore, drug inhibitors targeting specific entry pathways3 demonstrated that the Omicron spike inefficiently uses the cellular protease TMPRSS2, which promotes cell entry through plasma membrane fusion, with greater dependency on cell entry through the endocytic pathway. Consistent with suboptimal S1/S2 cleavage and inability to use TMPRSS2, syncytium formation by the Omicron spike was substantially impaired compared with the Delta spike. The less efficient spike cleavage of Omicron at S1/S2 is associated with a shift in cellular tropism away from TMPRSS2-expressing cells, with implications for altered pathogenesis.

Entry Deregulation, Market Turnover, and Efficiency: China’s Business Registration Reform

Entry Deregulation, Market Turnover, and Efficiency: China’s Business Registration Reform

Spatiotemporal tracking of the transport of RNA nano-drugs: from transmembrane to intracellular delivery.

The popularity of RNA nanoparticles (RNPs) has risen rapidly during the past decade due to the development of RNA nanotechnology. Understanding the fast dynamic process of cell entry and intracellular delivery of RNPs is essential for the design of intelligent therapeutic RNA nano-drugs and mRNA vaccines.How the interaction between the membrane and target ligand of RNPs influences the cell entry, and how the dynamic mechanism of RNPs takes place in different organelles remain ill-defined. Herein, the cell entry of Antimir21-RNP-Apt is monitored using a force tracing technique with a high spatiotemporal resolution at the single particle level, the specific interaction of Apt and EGFR promotes the cell entry efficiency and achieves long-lasting curative effects. Furthermore, the intracellular delivery pathway through different organelles is discovered using fluorescence tracking, and the low motility in early endosomes and the high motility in late endosomes are analyzed. This report provides key strategies for engineering RNA nanomedicines and facilitating clinical translation.

Social Efficiency of Entry in an Open Economy

Abstract We show that cost asymmetry between the domestic and foreign firms is not necessary for the occurrence of insufficient entry in the domestic country. This result provides a rationale for pro-competitive domestic policies even in the absence of cost asymmetries among the domestic and foreign firms. However, if significant demand comes from foreign countries, and the market structures are determined endogenously in the domestic and foreign countries, domestic-entry in an open economy might not be insufficient, implying that foreign competition might not reduce the importance of anti-competitive domestic policies.

Rapid assessment of SARS-CoV-2-evolved variants using virus-like particles.

Efforts to determine why new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants demonstrate improved fitness have been limited to analyzing mutations in the spike (S) protein with the use of S-pseudotyped particles. In this study, we show that SARS-CoV-2 virus-like particles (SC2-VLPs) can package and deliver exogenous transcripts, enabling analysis of mutations within all structural proteins and at multiple steps in the viral life cycle. In SC2-VLPs, four nucleocapsid (N) mutations found universally in more-transmissible variants independently increased messenger RNA delivery and expression ~10-fold, and in a reverse genetics model, the serine-202→arginine (S202R) and arginine-203→methionine (R203M) mutations each produced >50 times as much virus. SC2-VLPs provide a platform for rapid testing of viral variants outside of a biosafety level 3 setting and demonstrate N mutations and particle assembly to be mechanisms that could explain the increased spread of variants, including B.1.617.2 (Delta, which contains the R203M mutation).

Mitochondria-targeted nanoparticles in treatment of neurodegenerative diseases.

Neurodegenerative diseases (NDs) are a class of heterogeneous diseases that includes Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis. Mitochondria play an important role in oxidative balance and metabolic activity of neurons; therefore, mitochondrial dysfunction is associated with NDs and mitochondria are considered a potential treatment target for NDs. Several obstacles, including the blood-brain barrier (BBB) and cell/mitochondrial membranes, reduce the efficiency of drug entry into the target lesions. Therefore, a variety of neuron mitochondrial targeting strategies has been developed. Among them, nanotechnology-based treatments show especially promising results. Owing to their adjustable size, appropriate charge, and lipophilic surface, nanoparticles (NPs) are the ideal theranostic system for crossing the BBB and targeting the neuronal mitochondria. In this review, we discussed the role of dysfunctional mitochondria in ND pathogenesis as well as the physiological barriers to various treatment strategies. We also reviewed the use and advantages of various NPs (including organic, inorganic, and biological membrane-coated NPs) for the treatment and diagnosis of NDs. Finally, we summarized the evidence and possible use for the promising role of NP-based theranostic systems in the treatment of mitochondrial dysfunction-related NDs.

What Oncologists Want: Identifying Challenges and Preferences on Diagnosis Data Entry to Reduce EHR-Induced Burden and Improve Clinical Data Quality.

Accurate recording of diagnosis (DX) data in electronic health records (EHRs) is important for clinical practice and learning health care. Previous studies show statistically stable patterns of data entry in EHRs that contribute to inaccurate DX, likely because of a lack of data entry support. We conducted qualitative research to characterize the preferences of oncological care providers on cancer DX data entry in EHRs during clinical practice. We conducted semistructured interviews and focus groups to uncover common themes on DX data entry preferences and barriers to accurate DX recording. Then, we developed a survey questionnaire sent to a cohort of oncologists to verify the generalizability of our initial findings. We constrained our participants to a single specialty and institution to ensure similar clinical backgrounds and clinical experience with a single EHR system. A total of 12 neuro-oncologists and thoracic oncologists were involved in the interviews and focus groups. The survey developed from these two initial thrusts was distributed to 19 participants yielding a 94.7% survey response rate. Clinicians reported similar user interface experiences, barriers, and dissatisfaction with current DX entry systems including repetitive entry operations, difficulty in finding specific DX options, time-consuming interactions, and the need for workarounds to maintain efficiency. The survey revealed inefficient DX search interfaces and challenging entry processes as core barriers. Oncologists seem to be divided between specific DX data entry and time efficiency because of current interfaces and feel hindered by the burdensome and repetitive nature of EHR data entry. Oncologists' top concern for adopting data entry support interventions is ensuring that it provides significant time-saving benefits and increasing workflow efficiency. Future interventions should account for time efficiency, beyond ensuring data entry effectiveness.