A case of convergent evolution: Several viral and bacterial pathogens hijack RSK kinases through a common linear motif.

Microbes have been coevolving with their host for millions of years, exploiting host resources to their own benefit. We show that viral and bacterial pathogens convergently evolved to hijack cellular mitogen-activated protein kinase (MAPK) p90-ribosomal S6-kinases (RSKs). Theiler's virus leader (L) protein binds RSKs and prevents their dephosphorylation, thus maintaining the kinases active. Recruitment of RSKs enables L-protein-mediated inhibition of eukaryotic translation initiation factor 2 alpha kinase 2 (EIF2AK2 or PKR) and stress granule formation. Strikingly, ORF45 protein of Kaposi's sarcoma-associated herpesvirus (KSHV) and YopM protein of Yersinia use the same peptide motif as L to recruit and activate RSKs. All three proteins interact with a conserved surface-located loop of RSKs, likely acting as an allosteric regulation site. Some unrelated viruses and bacteria thus evolved to harness RSKs in a common fashion, yet to target distinct aspects of innate immunity. As documented for Varicella zoster virus ORF11, additional pathogens likely evolved to hijack RSKs, using a similar short linear motif.

Dual-energy CT angiography in detecting underlying causes of intracerebral hemorrhage: an observational cohort study.

BACKGROUND: CT angiography (CTA) is often used to detect underlying causes of acute intracerebral hemorrhage (ICH). Dual-energy CT (DECT) is able to distinguish materials with similar attenuation but different compositions, such as hemorrhage and contrast. We aimed to evaluate the diagnostic yield of DECT angiography (DECTA), compared to conventional CTA in detecting underlying ICH causes. METHODS: All non-traumatic ICH patients who underwent DECTA (both arterial as well as delayed venous phase) at our center between January 2014 and February 2020 were analyzed. Conventional CTA acquisitions were reconstructed ('merged') from DECTA data. Structural ICH causes were assessed on both reconstructed conventional CTA and DECTA. The final diagnosis was based on all available diagnostic and clinical findings during one-year follow up. RESULTS: Of 206 included ICH patients, 30 (14.6%) had an underlying cause as final diagnosis. Conventional CTA showed a cause in 24 patients (11.7%), DECTA in 32 (15.5%). Both false positive and false negative findings occurred more frequently on conventional CTA. DECTA detected neoplastic ICH in all seven patients with a definite neoplastic ICH diagnosis, whereas conventional CTA only detected four of these cases. Both developmental venous anomalies (DVA) and cerebral venous sinus thrombosis (CVST) were more frequently seen on DECTA. Arteriovenous malformations and aneurysms were detected equally on both imaging modalities. CONCLUSIONS: Performing DECTA at clinical presentation of ICH may be of additional diagnostic value in the early detection of underlying causes, especially neoplasms, CVST and DVAs.

Viral clade is associated with severity of symptomatic genotype 3 hepatitis E virus infections in Belgium, 2010-2018.

BACKGROUND & AIMS: HEV genotype (gt) 3 infections are prevalent in high-income countries and display a wide spectrum of clinical presentations. Host - but not viral - factors are reported to be associated with worse clinical outcomes. METHODS: Demographic, clinical, and biochemical data laboratory-confirmed HEV infections (by PCR and/or a combination of IgM and IgG serology) at the Belgian National Reference Centre between January 2010 and June 2018 were collected using standardised case report forms. Genotyping was based on HEV open reading frame 2 sequences. Serum CXCL10 levels were measured by a magnetic bead-based assay. H&E staining was performed on liver biopsies. RESULTS: A total of 274 HEV-infected individuals were included. Subtype assignment was possible for 179/218 viraemic cases, confirming gt3 as dominant with an almost equal representation of clades abchijklm and efg. An increased hospitalisation rate and higher peak serum levels of alanine aminotransferase, bilirubin, and alkaline phosphatase were found in clade efg-infected individuals in univariate analyses. In multivariable analyses, clade efg infections remained more strongly associated with severe disease presentation than any of the previously identified host risk factors, being associated with a 2.1-fold higher risk of hospitalisation (95% CI 1.1-4.4, p = 0.034) and a 68.2% higher peak of bilirubin levels (95% CI 13.3-149.9, p = 0.010), independently of other factors included in the model. In addition, acute clade efg infections were characterised by higher serum CXCL10 levels (p = 0.0005) and a more pronounced liver necro-inflammatory activity (p = 0.022). CONCLUSIONS: In symptomatic HEV gt3 infections, clade efg is associated with a more severe disease presentation, higher serum CXCL10 levels, and liver necro-inflammatory activity, irrespective of known host risk factors. CLINICAL TRIAL REGISTRATION: The protocol was submitted to clinicaltrials.gov (NCT04670419). IMPACT AND IMPLICATIONS: HEV genotype (gt) 3 infections display a wide spectrum of clinical presentations currently ascribed to host factors. Here we examined the role of viral factors on liver disease outcomes by combining viral phylogeny with clinical, biochemical, cytokine, and histological data from 274 Belgian adults infected with HEV presenting between 2010 and 2018. HEV gt 3 clade efg infections were associated with a more severe disease presentation, higher serum CXCL10 levels and liver necro-inflammatory activity, irrespective of known host risk factors. HEV gt3 clade-dependent clinical outcomes call for broad HEV gt3 subtyping in clinical practice and research to help identify those at higher risk for worse outcomes and to further unravel underlying virus-host interactions.

Hepatitis of unknown aetiology in children - epidemiological overview of cases reported in Europe, 1 January to 16 June 2022.

Following the report of an excess in paediatric cases of severe acute hepatitis of unknown aetiology by the United Kingdom (UK) on 5 April 2022, 427 cases were reported from 20 countries in the World Health Organization European Region to the European Surveillance System TESSy from 1 January 2022 to 16 June 2022. Here, we analysed demographic, epidemiological, clinical and microbiological data available in TESSy. Of the reported cases, 77.3% were 5 years or younger and 53.5% had a positive test for adenovirus, 10.4% had a positive RT-PCR for SARS-CoV-2 and 10.3% were coinfected with both pathogens. Cases with adenovirus infections were significantly more likely to be admitted to intensive care or high-dependency units (OR = 2.11; 95% CI: 1.18-3.74) and transplanted (OR = 3.36; 95% CI: 1.19-9.55) than cases with a negative test result for adenovirus, but this was no longer observed when looking at this association separately between the UK and other countries. Aetiological studies are needed to ascertain if adenovirus plays a role in this possible emergence of hepatitis cases in children and, if confirmed, the mechanisms that could be involved.

Use of reflective writing within interprofessional education: a mixed-methods analysis.

Nurturing student's development of interprofessional collaboration is fundamental. Assessment-For-Learning can use reflection as one technique to support students' growth. Thus, we investigated using reflective-writing within an interprofessional education (IPE) course using an exploratory mixed-methods design. In 2015, student-nurses, student-pharmacists, and student-physicians participated in an IPE course and completed self-assessments of student learning objectives (SLOs). In 2016, new cohorts of student-nurses, student-pharmacists, and student-physicians participated in the course and completed their self-assessments of SLOs; however, student-nurses and student-pharmacists also reflectively-wrote. Quantitatively comparing SLOs from 2015 cohorts with 2016 cohorts, we found that the effect-sizes (magnitude of difference) for those who reflectively-wrote (student-nurses and student-pharmacists) grew more than historical controls, whereas the effect-sizes remained unchanged for a control group (student-physicians) who did not reflectively-write. Qualitatively, initial and final reflective-writings were explored using content analysis. Initial reflective-writings helped students create a baseline for their final reflective-writings. In final reflective-writings, most students discussed their growth in understanding roles/responsibilities and communication, though limited growth was discussed for teams/teamwork and values/ethics. Thus, initial and final reflective-writings appeared useful within this IPE course. Initial reflective-writing further enhanced students' self-assessed IPE improvement and recorded students' baseline perceptions for later review, while final reflective-writings documented students' self-actualized IPE development.

Subtype-specific differences in the risk of hospitalisation among patients infected with hepatitis E virus genotype 3 in Belgium, 2010-2018.

Some European countries recently reported an increase in hepatitis E virus genotype 3 (HEV-3) of the subtype 3c. No link between HEV-3 subtypes and severity is established to date. Here, we report that patients infected with HEV-3c were at lower risk of hospitalisation, compared to those infected with HEV-3f, the other main subtype circulating in Belgium.

Constitutive downregulation protein kinase C epsilon in hSOD1G93A astrocytes influences mGluR5 signaling and the regulation of glutamate uptake.

Accumulating evidence indicates that motor neuron degeneration in amyotrophic lateral sclerosis (ALS) is a non-cell-autonomous process and that impaired glutamate clearance by astrocytes, leading to excitotoxicity, could participate in progression of the disease. In astrocytes derived from an animal model of ALS (hSOD1G93A rats), activation of type 5 metabotropic glutamate receptor (mGluR5) fails to increase glutamate uptake, impeding a putative dynamic neuroprotective mechanism involving astrocytes. Using astrocyte cultures from hSOD1G93A rats, we have demonstrated that the typical Ca2+ oscillations associated with mGluR5 activation were reduced, and that the majority of cells responded with a sustained elevation of intracellular Ca2+ concentration. Since the expression of protein kinase C epsilon isoform (PKCɛ) has been found to be considerably reduced in astrocytes from hSOD1G93A rats, the consequences of manipulating its activity and expression on mGluR5 signaling and on the regulation of glutamate uptake have been examined. Increasing PKCɛ expression was found to restore Ca2+ oscillations induced by mGluR5 activation in hSOD1G93A -expressing astrocytes. This was also associated with an increase in glutamate uptake capacity in response to mGluR5 activation. Conversely, reducing PKCɛ expression in astrocytes from wild-type animals with specific PKCɛ-shRNAs was found to alter the mGluR5 associated oscillatory signaling profile, and consistently reduced the regulation of the glutamate uptake-mediated by mGluR5 activation. These results suggest that PKCɛ is required to generate Ca2+ oscillations following mGluR5 activation, which support the regulation of astrocytic glutamate uptake. Reduced expression of astrocytic PKCɛ could impair this neuroprotective process and participate in the progression of ALS.

PKC epsilon-dependent calcium oscillations associated with metabotropic glutamate receptor 5 prevent agonist-mediated receptor desensitization in astrocytes.

A critical role has been assigned to protein kinase C (PKC)ε in the control of intracellular calcium oscillations triggered upon activation of type 5 metabotropic glutamate receptor (mGluR5) in cultured astrocytes. Nevertheless, the physiological significance of this particular signalling profile in the response of astrocytes to glutamate remains largely unknown. Considering that kinases are frequently involved in the regulation of G protein-coupled receptors, we have examined a putative link between the nature of the calcium signals and the response regulation upon repeated exposures of astrocytes to the agonist (S)-3,5-dihydroxyphenylglycine. We show that upon repeated mGluR5 activations, a robust desensitization was observed in astrocytes grown in culture conditions favouring the peak-plateau-type response. At variance, in cell cultures where calcium oscillations were predominating, the response was fully preserved even during repeated challenges with the agonist. Pharmacological inhibition of PKCε or genetic suppression of this isoform using shRNA was found to convert an oscillatory calcium profile to a sustained calcium mobilization and this latter profile was subject to desensitization upon repetitive mGluR5 activation. Our results suggest a yet undocumented scheme in which the activity of PKCε contributes to preserve the receptor sensitivity upon repeated or sustained activations. Cover Image for this issue: doi: 10.1111/jnc.13797.

Academic conference posters: Describing visual impression in pharmacy education.

Background: Academic conference posters are a key communication before journal articles. Attention to visual attributes can enhance academic poster communication. Objective: This investigation's purpose was to create a visual impression measurement instrument, and then to describe and compare visual impression among scientific posters from an academic conference. Methods: A mixed-approach rubric was created to quickly measure visual impression of academic posters. Then, posters from a pharmacy education conference were retrospectively reviewed and scored. Visual impression was compared for traditional versus contemporary poster-formats. Various poster characteristics (poster-format, summary statement presence, abstract presence, wordiness, QR-code presence, logical sequencing, visuals) that might have impacted visual communication were coded. These characteristics were regressed onto visual impression scores. Results: Three-hundred seventy-eight posters were scored with sound inter-rater reliability. Contemporary poster-format scored significantly higher than traditional. Poster-format, abstract absence, lack of wordiness, QR-code presence, logical sequencing, and number of visuals were significant when regressed. Conclusion: Posters at one academic conference had varied visual impression. While a contemporary poster-format appeared more helpful, it was not a panacea; variation from poor through exemplary was seen with both poster-formats. Posters are not text-filled articles; displaying a combination of visuals/text clearly and concisely can help effective communication with academic posters.

Leadership and Followership in Health Professions: A Systematic Review.

Objective: Leadership discussion, including leadership development programs, is common. However, discussion of followership as a component of leadership seems less frequently discussed. With a focus on leadership and followership, this investigation reviewed the health-professions education literature and characterized leadership-followership within health-professions education. Methods: Using PubMed, ERIC, and Google Scholar, two investigators independently and systematically searched health-professions education literature for articles related to leadership and followership. Reports were categorized based on the articles by type, application, profession, leadership, and followership qualities. Results: Eighty-one articles were included. More than half [59% (48/81)] were theoretical, 27% (22/81) empirical, 7% (6/81) commentaries, and 6% (5/81) letters-to-the-editor). Empirical studies did not share outcomes that could be meaningfully combined quantitatively by meta-analysis; however, the vast majority (96%) of theoretical articles discussed a healthcare-related application of leadership and followership (e.g., improving patient care, improving communication, improving organizational efficiency). Thus, a qualitative review was completed. Of the 81 articles, 57% (n=46) involved multiple professions, while 43% (n=35) focused on a specific profession [Nursing (n=16), Medicine (n=7), Others (n=5) Surgery (n=3), Pharmacy (n=2), Veterinary Medicine (n=2)]. While most articles (75%) discussed leadership qualities (with top qualities of effective communication, visionary, and delegating tasks), fewer (57%) discussed followership qualities (with top qualities of being responsible, committed, and supportive). Of note, some qualities overlapped in both leadership and followership (with top qualities of effective communication, being supportive, and providing/receiving feedback). Conclusions: Leadership-Followership was described in many health-professions' education literature. However, Pharmacy and Veterinary Medicine had substantially fewer articles published on this topic. Notably, followership did not receive nearly as much attention as leadership. Leadership has a dynamic and complex interaction with followership highlighting that an effective leader must know how to be an effective follower and vice versa. To improve leadership within healthcare teamwork, education should focus on both leadership-followership.

Interacting within an asynchronous online interprofessional education workshop focused on social determinants of health.

BACKGROUND: Meaningful interprofessional education (IPE) involves students from at least two professions interacting to learn with, about, and from one another. Our objective was to describe a novel online approach used to create meaningful IPE within a social determinants of health (SDoH) workshop. INTERPROFESSIONAL EDUCATION ACTIVITY: This online workshop integrated four different professions' perspectives on SDoH (social-work, public-health, nursing, and pharmacy). Each six-student interprofessional team was assigned a local neighborhood. This week-long workshop had numerous activities (pre- and post-workshop quizzes, a SDoH-primer video, video self-introduction to teammates, a windshield questionnaire with two subsequent clinical cases, a post-workshop reflection, and post-workshop evaluation). For discussion, asynchronous video-based responses were used instead of traditional text-based discussion-boards. DISCUSSION: Quantitatively comparing quiz scores, students' SDoH knowledge increased with this workshop. Qualitatively from evaluations, most students found this workshop helpful and meaningful. Supporting use of video-based responses, many students' favorite aspect was interacting and collaborating within their interprofessional teams, although some students desired synchronous activities instead. Faculty facilitators confirmed that meaningful IPE interactions occurred. IMPLICATIONS: In short, students from multiple health-professions learned SDoH-content and, using video-based responses, interacted asynchronously during this online workshop. This report demonstrated one tool available to help facilitate meaningful IPE asynchronously. This asynchronous, online IPE workshop appears to be a promising format to be integrated with other in-person IPE sessions.

Human papillomavirus negative high grade cervical lesions and cancers: Suggested guidance for HPV testing quality assurance.

BACKGROUND: Some high-grade cervical lesions and cervical cancers (HSIL+) test negative for human papillomavirus (HPV). The HPV-negative fraction varies between 0.03 % and 15 % between different laboratories. Monitoring and extended re-analysis of HPV-negative HSIL+ could thus be helpful to monitor performance of HPV testing services. We aimed to a) provide a real-life example of a quality assurance (QA) program based on re-analysis of HPV-negative HSIL+ and b) develop international guidance for QA of HPV testing services based on standardized identification of apparently HPV-negative HSIL+ and extended re-analysis, either by the primary laboratory or by a national HPV reference laboratory (NRL). METHODS: There were 116 initially HPV-negative cervical specimens (31 histopathology specimens and 85 liquid-based cytology samples) sent to the Swedish HPV Reference Laboratory for re-testing. Based on the results, an international QA guidance was developed through an iterative consensus process. RESULT: Standard PCR testing detected HPV in 55.2 % (64/116) of initially "HPV-negative" samples. Whole genome sequencing of PCR-negative samples identified HPV in an additional 7 samples (overall 61.2 % HPV positivity). Reasons for failure to detect HPV in an HSIL+ lesion are listed and guidance to identify cases for extended re-testing, including which information should be included when referring samples to an NRL are presented. CONCLUSION: Monitoring the proportion of and reasons for failure to detect HPV in HSIL+ will help support high performance and quality improvement of HPV testing services. We encourage implementation of QA strategies based on re-analysis of "HPV negative" HSIL+ samples.

Using Rasch measurement for instrument rating scale refinement.

OUR SITUATION: Rasch measurement is an analysis tool that can provide validity evidence for instruments that attempt to measure student learning or other psychosocial behaviors, regardless if tools are newly created, modified, or previously developed. Rating scales are exceedingly common among psychosocial instruments and properly functioning rating scales are critical to effective measurement. Rasch measurement can help investigate this. METHODOLOGICAL LITERATURE REVIEW: Aside from using Rasch measurement from the beginning to help create rigorous new measurement instruments, researchers can also benefit from employing Rasch measurement on previously developed instruments that had not included Rasch measurement during development. This article is focused on Rasch measurement's unique analysis of rating scales. That is, Rasch measurement can uniquely help examine if and how an instrument's rating scale is functioning among newly studied respondents (who will likely differ from the originally researched sample). OUR RECOMMENDATIONS AND THEIR APPLICATION: After reviewing this article, the reader should be able to describe Rasch measurement, including how it is focused on fundamental measurement and how it differs from classical test theory and item-response theory, and reflect on situations in their own research where a Rasch measurement analysis might be helpful for generating validation evidence with a previously developed instrument. POTENTIAL IMPACT: In the end, Rasch measurement can offer a helpful, unique, rigorous approach to further developing instruments that scientifically measure, accurately and precisely.

A Multifaceted Approach for Evaluating Hepatitis E Virus Infectivity In Vitro: Cell Culture and Innovative Molecular Methods for Integrity Assessment.

Hepatitis E virus is a prominent cause of viral hepatitis worldwide. In Western countries, most infections are asymptomatic. However, acute self-limiting hepatitis and chronic cases in immunocompromised individuals can occur. Studying HEV is challenging due to its difficulty to grow in cell culture. Consequently, the detection of the virus mainly relies on RT-qPCR, which cannot differentiate between infectious and non-infectious particles. To overcome this problem, methods assessing viral integrity offer a possible solution to differentiate between intact and damaged viruses. This study aims at optimizing existing HEV cell culture models and RT-qPCR-based assays for selectively detecting intact virions to establish a reliable model for assessing HEV infectivity. In conclusion, these newly developed methods hold promise for enhancing food safety by identifying approaches for inactivating HEV in food processing, thereby increasing food safety measures.

Hepatitis E virus in pork meat products and exposure assessment in Belgium.

Zoonotic hepatitis E virus (HEV) genotype 3 infections are the predominant cause of acute viral hepatitis in Europe, mostly associated with the consumption of HEV contaminated pork meat. In this study we looked at the HEV RNA positivity rate of pork meat products readily available from Belgian supermarkets and evaluated the overall HEV consumer exposure in a Belgian context. Two basic assessments were performed in a 'worst-case' scenario setting: one solely focusing on the contamination level of the product itself (ingredients and processing parameters) and another estimating the overall consumer exposure, taking into account consumption habits in Belgium. Non-thermal-processed ready-to-eat (i.e. ready for consumption without additional cooking step by consumer) pork meat products (e.g. raw dried sausages), had a high estimated HEV contamination level, while thermal-processed ready-to-eat pork meat products (e.g. pork liver pâté) had the highest overall consumer exposure estimates. Following these assessments, pork liver pâtés, raw dried hams and raw dried sausages (n = 54) were purchased from Belgian supermarkets (n = 3) and analyzed for HEV RNA by RT-PCR. In total, 31 % (n = 17) products tested positive. HEV RNA was found in 65 % of the pork liver pâtés, 15 % of raw dried hams and 0 % of raw dried sausages. Phylogenetic analysis of four isolates (all gt3c) from pork liver pâté samples showed similarities with human clinical cases from Germany and Belgium.

Decrease in incidence of oral anticoagulant-related intracerebral hemorrhage over the past decade in the Netherlands.

Background: Data on oral anticoagulant-related (OAC) intracerebral hemorrhage (ICH) incidence are scarce. Most studies on incidence time trends were performed before the introduction of Direct Oral Anticoagulants (DOACs). Between 2008 and 2018, the number of OAC-users in the Netherlands increased by 63%, with the number of DOAC-users almost equaling that of Vitamin K Antagonists (VKA)-users. We aimed to determine the recent total and OAC-related ICH incidence and assess changes over the last decade, including the effect of DOAC introduction. Methods: All adult non-traumatic ICH patients presenting in any of three hospitals in the enclosed region of South-Limburg, the Netherlands, were retrospectively included, during two 3-year time periods: 2007-2009 and 2017-2019. OAC-related ICH was defined as ICH in patients using VKAs or DOACs. We calculated the incidence rate ratio (IRR) between the two study periods. Results: In the 2007-2009 period, we registered 652 ICHs of whom 168 (25.8%) were OAC-related (all VKA). In the 2017-2019 period, we registered 522 ICHs, 121 (23.2%) were OAC-related (70 VKA and 51 DOAC). In 2007-2009, the annual incidence of total ICH and OAC-related ICH was 40.9 and 10.5 per 100,000 person-years, respectively, which decreased to 32.4 and 7.5 per 100,000 person-years in 2017-2019. The IRR for total ICH and OAC-related ICH was 0.67 (95%-CI: 0.60-0.75) and 0.58 (0.46-0.73), respectively. Conclusion: Both total ICH and OAC-related ICH incidence decreased over the past decade in South-Limburg, the Netherlands, despite the aging population and increasing number of OAC-users. The introduction of DOACs, and possibly an improved cardiovascular risk management and change in OAC prescription pattern, could explain these findings.

Moving beyond Cronbach's Alpha and Inter-Rater Reliability: A Primer on Generalizability Theory for Pharmacy Education.

BACKGROUND: When available, empirical evidence should help guide decision-making. Following each administration of a learning assessment, data becomes available for analysis. For learning assessments, Kane's Framework for Validation can helpfully categorize evidence by inference (i.e., scoring, generalization, extrapolation, implications). Especially for test-scores used within a high-stakes setting, generalization evidence is critical. While reporting Cronbach's alpha, inter-rater reliability, and other reliability coefficients for a single measurement error are somewhat common in pharmacy education, dealing with multiple concurrent sources of measurement error within complex learning assessments is not. Performance-based assessments (e.g., OSCEs) that use raters, are inherently complex learning assessments. PRIMER: Generalizability Theory (G-Theory) can account for multiple sources of measurement error. G-Theory is a powerful tool that can provide a composite reliability (i.e., generalization evidence) for more complex learning assessments, including performance-based assessments. It can also help educators explore ways to make a learning assessment more rigorous if needed, as well as suggest ways to better allocate resources (e.g., staffing, space, fiscal). A brief review of G-Theory is discussed herein focused on pharmacy education. MOVING FORWARD: G-Theory has been common and useful in medical education, though has been used rarely in pharmacy education. Given the similarities in assessment methods among health-professions, G-Theory should prove helpful in pharmacy education as well. Within this Journal and accompanying this Idea Paper, there are multiple reports that demonstrate use of G-Theory in pharmacy education.

A Teachable Moment Matters for: Striving for inter-rater reliability with a patient counseling assessment rubric: Lessons learned.

The Teachable Moments Matter (TMM) category of articles is designed to offer readers insight into a methodological issue identified within a companion article. Written in collaboration with one of these authors, these articles provide an opportunity to focus on a challenge experienced by the authors and, in the process, provide one or more perspectives as to how to successfully navigate this issue. The current TMM is focused on issues and pitfalls in validation. The Journal hopes this case-based approach will help highlight the nuance of a topic in context, something that might get "lost" in the entirety of a full-length article.