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Study Objectives: The coronavirus disease pandemic has highlighted disparities in health care. Parents assist in sickle cell disease management in children. Understanding sleep in parents of children who are diagnosed with sickle cell disease is an important facet of disease management. Our objectives were to identify sleep characteristics of parents of children with sickle cell disease age 18 years or younger during the coronavirus disease pandemic, to investigate measures used by parents to achieve restful sleep, and to discuss how sleep may be improved in parents of children diagnosed with sickle cell disease., Methods: This study conducted 14 face-to-face semistructured interviews with parents of children diagnosed with sickle cell disease. A majority of the interviews (93%) were conducted at a hematology clinic. Qualitative thematic analysis was used to analyze data., Results: All parents reported that their child experienced pain because of sickle cell disease. Most parents (86.7%) reported that their child was diagnosed with sickle cell anemia. Four themes were identified: difficulty obtaining restful sleep during their child's sickle cell crisis, sleep during the coronavirus disease pandemic, factors affecting sleep, and effects of sleep disturbance and deprivation on parental performance., Conclusions: This research has highlighted some of the difficulties experienced by parents of children who are diagnosed with sickle cell disease and allows for additional insight into the sleep experiences of parents as they attempt to manage their child's disease., Citation: Osborne JC, Odlum M, Sedrak A. Sleep experiences of parents of children 18 years or younger with sickle cell disease during the COVID-19 pandemic: a qualitative study. J Clin Sleep Med . 2024;20(5):777-782., (© 2024 American Academy of Sleep Medicine.)

Background: Every year, many thousands of travellers return to the United Kingdom (UK) from visits to other countries and some will become unwell due to infections acquired abroad. Many imported infections have similar clinical presentations, such as fever and myalgia, so diagnostic testing is an important tool to improve patient management and outcomes. The aim of this study was to examine the demographics, travel history, presenting symptoms and diagnostic outcomes of referrals to the UK's specialist diagnostic Rare & Imported Pathogens Laboratory (RIPL) for the period 2015-2020., Methods: Anonymised clinical and laboratory data were extracted from RIPL's Laboratory Information Management System and cleaned prior to descriptive analysis of the data. Travel history data were mapped to one of eight world regions, whereas symptom data were categorised into presenting syndromes. Diagnostic data were categorised as either positive, equivocal or negative., Results: During the period 2015-2020, RIPL received 73 951 samples from 53 432 patients suspected of having infections that are rare in the UK. The most common age group for unwell returning travellers was 30-39 years and the most commonly reported travel destination was Southern and SE Asia. Dengue virus was the most diagnosed infection overall, followed by chikungunya, Zika, leptospirosis and spotted fever group Rickettsia. Dengue virus was among the top three most frequent diagnoses for all world regions except Europe and represented 62.5% of all confirmed/probable diagnoses., Conclusions: None of the top five infections diagnosed by RIPL in travellers are vaccine-preventable, therefore understanding traveller demographics, destination-specific risk factors and encouraging preventative behaviours is the best available strategy to reduce the number of returning travellers who become infected. Prompt referral of acute samples with a detailed travel history, including purpose of travel and activities undertaken as well as dates and destinations can be a valuable tool in designing public health interventions and diagnostic algorithms., (© International Society of Travel Medicine 2023. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Substance use disorders (SUDs) represent a critical public and occupational health issue. Therefore, understanding the process of SUD recovery has become an issue of growing importance among substance use and recovery professionals. Nonetheless, despite the acknowledged importance of employment for SUD recovery, little conceptual or empirical work exists on how the workplace might support or undermine SUD recovery. In this article, we address this limitation in several ways. First, to promote a better understanding of SUD recovery for occupational health researchers, we provide a brief overview of the nature of a SUD, prior definitions of SUD recovery, and general themes associated with the recovery process. Second, we develop a working definition of workplace supported recovery . Third, we present a heuristic conceptual model showing how the workplace might impact the SUD recovery process. Fourth, using this model and research from the substance use and occupational health literatures, we develop a series of general research propositions. These propositions highlight broad directions requiring more detailed conceptualization and empirical research to understand better how work conditions may support or undermine the process of employee SUD recovery. Our overarching goal is to motivate innovative conceptualization and research on workplace supported recovery from SUDs. Such research may inform the development and evaluation of workplace interventions and policies supporting SUD recovery and highlight the benefits of workplace supported SUD recovery for employees, employers, and communities. Research on this issue may allow occupational health researchers to impact a significant societal and occupational health issue.

Background: Cases of human monkeypox are rarely seen outside of west and central Africa. There are few data regarding viral kinetics or the duration of viral shedding and no licensed treatments. Two oral drugs, brincidofovir and tecovirimat, have been approved for treatment of smallpox and have demonstrated efficacy against monkeypox in animals. Our aim was to describe the longitudinal clinical course of monkeypox in a high-income setting, coupled with viral dynamics, and any adverse events related to novel antiviral therapies., Methods: In this retrospective observational study, we report the clinical features, longitudinal virological findings, and response to off-label antivirals in seven patients with monkeypox who were diagnosed in the UK between 2018 and 2021, identified through retrospective case-note review. This study included all patients who were managed in dedicated high consequence infectious diseases (HCID) centres in Liverpool, London, and Newcastle, coordinated via a national HCID network., Findings: We reviewed all cases since the inception of the HCID (airborne) network between Aug 15, 2018, and Sept 10, 2021, identifying seven patients. Of the seven patients, four were men and three were women. Three acquired monkeypox in the UK: one patient was a health-care worker who acquired the virus nosocomially, and one patient who acquired the virus abroad transmitted it to an adult and child within their household cluster. Notable disease features included viraemia, prolonged monkeypox virus DNA detection in upper respiratory tract swabs, reactive low mood, and one patient had a monkeypox virus PCR-positive deep tissue abscess. Five patients spent more than 3 weeks (range 22-39 days) in isolation due to prolonged PCR positivity. Three patients were treated with brincidofovir (200 mg once a week orally), all of whom developed elevated liver enzymes resulting in cessation of therapy. One patient was treated with tecovirimat (600 mg twice daily for 2 weeks orally), experienced no adverse effects, and had a shorter duration of viral shedding and illness (10 days hospitalisation) compared with the other six patients. One patient experienced a mild relapse 6 weeks after hospital discharge., Interpretation: Human monkeypox poses unique challenges, even to well resourced health-care systems with HCID networks. Prolonged upper respiratory tract viral DNA shedding after skin lesion resolution challenged current infection prevention and control guidance. There is an urgent need for prospective studies of antivirals for this disease., Funding: None., Competing Interests: Declaration of interests JD reports an unremunerated role on a Data Safety and Monitoring Board (DSMB) for an extended access programme for tecovirimat. MGS reports non-remunerated roles in a DSMB for Pfizer's mRNA vaccine programme, the UK Scientific Advisory Group for Emergencies, and New Emerging Respiratory Virus Threats Advisory Group; and reports chairing a Scientific Advisory Board and holding stock or stock options in Integrum Scientific LLC, Greensboro, NC, USA. SHK reports grants (unrelated to the current work) from ViiV, Merck, and Gilead Sciences; advisory board membership for ViiV and Merck; honoraria from ViiV; and being an unremunerated chair of a DSMB for a Gates Foundation-funded trial., (Copyright © 2022 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY-NC-ND 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

Background: Underrepresented minority (URM) students in nursing education are essential to address health disparities., Purpose: To determine barriers and facilitators to success among URM students in nursing education., Methods: CINAHL, PubMed, Embase, and PsycINFO were searched for English language research studies published between 1980 and 2020, which focused on barriers and facilitators to success in nursing education in the United States among URM students. Using PRISMA guidelines, 2 reviewers independently reviewed and appraised the quality of relevant articles., Results: Among the 4435 articles surveyed, 14 met inclusion criteria. Most studies were focused on Black and Hispanic/Latinx students, and 71% were qualitative. Barriers included social isolation, caregiving demands, lack of mentorship, and inadequate financial resources. Facilitators included mentorship, positive class and curriculum, family support, financial support, and diverse/inclusive learning environments., Conclusions: URM students face complex barriers to success in nursing education. Addressing these barriers requires a multilevel approach., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Black and Hispanic individuals experience poorer outcomes related to coronavirus disease (COVID-19), yet have alarmingly lower uptake of the COVID-19 vaccine compared to non-Hispanic White individuals., Objective: To explore the perceptions of barriers and facilitators of COVID-19 vaccine acceptance among Black and Hispanic adults in the New York., Methods: A qualitative study consisting of one-on-one semistructured interviews with Black and/ or Hispanic adults in New York state was conducted. Participants were recruited from local businesses, community-based social service agencies and Black and Hispanic churches and a college. All data were collected between February and March 2021. Transcripts were analyzed using qualitative thematic analysis., Results: A total of 50 individuals (32 [64%] women; mean [SD] age, 42.04 [15.99] years) participated. The majority of participants identified as Black (n = 34, 68%), 9 (18%) identified as Hispanic, 3 (6%) as Black and Hispanic, and 4 (8%) as White and Hispanic. Twenty-two participants (44%) had high-school level education or less. Mistrust emerged as a central barrier to COVID-19 vaccine acceptance. Facilitators of effective COVID-19 vaccine messages include (1) key informants: like me, (2) personalized community outreach, (3) present the facts, and (4) communicate across multiple media platforms., Conclusions: This qualitative study found that among Black and Hispanic participants, receipt of reliable vaccine related information, social networks, seeing people like themselves receive the vaccination, and trusted doctors are key drivers of vaccine acceptance., (Copyright © 2021 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.)

Home health aides (HHAs) are a vital workforce essential to meet the complex care needs of the persons living with dementia (PLWD) who remain at home. Care plans for PLWD in the home healthcare setting should incorporate HHAs perspectives. We sought to understand HHAs' perspectives about their information needs in caring for PLWD, from June to August 2020, semi-structured interviews telephone interviews (n = 25) with English and Spanish-speaking HHAs with limited English proficiency in the New York metropolitan area. Interviews were audio-recorded, transcribed verbatim and transcripts were analyzed using conventional content analysis. Four key themes emerged reflective of information needs of HHAs caring for PLWD: (1) ambiguities of scope of HHA tasks related to medication management; (2) clinical information needs of HHAs; (3) dementia-related concerns; and (4) going above and beyond. Findings from this research can guide efforts to develop dementia-specific care plans, and training to support the HHA workforce caring for the growing population of PLWD., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Objective: To investigate medication factors and patient characteristics associated with readmissions following alcohol-related hospitalizations., Patients and Methods: Adult patients admitted from September 1, 2016, through August 31, 2019, who had an alcohol-related hospitalization were identified through electronic health records. Patient characteristics and medications of interest administered during hospitalization or prescribed at discharge were identified. Medications of interest included US Food and Drug Administration-approved medications for alcohol use disorder, benzodiazepines, barbiturates, gabapentin, opioids, and muscle relaxants. The primary outcome was to identify medications and patient factors associated with 30-day alcohol-related readmission. Secondary outcomes included medications and patient characteristics associated with multiple alcohol-related readmissions within a year from the index admission (ie, two or more readmissions) and factors associated with 30-day all-cause readmission., Results: Characteristics of the 932 patients included in this study associated with a 30-day alcohol-related readmission included younger age, severity of alcohol withdrawal, history of psychiatric disorder, marital status, and the number of prior alcohol-related admission in the previous year. Benzodiazepine or barbiturate use during hospitalization or upon discharge was associated with 30-day alcohol-related readmission ( P =.006). Gabapentin administration during hospitalization or upon discharge was not associated with 30-day alcohol-related readmission ( P =.079)., Conclusion: The findings reinforce current literature identifying patient-specific factors associated with 30-day readmissions. Gabapentin use was not associated with readmissions; however, there was an association with benzodiazepine/barbiturate use., (© 2021 The Authors.)

The United States is experiencing an evolving and worsening drug overdose epidemic. Although the rate of drug use among workers has remained relatively stable, the risk of overdose and death among drug users has not, as illicit drugs have increased in potency and lethality. The cumulative impacts of COVID-19 and the opioid crisis increase the likelihood of illness and death among workers with opioid use disorder. Workplaces represent a critical point of contact for people living in the United States who are struggling with or recovering from a substance use disorder, and employment is a vital source of recovery "capital." The benefits of addressing substance use in the workplace, supporting treatment, and employing workers in recovery are evident. The National Institute for Occupational Safety and Health has published research to inform policy and practice toward prevention efforts and has developed accessible resources and toolkits to support workers, employers, and workplaces in combatting the opioid overdose crisis and creating safer, healthier communities.

Background: Despite their integral role, Home Health Aides (HHAs) are largely unrecognized as essential to implementing effective infection prevention and control practices in the home healthcare setting. We sought to understand the infection prevention and control needs and challenges associated with caring for patients during the pandemic from the perspective of HHAs., Methods: From June to August 2020, data were collected from HHAs in the New York metropolitan area using semi-structured qualitative interviews by telephone; 12 HHAs were interviewed in Spanish. Audio-recorded interviews were transcribed, translated and analyzed using conventional content analysis., Results: In total, 25 HHAs employed by 4 unique home care agencies participated. HHAs had a mean age of 49.8 (± 9.1), 24 (97%) female, 11 (44%) Black, 12 (48%) Hispanic. Three major themes related to the experience of HHA's working during the COVID-19 pandemic emerged: (1) all alone, (2) limited access to information and resources, and (3) dilemmas related to enhanced COVID-19 precautions. Hispanic HHAs with limited English proficiency faced additional difficulties related to communication., Conclusions: We found that HHA communication with nursing staff, plays a key role in infection control efforts in home care. Efforts to manage COVID-19 in home care should include improving communication between HHAs and nursing staff., (Copyright © 2021 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.)

Currently, there is minimal guidance to antiepileptic dose adjustment for a patient requiring either venoarterial (VA) extracorporeal membrane oxygenation (ECMO) or plasma exchange (PLEX) therapy, and to our knowledge, there are rare guidances for a patient requiring both. Given the dangers with non-therapeutic concentrations of phenytoin, it is critical for the intensive care unit (ICU) practitioner to understand how the pharmacokinetic parameters of phenytoin change in critically ill patients requiring extracorporeal support. This case study presents a 41-year-old female transferred to the cardiovascular ICU requiring VA ECMO and PLEX for the treatment of systemic lupus erythematosus (SLE)-induced catastrophic antiphospholipid syndrome (CAPS). Free phenytoin concentrations were measured to assess the removal of phenytoin. There was no significant decrease in the free phenytoin concentrations post-PLEX and while on ECMO. Free phenytoin concentrations are not influenced in the setting of PLEX and while on ECMO., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2021, Osborne et al.)

Millions of older Americans receive nurse practitioner (NP)-provided home based primary care (HBPC). Little is known about how state scope-of- practice (SOP) laws may impact use of NP-home visits. Using 2017 Centers for Medicare and Medicaid Services Provider Utilization and Payment Data Public Use File (PUF), we examined the impact of state SOP laws on the use of NP-home visits. The PUF file was merged with the 2017 American Community Survey to assess area-level median income. Over 4.4 million home visits were provided to 1.6 million Medicare beneficiaries. NPs represented the largest share of providers (47.5%). In states with restricted SOP laws, compared to NPs, physicians and physician assistants had higher odds of providing HBPC. In states with reduced SOP laws, compared to NPs, physicians and PAs had decreased odds of providing HBPC. Our study provides evidence that SOP restrictions are associated with decreased utilization of NP-provided HBPC., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: While pain is the hallmark of sickle cell disease (SCD), healthcare personnel are often ill-equipped to adequately treat patients who present in vaso-occlusive crisis (VOC). Although symptom severity varies from individual to individual, SCD is characterized by intervallic pain as a result of oxygen deprivation in tissues and organs. Regardless of pain severity, SCD patients are often viewed as drug seekers by healthcare personnel who have concerns regarding patients' dependence on opioids which may lead to addiction. The objective was to assess the types and amount of opioids used to treat VOC in comparison to Centers for Disease Control opioid prescription guidelines., Methods: Literature search was conducted using CINAHL, PubMed, the Cochrane Library, Web of Science and hand search. Data were analyzed from 1999 to 2018. Randomized trials, observational, and case studies involved hospitalized adults with SCD who were prescribed opioids to treat VOC. Quality assessment was conducted using Downs and Black checklist. Meta-analysis was not conducted., Results: Five studies were conducted in the USA, Arabia and the Netherlands, and the USA and Canada were included. Participants were treated with either morphine or morphine milligram equivalent (MME). No study used the same method of opioid administration., Conclusions: Patients with SCD who are hospitalized secondary to VOC mostly received opioids for pain well within the Centers for Disease Control and Prevention prescription guidelines. No uniform method exists. Additional research is warranted., Competing Interests: None to declare., (Copyright 2021, Osborne et al.)

Introduction: Approximately 10% to 15% of people affected by sickle cell disease (SCD) in the United States are Latino, many of whom are primary Spanish speakers. A key tool for self-reported outcome measures, the Pediatric QOL Inventory (PedsQL) SCD module, was not available in Spanish. Here, we assess the linguistic validity of a Spanish translation and compare perception of disease-specific and generic quality of life (QOL) in a sample of Latino and non-Latino children with SCD and their parents., Method: Following forward and backward translation, Spanish-speaking child-parent dyads linguistically validated the translated instruments. Disease-specific and generic QOL perception of 28 child-parent dyads who participated in a clinical feasibility trial, HABIT (Hydroxyurea Adherence for Personal Best in Sickle Cell Disease), were compared by ethnicity. Data were analyzed by descriptive statistics, Mann-Whitney U test, absolute score differences, and minimal clinically important differences (MCID)., Results: The translated questionnaire required no further language changes. QOL scores were higher for Latino children and parents compared with non-Latinos, with score differences exceeding MCIDs for total scores and the majority of subscale scores., Conclusion: Spanish language PedsQL SCD instruments allow measurement of QOL in Spanish-speaking Latino children with SCD and their parents. Score differences for Latinos mostly exceeded MCIDs, suggesting that these differences are clinically meaningful. Confirmation of these findings is warranted.

We report a case of Toscana virus encephalitis. This emerging pathogen is among the three most common causes of meningoencephalitis in Europe during the warm season, yet remains under-recognised. Doctors should consider Toscana virus infection in patients presenting with neurological symptoms who have a relevant exposure history during the summer months., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

Background: Mobile communication technologies provide novel opportunities to support clinic-based health initiatives. Adoption of technologies for daily use and for health communication can differ between communities, depending upon demographic and cultural characteristics., Design and Methods: A survey was administered in adolescent primary care and subspecialty clinics to assess parent-adolescent preferences in use of mobile technologies and social media to support provider-patient communication in an urban Latino community., Results: Of 130 respondents (65 parent-adolescent pairs), approximately half frequently sent and received text messages but lacked agreement regarding the other's text messaging use. In contrast, adolescents only rarely used email compared to parents (15.4% versus 37.5%, P=0.006). Of social media, Facebook™/MySpace™ was most frequently used by parents and youth (60% and 55.4%, P=0.59); however, most lacked interest in using social media for health communication. Parents reported more interest than adolescents in receiving email (73.4% versus 35.9%, P<0.001) and text messages (58.5% versus 33.9%, P=0.005) for health, but had more concerns about privacy issues (26.2% versus 9.2%, P=0.01). Respondents who were American born (aOR 5.7, 95%CI 1.2-28.5) or regularly used Instant Messaging or Facebook™/MySpace™ (aOR 4.6, 95%CI 1.4-14.7) were more likely to be interested in using social media for health communication., Conclusions: These findings underscore the importance of targeted assessment for planning the utilization of communication technologies and social media in clinical care or research for underserved youth. Significance for public healthCommunication technologies provide novel opportunities to support clinic-based health initiatives for underserved youth. However, adoption of technologies among communities may differ depending upon demographic and cultural characteristics. We surveyed a sample of urban Latino parents and youth regarding their current use of mobile and social media technologies and preferences for use of these technologies for health communication. This is the first study to compare the perspective of underserved parents and their youth regarding use of a wide variety of mobile and social communication technologies, concordance between youth-parent pairs in perceived use of texting and preferences for the purpose of health communication. Our findings differ from those from adults surveyed in other under-served communities, highlighting heterogeneity between communities. Variations in use of communication technologies and social media and preferences between parent-youth pairs suggest that understanding these factors within target populations is crucial for successful use to support health and health services.

Objectives: Undifferentiated febrile illnesses have been a threat to British expeditionary forces ever since the Crusades. The infections responsible were identified during the Colonial Era, both World Wars and smaller conflicts since, but nearly all remain a significant threat today. Undiagnosed febrile illnesses have occurred amongst British troops in Helmand, Afghanistan since 2006 and so a fever study was performed to identify them., Methods: From May to October 2008, all undifferentiated fever cases seen at the British field hospital in Helmand, Afghanistan were assessed using a standard protocol. Demographic details, clinical features and laboratory results were recorded and paired serum samples were sent for testing at the UK Special Pathogens Reference Unit (SPRU)., Results: Over 6 months, there were 26 cases of"Helmand Fever" assessed and 23 diagnoses were made of which 12 (52%) were sandfly fever, 6 (26%) were acute Qfever and 5 (22%) were rickettsial infections. Four cases had co-infections and 7 cases were not diagnosed (mostly due to inadequate samples). The clinical features and laboratory results available at the British field hospital did not allow these diseases to be distinguished from each other. The exact type of rickettsial infection could not be identified at SPRU., Conclusions: These cases probably represent the "tip of an iceberg" for British and Allied forces. More resources for diagnostic facilities and follow-up of patients are required to improve the management and surveillance of "Helmand Fever" cases; until then doxycycline 100 mg twice daily for 2 weeks should be given to all troops who present with an undifferentiated febrile illness in Helmand, Afghanistan. Patients with acute Q fever should be followed-up for at least 2 years to exclude chronic Q fever. Prevention of these diseases requires a better understanding of their epidemiology, but prophylaxis with doxycycline and possibly Q fever vaccine should be considered.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus best known for causing fever, rash, arthralgia, and occasional neurologic disease. By using real-time reverse transcription-PCR, we detected CHIKV in plasma samples of 8 (14%) of 58 children with suspected central nervous system infection in Bellary, India. CHIKV was also detected in the cerebrospinal fluid of 3 children.

The bunyavirus nucleocapsid protein, N, plays a central role in viral replication in encapsidating the three genomic RNA segments to form functional templates for transcription and replication by the viral RNA-dependent RNA polymerase. Here we report functional mapping of interacting domains of the Bunyamwera orthobunyavirus N protein by yeast and mammalian two-hybrid systems, immunoprecipitation experiments, and chemical cross-linking studies. N forms a range of multimers from dimers to high-molecular-weight structures, independently of the presence of RNA. Deletion of the N- or C-terminal domains resulted in loss of activity in a minireplicon assay and a decreased capacity for N to form higher multimers. Our data suggest a head-to-head and tail-to-tail multimerization model for the orthobunyavirus N protein.

Background: In our cohort of patients with hepatitis-C virus, which is the most common indication for liver transplantation, we have noted higher relative blood levels of tacrolimus compared to patients without hepatitis-C virus., Aim: To verify this observation and determine its clinical significance, we performed a comparison of doses and blood levels of tacrolimus in hepatitis-C virus and non- hepatitis-C virus liver transplantation recipients., Methods: Tacrolimus dose and trough level, as well as mean alanine aminotransferase, for all patients transplanted at our center with a deceased donor between 1/1995 and 12/1999 with hepatitis-C virus were recorded at monthly intervals during the first 24 months following transplantation and compared to patients without hepatitis-C virus., Results: The tacrolimus levels for hepatitis-C virus and non-hepatitis-C virus patients were not significantly different at any of the monthly intervals, except month 9. In addition, the overall mean tacrolimus levels for hepatitis-C virus and non-hepatitis-C virus patients were not significantly different (P = ns). However, the mean tacrolimus dose (mg/kg) was significantly higher for hepatitis-C virus patients at 12, 15, 18, 21 and 24 months, P < 0.01. The total mean tacrolimus dose in hepatitis-C virus patients was lower during year one by 39% (P = 0.018) and by 73% (P = 0.001) during year two. The total difference in cost of tacrolimus (for year one and two) administered to hepatitis-C virus patients was $4920, P = 0.03. The serum alanine aminotransferase was significantly higher in hepatitis-C virus patients at each monthly interval except month 1, P < or = 0.01., Conclusions: Liver transplant recipients with hepatitis-C virus require significantly lower oral doses of tacrolimus to achieve the same blood levels compared to non-hepatitis-C virus patients. This difference may result in a significant reduction in the cost of tacrolimus in hepatitis-C virus patients. The most likely explanation for these findings is decreased hepatic clearance of tacrolimus caused by mild hepatic injury from recurrent hepatitis-C virus.

Sequence analysis of the L RNA genome segment and predicted encoded L polymerase protein of Crimean-Congo hemorrhagic fever (CCHF) virus (genus Nairovirus, family Bunyaviridae) demonstrates that they are approximately twice the size of those found in viruses of other bunyavirus genera. The CCHF virus L segment and encoded protein (12,164 nucleotides and 3944 amino acids, respectively) are similar in size and sequence to those of the nairovirus Dugbe virus (12,255/62% and 4036/62% nucleotide and amino acid length/identity, respectively). The identification of an ovarian tumor (OTU)-like protease motif in the L protein amino termini of the nairoviruses Dugbe, CCHF, and Nairobi sheep disease (NSD) indicates these proteins are members of the recently described OTU-like protease family and suggests that these large proteins may be polyproteins that are autoproteolytically cleaved or involved in deubiquitination.

The genus Nairovirus (family Bunyaviridae) contains seven serogroups consisting of 34 predominantly tick-borne viruses, including several associated with severe human and livestock diseases [e.g., Crimean Congo hemorrhagic fever (CCHF) and Nairobi sheep disease (NSD), respectively]. Before this report, no comparative genetic studies or molecular detection assays had been developed for this virus genus. To characterize at least one representative from each of the seven serogroups, reverse transcriptase-polymerase chain reaction (RT-PCR) primers targeting the L polymerase-encoding region of the RNA genome of these viruses were successfully designed based on conserved amino acid motifs present in the predicted catalytic core region. Sequence analysis showed the nairoviruses to be a highly diverse group, exhibiting up to 39.4% and 46.0% nucleotide and amino acid identity differences, respectively. Virus genetic relationships correlated well with serologic groupings and with tick host associations. Hosts of these viruses include both the hard (family Ixodidae) and soft (family Argasidae) ticks. Virus phylogenetic analysis reveals two major monophyletic groups: hard tick and soft tick-vectored viruses. In addition, viruses vectored by Ornithodoros, Carios, and Argas genera ticks also form three separate monophyletic lineages. The striking similarities between tick and nairovirus phylogenies are consistent with possible coevolution of the viruses and their tick hosts. Fossil and phylogenetic data placing the hard tick-soft tick divergence between 120 and 92 million years ago suggest an ancient origin for viruses of the genus Nairovirus.

A virus isolated from dead Chaerephon plicata bats collected near Kampot, Cambodia, was identified as a member of the family Bunyaviridae by electron microscopy. The only bunyavirus previously isolated from Chaerephon species bats in South-East Asia is Kaeng Khoi (KK) virus (genus Orthobunyavirus), detected in Thailand over 30 years earlier and implicated as a public health problem. Using RT-PCR, nucleotide sequences from the M RNA segment of several virus isolates from the Cambodian C. plicata bats were found to be almost identical and to differ from those of the prototype KK virus by only 2.6-3.2 %, despite the temporal and geographic separation of the viruses. These results identify the Cambodian bat viruses as KK virus, extend the known virus geographic range and document the first KK virus isolation in 30 years. These genetic data, together with earlier serologic data, show that KK viruses represent a distinct group within the genus Orthobunyavirus.

The genome of Bunyamwera virus (BUN) (family Bunyaviridae, genus Bunyavirus) comprises three negative-sense RNA segments which act as transcriptional templates for the viral polymerase only when encapsidated by the nucleocapsid protein (N). Previous studies have suggested that the encapsidation signal may reside within the 5' terminus of each segment. The BUN N protein was expressed as a 6-histidine-tagged fusion protein in Escherichia coli and purified by metal chelate chromatography. An RNA probe containing the 5'-terminal 32 and 3'-terminal 33 bases of the BUN S (small) genome segment was used to investigate binding by the N protein in vitro using gel mobility shift and filter binding assays. On acrylamide gels a number of discrete RNA-N complexes were resolved, and analysis of filter binding data indicated a degree of cooperativity in N protein binding. RNA-N complexes were resistant to digestion with up to 1 microg of RNase A per ml. Competition assays with a variety of viral and nonviral RNAs identified a region within the 5' terminus of the BUN S segment for which N had a high preference for binding. This site may constitute the signal for initiation of encapsidation by N.

Radiation target analysis is based on the action of ionizing radiation directly on macromolecules. Interactions of this radiation with the molecules leads to considerable structural damage and consequent loss of biological activity. The radiation sensitivity is dependent on the size of the macromolecules. There has been confusion and discrepancy as to whether the molecular mass or the molecular volume was the determinant factor in the sensitivity. Some proteins are known to change their hydrodynamic volume at low pH, and this characteristic can be utilized to compare the radiation sensitivities of these proteins in the two states. The results show that the radiation sensitivity of proteins depends on the mass of the molecule and is independent of the molecular volume/shape.

Radiation inactivation and sedimentation equilibrium analysis were used to determine the functional and physical size of the chicken hepatic membrane receptor that binds N-acetylglucosamine-terminated glycoproteins. Purified plasma membranes from chicken liver were irradiated with high energy electrons and assayed for 125I-agalactoorosomucoid binding. Increasing the dose of ionizing radiation resulted in a monoexponential decay in binding activity due to a progressive loss of binding sites. The molecular mass of the chicken lectin, determined in situ by target analysis, was 69,000 +/- 9,000 Da. When the same irradiated membranes were solubilized in Brij 58 and assayed, the binding protein exhibited a target size of 62,000 +/- 4,000 Da; in Triton X-100, the functional size of the receptor was 85,000 +/- 10,000 Da. Sedimentation equilibrium measurements of the purified binding protein yielded a lower limit molecular weight of 79,000 +/- 7,000. However, the solubilized lectin was detected as a heterogeneous population of oligomers with molecular weights as high as 450,000. Addition of calcium or calcium plus N-acetylglucosamine decreased the higher molecular weight species, but the lower limit molecular weights remained invariant. Similar results were determined when the chicken lectin was solubilized in Brij 58, C12E9, or 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonic acid (CHAPS). Results from the present study suggest that in the plasma membrane, the functional species of the chicken hepatic lectin exists as a trimer. However, in detergent solution, the purified receptor forms a heterogeneous population of irreversible oligomers that exhibit binding activity proportional to size.

Pancreatic cholesterol esterase (CEase) regulates dietary cholesterol absorption and is activated in the presence of trihydroxy bile salts while remaining inactive monohydroxy bile salts. CEase from rat pancreas has been purified by ammonium sulfate precipitation, hydroxylapatite chromatography, and gel filtration on Sephacryl S-200/S-300 columns connected in series, and its homogeneity and Mr (55,418 +/- 288) have been determined by sedimentation equilibrium centrifugation. The effects of tri-, di-, and monohydroxy bile salts on the conformation of the purified enzyme in buffer solution and in an in vitro assay system were studied by circular dichroism spectropolarimetry. The CD spectrum of the enzyme in solution shows a curve shape suggestive of an alpha-helicity, but low mean residue ellipticity (MRE) values may indicate an important beta-turn contribution. Sodium cholate, a trihydroxy bile salt, induces a decrease in the negative MRE values of the enzyme in solution at bile salt concentrations of 70-100 nM, with no further spectral changes at concentrations as high as 1 mM. Sodium cholate concentrations higher than 1 microM also induce an increase in the enzyme's negative MRE values under activity assay conditions, which reverts toward its original value once the reaction reaches equilibrium. These latter changes are interpreted as induced by substrate binding to the enzyme followed by partial substrate depletion after the reaction reaches equilibrium. Sodium deoxycholate, a dihydroxy bile salt, induces unstable transient increases and decreases in the MRE values of CEase in buffer solution and under activity assay conditions. These changes are bile salt concentration-dependent and may reflect self-association of the protein. Sodium taurolithocholate, a monohydroxy bile salt, does not affect the CD spectrum of CEase, and neither the di- or the monohydroxy bile salt activates the enzyme.

Radioiodinated apolipoprotein A-I was separated into two pools by gel chromatography on Sephadex G-150 superfine resin or by high performance liquid chromatography using a TSK-2000 column. The first pool, pool 1, was indistinguishable from unlabeled apo-A-I as judged by sedimentation equilibrium, circular dichroic measurements, and radial immunodiffusion. This pool self-associated according to a monomer-dimer-tetramer-octamer scheme and underwent the expected conformation changes with dissociation as reported previously for unlabeled apo-A-I. The radioiodinated material in pool 2 had less secondary structure as compared to the unlabeled material and did not self-associate. Protein in this pool was immunochemically less reactive than unlabeled apo-A-I. Kinetic studies, in vivo, demonstrated that these two pools of radioiodinated apo-A-I are also distinguished metabolically. Discussion centers around possible mechanisms of formation of the incompetent protein in pool 2 and factors that should be taken into consideration when using unfractionated radioiodinated apo-A-I for experimental studies.

The syntheses of four new sulfobetaine derivatives of bile salts are presented, along with a general set of criteria for useful detergents in membrane biochemistry. Physical properties including the critical micelle concentration, aggregation number, partial specific volume, critical micellar temperature, uv-vis spectrum, and circular dichroism spectrum are examined for the new compounds. To examine the interaction of this class of compounds with macromolecules, one of these (CHAPS) was further studied. Circular dichroism spectra of apolipoprotein C-III2 were measured in the presence of varying concentrations of CHAPS to determine the effect of this compound on secondary structure. Gel-exclusion chromatography and sedimentation equilibrium studies of cytochrome P-450 in the presence of CHAPS were also performed to establish the ability of this detergent to disaggregate cytochrome P-450 to a monomeric/dimeric state.

To investigate the risk of cytomegalovirus transmission by corneal transplantation, we quantitated anticytomegalovirus IgG levels of donor, preoperative, and postoperative serum samples. Of 118 patients, 79 (67%) were seropositive preoperatively. Twenty-five patients who were seronegative preoperatively received a graft from a positive donor and two (8%) seroconverted. Eleven patients who were seronegative preoperatively received a graft from a negative donor and one (9%) seroconverted. None of the patients who seroconverted had a febrile illness and all three grafts were clear.

The soluble form of dopamine beta-hydroxylase from bovine adrenal medulla has previously been shown to exist as a tetrameric species of Mr = 290,000 composed of two disulfide-linked dimers. Here we report that this enzyme can also undergo a reversible tetramerdimer dissociation which is dependent on pH. Gel permeation chromatography of dopamine beta-hydroxylase at pH 5.0 demonstrates a Stokes radius of 5.8 nm. When the pH is shifted to 5.7, the Stokes radius changes to 6.9 nm. Sedimentation equilibrium analysis of the purified enzyme demonstrates that this change in molecular size is due to a change in molecular weight. At low protein concentration, the estimated Mr of the enzyme is 145,000 at pH 5.0 and at high protein concentration approaches 290,000 at pH 5.7. This change in Mr is consistent with the existence of a tetramer-dimer dissociation and a change in the equilibrium constant from 1.8 X 10(-6) M to 1.16 X 10(-9) M when the pH is increased from 5.0 to 5.7. This pH-dependent subunit dissociation is correlated with pH-dependent changes in enzyme activity. Purified bovine-soluble dopamine beta-hydroxylase activity is a hyperbolic function of tyramine concentration at pH 5.0. However, the hydroxylase activity displays non-hyperbolic kinetics at pH 6.0. The kinetic data obtained at pH 6.0 can be accounted for by fitting to a model containing two nonidentical catalytic forms of enzyme generated by the pH-dependent partial dissociation of tetrameric enzyme to dimeric subunits. The two catalytic forms have apparently identical maximal velocities; however, they differ in their Michaelis constants for the substrate; the dimeric form having a low Km and the tetrameric form having a high Km. Since the pH inside bovine adrenal medullary chromaffin granules is approximately 5.5, we conclude that the subunits of dopamine beta-hydroxylase are in dynamic dissociation in a physiologically important pH range.

We have investigated effects of temperature on the catalytic and allosteric properties of the cGMP-stimulated cyclic nucleotide phosphodiesterase from calf liver. Vmax for cAMP and cGMP increased as assay temperature increased from 5 to 45 degrees C. At substrate concentrations below Kmapp, however, hydrolysis increased as temperature decreased from 45 to 5 degrees C and was much greater at 5 degrees C than at 45 degrees C. As assay temperature decreased, Kmapp for cAMP and cGMP decreased. Hill coefficients for cAMP and cGMP were approximately 1.9 at 45 degrees C and 1.2-1.0 at 5 degrees C. cGMP stimulated hydrolysis of 0.5 microM [3H]cAMP at all assay temperatures. Although maximal activity stimulated by cGMP, like Vmax, was lowest at 5 degrees C, presumably because of the effect of temperature on catalytic activity, the apparent activation constant (K alpha app) for cGMP stimulation was lower at 5 degrees C than at 45 degrees C. Thus, affinity for both substrate and effector was increased at 5 degrees C, suggesting that low temperature promotes transitions of the cGMP-stimulated phosphodiesterase to a "high affinity" state. That cGMP stimulated cAMP hydrolysis at 5 degrees C suggests that temperature-induced transitions are incomplete and/or readily reversible. In assays at 30 degrees C competitive inhibitors, like substrates, induce allosteric transitions which result in enhanced hydrolysis of low substrate (1.0 microM [3H] cAMP) concentrations. At higher substrate concentrations (50 microM [3H]cAMP), with the enzyme in the "activated" state, inhibitors compete with substrate at catalytic sites and reduce hydrolysis. At 45 degrees C, as at 30 degrees C, 1-methyl-3-isobutylxanthine (IBMX) and papaverine increased hydrolysis of 1.0 microM [3H]cAMP and reduced hydrolysis of 50 microM [3H]cAMP. At 5 degrees C, however, IBMX and papaverine inhibited hydrolysis of both 1.0 and 50 microM [3H]cAMP. Enzyme activity was relatively more sensitive to inhibition by IBMX at 5 degrees C than at 45 degrees C. Taken together, these observations support the notion that low temperature induces incomplete or readily reversible transitions to the high affinity state for substrates, effectors, and inhibitors. These observed effects of temperature also point out that enzyme determinants and topographical features responsible for transitions to the high affinity state and expression of catalytic activity can be regulated independently.

We have investigated effects of pH on the catalytic and allosteric properties of the cGMP-stimulated cyclic nucleotide phosphodiesterase purified from calf liver. In the "activated" state, i.e., with 0.5 microM [3H]cAMP plus 1 microM cGMP or at saturating substrate concentrations (250 microM [3H]cAMP or [3H]cGMP), hydrolysis was maximal at pH 7.5-8.0 in assays of different pH. Hydrolysis of concentrations of substrate not sufficient to saturate regulatory sites and below the apparent Michaelis constant (Kmapp), i.e., 0.5 microM [3H]cAMP or 0.01 microM [3H]cGMP, was maximal at pH 9.5. Although hydrolysis of 0.5 microM [3H]cAMP increased with pH from 7.5 to 9.5, cGMP stimulation of cAMP hydrolysis decreased. As pH increased or decreased from 7.5, Hill coefficients (napp) and Vmax for cAMP decreased. Thus, assay pH affects both catalytic (Vmax) and allosteric (napp) properties. Enzyme was therefore incubated for 5 min at 30 degrees C in the presence of MgCl2 at various pHs before assay at pH 7.5. Prior exposure to different pHs from pH 6.5 to 10.0 did not alter the Vmax or cGMP-stimulated activity (assayed at pH 7.5). Incubation at high (9.0-10.0) pH did, in assays at pH 7.5, markedly increase hydrolysis of 0.5 microM [3H]cAMP and reduce Kmapp and napp. After incubation at pH 10, hydrolysis of 0.5 microM [3H]cAMP was maximally increased and was similar in the presence or absence of cGMP. Thus, after incubation at high pH, the phosphodiesterase acquires characteristics of the cGMP-stimulated form. Activation at high pH occurs at 30 degrees C but not 5 degrees C, requires MgCl2, and is prevented but not reversed by ethylenediaminetetraacetic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

A new, rapid method for purification of calmodulin-stimulated phosphodiesterase from bovine, ovine, and porcine brain using only DEAE-agarose and calmodulin-Sepharose chromatography is described. Purified enzymes from the three species each exhibited a single polypeptide of Mr approximately 59,000 on gel electrophoresis under denaturing conditions. Proteolysis of ovine and bovine enzymes with alpha-chymotrypsin, however, yielded different peptides, indicating that these proteins differ in primary sequence. Homogeneous preparations of bovine and ovine enzymes (purified approximately 5,000- and 2,000-fold, respectively) had different specific activities, although their substrate affinities and activation by calmodulin (8- to 14-fold activation, Kact approximately 1 nM) were very similar. The total amount in ovine was almost twice that in bovine brain. The hydrodynamic properties of bovine and ovine enzymes were indistinguishable with a Stokes radius of 4.35 nm and s20,w of 5.95 S. The calculated frictional ratios of 1.30 to 1.38 suggest a slightly asymmetric molecule. Equilibrium sedimentation data yielded apparent Mr approximately 57,000 in the presence of 6 M guanidine and 124,000 and 112,000 for the native bovine and ovine enzymes, respectively. In addition to the enzyme that was purified to homogeneity (pI approximately 5.6), a major fraction of calmodulin-activated phosphodiesterase with a lower isoelectric point was found in bovine and ovine brain. Whether these represent isozymes, perhaps localized in different types of cells, or whether one is a post-translationally modified form, remains to be determined. The existence of these two otherwise very similar forms of the enzyme has apparently not been previously recognized.

The molecular properties of native and modified pregnancy-specific beta 1-glycoprotein (SP1) from human placenta were evaluated by sedimentation equilibrium, gel electrophoresis, and circular dichroic measurements. Native SP1 contained 6.2% N-acetylneuraminic acid (NANA), 5.8% galactose (Gal), 13% N-acetylglucosamine, 6.5% mannose, and 1.1% fucose but no detectable N-acetylgalactosamine. Treatment with mixed exoglycosidases and alpha-mannosidase removed 79% of the carbohydrate including all of the NANA and Gal. The intensity of the circular dichroic spectrum of SP1 in the far ultraviolet was quite low with a positive maximum at 235 nm and a negative maximum at 215 nm. The 235-nm band was lost upon treatment with reducing agents or with guanidinium chloride (GdmCl), but not by treatment with neuraminidase. Treatment of SP1 with neuraminidase, or with mixed exoglycosidases and alpha-mannosidase, resulted in decreases of the apparent molecular weight obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Neither exposure of SP1 to GdmCl nor its reduction and alkylation resulted in the appearance of subunits on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The partial specific volume of SP1 determined experimentally by comparing sedimentation equilibrium profiles in H2O and D2O was 0.695 +/- 0.007 mL/g. The molecular weight of SP1 in 6 M GdmCl (in the presence or absence of reducing agents) by equilibrium sedimentation was 42 300 +/- 400. In the absence of denaturing agents, SP1 existed in the form of aggregates (at least as high as trimeric SP1) that dissociated only slowly upon dilution. The presence of these aggregates may contribute to the reported molecular heterogeneity of SP1.

Choleragen and its A protomer catalyzed the hydrolysis of NAD to ADP-ribose and nicotinamide. NADase activity was inhibited by gangliosides GM1 (galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosylceramide), GM2 (N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosylceramide), GM3 (N-acetylneuraminyl-galactosylglucosylceramide), and GD1a (N-acetylneuraminylgalactosyl-N-acetylgalactosaminyl-E1N-acetylneuraminyl]-galactosylglucosylceramide). These gangliosides also increased the intensity of the tryptophanyl fluorescence of the isolated A protomer (lambda max = 328 nm). GM1 but not GM2, GM3, and GD1a caused a "blue shift" in the fluorescence spectrum of the B protomer. These results are consistent with other evidence that the specificity of GM1 as the choleragen receptor resides in its carbohydrate moiety. The NADase activity of choleragen was similar to that of diphtheria toxin previously described [J. Kandel, R. J. Collier & D. W. Chung (1974) J. Biol. Chem. 249, 2088-2097]. As with diphtheria toxin, analogues of NAD were inhibitory, adenine being the most effective. Significant inhibition was also noted with adenosine, AMP, ADP-ribose, nicotinamide, nicotinamide mononucleotide, and NADP. NADP was hydrolyzed only slowly by choleragen. In the NADase reaction catalyzed by diphtheria toxin, water serves as an acceptor for the ADP-ribose moiety of NAD in lieu of the natural acceptor molecule, which is elongation factor II (Kandel et al., 1974). It seems probable that the natural protein acceptor for ADP-ribose in the reaction catalyzed by choleragen is adenylate cyclase or a protein component of a cyclase complex that regulates enzymatic activity.

In a previous study, it was shown that purified soluble bovine dopamine beta-hydroxylase exhibits pH-dependent reversible tetramer-dimer dissociation (Saxena, A., Hensley, P., Osborne, J. C., Jr., and Fleming, P. J. (1985) J. Biol. Chem. 260, 3386-3392). Here we report evidence for the dissociation of this enzyme by magnesium-adenosine diphosphate independent of pH in the pH range 5-7. Quantitative binding of ADP to dopamine beta-hydroxylase revealed that there are two binding sites/dimeric species of hydroxylase and that ADP is tightly bound with a KD less than 10(-8) M. Kinetic data obtained at pH 5.5, the pH inside the chromaffin granule, shows that the apparent Km values for both the substrates tyramine and ascorbate are lowered by the presence of ADP without affecting the Vmax of the enzyme. The ADP-dependent lowering of apparent Km values results from a dissociation of the enzyme to the dimeric species which has inherently lower apparent Km values for substrates.

The molecular properties of the single linear chain form of human apoA-II, i.e., Cm apoA-II, have been evaluated by circular dichroism, polarization of fluorescence, difference absorption, and sedimentation equilibrium. The self-association of Cm apoA-II to a dimer resembles closely that of apoA-II though the free energy change is somewhat smaller. The dimerization of Cm apoA-II is accompanied by major changes in secondary and tertiary structure. The apoA-II molecule, therefore, represents a molecular association where the intramolecular structure is strongly dependent on the quaternary structure.