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Autophagosomes fuse with lysosomes, forming autolysosomes that degrade engulfed cargo. To maintain lysosomal capacity, autolysosome reformation (ALR) must regenerate lysosomes from autolysosomes using a membrane tubule-based process. Maintaining lysosomal capacity is required to maintain proteostasis and cellular health, especially in neurons where lysosomal dysfunction has been repeatedly implicated in neurodegenerative disease. Cell biological studies have linked the DNA-J domain Hsc70 co-chaperone RME-8/DNAJC13 to endosomal coat protein regulation, while human genetics studies have linked RME-8/DNAJC13 to neurological disease, including Parkinsonism and Essential Tremor. We report new analysis of the requirements for the RME-8/DNAJC13 protein in neurons, focusing onC. elegansmechanosensory neurons in the intact animal, and in primary mouse cortical neurons in culture. We find that loss of RME-8/DNAJC13 in both systems results in accumulation of grossly elongated autolysosomal tubules. FurtherC. elegansanalysis revealed a similar autolysosome tubule accumulation defect in mutants known to be required for ALR in mammals, including bec-1/beclin and vps-15/PIK3R4/p150 that regulate type-III PI3-kinase VPS-34, and dyn-1/dynaminthat severs ALR tubules. Clathrin is also an important ALR regulator implicated in autolysosome tubule formation and release. InC. eleganswe found that loss of RME-8 causes severe depletion of clathrin from neuronal autolysosomes, a phenotype shared withbec-1andvps-15mutants. We conclude that RME-8/DNAJC13 plays a conserved but previously unrecognized role in autolysosome reformation, likely affecting ALR tubule initiation and/or severing. Additionally, in both systems, we found that loss of RME-8/DNAJC13 appeared to reduce autophagic flux, suggesting feedback regulation from ALR to autophagy. Our results connecting RME-8/DNAJC13 to ALR and autophagy provide a potential mechanism by which RME-8/DNAJC13 could influence neuronal health and the progression of neurodegenerative disease.

Patients with end-stage renal disease (ESRD) requiring intermittent hemodialysis (IHD) are at increased risk of infection, which represents a leading cause of mortality in this population. The use of additional vascular access devices such as peripherally inserted central catheters to treat such infections should be minimized in patients with ESRD requiring IHD in order to mitigate complications such as infection and thrombosis and to maintain venous patency for hemodialysis access. Intravenous antimicrobial dosing following IHD has the advantages of avoiding additional access devices and providing convenience for patients and providers. Vancomycin, cefazolin, and aminoglycosides have historically been regarded as the primary intravenous antimicrobials administered with IHD given their relatively low cost, convenient dosing, and longevity of clinical use. Despite this, a growing body of literature is evaluating the use of an expanded list of antimicrobials that may be employed using post-dialysis dosing for patients requiring IHD; however, the available data are largely limited to pharmacokinetic studies and small cohorts of infected patients or uninfected subjects. Post-dialytic dosing of intravenous antimicrobials may be considered on a patient-by-patient basis after careful consideration of clinical, microbiological, and logistical factors that may influence the probability of treatment success. This document reviews and evaluates currently available information on the post-dialytic administration of an expanded list of intravenous antimicrobials in the setting of thrice-weekly, high-flux IHD.

Adverse childhood experiences (ACEs) are associated with negative health outcomes; however, screening for ACEs is not routinely performed among people living with HIV (PLWH). We conducted a single-center, cross-sectional pilot study to define the (1) prevalence of ACEs in PLWH and (2) acceptability of ACEs screening in routine out-patient clinical care. One hundred participants completed screening: median age of participants was 49 years (interquartile range: 38.5-59.5), 73% male, 66% Non-Hispanic Black/African American, and 47% gay/lesbian. Clinically significant ACEs score, defined as ≥4, was reported in 51%. High ACEs score was more common among participants

Objective To evaluate whether opt out framing, messaging incorporating behavioral science concepts, or electronic communication increases the uptake of hepatitis C virus (HCV) screening in patients born between 1945 and 1965. Design Pragmatic randomized controlled trial. Setting 43 primary care practices from one academic health system (Philadelphia, PA, USA) between April 2019 and May 2020. Participants Patients born between 1945 and 1965 with no history of screening and at least two primary care visits in the two years before the enrollment period. Interventions This multilevel trial was divided into two studies. Substudy A included 1656 eligible patients of 17 primary care clinicians who were randomized in a 1:1 ratio to a mailed letter about HCV screening (letter only), or a similar letter with a laboratory order for HCV screening (letter+order). Substudy B included the remaining 19 837 eligible patients followed by 417 clinicians. Active electronic patient portal users were randomized 1:5 to receive a mailed letter about HCV screening (letter), or an electronic patient portal message with similar content (patient portal); inactive patient portal users were mailed a letter. In a factorial design, patients in substudy B were also randomized 1:1 to receive standard content (usual care), or content based on principles of social norming, anticipated regret, reciprocity, and commitment (behavioral content). Main outcome measures Proportion of patients who completed HCV testing within four months. Results 21 303 patients were included in the intention-to-treat analysis. Among the 1642 patients in substudy A, 19.2% (95% confidence interval 16.5% to 21.9%) completed screening in the letter only arm and 43.1% (39.7% to 46.4%) in the letter+order arm (P Conclusions Opt out framing and effort reduction by including a signed laboratory order with outreach increased screening for HCV. Behavioral science messaging content did not increase uptake, and mailed letters achieved a greater response rate than patient portal messages. Trial registration ClinicalTrials.gov NCT03712553 .

Tetraspanins are a unique family of 4-pass transmembrane proteins that play important roles in a variety of cell biological processes. We have previously shown that 2 paralogous tetraspanins in Caenorhabditis elegans, TSP-12 and TSP-14, function redundantly to promote bone morphogenetic protein (BMP) signaling. The underlying molecular mechanisms, however, are not fully understood. In this study, we examined the expression and subcellular localization patterns of endogenously tagged TSP-12 and TSP-14 proteins. We found that TSP-12 and TSP-14 share overlapping expression patterns in multiple cell types, and that both proteins are localized on the cell surface and in various types of endosomes, including early, late, and recycling endosomes. Animals lacking both TSP-12 and TSP-14 exhibit reduced cell-surface levels of the BMP type II receptor DAF-4/BMPRII, along with impaired endosome morphology and mislocalization of DAF-4/BMPRII to late endosomes and lysosomes. These findings indicate that TSP-12 and TSP-14 are required for the recycling of DAF-4/BMPRII. Together with previous findings that the type I receptor SMA-6 is recycled via the retromer complex, our work demonstrates the involvement of distinct recycling pathways for the type I and type II BMP receptors and highlights the importance of tetraspanin-mediated intracellular trafficking in the regulation of BMP signaling in vivo. As TSP-12 and TSP-14 are conserved in mammals, our findings suggest that the mammalian TSP-12 and TSP-14 homologs may also function in regulating transmembrane protein recycling and BMP signaling.

After endocytosis, transmembrane cargoes such as signaling receptors, channels, and transporters enter endosomes where they are sorted to different destinations. Retromer and ESCRT (endosomal sorting complex required for transport) are functionally distinct protein complexes on endosomes that direct cargo sorting into the recycling retrograde transport pathway and the degradative multivesicular endosome pathway (MVE), respectively. Cargoes destined for degradation in lysosomes are decorated with K63-linked ubiquitin chains, which serve as an efficient sorting signal for entry into the MVE pathway. Defects in K63-linked ubiquitination disrupt MVE sorting and degradation of membrane proteins. Here, we unexpectedly found that UBC-13, the E2 ubiquitin-conjugating enzyme that generates K63-linked ubiquitin chains, is essential for retrograde transport of multiple retromer-dependent cargoes including MIG-14/Wntless. Loss of ubc-13 disrupts MIG-14/Wntless trafficking from endosomes to the Golgi, causing missorting of MIG-14 to lysosomes and impairment of Wnt-dependent processes. We observed that retromer-associated SNX-1 and the ESCRT-0 subunit HGRS-1/Hrs localized to distinct regions on a common endosome in wild type but overlapped on ubc-13(lf) endosomes, indicating that UBC-13 is important for the separation of retromer and ESCRT microdomains on endosomes. Our data suggest that cargo ubiquitination mediated by UBC-13 plays an important role in maintaining the functionally distinct subdomains to ensure efficient cargo segregation on endosomes.

Background Hepatitis C virus (HCV) infects 4.1 million people in the United States, of whom 50% are unaware of their status. In 2016, Pennsylvania introduced a law mandating HCV screening for patients born between 1945-1965 in inpatient settings. However, HCV screening during hospital admissions has remained low in part due to limited knowledge on HCV testing requirements, interpretation of results, and treatment approaches. To overcome these barriers, we implemented a quality improvement initiative to automate HCV screening as part of hospital admission order sets, facilitate linkage to HCV treatment, and sought to evaluate its effectiveness. Methods Between September 2020 and May 2021, the automated inpatient HCV screening strategy was implemented at a single 328-bed academic hospital in Philadelphia, PA. Patients born between 1945-1965 without documentation of HCV screening or diagnosis in the electronic medical record had a HCV antibody with reflexive confirmatory RNA assay automatically populated in the admission order set. Admitting providers could opt out of the screening as appropriate. All patients with reactive HCV antibody were approached by the Hepatitis Linkage Team for result disclosure, counseling, and linkage to treatment for those with HCV viremia. Cascade of care was detailed for those linked to providers within the health system. Results During the initial 8 months of the program, 2,203 patients were screened for HCV, identifying 156 with reactive HCV antibody (7.1% seroprevalence). Among 147 with completed HCV RNA assay, 51 were viremic (34.7%). Fourteen viremic patients were not linked to care, including six with a terminal illness, two who declined linkage, and six who did not respond to linkage attempts. Nine were linked to care at other health systems. Among the 28 patients linked to providers in the health system, 50% completed initial visits, 42.8% were prescribed direct acting antivirals (DAA), and 21.4% completed therapy by May 2021. One person achieved sustained virologic response 12 weeks after treatment as of May 2021 (Figure 1). Figure 1. Cascade of HCV Care Among Patients Screened During Hospital Admission from September 2020 to May 2021 Conclusion Automated inpatient HCV screening is a viable strategy to identify people with HCV and facilitate linkage to care. Optimal strategies to ensure patients access and maintain care require further study. Disclosures All Authors: No reported disclosures

Background A consensus on a central line-associated bloodstream infection (CLABSI) surveillance definition in home infusion is needed to standardize measurement and benchmark CLABSI to provide data to drive improvement initiatives Methods Experts across fields including home infusion therapy, infectious diseases, and healthcare epidemiology convened to perform a 3-step modified Delphi approach to obtain input and achieve consensus on a candidate home infusion CLABSI definition. Results The numerator criterion was identified by participants as involving one of the 2 following: (1) recognized pathogen isolated from blood culture and pathogen is not related to infection at another site, or (2) one of the following signs or symptoms: fever of 38°C (100.4°F), chills, or hypotension (systolic blood pressure ≤90 mm Hg), and one of the 2 following: (A) common skin contaminant isolated from 2 blood cultures drawn on separate occasions and organism is not related to infection at another site, or (B) common skin contaminant isolated from blood culture from patient with intravascular access device and provider institutes appropriate antimicrobial therapy. The criteria for a denominator included days from the day of admission with a central venous catheter to day of removal of central venous catheter. In addition, 11 inclusion criteria and 4 exclusion criteria were included. Discussion Home infusion therapy and healthcare epidemiology experts developed candidate criteria for a home infusion CLABSI surveillance definition. Conclusions Home care and home infusion agencies can use this definition to monitor their own CLABSI rates and implement preventative strategies.

Background Peripherally inserted central catheters (PICCs) and midlines are often used in hospitalized patients who require outpatient parenteral antimicrobial therapy (OPAT) upon discharge. PICCs/midlines offer ease of insertion but still carry the risks of venous thrombosis, phlebitis, and catheter-associated infection. We report the results of a prospective audit and feedback (PAF) intervention targeting the placement of PICCs/midlines for OPAT at our institution. Methods We prospectively identified a cohort of patients identified by a real-time PICC/midline alert from 5/20/2019 through 5/29/2020 at two large academic medical centers. Alerts were generated by a third-party interface with the electronic health record and identified new line orders with an antimicrobial indication selected. Patients without infectious diseases (ID) consult underwent PAF by the antimicrobial stewardship team. Descriptive statistics were used to characterize patients, interventions, and outcomes. Results During the study period, 1267 PICC/midline alerts were identified. Most were excluded due to ID consult (85.4%). After exclusions, 113 alerts underwent full review. Median patient age was 64 years with female predominance (54.2%). Reviewable alerts most commonly originated from Pulmonary (36.5%) and Hospitalist (26.0%) services. The most frequent antimicrobial indications were pneumonia (37.5%) and bloodstream infection (28.1%), and the most frequently ordered antimicrobials were cefepime (27.1%) and piperacillin-tazobactam (17.7%). Median time from line order to insertion was 22 hours and from line insertion to discharge was 48 hours. Of 113 alerts reviewed by the stewardship team, 26 (23.0%) resulted in a recommendation to avoid line placement and 45 (39.8%) resulted in at least one specific stewardship recommendation (Table 1). Recommendations were fully or partially accepted in 58.3% of instances. TABLE 1. Interventions Resulting from Prospective Venous Catheter Stewardship Conclusion Prospective audit of PICC/midline orders for OPAT identified a line-sparing opportunity in nearly 1 in 4 cases. Where line avoidance was not possible, other opportunities for antimicrobial optimization were common. This high-yield intervention should be considered for institutions that do not mandate infectious diseases consult for all OPAT discharges. Disclosures All Authors: No reported disclosures

After endocytosis, transmembrane cargo reaches endosomes, where it encounters complexes dedicated to opposing functions: recycling and degradation. Microdomains containing endosomal sorting complexes required for transport (ESCRT)-0 component Hrs [hepatocyte growth factor-regulated tyrosine kinase substrate (HGRS-1) in Caenorhabditis elegans] mediate cargo degradation, concentrating ubiquitinated cargo and organizing the activities of ESCRT. At the same time, retromer associated sorting nexin one (SNX-1) and its binding partner, J-domain protein RME-8, sort cargo away from degradation, promoting cargo recycling to the Golgi. Thus, we hypothesized that there could be important regulatory interactions between retromer and ESCRT that balance degradative and recycling functions. Taking advantage of the naturally large endosomes of the C. elegans coelomocyte, we visualized complementary ESCRT-0 and RME-8/SNX-1 microdomains in vivo and assayed the ability of retromer and ESCRT microdomains to regulate one another. We found in snx-1(0) and rme-8(ts) mutants increased endosomal coverage and intensity of HGRS-1-labeled microdomains, as well as increased total levels of HGRS-1 bound to membranes. These effects are specific to SNX-1 and RME-8, as loss of other retromer components SNX-3 and vacuolar protein sorting-associated protein 35 (VPS-35) did not affect HGRS-1 microdomains. Additionally, knockdown of hgrs-1 had little to no effect on SNX-1 and RME-8 microdomains, suggesting directionality to the interaction. Separation of the functionally distinct ESCRT-0 and SNX-1/RME-8 microdomains was also compromised in the absence of RME-8 and SNX-1, a phenomenon we observed to be conserved, as depletion of Snx1 and Snx2 in HeLa cells also led to greater overlap of Rme-8 and Hrs on endosomes.

BACKGROUND: As electronic discharge summaries (EDS) become more prevalent and health care systems increase their focus on transitions of care, analysis of EDS quality is important. The objective of this study was to assess the timeliness and quality of EDS compared with dictated summaries for surgical patients, which has not previously been evaluated. METHODS: A retrospective study was conducted of a sample of discharge summaries from surgical patients at an urban university teaching hospital before and after the implementation of an EDS program. Summaries were evaluated on several dimensions, including time to summary completion, summary length, and summary quality, which was measured on a 13-item scoring tool. RESULTS: After the exclusion of 5 patients who died, 195 discharge summaries were evaluated. Discharge summaries before and after EDS implementation were similar in admission types and discharge destinations of the patients. Compared with dictated summaries, EDS had equivalent overall quality (P 5 .11), with higher or equivalent scores on all specific quality aspects except readability. There was a highly significant statistical and clinical improvement in timeliness for electronic summaries (P , .01). Obvious use of copying and pasting was identified in 8% of discharge summaries and was associated with decreased readability (P 5 .02). CONCLUSIONS: The implementation of EDS can improve the timeliness of summary completion without sacrificing quality for surgical patients. Excessive copying and pasting can reduce the readability of discharge summaries, and strategies to discourage this practice without the use of appropriate editing should be used.

Validation of a Swedish version of the Helsinki Chronic Pain Index for the measure of chronic pain behaviors in canine osteoarthritis

In a retrospective study of home infusion patients with central line-associated bloodstream infection, use of a central venous port, cancer diagnosis, and the absence of systemic inflammatory response syndrome were associated with use of catheter-salvage. Relapse of infection was uncommon.

Background: Many hospitalized patients with complicated infections are discharged on outpatient parenteral antimicrobial therapy (OPAT). However, little is known about how to improve the postdischarge care of OPAT patients. Objective: The impact of an infectious diseases transitions service (IDTS) on OPAT patient readmissions, as well as on processes of care, was evaluated. Methods: We performed a controlled, quasi-experimental evaluation over 15 months in an academic medical center. Intervention-arm patients, before and after the introduction of an IDTS, were seen by the general infectious diseases consult teams, while control-arm patients (discharged on OPAT after hospitalization with bacteremia) were not. The IDTS prospectively tracked all OPAT patients and coordinated follow-up. The impact of the IDTS was calculated using a differences-in-differences approach where the interaction between time (before vs after the IDTS intervention) and study arm (intervention vs control arm) was the variable of interest. The control arm was used only in primary outcome analyses (readmissions and emergency department visits). Secondary outcomes included process of care measures and non-readmission clinical outcomes. Results: Of 488 consecutive patients requiring OPAT, 362 were in the intervention arm (215 pre-intervention and 147 post-intervention) and 126 in the control arm (70 pre-intervention and 56 post-intervention). Compared to the control arm, the IDTS was not associated with changes in 60-day readmissions and/or emergency department visits (adjusted odds ratio [OR] = 0.48; 95% confidence interval [CI] = 0.13-1.79). In the intervention arm, implementation of the IDTS was associated with fewer antimicrobial therapy errors (OR = 0.062; 95% CI = 0.015-0.262), increased laboratory test receipt (OR = 27.85; 95% CI = 12.93-59.99), and improved outpatient follow-up (OR = 2.44; 95% CI = 1.50-3.97). Conclusions: In a controlled evaluation, the IDTS did not affect readmissions despite improving process of care measures for targeted patients. Care coordination services may improve OPAT quality of care, but their relationship to readmissions is unclear.

A recent explosion of work surrounds the interactions between Sir3p (Silent Information Regulator 3) and chromatin. We review here the Sir3p functions related to its role in silencing in Saccharomyces cerevisiae. This unusual protein, which is absolutely required for silencing, is distantly related to the highly conserved replication initiator Orc1p, but is itself phylogenetically limited to “post-genome-duplicated” budding yeasts. Several recent studies revise earlier models for Sir3p action. Specifically, the N-terminal bromo-adjacent homology (BAH) domain plays a now well-defined role in silencing, and a picture is emerging in which both termini of Sir3p bind two locations on the nucleosome: (1) the loss of ribosomal DNA silencing (LRS) surface in the nucleosome core, and (2) the N-terminal histone tails for effective silencing at telomeres. We relate Sir3p structure and function, and summarize recent molecular studies of Sir3p/chromatin binding, Sir3p/Dot1p competition, and the possible role of O-Acetyl ADP ribose (O-AADPR) in Sir3p/chromatin binding. We emphasize recent genetic data that provide important new insights and settle controversies created by in vitro work. Finally, we synthesize these ideas to revise the model for how Sir3p mediates silent chromatin formation in yeast, in part through its affinity for the LRS region of the nucleosome, which must be “just right.”

SummaryThe reduction of all-cause hospital readmission among heart failure (HF) patients is a national priority. Telehealth is one strategy employed to impact this sought-after patient outcome. Prior research indicates varied results on all-cause hospital readmission highlighting the need to understand telehealth processes and optimal strategies in improving patient outcomes.The purpose of this paper is to describe how one Medicare-certified home health agency launched and maintains a telehealth program intended to reduce all-cause 30-day hospital readmissions among HF patients receiving skilled home health and report its impact on patient outcomes.Using the Transitional Care Model as a guide, the telehealth program employs a 4G wireless tablet-based system that collects patient vital signs (weight, heart rate, blood pressure and blood oxygenation) via wireless peripherals, and is preloaded with subjective questions related to HF and symptoms and instructional videos.Year one all-cause 30-day readmission rate was 19.3%. Fiscal year 2015 ended with an all-cause 30-day readmission rate of 5.2%, a reduction by 14 percentage points (a 73% relative reduction) in three years. Telehealth is now an integral part of the University of Pennsylvania Health System’s readmission reduction program.Telehealth was associated with a reduction in all-cause 30-day readmission for one mid-sized Medicare-certified home health agency. A description of the program is presented as well as lessons learned that have significantly contributed to this program’s success. Future expansion of the program is planned. Telehealth is a promising approach to caring for a chronically ill population while improving a patient’s ability for self-care.

Previous studies of visitors to Australia's Great Barrier Reef (GBR) reveal that, for some, a visit to the reef is an active learning experience, while for others it is merely a chance to relax or socialize in a pleasant natural setting. The aim of this study was to further explore these findings by conducting a detailed benefit-based segmentation study of tourists on commercial tours to the reef. The resulting segments differed with respect to the desired level of active involvement with the reef and the desire for information and learning. Dive Learners (n = 442) exhibited the greatest desire for a hands-on reef learning experience, while Nature Learners (n = 613) showed a preference for a more passive reef learning experience. In contrast, the Nature Relaxers (n = 345) and Nature Family Visitors (n = 411) were more interested in a relaxing and/or social reef experience. Visitor management implications are discussed.

It has long been recognised in the field of education that there is no better teaching method than participation. Convention and event management is a relatively new area of hospitality and tourism education that offers an opportunity to provide students with the real world practical and applied experience that many seek. This paper will report on the use of real world activities in a class in Convention and Events Management. Students in this class were required to plan, market, manage and evaluate a real event. This paper will firstly describe the process used to plan and run the events. It will then review the outcomes of two types of evaluation, an evaluation of the success of each event and an evaluation of the success of using real world events as a teaching technique. The results have implications both for the general area of event management and more specifically for the teaching of convention and events management.

The counterbalancing action of the endocytosis and secretory pathways maintains a dynamic equilibrium that regulates the composition of the plasma membrane, allowing it to maintain homeostasis and to change rapidly in response to alterations in the extracellular environment and/or intracellular metabolism. These pathways are intimately integrated with intercellular signaling systems and play critical roles in all cells. Studies in Caenorhabditis elegans have revealed diverse roles of membrane trafficking in physiology and development and have also provided molecular insight into the fundamental mechanisms that direct cargo sorting, vesicle budding, and membrane fisson and fusion. In this review, we summarize progress in understanding membrane trafficking mechanisms derived from work in C. elegans, focusing mainly on work done in non-neuronal cell-types, especially the germline, early embryo, coelomocytes, and intestine.

Recoverin is a small calcium binding protein involved in regulation of the phototransduction cascade in retinal rod cells. It functions as a calcium sensor by undergoing a cooperative, ligand-dependent conformational change, resulting in the extrusion of the N-terminal myristoyl group from a hydrophobic pocket. To test the role of certain core residues in tuning this allosteric switch, we have made and characterized two mutants: W31K, which replaces Trp31 with Lys; and a double mutant, I52A/Y53A, in which Ile52 and Tyr53 are both replaced by Ala. These mutations decrease the hydrophobicity of the myristoyl binding pocket. They are thus expected to make sequestering of the myristoyl group less favorable and destabilize the Ca2+-free state. As predicted, the myristoylated forms of the mutants exhibit increased affinity for Ca2+, whether monitored by equilibrium binding of 45Ca2+ (Kd = 17.2, 7.9, and 8.1 microM for wild type, W31K, and I52A/Y53A, respectively) or by the change in tryptophan fluorescence associated with the conformational change (Kd = 17.9, 3.6, and 4.4 microM for wild type, W31K, and I52A/Y53A, respectively). The mutants also exhibit decreased cooperativity of binding (Hill coefficient = 1.2 and 1.0 for W31K and I52A/Y53A vs 1. 4 for wild type). Binding of the mutant proteins to rod outer segment membranes occurs at lower Ca2+ concentrations compared to wild-type protein (K1/2 = 5.6, 2.2, and 1.0 microM for wild type, W31K, and I52A/Y53A, respectively). The unmyristoylated forms of the mutants exhibit biphasic Ca2+ binding curves, nearly identical to that observed for wild type. The binding data for the two mutants can be explained by a concerted allosteric model in which the mutations affect only the equilibrium constant L between the two allosteric forms, T (the Ca2+-free form) and R (the Ca2+-bound form), without affecting the intrinsic binding constants for the two Ca2+ sites. Two-dimensional NMR spectra of the Ca2+-free forms of the mutants have been compared to the wild-type spectrum, whose peaks have been assigned to specific residues (1). Many resonances assigned to residues in the C-terminal domain (residues 100-202) in the wild-type spectrum are identical in the mutant spectra, suggesting that the backbone structure of the C-terminal domain is probably unchanged in both mutants. The N-terminal domain, in which both mutations are located, reveals in each case numerous changes of undetermined spatial extent.

The binding of NGF (nerve growth factor) to the rat low affinity nerve growth factor receptor (p75NGFR) has been studied by site-directed mutagenesis of the receptor. Introduction of non-native N-glycosylation sites within the binding domain indicates that the second of the characteristic cysteine-rich repeats may be particularly important to NGF binding. Two mutants of the second repeat, S42N and S66N, are glycosylated and bind NGF at a drastically reduced level, while still maintaining a conformation recognized by the monoclonal antibody against p75, MC192. Alanine substitution at these sites does not affect NGF binding. Two other mutations that result in local structural changes in the second repeat also greatly decrease binding. One of these altered residues, Ser50, appears to play an essential structural role, since it cannot be replaced by Asn, Ala, or Thr without loss of both NGF binding and MC192 recognition on a Western. Glycosylation of selected sites in the other repeats has little effect on NGF binding or antibody recognition. The introduction of non-native N-glycosylation sites may provide a generally useful scanning technique for the study of protein-protein interactions.

Objective To describe the clinical and radiographic presentation, risk factors, response to therapy and outcome of 16 patients with HIV infection and pulmonary infections caused by Pseudomonas aeruginosa. Design Retrospective review of medical records. Setting An academic tertiary-care hospital. Patients Sixteen patients who met the case definition were included for retrospective review. Results P. aeruginosa pneumonia was community-acquired in 15 patients (94%). The majority of patients (94%) had a diagnosis of AIDS with a mean CD4 cell count of 27 x 10(6)/l cells. Traditional risk factors for the development of P. aeruginosa were missing in most patients. Cavitary infiltrates were present on admission chest radiograph in eight patients (50%). An additional three patients (19%) presented with pulmonary infiltrates that cavitated subsequently. Clinical course was extremely varied with an in-hospital mortality of only 19%, but with an additional 25% of patients developing chronic or recurrent disease. Conclusions Community-acquired pneumonia caused by P. aeruginosa occurs in patients with end-stage HIV infection. The presence of cavitary pulmonary infiltrates on chest radiograph in a patient with a low CD4 count should raise suspicion of P. aeruginosa infection. Obvious risk factors for P. aeruginosa infection may be absent. While the initial mortality rate is lower than that observed in other immunocompromised hosts, the potential for chronic or recurrent infection should be recognized and patients should be followed closely.

The binding domain of the low affinity nerve growth factor receptor (p75NGFR) is built from four "cysteine repeats," with almost identical patterns of half-cystine residues. To study the pattern of disulfide bridging within each cysteine repeat, we have mutated pairs of cysteine residues, primarily in the fourth repeat, and have tested the relative ability of each mutant to be cross-linked to the nerve growth factor after transient expression in COS 7 cells. The results give an indication of the relative importance of different disulfide bonds for the stability of the active conformation. Immunofluorescent staining shows that all of these proteins, even those that may be partially misfolded, are expressed to varying degrees on the surface of COS cells. They can also be detected by the monoclonal antibody MC192 on a Western blot following electrophoresis on a nondenaturing gel. Some cannot, however, be detected by MC192 on a Western blot after heat denaturation in SDS. The ability to refold under these conditions correlates in general with the ability of the mutant protein to bind nerve growth factor on the surface of COS cells.