Development and validation of the HMS-Sepsis mortality model.

BACKGROUND: When comparing outcomes after sepsis, it is essential to account for patient case mix to make fair comparisons. We developed a model to assess risk-adjusted 30-day mortality in the Michigan Hospital Medicine Safety's sepsis initiative (HMS-Sepsis). QUESTION: Can HMS-Sepsis registry data adequately predict risk of 30-day mortality? Do performance assessments using adjusted vs unadjusted data differ? STUDY DESIGN AND METHODS: Retrospective cohort of community-onset sepsis hospitalizations in HMS-Sepsis registry (4/2022-9/2023), with split derivation (70%) and validation (30%) cohorts. We fit a risk-adjustment model (HMS-Sepsis mortality model) incorporating acute physiology, demographic, and baseline health data and assessed model performance using c-statistics, Brier's scores, and comparisons of predicted vs observed mortality by deciles of risk. We compared hospital performance (1st quintile, middle quintiles, 5th quintile) using observed versus adjusted mortality to understand the extent to which risk-adjustment impacted hospital performance assessment. RESULTS: Among 17,514 hospitalizations from 66 hospitals during the study period, 12,260 (70%) were used for model derivation and 5,254 (30%) for model validation. 30-day mortality for the total cohort was 19.4%. The final model included 13 physiologic variables, two physiologic interactions, and 16 demographic and chronic health variables. The most significant variables were age, metastatic solid tumor, temperature, altered mental status, and platelet count. The model c-statistic was 0.82 for the derivation cohort, 0.81 for the validation cohort, and ≥0.78 for all subgroups assessed. Overall calibration error was 0.0% and mean calibration error across deciles of risk was 1.5%. Standardized mortality ratios yielded different assessments than observed mortality for 33.9% of hospitals. CONCLUSIONS: The HMS-Sepsis mortality model had strong discrimination, adequate calibration, and reclassified one-third of hospitals to a different performance category from unadjusted mortality. Based on its strong performance, the HMS-Sepsis mortality model can aid in fair hospital benchmarking, assessment of temporal changes, and observational causal inference analysis.

Awareness of peripheral IV catheters among nurses, physicians, and trainees: A prospective cohort study.

BACKGROUND: Peripheral intravenous catheters (PIVs) are the most frequently used invasive device in hospitalized patients. These devices are not benign and are associated with complications. However, clinician awareness of them is variable and poorly understood. METHODS: We conducted a prospective, multicenter, observational point prevalence study to assess awareness of PIV presence among clinicians caring for hospitalized patients in 4 hospitals between May 2018 and February 2019 located in Michigan, USA. We first assessed patients for the presence of a PIV then interviewed their providers. Differences in awareness by provider type were assessed via χ² tests; P < .05 was considered statistically significant. Analyses were performed on Stata MP v16. RESULTS: A total of 1,385 patients and 4,003 providers were interviewed. Nurses had the greatest awareness of overall PIV presence, 98.6%, while attendings were correct 88.1% of the time. Nurses were more likely to correctly assess PIV presence and exact location than physicians (67.7% vs <30% for all others). Awareness of PIV presence did not significantly vary in patients on contact precautions or those receiving infusions. CONCLUSIONS: Given the ubiquity of PIVs and known complications, methods to increase awareness to ensure appropriate care and removal are necessary.

Improving Appropriate Use of Peripherally Inserted Central Catheters Through a Statewide Collaborative Hospital Initiative: A Cost-Effectiveness Analysis.

BACKGROUND: Quality improvement (QI) programs require significant financial investment. The authors evaluated the cost-effectiveness of a physician-led, performance-incentivized, QI intervention that increased appropriate peripherally inserted central catheter (PICC) use. METHODS: The authors used an economic evaluation from a health care sector perspective. Implementation costs included incentive payments to hospitals and costs for data abstractors and the coordinating center. Effectiveness was calculated from propensity score-matched observations across two time periods for complications (venous thromboembolism [VTE], central line-associated bloodstream infection [CLABSI], and catheter occlusion): preintervention period (January 2015 through December 2016) and intervention period (January 2017 through December 2021). Cost-effectiveness was presented as the cost-offset per averted complication, reflecting the health care costs avoided due to having lower complication rates. RESULTS: Across 35 hospitals, this study sampled 17,418 PICCs placed preintervention and 26,004 placed during the intervention period. PICC complications decreased significantly following the intervention. CLABSIs decreased from 2.1% to 1.5%, VTEs from 3.2% to 2.3%, and catheter occlusions from 10.8% to 7.0% (all p < 0.01). Estimated number of complications prevented included 871 CLABSIs, 2,535 VTEs, and 8,743 catheter occlusions. Project implementation costs were $31.8 million, and the cost-offset related to avoided complications was $64.4 million. Each participating hospital averaged $932,073 in cost-offset over seven years, and the average cost-offset per complication averted was $2,614 (95% CI [confidence interval] $2,314-$3,003). CONCLUSION: A large-scale, multihospital QI initiative to improve appropriate PICC use yielded substantial return on investment from cost-offset of prevented complications.

UV reflectance in crop remote sensing: Assessing the current state of knowledge and extending research with strawberry cultivars.

Remote sensing of spectral reflectance is a crucial parameter in precision agriculture. In particular, the visual color produced from reflected light can be used to determine plant health (VIS-IR) or attract pollinators (Near-UV). However, the UV spectral reflectance studies largely focus on non-crop plants, even though they provide essential information for plant-pollinator interactions. This literature review presents an overview of UV-reflectance in crops, identifies gaps in the literature, and contributes new data based on strawberry cultivars. The study found that most crop spectral reflectance studies relied on lab-based methodologies and examined a wide spectral range (Near UV to IR). Moreover, the plant family distribution largely mirrored global food market trends. Through a spectral comparison of white flowering strawberry cultivars, this study discovered visual differences for pollinators in the Near UV and Blue ranges. The variation in pollinator visibility within strawberry cultivars underscores the importance of considering UV spectral reflectance when developing new crop breeding lines and managing pollinator preferences in agricultural fields.

Use and Outcomes of Peripheral Vasopressors in Early Sepsis-Induced Hypotension Across Michigan Hospitals: A Retrospective Cohort Study.

BACKGROUND: Vasopressors traditionally are administered via central access, but newer data suggest that peripheral administration may be safe and may avoid delays and complications associated with central line placement. RESEARCH QUESTION: How commonly are vasopressors initiated through peripheral IV lines in routine practice? Is vasopressor initiation route associated with in-hospital mortality? STUDY DESIGN AND METHODS: This retrospective cohort study included adults hospitalized with sepsis (November 2020-September 2022) at 29 hospitals in the Michigan Hospital Medicine Safety Consortium, a Collaborative Quality Initiative sponsored by Blue Cross Blue Shield of Michigan. We assessed route of early vasopressor initiation, factors and outcomes associated with peripheral initiation, and timing of central line placement. RESULTS: Five hundred ninety-four patients received vasopressors within 6 h of hospital arrival and were included in this study. Peripheral vasopressor initiation was common (400/594 [67.3%]). Patients with peripheral vs central initiation were similar; BMI was the only patient factor associated independently with initiation route (adjusted OR [aOR] of peripheral initiation [per 1-kg/m2 increase], 0.98; 95% CI, 0.97-1.00; P = .015). The specific hospital showed a large impact on initiation route (median OR, 2.19; 95% CI, 1.31-3.07). Compared with central initiation, peripheral initiation was faster (median, 2.5 h vs 2.7 h from hospital arrival; P = .002), but was associated with less initial norepinephrine use (84.3% vs 96.8%; P = .001). We found no independent association between initiation route and in-hospital mortality (32.3% vs 42.2%; aOR, 0.66; 95% CI, 0.39-1.12). No tissue injury from peripheral vasopressors was documented. Of patients with peripheral initiation, 135 of 400 patients (33.8%) never received a central line. INTERPRETATION: Peripheral vasopressor initiation was common across Michigan hospitals and had practical benefits, including expedited vasopressor administration and avoidance of central line placement in one-third of patients. However, the findings of wide practice variation that was not explained by patient case mix and lower use of first-line norepinephrine with peripheral administration suggest that additional standardization may be needed.

Institutional Structures and Processes to Support Sepsis Care: A Multihospital Study.

OBJECTIVES: To identify opportunities for improving hospital-based sepsis care and to inform an ongoing statewide quality improvement initiative in Michigan. DESIGN: Surveys on hospital sepsis processes, including a self-assessment of practices using a 3-point Likert scale, were administered to 51 hospitals participating in the Michigan Hospital Medicine Safety Consortium, a Collaborative Quality Initiative sponsored by Blue Cross Blue Shield of Michigan, at two time points (2020, 2022). Forty-eight hospitals also submitted sepsis protocols for structured review. SETTING: Multicenter quality improvement consortium. SUBJECTS: Fifty-one hospitals in Michigan. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Of the included hospitals, 92.2% (n = 47/51) were nonprofit, 88.2% (n = 45/51) urban, 11.8% (n = 6/51) rural, and 80.4% (n = 41/51) teaching hospitals. One hundred percent (n = 51/51) responded to the survey, and 94.1% (n = 48/51) provided a sepsis policy/protocol. All surveyed hospitals used at least one quality improvement approach, including audit/feedback (98.0%, n = 50/51) and/or clinician education (68.6%, n = 35/51). Protocols included the Sepsis-1 (18.8%, n = 9/48) or Sepsis-2 (31.3%, n = 15/48) definitions; none (n = 0/48) used Sepsis-3. All hospitals (n = 51/51) used at least one process to facilitate rapid sepsis treatment, including order sets (96.1%, n = 49/51) and/or stocking of commonly used antibiotics in at least one clinical setting (92.2%, n = 47/51). Treatment protocols included guidance on antimicrobial therapy (68.8%, n = 33/48), fluid resuscitation (70.8%, n = 34/48), and vasopressor administration (62.5%, n = 30/48). On self-assessment, hospitals reported the lowest scores for peridischarge practices, including screening for cognitive impairment (2.0%, n = 1/51 responded "we are good at this") and providing anticipatory guidance (3.9%, n = 2/51). There were no meaningful associations of the Centers for Medicare and Medicaid Services' Severe Sepsis and Septic Shock: Management Bundle performance with differences in hospital characteristics or sepsis policy document characteristics. CONCLUSIONS: Most hospitals used audit/feedback, order sets, and clinician education to facilitate sepsis care. Hospitals did not consistently incorporate organ dysfunction criteria into sepsis definitions. Existing processes focused on early recognition and treatment rather than recovery-based practices.

Midline catheters in patients with advanced chronic kidney disease.

BACKGROUND: Midline catheters (midlines) are increasingly used in patients with advanced chronic kidney disease (CKD). OBJECTIVE: This study describes current practice and acute complications associated with midlines in CKD patients. DESIGNS, SETTING, AND PARTICIPANTS: Trained abstractors at 66 hospitals from the Michigan Hospital Medicine Safety (HMS) Consortium collected data on a sample of patients who received a midline during hospitalization. Patients were classified as having advanced CKD if their estimated glomerular filtration rate was <45 mL/min/1.73 m2 . MAIN OUTCOME AND MEASURES: Midline recipients with advanced CKD were compared to those without advanced CKD by patient, provider, and device characteristics, and by the occurrence of acute complications including major (e.g., upper extremity deep vein thrombosis [UE-DVT] and catheter-related bloodstream infection [CRBSI]) or minor (e.g., catheter occlusion, catheter dislodgement, infiltration, superficial thrombophlebitis, and leaking at insertion site) events. Multivariable mixed effects logistic regression was used to evaluate the association between catheter-related complications and stage of CKD. RESULTS: Of 21,415 midline recipients, 5272 (24.6%) had advanced CKD, while 16,143 (75.4%) did not. Most midlines were single lumen (90.5%) and remained in place for a median of 6 days. A major or minor midline complication occurred in 804 (15.3%) patients with and 2239 (14.4%) patients without advanced CKD (adjusted odds ratios = 1.04; 95% confidence interval: 0.94-1.14). Among patients with advanced CKD, CRBSI occurred in 13 patients (0.2%) and UE-DVT occurred in 65 patients (1.2%). The proportion of advanced CKD among midline recipients and the frequency of midline-related complications varied across hospitals (interquartile range [IQR] = 19.2% to 29.8% [median = 25.0%] and IQR = 11.0%-18.9% [median = 15.4%], respectively).

Decentralised, patient-centric, site-less, virtual, and digital clinical trials? From confusion to consensus.

There is increasing interest in clinical trials that use technologies and other innovative operational approaches to organise trial activities around trial participants instead of investigator sites. A range of terms has been introduced to refer to this operational clinical trial model, including virtual, digital, remote, and decentralised clinical trials (DCTs). However, this lack of standardised terminology can cause confusion over what a particular trial model entails and for what purposes it can be used, hampering discussions by stakeholders on its acceptability and suitability. Here, we review the different terms described in the scientific literature, advocate the consistent use of a unified term, 'decentralised clinical trial,' and provide a detailed definition of this term.

A cross-sectional survey on the early impact of COVID-19 on the uptake of decentralised trial methods in the conduct of clinical trials.

BACKGROUND: The COVID-19 pandemic significantly impacted the conduct of clinical trials through delay, interruption or cancellation. Decentralised methods in clinical trials could help to continue trials during a pandemic. This paper presents the results of an exploratory study conducted early in the pandemic to gain insight into and describe the experiences of organisations involved in clinical trials, with regard to the impact of COVID-19 on the conduct of trials, and the adoption of decentralised methods prior to, and as mitigation for the impact, of COVID-19. METHODS: A survey with 11 open-ended and four multiple choice questions was conducted in June 2020 among member organisations of the public-private "Trials@Home" consortium. The survey investigated (1) the impact and challenges of COVID-19 on the continuation of ongoing clinical trials, (2) the adoption of decentralised methods in clinical trials prior to and as a mitigation strategy for COVID-19, (3) the challenges of conducting clinical trials during COVID-19, (4) the expected permanency of COVID-19-driven changes to the adoption of decentralised methods in clinical trials, and (5) lessons learned from conducting clinical trials during the COVID-19 pandemic. A thematic, inductive analysis of open survey questions was performed, complemented with descriptive statistics (frequencies and distributions). RESULTS: The survey had a response rate of 81%. All organisations included in the analysis (n = 18) implemented (some) decentralised methods in their clinical trials prior to COVID-19, and 15 (83%) implemented decentralised methods as mitigation for COVID-19. Decentralised methods for IMP supply, patient-health care provider interaction and communication, clinic visits and source document verification were used more often as mitigation strategies than they were used prior to COVID-19. Many respondents expect to maintain those decentralised methods they implemented during COVID-19 in ongoing trials, as well as implement them in future trials. CONCLUSIONS: Decentralised methods are a widely implemented mitigation strategy for trial conduct in the face of the COVID-19 pandemic. The results of this survey show that there is an interest to continue the use of decentralised methods in future trials, but important points of attention have been identified that need solutions to help guide the transition from the traditional trial model to a more decentralised trial model.

Sodium zirconium cyclosilicate to increase lithium elimination.

Lithium is considered a mood stabilizer for bipolar affective disorders, but it has a narrow therapeutic index of 0.6-1.2 mEq/L. This can easily result in toxic levels after minimal changes in renal function or individual patient's pharmacokinetics. Lithium toxicity can arise with levels as low as 1.5 mEq, and there are limited therapeutic options to treat these patients presenting to the emergency department (ED). At therapeutic levels 95% of lithium is eliminated unchanged by the kidneys. However, previous literature has examined sodium polystyrene sulfonate (SPS) as an option to reduce lithium levels by binding the lithium cation and enhancing its excretion via the gastrointestinal tract. This suggests there may be an increased degree of non-renal clearance and altered toxicokinetics at supratherapeutic levels. However, SPS has been associated with intestinal necrosis and may cause treatment limiting hypokalemia, and is therefore not commonly recommended in treatment algorithms for lithium toxicity. A newer cation exchange resin, sodium zirconium cyclosilicate (SZC), may provide a safer alternative to SPS while also aiding in the clearance of lithium. We present a patient case where a patient with symptomatic acute-on-chronic lithium toxicity had increased clearance of lithium after a dose of SZC.

Comparative hemostatic efficacy of 4F-PCC in patients with intracranial hemorrhage on factor Xa inhibitors versus warfarin.

OBJECTIVE: Patients experiencing an intracranial hemorrhage (ICH) on oral anticoagulants often require rapid reversal. This study evaluated patients taking factor Xa inhibitors or warfarin that received reversal with 4-factor prothrombin complex concentrate (4F-PCC) for an ICH. The objective of the study was to determine if the efficacy of 4F-PCC for the reversal of factor Xa inhibitors is noninferior to its use in warfarin reversal in patients with ICH. METHODS: This was a retrospective, single center, noninferiority trial. Patients presenting to the emergency department with ICH were divided into two cohorts: those taking factor Xa inhibitors versus those taking warfarin. In each cohort, patients received anticoagulation reversal with weight-based 4F-PCC. The primary endpoint was hemostatic efficacy defined as ≤20% expansion in hematoma volume on repeat computed tomography imaging. A pre-specified noninferiority margin of -10% was selected to evaluate the difference between groups for the primary endpoint. RESULTS: A total of 221 patients were included in the study (factor Xa inhibitors, n = 87; warfarin, n = 134). Effective hemostasis was achieved in 70 patients (81%) on factor Xa inhibitors compared to 111 patients (83%) on warfarin, (-2.4% difference, [95% confidence interval, -12.87 to 8.12]; p = 0.654). There was no statistically significant difference between groups with regards to the primary outcome; however, the use of 4F-PCC in factor Xa inhibitor reversal was not noninferior when compared to 4F-PCC use for warfarin reversal. Hospital length of stay and discharge disposition were similar between cohorts. CONCLUSIONS: The efficacy of 4F-PCC in reversing factor Xa inhibitor-related ICH compared to warfarin-related ICH was not significantly different between groups; however, these results did not prove noninferiority. Further study is warranted to delineate 4F-PCC's role in reversing factor Xa inhibitors in patients with ICH.

Activation of neural stem cells from quiescence drives reactive hippocampal neurogenesis after alcohol dependence.

Neural stem cell-driven adult neurogenesis contributes to the integrity of the hippocampus. Excessive alcohol consumption in alcoholism results in hippocampal degeneration that may recover with abstinence. Reactive, increased adult neurogenesis during abstinence following alcohol dependence may contribute to recovery, but the mechanism driving reactive neurogenesis is not known. Therefore, adult, male rats were exposed to alcohol for four days and various markers were used to examine cell cycle dynamics, the percentage and number of neural progenitor cell subtypes, and the percentage of quiescent versus activated progenitors. Using a screen for cell cycle perturbation, we showed that the cell cycle is not likely altered at 7 days in abstinence. As the vast majority of Bromodeoxyuridine-positive (+) cells were co-labeled with progenitor cell marker, Sox2, we then developed a quadruple fluorescent labeling scheme to examine Type-1, -2a, -2b and -3 progenitor cells simultaneously. Prior alcohol dependence indiscriminately increased all subtypes at 7 days, the peak of the reactive proliferation. An evaluation of the time course of reactive cell proliferation revealed that cells begin proliferating at 5 days post alcohol, where only actively dividing Type 2 progenitors were increased by alcohol. Furthermore, prior alcohol increased the percentage of actively dividing Sox2+ progenitors, which supported that reactive neurogenesis is likely due to the activation of progenitors out of quiescence. These observations were associated with granule cell number returning to normal at 28 days. Therefore, activating stem and progenitor cells out of quiescence may be the mechanism underlying hippocampal recovery in abstinence following alcohol dependence.

Comparative study of Tat vaccine regimens in Mauritian cynomolgus and Indian rhesus macaques: influence of Mauritian MHC haplotypes on susceptibility/resistance to SHIV(89.6P) infection.

Protection afforded by HIV Tat-based vaccines has differed in Indian rhesus and Mauritian cynomolgus macaques. We evaluated native Tat and Ad-HIVtat priming/Tat-boosting regimens in both species. Both vaccines were immunogenic. Only the Ad-tat regimen modestly reduced acute viremia in rhesus macaques after SHIV(89.6P) challenge. Confounding variables uncovered in Mauritian macaques included significant associations of susceptibility to infection with MHC class IB and class II H2 and H5 haplotypes, and resistance to infection with class IB haplotypes H3 and H6. Although protection here was limited, Tat-based vaccines incorporating other HIV components have shown greater efficacy. Combination strategies should be further explored.

A replication-competent adenovirus-human immunodeficiency virus (Ad-HIV) tat and Ad-HIV env priming/Tat and envelope protein boosting regimen elicits enhanced protective efficacy against simian/human immunodeficiency virus SHIV89.6P challenge in rhesus macaques.

We previously demonstrated that replication-competent adenovirus (Ad)-simian immunodeficiency virus (SIV) recombinant prime/protein boost regimens elicit potent immunogenicity and strong, durable protection of rhesus macaques against SIV(mac251). Additionally, native Tat vaccines have conferred strong protection against simian/human immunodeficiency virus SHIV(89.6P) challenge of cynomolgus monkeys, while native, inactivated, or vectored Tat vaccines have failed to elicit similar protective efficacy in rhesus macaques. Here we asked if priming rhesus macaques with replicating Ad-human immunodeficiency virus (HIV) tat and boosting with the Tat protein would elicit protection against SHIV(89.6P). We also evaluated a Tat/Env regimen, adding an Ad-HIV env recombinant and envelope protein boost to test whether envelope antibodies would augment acute-phase protection. Further, expecting cellular immunity to enhance chronic viremia control, we tested a multigenic group: Ad-HIV tat, -HIV env, -SIV gag, and -SIV nef recombinants and Tat, Env, and Nef proteins. All regimens were immunogenic. A hierarchy was observed in enzyme-linked immunospot responses (with the strongest response for Env, followed by Gag, followed by Nef, followed by Tat) and antibody titers (with the highest titer for Env, followed by Tat, followed by Nef, followed by Gag). Following intravenous SHIV(89.6P) challenge, all macaques became infected. Compared to controls, no protection was seen in the Tat-only group, confirming previous reports for rhesus macaques. However, the multigenic group blunted acute viremia by approximately 1 log (P = 0.017), and both the multigenic and Tat/Env groups reduced chronic viremia by 3 and 4 logs, respectively, compared to controls (multigenic, P = 0.0003; Tat/Env, P < 0.0001). The strikingly greater reduction in the Tat/Env group than in the multigenic group (P = 0.014) was correlated with Tat and Env binding antibodies. Since prechallenge anti-Env antibodies lacked SHIV(89.6P)-neutralizing activity, other functional anti-Env and anti-Tat activities are under investigation, as is a possible synergy between the Tat and Env immunogens.

A complementary single-stranded docking site is required for enhancement of strand exchange by human immunodeficiency virus nucleocapsid protein on substrates that model viral recombination.

Enhancement of strand exchange by nucleocapsid protein (NC) is proposed to occur during retroviral recombination. The mechanism was examined using an RNA (donor)-DNA hybrid that mimicked a retrovirus replication intermediate. This consisted of a 25 base pair hybrid region flanked on each side by single-stranded RNA or DNA. A second set of acceptor RNAs that could bind to the 25-base hybrid region and to various lengths of additional bases on the DNA was used to displace the donor by hybridizing with the DNA. Displacement required a complementary single-stranded DNA region outside the donor-DNA 25-nucleotide hybrid region. NC enhanced displacement slightly when the acceptor could bind 10 nucleotides and significantly when binding 22 or more nucleotides in the single-stranded region. Two mutated acceptors that bound over 47 total nucleotides on the DNA (22 in the single-stranded region plus 25 in the hybrid region) were constructed. One had three mismatches in the hybrid region; the other, three in the single-stranded region and one in the hybrid region. Each acceptor bound the DNA with approximately equal thermodynamic stability, yet NC stimulated exchange with the former and actually inhibited with the latter. This emphasized the importance of the single-stranded region in NC stimulation. The results support a mechanism where NC enhances the docking of the acceptor to the single-stranded region and then the acceptor "zippers" through the hybrid and displaces the donor. Results with the mutated acceptors indicate that NC may actually inhibit strand exchange between genomes in nonhomologous regions.

Differing roles of the N- and C-terminal zinc fingers in human immunodeficiency virus nucleocapsid protein-enhanced nucleic acid annealing.

The replication process of human immunodeficiency virus requires a number of nucleic acid annealing steps facilitated by the hybridization and helix-destabilizing activities of human immunodeficiency virus nucleocapsid (NC) protein. NC contains two CCHC zinc finger motifs numbered 1 and 2 from the N terminus. The amino acids surrounding the CCHC residues differ between the two zinc fingers. Assays were preformed to investigate the activities of the fingers by determining the effect of mutant and wild-type proteins on annealing of 42-nucleotide RNA and DNA complements. The mutants 1.1 NC and 2.2 NC had duplications of the N- and C-terminal zinc fingers in positions 1 and 2. The mutant 2.1 NC had the native zinc fingers with their positions switched. Annealing assays were completed with unstructured and highly structured oligonucleotide complements. 2.2 NC had a near wild-type level of annealing of unstructured nucleic acids, whereas it was completely unable to stimulate annealing of highly structured nucleic acids. In contrast, 1.1 NC was able to stimulate annealing of both unstructured and structured substrates, but to a lesser degree than the wild-type protein. Results suggest that finger 1 has a greater role in unfolding of strong secondary structures, whereas finger 2 serves an accessory role that leads to a further increase in the rate of annealing.

Evidence for the differential effects of nucleocapsid protein on strand transfer in various regions of the HIV genome.

An in vitro strand transfer assay that mimicked recombinational events occurring during reverse transcription in HIV-1 was used to assess the role of nucleocapsid protein (NC) in strand transfer. Strand transfer in highly structured nucleic acid species from the U3 3' long terminal repeats, gag-pol frameshift region, and Rev response element were strongly enhanced by NC. In contrast, weakly structured templates from the env and pol-vif regions transferred well without NC and showed lower enhancement. The lack of strong polymerase pause sites in the latter regions demonstrated that non-pause driven mechanisms could also promote transfer. Assays conducted using NC zinc finger mutants supported a differential role for the two fingers in strand transfer with finger 1 (N-terminal) being more important on highly structured RNAs. Overall this report suggests a role for structural intricacies of RNA templates in determining the extent of influence of NC on recombination and illustrates that strand transfer may occur by several different mechanisms depending on the structural nature of the RNA.