Genetic determinants of antimicrobial resistance in polymyxin B resistant Pseudomonas aeruginosa isolated from airways of patients with cystic fibrosis.

Pseudomonas aeruginosa is the main pathogen associated with pulmonary exacerbation in patients with cystic fibrosis (CF). CF is a multisystemic genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator gene, which mainly affects pulmonary function. P. aeruginosa isolated from individuals with CF in Brazil is not commonly associated with multidrug resistance (MDR), especially when compared to global occurrence, where the presence of epidemic clones, capable of expressing resistance to several drugs, is often reported. Due to the recent observations of MDR isolates of P. aeruginosa in our centers, combined with these characteristics, whole-genome sequencing was employed for analyses related to antimicrobial resistance, plasmid identification, search for phages, and characterization of CF clones. All isolates in this study were polymyxin B resistant, exhibiting diverse mutations and reduced susceptibility to carbapenems. Alterations in mexZ can result in the overexpression of the MexXY efflux pump. Mutations in oprD, pmrB, parS, gyrA and parC may confer reduced susceptibility to antimicrobials by affecting permeability, as observed in phenotypic tests. The phage findings led to the assumption of horizontal genetic transfer, implicating dissemination between P. aeruginosa isolates. New sequence types were described, and none of the isolates showed an association with epidemic CF clones. Analysis of the genetic context of P. aeruginosa resistance to polymyxin B allowed us to understand the different mechanisms of resistance to antimicrobials, in addition to subsidizing the understanding of possible relationships with epidemic strains that circulate among individuals with CF observed in other countries.

Pavement ant extract is a chemotaxis repellent for C. elegans.

Ant behavior relies on a collection of natural products, from following trail pheromones during foraging to warding off potential predators. How nervous systems sense these compounds to initiate a behavioral response remains unclear. Here, we used Caenorhabditis elegans chemotaxis assays to investigate how ant compounds are detected by heterospecific nervous systems. We found that C. elegans avoid extracts of the pavement ant ( Tetramorium immigrans ) and either osm-9 or tax-4 ion channels are required for this response. These experiments were conducted in an undergraduate laboratory course, demonstrating that new insights into interspecies interactions can be generated through genuine research experiences in a classroom setting.

Carvacrol in ovo delivery optimization and flow dynamics in broiler chicken eggs.

In ovo delivery of carvacrol, the primary active compound in oregano essential oil (OEO) has the potential to enhance gut development in broilers. This study aimed to optimize in ovo application of OEO by investigating day and site of injection and delivery of carvacrol to different embryonic tissues. In Experiment 1, 2 d of injection (embryonic day (E) 12 or 17.5) and 3 sites of injection for OEO (air cell, amniotic fluid, or yolk) were evaluated based on hatchability and posthatching performance. Experiment 2 aimed to examine the impact of combining OEO with the nonionic surfactant polysorbate 80 (p80) at ratios to carvacrol of 0:0, 0:1, 0.5:1, and 1:1 on carvacrol concentration in amniotic fluid, blood, and yolk. The concentration of carvacrol was measured at 3, 6, and 9 h after OEO injection either without (0:1) or with (1:1) p80. Injection of OEO on E12 led to a significant lower hatchability compared to E17.5 (P ≤ 0.01; Δ = 9.2%). Injecting OEO into the air cell, amniotic fluid, or yolk at E17.5 did not significantly affect hatchability and posthatching performance. The highest concentrations of carvacrol found in egg tissues were observed when injected together with surfactant at the 1:1 ratio (P ≤ 0.001; 14.45 µM, 16.64 µM, and 124.82 µM, for air cell, amniotic fluid, and yolk, respectively) compared to the 0:0, 0:1 or 0.5:1 ratios. Carvacrol was highest in the amniotic fluid and blood at the first time point (3 h postinjection) and decreased afterward (P ≤ 0.001), whereas the concentration in yolk remained elevated up to 9 h postinjection. In conclusion, the optimization of the in ovo delivery of carvacrol resulted in that early injection (E12) had negative effects on hatchability and should be avoided. The findings also suggest that using a nonionic surfactant was crucial for an effective delivery of carvacrol in ovo and the migration from amniotic fluid to yolk within 3 h. In addition, carvacrol's persistence in yolk may serve as a route for delivery into the gastrointestinal tract via the yolk stalk during the peri-hatching phase, potentially influencing gut development.

In ovo delivery of oregano essential oil activated xenobiotic detoxification and lipid metabolism at hatch in broiler chickens.

In ovo interventions are used to improve embryonic development and robustness of chicks. The objective of this study was to identify the optimal dose for in ovo delivery of oregano essential oil (OEO), and to investigate metabolic impacts. Broiler chickens Ross 308 fertile eggs were injected with 7 levels of OEO (0, 5, 10, 20, 30, 40, and 50 µL) into the amniotic fluid at embryonic d 17.5 (E17.5) (n = 48). Chick quality was measured by navel score (P < 0.05) and/or hatchability rates (P < 0.01) were significantly decreased at doses at or above 10 or 20 µL/egg, respectively, indicating potential toxicity. However, no effects were observed at the 5 µL/egg, suggesting that compensatory mechanisms were effective to maintain homeostasis in the developing embryo. To pursue a better understanding of these mechanisms, transcriptomic analyses of the jejunum were performed comparing the control injected with saline and the group injected with 5 µL of OEO. The transcriptomic analyses identified that 167 genes were upregulated and 90 were downregulated in the 5 µL OEO compared to the control group injected with saline (P < 0.01). Functional analyses of the differentially expressed genes (DEG) showed that metabolic pathways related to the epoxygenase cytochrome P450 pathway associated with xenobiotic catabolic processes were significantly upregulated (P < 0.05). In addition, long-chain fatty acid metabolism associated with ATP binding transporters was also upregulated in the OEO treated group (P < 0.05). The results indicated that low doses of OEO in ovo have the potential to increase lipid metabolism in late stages (E17.5) of embryonic development. In conclusion, in ovo delivery of 5 µL OEO did not show any negative impact on hatchability and chick quality. OEO elevated expression of key enzymes and receptors involved in detoxification pathways and lipid metabolism in the jejunum of hatchling broiler chicks.

[Analysis of a differentiated resuscitation room activation at a national trauma center]. / Analyse einer differenzierten Schockraumalarmierung an einem überregionalen Traumazentrum.

BACKGROUND: In order to continue to efficiently provide both personnel-intensive and resource-intensive care to severely injured patients, some hospitals have introduced individually differentiated systems for resuscitation room treatment. The aim of this study was to evaluate the concept of the A and B classifications in terms of practicability, indications, and potential complications at a national trauma center in Bavaria. METHODS: In a retrospective study, data from resuscitation room trauma patients in the year 2020 were collected. The assignment to A and B was made by the prehospital emergency physician. Parameters such as the injury severity score (ISS), Glasgow outcome scale (GOS), upgrade rate, and the indication criteria according to the S3 guidelines were recorded. Statistical data comparisons were made using t­tests, χ2-tests, or Mann-Whitney U­tests. RESULTS: A total of 879 resuscitation room treatments (A 473, B 406) met the inclusion criteria. It was found that 94.5% of resuscitation room A cases had physician accompaniment, compared to 48% in resuscitation room B assignments. In addition to significantly lower ISS scores (4.1 vs. 13.9), 29.8% of B patients did not meet the treatment criteria defined in the S3 guidelines. With a low upgrade rate of 4.9%, 98% of B patients had a GOS score of 4 or 5. CONCLUSION: The presented categorization is an effective and safe way to manage the increasing number of resuscitation room alerts in a resource-optimized manner.

Use of the Pfizer Respiratory Syncytial Virus Vaccine During Pregnancy for the Prevention of Respiratory Syncytial Virus-Associated Lower Respiratory Tract Disease in Infants: Recommendations of the Advisory Committee on Immunization Practices - United States, 2023.

Respiratory syncytial virus (RSV) is the leading cause of hospitalization among U.S. infants. Nirsevimab (Bevfortus, Sanofi and AstraZeneca) is recommended to prevent RSV-associated lower respiratory tract infection (LRTI) in infants. In August 2023, the Food and Drug Administration (FDA) approved RSVpreF vaccine (Abrysvo, Pfizer Inc.) for pregnant persons as a single dose during 32-36 completed gestational weeks (i.e., 32 weeks and zero days' through 36 weeks and 6 days' gestation) to prevent RSV-associated lower respiratory tract disease in infants aged <6 months. Since October 2021, CDC's Advisory Committee on Immunization Practices (ACIP) RSV Vaccines Pediatric/Maternal Work Group has reviewed RSV epidemiology and evidence regarding safety, efficacy, and potential economic impact of pediatric and maternal RSV prevention products, including RSVpreF vaccine. On September 22, 2023, ACIP and CDC recommended RSVpreF vaccine using seasonal administration (i.e., during September through end of January in most of the continental United States) for pregnant persons as a one-time dose at 32-36 weeks' gestation for prevention of RSV-associated LRTI in infants aged <6 months. Either maternal RSVpreF vaccination during pregnancy or nirsevimab administration to the infant is recommended to prevent RSV-associated LRTI among infants, but both are not needed for most infants. All infants should be protected against RSV-associated LRTI through use of one of these products.

Nanoscaled RIM clustering at presynaptic active zones revealed by endogenous tagging.

Chemical synaptic transmission involves neurotransmitter release from presynaptic active zones (AZs). The AZ protein Rab-3-interacting molecule (RIM) is important for normal Ca2+-triggered release. However, its precise localization within AZs of the glutamatergic neuromuscular junctions of Drosophila melanogaster remains elusive. We used CRISPR/Cas9-assisted genome engineering of the rim locus to incorporate small epitope tags for targeted super-resolution imaging. A V5-tag, derived from simian virus 5, and an HA-tag, derived from human influenza virus, were N-terminally fused to the RIM Zinc finger. Whereas both variants are expressed in co-localization with the core AZ scaffold Bruchpilot, electrophysiological characterization reveals that AP-evoked synaptic release is disturbed in rimV5-Znf but not in rimHA-Znf In addition, rimHA-Znf synapses show intact presynaptic homeostatic potentiation. Combining super-resolution localization microscopy and hierarchical clustering, we detect ∼10 RIMHA-Znf subclusters with ∼13 nm diameter per AZ that are compacted and increased in numbers in presynaptic homeostatic potentiation.

Use of Nirsevimab for the Prevention of Respiratory Syncytial Virus Disease Among Infants and Young Children: Recommendations of the Advisory Committee on Immunization Practices - United States, 2023.

Respiratory syncytial virus (RSV) is the leading cause of hospitalization among U.S. infants. In July 2023, the Food and Drug Administration approved nirsevimab, a long-acting monoclonal antibody, for passive immunization to prevent RSV-associated lower respiratory tract infection among infants and young children. Since October 2021, the Advisory Committee on Immunization Practices (ACIP) Maternal and Pediatric RSV Work Group has reviewed evidence on the safety and efficacy of nirsevimab among infants and young children. On August 3, 2023, ACIP recommended nirsevimab for all infants aged <8 months who are born during or entering their first RSV season and for infants and children aged 8-19 months who are at increased risk for severe RSV disease and are entering their second RSV season. On the basis of pre-COVID-19 pandemic patterns, nirsevimab could be administered in most of the continental United States from October through the end of March. Nirsevimab can prevent severe RSV disease among infants and young children at increased risk for severe RSV disease.

Impact of a Femoral Fracture on Outcome after Traumatic Brain Injury-A Matched-Pair Analysis of the TraumaRegister DGU®.

Traumatic brain injury (TBI) is the leading cause of death and disability in polytrauma and is often accompanied by concomitant injuries. We conducted a retrospective matched-pair analysis of data from a 10-year period from the multicenter database TraumaRegister DGU® to analyze the impact of a concomitant femoral fracture on the outcome of TBI patients. A total of 4508 patients with moderate to critical TBI were included and matched by severity of TBI, American Society of Anesthesiologists (ASA) risk classification, initial Glasgow Coma Scale (GCS), age, and sex. Patients who suffered combined TBI and femoral fracture showed increased mortality and worse outcome at the time of discharge, a higher chance of multi-organ failure, and a rate of neurosurgical intervention. Especially those with moderate TBI showed enhanced in-hospital mortality when presenting with a concomitant femoral fracture (p = 0.037). The choice of fracture treatment (damage control orthopedics vs. early total care) did not impact mortality. In summary, patients with combined TBI and femoral fracture have higher mortality, more in-hospital complications, an increased need for neurosurgical intervention, and inferior outcome compared to patients with TBI solely. More investigations are needed to decipher the pathophysiological consequences of a long-bone fracture on the outcome after TBI.

Effectiveness of COVID-19 mRNA Vaccines in Preventing COVID-19-Associated Outpatient Visits and Hospitalizations Among American Indian and Alaska Native Persons, January-November 2021: A Test-Negative Case-Control Analysis Using Surveillance Data.

Background: Despite the disproportionate morbidity and mortality experienced by American Indian and Alaska Native (AI/AN) persons during the coronavirus disease 2019 (COVID-19) pandemic, few studies have reported vaccine effectiveness (VE) estimates among these communities. Methods: We conducted a test-negative case-control analysis among AI/AN persons aged ≥12 years presenting for care from January 1, 2021, through November 30, 2021, to evaluate the effectiveness of mRNA COVID-19 vaccines against COVID-19-associated outpatient visits and hospitalizations. Cases and controls were patients with ≥1 symptom consistent with COVID-19-like illness; cases were defined as those test-positive for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and controls were defined as those test-negative for SARS-CoV-2. We used unconditional multivariable logistic regression to estimate VE, defined as 1 minus the adjusted odds ratio for vaccination among cases vs controls. Results: The analysis included 207 cases and 267 test-negative controls. Forty-four percent of cases and 78% of controls received 2 doses of either BNT162b2 or mRNA-1273 vaccine. VE point estimates for 2 doses of mRNA vaccine were higher for hospitalized participants (94.6%; 95% CI, 88.0-97.6) than outpatient participants (86.5%; 95% CI, 63.0-95.0), but confidence intervals overlapped. Conclusions: Among AI/AN persons, mRNA COVID-19 vaccines were highly effective in preventing COVID-associated outpatient visits and hospitalizations. Maintaining high vaccine coverage, including booster doses, will reduce the burden of disease in this population.

Seasonality of Respiratory Syncytial Virus - United States, 2017-2023.

In the United States, respiratory syncytial virus (RSV) infections cause an estimated 58,000-80,000 hospitalizations among children aged <5 years (1,2) and 60,000-160,000 hospitalizations among adults aged ≥65 years each year (3-5). U.S. RSV epidemics typically follow seasonal patterns, peaking in December or January (6,7), but the COVID-19 pandemic disrupted RSV seasonality during 2020-2022 (8). To describe U.S. RSV seasonality during prepandemic and pandemic periods, polymerase chain reaction (PCR) test results reported to the National Respiratory and Enteric Virus Surveillance System (NREVSS)* during July 2017-February 2023 were analyzed. Seasonal RSV epidemics were defined as the weeks during which the percentage of PCR test results that were positive for RSV was ≥3% (9). Nationally, prepandemic seasons (2017-2020) began in October, peaked in December, and ended in April. During 2020-21, the typical winter RSV epidemic did not occur. The 2021-22 season began in May, peaked in July, and ended in January. The 2022-23 season started (June) and peaked (November) later than the 2021-22 season, but earlier than prepandemic seasons. In both prepandemic and pandemic periods, epidemics began earlier in Florida and the Southeast and later in regions further north and west. With several RSV prevention products in development,† ongoing monitoring of RSV circulation can guide the timing of RSV immunoprophylaxis and of clinical trials and postlicensure effectiveness studies. Although the timing of the 2022-23 season suggests that seasonal patterns are returning toward those observed in prepandemic years, clinicians should be aware that off-season RSV circulation might continue.

Single-Molecule Localization Microscopy of Presynaptic Active Zones in Drosophila melanogaster after Rapid Cryofixation.

Single-molecule localization microscopy (SMLM) greatly advances structural studies of diverse biological tissues. For example, presynaptic active zone (AZ) nanotopology is resolved in increasing detail. Immunofluorescence imaging of AZ proteins usually relies on epitope preservation using aldehyde-based immunocompetent fixation. Cryofixation techniques, such as high-pressure freezing (HPF) and freeze substitution (FS), are widely used for ultrastructural studies of presynaptic architecture in electron microscopy (EM). HPF/FS demonstrated nearer-to-native preservation of AZ ultrastructure, e.g., by facilitating single filamentous structures. Here, we present a protocol combining the advantages of HPF/FS and direct stochastic optical reconstruction microscopy (dSTORM) to quantify nanotopology of the AZ scaffold protein Bruchpilot (Brp) at neuromuscular junctions (NMJs) of Drosophila melanogaster. Using this standardized model, we tested for preservation of Brp clusters in different FS protocols compared to classical aldehyde fixation. In HPF/FS samples, presynaptic boutons were structurally well preserved with ~22% smaller Brp clusters that allowed quantification of subcluster topology. In summary, we established a standardized near-to-native preparation and immunohistochemistry protocol for SMLM analyses of AZ protein clusters in a defined model synapse. Our protocol could be adapted to study protein arrangements at single-molecule resolution in other intact tissue preparations.

Surveillance for Acute Respiratory Illnesses in Pediatric Chronic Care Facilities.

Overall, 119 (33%) of 364 pediatric chronic care facility residents experienced 182 acute respiratory illnesses (ARIs) that met the surveillance definition which led to 31 (17%) emergency room visits, 34 (19%) acute care hospitalizations, and/or 25 (14%) ICU admissions. Continued PCR-positivity was observed in 35% of ARIs during follow-up testing.

Structural Basis for Rabbit Hemorrhagic Disease Virus Antibody Specificity.

Rabbit hemorrhagic disease virus (RHDV) typically causes a fatal disease in rabbits. In Australia, RHDV was imported to control the feral rabbit population, while it poses a severe threat to native rabbits in other countries. RHDV variants are genetically diverse and serological studies using antibodies isolated from infected rabbits or raised against RHDV virus-like particles (VLPs) have found RHDV variants antigenically distinct. In this study, we determined the X-ray crystal structure of an RHDV GI.2 (N11 strain) protruding (P) domain in complex with a diagnostic monoclonal antibody (2D9) Fab. We showed that 2D9 interacted with conserved and variable residues on top of the P domain with nanomolar affinity. To better illustrate 2D9 specificity, we determined the X-ray crystal structure of an RHDV GI.1b (Ast89 strain) that was a 2D9 non-binder. Structural analysis indicated that amino acid substitutions on the GI.1b P domain likely restricted 2D9 binding. Interestingly, a model of the GI.2 P domain-Fab complex superimposed onto a cryo-EM structure of an RHDV VLP revealed that 2D9 Fab molecules clashed with neighboring Fabs and indicated that there was a reduced antibody binding occupancy. Moreover, the RHDV GI.2 histo-blood group antigen (HBGA) co-factor binding site appeared obstructed when 2D9 was modeled on the VLP and suggested that 2D9 might also function by blocking HBGA attachment. Overall, this new data provides the first structural basis of RHDV antibody specificity and explains how amino acid variation at the binding site likely restricts 2D9 cross-reactivity. IMPORTANCE Isolated RHDV antibodies have been used for decades to distinguish between antigenic variants, monitor temporal capsid evolution, and examine neutralizing capacities. In this study, we provided the structural basis for an RHDV GI.2 specific diagnostic antibody (2D9) binding and reveal that a small number of amino acid substitutions at the binding site could differentiate between RHDV GI.2 and GI.1b. This novel structural information provides a framework for understanding how RHDV displays a specific antigenic epitope and engages an antibody at the atomic level. Importantly, part of the 2D9 binding region was earlier reported to contain a neutralizing epitope and our structural modeling as well as recent human norovirus antibody-mediated neutralization studies, suggest that the 2D9 antibody has the potential to block HBGA attachment. These new findings should aid in characterizing antigenic variants and advance the development of novel monoclonal antibodies for diagnostics and therapeutics.

Pre-operative Colonization by Staphylococcus aureus and Cephalosporin Non-susceptible Bacteria in Patients with Proximal Femoral Fractures.

Objective To estimate the frequency of Staphylococcus aureus and cephalosporin nonsusceptible bacteria colonization in patients with proximal femoral fracture during preoperative hospitalization. Methods Prevalence and incidence assessment in 63 hospitalized patients over 1 year. The median time of pretreatment hospitalization was 12 days. Samples were collected from the nostrils, groin skin and anal mucosa during the pretreatment hospitalization and were tested by the disc-diffusion technique. Results The hospital colonization incidence and the prevalence of positive results were 14.3 and 44.4% for S. aureus ; 3.2 and 6.4% for meticillin-resistant S. aureus ; 28.6 and 85.7% for meticillin-resistant coagulase-negative Staphylococcus ; 28.6 and 61.9% for cefazolin nonsusceptible Enterobacteriaceae (KFNSE); and 20.6 and 28.6% for cefuroxime nonsusceptible Enterobacteriaceae (CXNSE). In addition, factors such as to the duration of the pretreatment hospitalization period, being non-walker before fracture, antimicrobial use, American Society of Anesthesiologists (ASA) 4 surgical risk, and previous hospitalization, were related to an increase in the incidence of hospital acquisition and prevalence of colonization by the evaluated strains. The prevalence of colonization by KFNSE was three times higher than by CXNSE on admission, and twice as high at the time of fracture treatment. Conclusion There was a high incidence of hospital colonization and prevalence of colonization by all strains studied, which may guide the indication of prophylactic measures for infection.

Pre-operative Colonization by Staphylococcus aureus and Cephalosporin Non-susceptible Bacteria in Patients with Proximal Femoral Fractures / Colonização pré-operatória por Staphylococcus aureus e as bactérias não suscetíveis à cefalosporina, em pacientes com fratura proximal do fêmur

Abstract Objective To estimate the frequency of Staphylococcus aureus and cephalosporin nonsusceptible bacteria colonization in patients with proximal femoral fracture during preoperative hospitalization. Methods Prevalence and incidence assessment in 63 hospitalized patients over 1 year. The median time of pretreatment hospitalization was 12 days. Samples were collected from the nostrils, groin skin and anal mucosa during the pretreatment hospitalization and were tested by the disc-diffusion technique. Results The hospital colonization incidence and the prevalence of positive results were 14.3 and 44.4% for S. aureus; 3.2 and 6.4% for meticillin-resistant S. aureus; 28.6 and 85.7% for meticillin-resistant coagulase-negative Staphylococcus; 28.6 and 61.9% for cefazolin nonsusceptible Enterobacteriaceae (KFNSE); and 20.6 and 28.6% for cefuroxime nonsusceptible Enterobacteriaceae (CXNSE). In addition, factors such as to the duration of the pretreatment hospitalization period, being non-walker before fracture, antimicrobial use, American Society of Anesthesiologists (ASA) 4 surgical risk, and previous hospitalization, were related to an increase in the incidence of hospital acquisition and prevalence of colonization by the evaluated strains. The prevalence of colonization by KFNSE was three times higher than by CXNSE on admission, and twice as high at the time of fracture treatment. Conclusion There was a high incidence of hospital colonization and prevalence of colonization by all strains studied, which may guide the indication of prophylactic measures for infection.

Resumo Objetivo Estimar a frequência da colonização por Staphylococcus aureus e as bactérias não suscetíveis à cefalosporina, em pacientes com fratura proximal do fêmur durante a internação pré-operatória. Métodos Avaliação da prevalência e incidência em 63 pacientes hospitalizados ao longo de um ano. O tempo médio de internação pré-tratamento foi de 12 dias. As amostras foram coletadas das narinas, pele da virilha e mucosa anal, durante a internação prévia ao tratamento e testadas pela técnica de disco-difusão. Resultados A incidência da colonização hospitalar e a prevalência de resultados positivos foram de 14,3% e 44,4% para Staphylococcus aureus; 3,2% e 6,4% para S. aureus resistente à meticilina; 28,6% e 85,7% para Staphylococcus coagulase-negativo resistente à meticilina; 28,6% e 61,9% para Enterobacteriaceae não suscetível à cefazolina (KFNSE); e 20,6% e 28,6% para Enterobacteriaceae não suscetível à cefuroxima (CXNSE). Além da duração do período de internação pré-tratamento, os pacientes não deambularam previamente à ocorrência da fratura e nem fizeram uso de antimicrobiano. Além disso, a duração do período de internação pré-tratamento cirúrgico, ser não-deambulador antes da fratura, uso de antimicrobianos, risco cirúrgico IV pela American Society of Anesthesiologists (ASA) e internação anterior, estiveram relacionados a um aumento na incidência de aquisição hospitalar e prevalência de colonização pelas cepas avaliadas. A prevalência de colonização pela KFNSE foi três vezes maior do que pela CXNSE na admissão e duas vezes maior no momento do tratamento da fratura. Conclusão Observou-se uma alta incidência da colonização hospitalar e prevalência da colonização por todas as cepas estudadas, o que pode orientar a indicação de medidas profiláticas contra a infecção.

Dual role of neddylation in transcription of hepatitis B virus RNAs from cccDNA and production of viral surface antigen.

Background & Aims: HBV persistence is maintained by both an episomal covalently closed circular (ccc)DNA reservoir and genomic integration of HBV DNA fragments. While cccDNA transcription is regulated by Cullin4A-DDB1-HBx-mediated degradation of the SMC5/6 complex, HBsAg expression from integrants is largely SMC5/6 independent. Inhibiting neddylation of Cullin-RING ubiquitin ligases impairs degradation of substrates. Herein, we show that targeting neddylation pathway components by small-interfering (si)RNAs or the drug MLN4924 (pevonedistat) suppresses expression of HBV proteins from both cccDNA and integrants. Methods: An siRNA screen targeting secretory pathway regulators and neddylation genes was performed. Activity of MLN4924 was assessed in infection and integration models. Trans-complementation assays were used to study HBx function in cccDNA-driven expression. Results: siRNA screening uncovered neddylation pathway components (Nedd8, Ube2m) that promote HBsAg production post-transcriptionally. Likewise, MLN4924 inhibited production of HBsAg encoded by integrants and reduced intracellular HBsAg levels, independent of HBx. MLN4924 also profoundly inhibited cccDNA transcription in three infection models. Using the HBV inducible cell line HepAD38 as a model, we verified the dual action of MLN4924 on both cccDNA and integrants with sustained suppression of HBV markers during 42 days of treatment. Conclusions: Neddylation is required both for transcription of a cccDNA reservoir and for the genomic integration of viral DNA. Therefore, blocking neddylation might offer an attractive approach towards functional cure of chronic hepatitis B. Lay summary: Current treatments for chronic hepatitis B are rarely able to induce a functional cure. This is partly because of the presence of a pool of circular viral DNA in the host nucleus, as well as viral DNA fragments that are integrated into the host genome. Herein, we show that a host biological pathway called neddylation could play a key role in infection and viral DNA integration. Inhibiting this pathway could hold therapeutic promise for patients with chronic hepatitis B.

Plate osteosynthesis combined with bone cement provides the highest stability for tibial head depression fractures under high loading conditions.

Older patients sustaining tibial head depression fractures often cannot follow the post-operative rehabilitation protocols with partial weight-bearing of the affected limb, leading to osteosynthesis failure, cartilage step-off and arthritis development. Therefore, the aim of this study was to analyse the biomechanical performance of different types of osteosyntheses alone and in combination with bone cement simulating cyclically high loading conditions of tibial head depression fractures. Lateral tibial head depression fractures (AO: 41-B2.2; Schatzker type III) were created in synthetic bones and stabilized using three different osteosyntheses alone and in combination with a commonly used bone cement (chronOS™): 2 screws, 4 screws in the jail technique and a lateral angle-stable buttress plate. After fixation, the lateral tibial plateau was axially loaded in two, from each other independent testing series: In the first test protocol, 5000 cycles with 500 N and in the end load-to-failure tests were performed. In the second test protocol, the cyclic loading was increased to 1000 N. Parameters of interest were the displacement of the articular fracture fragment, the stiffness and the maximum load. The osteosyntheses revealed a higher stiffness in combination with bone cement compared to the same type of osteosynthesis alone (e.g., 500 N level: 2 screws 383 ± 43 N/mm vs. 2 screws + chronOs 520 ± 108 N/mm, increase by 36%, p < 0.01; 4 screws 368 ± 97 N/mm vs. 4 screws + chronOS 516 ± 109 N/mm, increase by 40%, p < 0.01; plate: 509 ± 73 N/mm vs. plate + chronOs 792 ± 150 N/mm, increase by 56%, p < 0.01). Bone cement reduced the displacement of the plate significantly (500 N level: plate: 8.9 ± 2.8 mm vs. plate + chronOs: 3.1 ± 1.4 mm, reduction by 65%, p < 0.01; 1000 N level: 16.9 ± 3.6 mm vs 5.6 ± 1.3 mm, reduction by 67%, p < 0.01). Thus, the highest stiffness and lowest displacement values were found when using the plate with bone cement in both loading conditions (500 N level: 2 screws + chronOs 3.7 ± 1.3 mm, 4 screws + chronOs 6.2 ± 2.4 mm; 1000 N level: 2 screws + chronOs 6.5 ± 1.2 mm, 4 screws + chronOs 5.7 ± 0.8 mm). From a biomechanical perspective, plate osteosynthesis of tibial head depression fractures should always be combined with bone cement, provides higher stability than 2-screw and 4-screw fixation and is a valid treatment option in cases where extraordinary stability is required.

Surgical Fixation of Calcaneal Beak Fractures-Biomechanical Analysis of Different Osteosynthesis Techniques.

The calcaneal beak fracture is a rare avulsion fracture of the tuber calcanei characterized by a solid bony fragment at the Achilles tendon insertion. Treatment usually requires osteosynthesis. However, lack of biomechanical understanding of the ideal fixation technique persists. A beak fracture was simulated in synthetic bones and assigned to five different groups of fixation: A) 6.5-mm partial threaded cannulated screws, B) 4.0-mm partial threaded cannulated screws, C) 5.0-mm headless cannulated compression screws, D) 2.3-mm locking plate, and E) 2.8-mm locking plate. Different traction force levels were applied through an Achilles tendon surrogate in a material-testing machine on all stabilized synthetic bones. Outcome measures were peak-to-peak displacement, total displacement, plastic deformation, stiffness, visual-fracture-line displacement, and mode of implant failure. The 2.3- and 2.8-mm plating groups showed a high drop-out rate at 100 N tension force and failed under higher tension levels of 200 N. The fracture fixation using 4.0-mm partial threaded screws showed a significantly higher repair strength and was able to withhold cyclic loading up to 300 N. The lowest peak-to-peak displacement and the highest load-to-failure and stiffness were provided by fracture fixation using 6.5-mm partial threaded cannulated screws or 5.0-mm headless cannulated compression screws. As anticipated, large 6.5-mm screw diameters provide the best biomechanical fixation. Surprisingly, the 5.0-mm headless cannulated compression screws yield reliable stability despite the absent screw head and washer. When such large screws cannot be applied, 4.0-mm screws also allow reasonable fixation strength. Plate fixation should be implemented with precaution and in combination with a restrictive postoperative motion protocol. Finally, clinical cases about the surgical application and recovery are included.