Targeting Recycling Endosomes to Potentiate mRNA Lipid Nanoparticles.

mRNA lipid nanoparticles (LNPs) have emerged as powerful modalities for gene therapies to control cancer and infectious and immune diseases. Despite the escalating interest in mRNA-LNPs over the past few decades, endosomal entrapment of delivered mRNAs vastly impedes therapeutic developments. In addition, the molecular mechanism of LNP-mediated mRNA delivery is poorly understood to guide further improvement through rational design. To tackle these challenges, we characterized LNP-mediated mRNA delivery using a library of small molecules targeting endosomal trafficking. We found that the expression of delivered mRNAs is greatly enhanced via inhibition of endocytic recycling in cells and in live mice. One of the most potent small molecules, endosidine 5 (ES5), interferes with recycling endosomes through Annexin A6, thereby promoting the release and expression of mRNA into the cytoplasm. Together, these findings suggest that targeting endosomal trafficking with small molecules is a viable strategy to potentiate the efficacy of mRNA-LNPs.

Quality of Life Impacts Following Surgery for Advanced Head and Neck Cancer.

Background: Understanding the impact of surgery on patients will enable clinicians to provide evidence-based perioperative management. This study aimed to investigate the quality of life (QoL) impacts following head and neck surgery for advanced stage head and neck cancer. Methods: Head and neck cancer survivors were invited to complete five validated questionnaires to investigate QoL. Associations between QoL and patient variables were analyzed. Variables included age, time since operation, length of surgery, length of stay, Comorbidity Index, estimated 10-year survival, sex, flap type, treatment and cancer type. Outcome measures were also compared to normative outcomes. Results: The majority of participants (N = 27; 55% male; mean (standard deviation) age: 62.6 (13.8) years; mean time since operation: 801 days) had a squamous cell carcinoma (88.9%) and free flap repair (100%). Time since operation was significantly (P < 0.05) associated with higher rates of depression (r = -0.533), psychological needs (r = -0.0415) and physical/daily living needs (r = -0.527). Length of surgery and length of stay were significantly associated with depression (r = 0.442; r = 0.435) and length of stay was significantly associated with speaking difficulties (r = -0.456). There was a significant association between work and education scores with age (r = 0.471), length of surgery (r = 0.424), Comorbidity Index (r = 0.456) and estimated 10-year survival (r = -0.523). Conclusions: Age, time since operation, length of surgery, length of stay, Comorbidity Index and estimated 10-year survival were the outcomes associated with QoL. Patient-reported outcome measures and psychological support could be included in the standard care pathway for head and neck cancer patients to ensure holistic management of their condition.

The impact of additional pathology analysis on the diagnosis and management of soft tissue tumours: a 10-year retrospective study.

In order to characterise soft tissue tumours, pathologists often utilise specialised additional tests, or may seek opinions from subspecialist pathologists due to rarity or complex morphology. Additionally, further review may be sought by subspecialist sarcoma pathologists, such as those at our tertiary referral centre in Sydney, Australia. The aim of this study was to examine the impact on diagnosis and management of this external review, following diagnosis at a specialised sarcoma unit. We collated the results of all additional external ancillary tests and specialist reviews over a 10-year period and characterised the impact on the preliminary diagnosis as 'confirmed', 'new' or 'no clear diagnosis'. We subsequently noted whether the additional findings resulted in a clinically significant change in management. Of the 136 cases sent away, 103 patients had their initial diagnosis confirmed, 29 patients received a new diagnosis and, for four patients, the diagnosis remained uncertain. Nine of the 29 patients receiving a new diagnosis had their management altered. This study demonstrated that within our specialised sarcoma unit, the majority of diagnoses provided by our specialist pathologists are confirmed on additional external testing and review, but external review does provide additional assurance and benefit to the patient.

Breast Radiation Recall Phenomenon After Astra-Zeneca COVID-19 Vaccine: A Case Series.

We report three cases of AstraZeneca vaccine (AZV)-induced radiation recall phenomenon (RRP) in three women who had previously undergone radiotherapy for breast cancer. RRP is a rare complication of vaccination that can mimic the more common pathology of breast cellulitis. Emergency physicians, primary care specialists, and surgeons should be aware of RRP when treating patients in the current coronavirus disease 2019 (COVID-19) climate.

Pop goes the tumour! Spontaneous haemorrhage of a hepatocellular carcinoma tumour.

An 84-year-old man presented to a rural hospital in Australia with haemodynamic instability and abdominal pain. Investigation revealed haemorrhage from a lesion in his liver-an incidental finding of a hepatocellular carcinoma. Initial resuscitation and damage control surgery was performed at the peripheral hospital prior to transfer to a tertiary centre 386 km away for the second stage of management. The second stage of management included interventional radiological embolization of the bleeding liver vessel and subsequent resection of the liver tumour. This was all undertaken with new policies in place to limit the spread of infection at the peak of the COVID-19 epidemic.

Letermovir Resistance Analysis in a Clinical Trial of Cytomegalovirus Prophylaxis for Hematopoietic Stem Cell Transplant Recipients.

BACKGROUND: Letermovir (LET), a cytomegalovirus (CMV) deoxyribonucleic acid (DNA) terminase inhibitor, was recently approved for prophylaxis of CMV infection in adult CMV-seropositive recipients of allogeneic hematopoietic stem cell transplantation. Cytomegalovirus genotyping was performed to identify LET-resistance-associated variants (RAVs) among subjects in a Phase 3 trial. METHODS: The CMV UL56 and UL89 genes, encoding subunits of CMV DNA terminase, were sequenced from plasma collected from subjects with clinically significant CMV infection (CS-CMVi). Novel variants were evaluated by recombinant phenotyping to assess their potential to confer resistance to LET. RESULTS: Genotyping was successful for 50 of 79 LET subjects with CS-CMVi. Resistance-associated variants (encoding pUL56 V236M and C325W) were detected independently in subjects 1 and 3 who experienced CS-CMVi while receiving LET prophylaxis, and 2 other variants (encoding pUL56 E237G and R369T) were detected >3 weeks after subjects 2 and 3, respectively, had discontinued LET prophylaxis and received preemptive therapy with ganciclovir. CONCLUSIONS: The detected incidence of CMV resistance among subjects who received LET as prophylaxis in this Phase 3 trial was low. The LET RAVs that were detected mapped to the CMV UL56 gene at positions associated with reduced susceptibility to LET based on resistance selections in cell culture.

Definitions of Resistant and Refractory Cytomegalovirus Infection and Disease in Transplant Recipients for Use in Clinical Trials.

Despite advances in preventive strategies, cytomegalovirus (CMV) infection remains a major complication in solid organ and hematopoietic cell transplant recipients. CMV infection may fail to respond to commercially available antiviral therapies, with or without demonstrating genotypic mutation(s) known to be associated with resistance to these therapies. This lack of response has been termed "resistant/refractory CMV" and is a key focus of clinical trials of some investigational antiviral agents. To provide consistent criteria for future clinical trials and outcomes research, the CMV Resistance Working Group of the CMV Drug Development Forum (consisting of scientists, clinicians, regulatory officials, and industry representatives from the United States, Canada, and Europe) has undertaken establishing standardized consensus definitions of "resistant" and "refractory" CMV. These definitions have emerged from the Working Group's review of the available virologic and clinical literature and will be subject to reassessment and modification based on results of future studies.

Blood Aspergillus RNA is a promising alternative biomarker for invasive aspergillosis.

A critical challenge for the successful application of antifungal therapies for invasive aspergillosis (IA) is a lack of reliable biomarkers to assess early treatment response. Patients with proven or probable IA were prospectively enrolled, and serial blood samples were collected at 8 specified time points during 12-week antifungal therapy. Total nucleic acid was extracted from 2.5 ml blood and tested for Aspergillus-specific RNA by a pan-Aspergillus real-time nucleic acid sequence-based amplification (NASBA) assay. Serum 1, 3-ß-D-glucan (BG) and galactomannan (GM) were measured in parallel. Clinical outcome was evaluated at 6 and 12 weeks. Overall, 48/328 (14.6%) blood samples from 29/46 (63%) patients had positive NASBA detection at baseline and/or some point during the study. Positive NASBA results during the first 4 and 6 weeks of treatment are significantly associated with the 12-week outcome. Blood RNA load change during weeks 4-6 may be informative to predict outcome at 12 weeks. While independent of serum GM, the kinetic change of circulating Aspergillus RNA appears to be well correlated with that of BG on some patient individuals. Monitoring blood Aspergillus RNA during the first 4-6 weeks of antifungal treatment may help assess therapeutic response. Combination of circulating Aspergillus RNA and BG may be a useful adjunct to assess response.

Macrophage reporter cell assay for screening immunopharmacological activity of cell wall-active antifungals.

Antifungal exposure can elicit immunological effects that contribute to activity in vivo, but this activity is rarely screened in vitro in a fashion analogous to MIC testing. We used RAW 264.7 murine macrophages that express a secreted embryonic alkaline phosphatase (SEAP) gene induced by transcriptional activation of NF-κB and activator protein 1 (AP-1) to develop a screen for immunopharmacological activity of cell wall-active antifungal agents. Isolates of Candida albicans and Aspergillus fumigatus that conditionally express genes involved in cell wall synthesis were also tested with the reporter macrophages. We found that growth of fungi in subinhibitory concentrations of glucan synthesis inhibitors (caspofungin and enfumafungin A) or repression of the ß-glucan catalytic subunit of glucan synthase, FKS1, increased macrophage NF-κB/AP-1 activation in a dectin-1-dependent manner. This pattern of activation was also transiently observed with repression of chitin synthesis in C. albicans or when yeast cells were incubated in low concentrations of the chitin synthesis inhibitor nikkomycin Z.

Caspofungin: the first in a new class of antifungal agents.

Caspofungin is the first approved agent from a new class of antifungals, the echinocandins. By targeting the fungal cell wall (as opposed to the fungal cell membrane), the echinocandins exhibit a unique mechanism of action relative to the other currently approved antifungal agents. Preclinical (in vitro and in vivo) studies have demonstrated activity for caspofungin against the most commonly encountered fungi in the hospital setting, namely Candida and Aspergillus species. Caspofungin is administered as a once-a-day, intravenous formulation. Notably, caspofungin is neither an inhibitor, inducer, nor metabolite of the cytochrome p450 system. To date, few drug-drug interactions have been seen for this echinocandin. A number of Phase II and III clinical studies in documented invasive candidiasis, esophageal candidiasis, and invasive aspergillosis have been completed and have demonstrated efficacy for caspofungin against all three diseases. In all studies, caspofungin manifested an excellent safety profile with few serious, drug-related adverse events or discontinuations due to drug-related adverse events. Isolated symptoms compatible with histamine release have been infrequently reported. In clinical studies, drug-related nephrotoxicity with caspofungin has been rare, and the incidence of liver transaminase elevations has been similar to the incidence seen with comparator agents. Results from a Phase III study as empirical therapy in patients with febrile neutropenia are anticipated in late 2003. Overall, caspofungin represents an important addition to the current antifungal armamentarium.

A peptide-based fluorescence resonance energy transfer assay for Bacillus anthracis lethal factor protease.

A fluorescence resonance energy transfer assay has been developed for monitoring Bacillus anthracis lethal factor (LF) protease activity. A fluorogenic 16-mer peptide based on the known LF protease substrate MEK1 was synthesized and found to be cleaved by the enzyme at the anticipated site. Extension of this work to a fluorogenic 19-mer peptide, derived, in part, from a consensus sequence of known LF protease targets, produced a much better substrate, cleaving approximately 100 times more efficiently. This peptide sequence was modified further on resin to incorporate donor/quencher pairs to generate substrates for use in fluorescence resonance energy transfer-based appearance assays. All peptides cleaved at similar rates with signal/background ranging from 9-16 at 100% turnover. One of these substrates, denoted (Cou)Consensus(K(QSY-35)GG)-NH(2), was selected for additional assay optimization. A plate-based assay requiring only low nanomolar levels of enzyme was developed for screening and inhibitor characterization.