Live imaging of SARS-CoV-2 infection in mice reveals that neutralizing antibodies require Fc function for optimal efficacy.

Neutralizing antibodies (NAbs) are effective in treating COVID-19, but the mechanism of immune protection is not fully understood. Here, we applied live bioluminescence imaging (BLI) to monitor the real-time effects of NAb treatment during prophylaxis and therapy of K18-hACE2 mice intranasally infected with SARS-CoV-2-nanoluciferase. Real-time imaging revealed that the virus spread sequentially from the nasal cavity to the lungs in mice and thereafter systemically to various organs including the brain, culminating in death. Highly potent NAbs from a COVID-19 convalescent subject prevented, and also effectively resolved, established infection when administered within three days. In addition to direct neutralization, depletion studies indicated that Fc effector interactions of NAbs with monocytes, neutrophils, and natural killer cells were required to effectively dampen inflammatory responses and limit immunopathology. Our study highlights that both Fab and Fc effector functions of NAbs are essential for optimal in vivo efficacy against SARS-CoV-2.

The deSUMOylase SENP2 coordinates homologous recombination and nonhomologous end joining by independent mechanisms.

SUMOylation (small ubiquitin-like modifier) in the DNA double-strand break (DSB) response regulates recruitment, activity, and clearance of repair factors. However, our understanding of a role for deSUMOylation in this process is limited. Here we identify different mechanistic roles for deSUMOylation in homologous recombination (HR) and nonhomologous end joining (NHEJ) through the investigation of the deSUMOylase SENP2. We found that regulated deSUMOylation of MDC1 prevents excessive SUMOylation and its RNF4-VCP mediated clearance from DSBs, thereby promoting NHEJ. In contrast, we show that HR is differentially sensitive to SUMO availability and SENP2 activity is needed to provide SUMO. SENP2 is amplified as part of the chromosome 3q amplification in many cancers. Increased SENP2 expression prolongs MDC1 focus retention and increases NHEJ and radioresistance. Collectively, our data reveal that deSUMOylation differentially primes cells for responding to DSBs and demonstrates the ability of SENP2 to tune DSB repair responses.

Isomerization of BRCA1-BARD1 promotes replication fork protection.

The integrity of genomes is constantly threatened by problems encountered by the replication fork. BRCA1, BRCA2 and a subset of Fanconi anaemia proteins protect stalled replication forks from degradation by nucleases, through pathways that involve RAD51. The contribution and regulation of BRCA1 in replication fork protection, and how this role relates to its role in homologous recombination, is unclear. Here we show that BRCA1 in complex with BARD1, and not the canonical BRCA1-PALB2 interaction, is required for fork protection. BRCA1-BARD1 is regulated by a conformational change mediated by the phosphorylation-directed prolyl isomerase PIN1. PIN1 activity enhances BRCA1-BARD1 interaction with RAD51, thereby increasing the presence of RAD51 at stalled replication structures. We identify genetic variants of BRCA1-BARD1 in patients with cancer that exhibit poor protection of nascent strands but retain homologous recombination proficiency, thus defining domains of BRCA1-BARD1 that are required for fork protection and associated with cancer development. Together, these findings reveal a BRCA1-mediated pathway that governs replication fork protection.

A qualitative exploration of the role of a palliative care pharmacist providing home-based care in the rural setting, from the perspective of health care professionals.

INTRODUCTION: Pharmacists are often not recognised as a core part of palliative care teams, despite their ideal placement to assist with the burden of medication management. OBJECTIVE: This study explored the role of pharmacists working in the rural palliative care team, in the home-based setting. DESIGN: Health care professionals working with palliative care patients in rural South Australia participated in semi-structured interviews. Data were analysed using thematic analysis. FINDINGS: Data from 20 participants identified 10 themes. Theme 1: This model of care gives patients a choice. Theme 2: The pharmacist is a trusted source of support and information. Theme 3: Patient, carer and family distress is reduced. Theme 4: Enables patients to stay at home by improving medication knowledge and decreasing burden; 4.1-Patient, carer and family's understanding about medication management is improved, 4.2-Patient, carer and family travel is decreased, 4.3-Burden associated with getting to the doctor is decreased. Theme 5: Communication between all parties is enhanced; 5.1-Enhanced communication between the patient and health care team, 5.2-Enhanced communication within the health care team. Theme 6: Patient, carer and family burden of coordinating prescriptions and medications is reduced. Theme 7: Benefits health care professionals by improving medication knowledge, reducing workload and stress; 7.1-Understanding about medications and their management is improved, 7.2-Workload is reduced, 7.3-Work-related stress is reduced. Theme 8: The disparity of care between rural and urban patients is reduced. Theme 9: Helps to address rural workforce shortages. Theme 10: Challenges of this model of care; 10.1-A need for greater pharmacist capacity to meet demand, 10.2-A need for increased and sustained funding for the pharmacist role, 10.3-Large amount of travel to get to patients. CONCLUSION: Rural health care professionals are supportive of pharmacists working as part of the palliative care team in home-based settings and identified many benefits of this model of care.

The proteasomal de-ubiquitinating enzyme POH1 promotes the double-strand DNA break response.

The regulation of Ubiquitin (Ub) conjugates generated by the complex network of proteins that promote the mammalian DNA double-strand break (DSB) response is not fully understood. We show here that the Ub protease POH1/rpn11/PSMD14 resident in the 19S proteasome regulatory particle is required for processing poly-Ub formed in the DSB response. Proteasome activity is required to restrict tudor domain-dependent 53BP1 accumulation at sites of DNA damage. This occurs both through antagonism of RNF8/RNF168-mediated lysine 63-linked poly-Ub and through the promotion of JMJD2A retention on chromatin. Consistent with this role POH1 acts in opposition to RNF8/RNF168 to modulate end-joining DNA repair. Additionally, POH1 acts independently of 53BP1 in homologous recombination repair to promote RAD51 loading. Accordingly, POH1-deficient cells are sensitive to DNA damaging agents. These data demonstrate that proteasomal POH1 is a key de-ubiquitinating enzyme that regulates ubiquitin conjugates generated in response to damage and that several aspects of the DSB response are regulated by the proteasome.

Lung Transplantation in Alpha-1-Antitrypsin Deficiency.

BACKGROUND: Lung transplantation is a therapeutic option for patients with end-stage lung disease and a survival benefit has been described in patients with alpha-1-antitrypsin deficiency (A1ATD). The aims of the current study were to determine the survival and health benefits of lung transplantation in UK patients with A1ATD compared to carefully matched non-transplant patients. METHODS: Patients with the PiZZ (alpha-1-antitrypsin deficiency) genotype who had undergone lung transplantation between 1996 and 2011 were identified from the UK A1ATD registry. Lung physiology, health status and survival were compared pre- and post-transplant using carefully matched non-transplant patients. RESULTS: Thirty-two A1ATD patients who had undergone lung transplant were identified. Lung function decline pre-transplant was not different to the closely matched non-transplanted cohort. The transplant group pre-transplant, although matched for FEV1, had lower gas transfer measurements, (mean KCO% predicted 41.0% SE ± 3.86 vs 55.6% SE ± 3.10 p < 0.001) and worse health status (SGRQ mean score 64.2 SE ± 2.5 vs 55.3 SE ± 2.0, p < 0.001). Post-transplant, physiology and health status improved significantly (p < 0.002). However, the post-operative mortality over 5 years was no better than for a second group of non-transplant patients further matched for gas transfer or a third group also matched for SGRQ. CONCLUSION: Patients who underwent lung transplant had lower gas transfer and quality-of-life pre-transplant compared to non-transplant patients matched for FEV1, age and sex, suggesting that these parameters provide extra information helpful in decision making. Lung transplantation for A1ATD patients significantly improves quality-of-life but not survival.

The deSUMOylase SENP7 promotes chromatin relaxation for homologous recombination DNA repair.

SUMO conjugation is known to occur in response to double-stranded DNA breaks in mammalian cells, but whether SUMO deconjugation has a role remains unclear. Here, we show that the SUMO/Sentrin/Smt3-specific peptidase, SENP7, interacts with the chromatin repressive KRAB-associated protein 1 (KAP1) through heterochromatin protein 1 alpha (HP1α). SENP7 promotes the removal of SUMO2/3 from KAP1 and regulates the interaction of the chromatin remodeler CHD3 with chromatin. Consequently, in the presence of CHD3, SENP7 is required for chromatin relaxation in response to DNA damage, for homologous recombination repair and for cellular resistance to DNA-damaging agents. Thus, deSUMOylation by SENP7 is required to promote a permissive chromatin environment for DNA repair.

Signaling between mammalian adiponectin and a mosquito adiponectin receptor reduces Plasmodium transmission.

IMPORTANCE: When a female mosquito takes a blood meal from a mammalian host, components of the blood meal can affect mosquito fitness and indirectly influence pathogen infectivity. We identified a pathway involving an Anopheles gambiae adiponectin receptor, which, triggered by adiponectin from an incoming blood meal, decreases Plasmodium infection in the mosquito. Activation of this pathway negatively regulates lipophorin expression, an important lipid transporter that both enhances egg development and Plasmodium infection. This is an unrecognized cross-phyla interaction between a mosquito and its vertebrate host. These processes are critical to understanding the complex life cycle of mosquitoes and Plasmodium following a blood meal and may be applicable to other hematophagous arthropods and vector-borne infectious agents.

Barriers and facilitators experienced in delivering alcohol screening and brief interventions in community pharmacy: a qualitative evidence synthesis.

BACKGROUND: Following increases in deaths due to alcohol during the COVID-19 pandemic, there have been renewed calls to increase resources in alcohol screening and brief intervention (SBI). Research has shown that community pharmacy could be a promising setting for SBI. This review aimed to investigate the barriers and facilitators to SBI delivery in community pharmacy to inform its further development. METHODS: A systematic search of four databases (MEDLINE, EMBASE, CINAHL, and PsycINFO) was conducted in October 2021 to identify relevant published qualitative or mixed-method studies. Relevant qualitative data were extracted from the included studies and a framework synthesis was performed using the Capability-Opportunity-Motivation-Behaviour (COM-B) model. RESULTS: Two thousand two hundred and ten articles were screened and nine studies were included in the review (seven in the United Kingdom and two in Australia). Identified barriers and facilitators to delivering SBI corresponded to all components of the COM-B model. Facilitators included non-confrontational communication skills, aligning SBI with existing pharmacy services and pharmacist role legitimacy. Barriers included multiple demands on staff time, a lack of staff experience with screening tools, and staff concerns of causing offence. Using the Behaviour Change Wheel (BCW), we propose five elements of a pharmacy SBI to address identified barriers. CONCLUSIONS: Research into SBI in community pharmacy is limited in comparison to other healthcare settings and this review provides an understanding of the barriers and facilitators to the delivery of SBI in community pharmacy from a behavioural perspective. Through the use of COM-B and BCW, our findings could inform the development of future pharmacy-based SBI.

A Digitally Enabled, Pharmacist service to detecT medicine harms in residential aged care (nursing home) (ADEPT): protocol for a feasibility study.

INTRODUCTION: This feasibility study aims to develop and test a new model of practice in Australia using digital technologies to enable pharmacists to monitor early signs and symptoms of medicine-induced harms in residential aged care. METHODS AND ANALYSIS: Thirty residents will be recruited from an aged care facility in South Australia. The study will be conducted in two phases. In phase I, the study team will work with aged care software providers and developers of digital technologies (a wearable activity tracker and a sleep tracking sensor) to gather physical activity and sleep data, as well as medication and clinical data from the electronic medication management system and aged care clinical software. Data will be centralised into a cloud-based monitoring platform (TeleClinical Care (TCC)). The TCC will be used to create dashboards that will include longitudinal visualisations of changes in residents' health, function and medicine use over time. In phase II, the on-site pharmacist will use the centralised TCC platform to monitor each resident's medicine, clinical, physical activity and sleep data to identify signs of medicine-induced harms over a 12-week period.A mixed methods process evaluation applying the RE-AIM (Reach, Effectiveness, Adoption, Implementation, Maintenance) evaluation framework will be used to assess the feasibility of the service. Outcome measures include service reach, changes in resident symptom scores (measured using the Edmonton Symptom Assessment System), number of medication adverse events detected, changes in physical activity and sleep, number of pharmacist recommendations provided, cost analysis and proportion of all pharmacists' recommendations implemented at 4-week, 8-week and 12-week postbaseline period. ETHICS AND DISSEMINATION: Ethical approval has been obtained from the University of South Australia's Human Research Ethics Committee (205098). Findings will be disseminated through published manuscripts, conference presentations and reporting to the study funder. TRIAL REGISTRATION NUMBER: ACTRN12623000506695.

USP50 suppresses alternative RecQ helicase use and deleterious DNA2 activity during replication.

Mammalian DNA replication employs several RecQ DNA helicases to orchestrate the faithful duplication of genetic information. Helicase function is often coupled to the activity of specific nucleases, but how helicase and nuclease activities are co-directed is unclear. Here we identify the inactive ubiquitin-specific protease, USP50, as a ubiquitin-binding and chromatin-associated protein required for ongoing replication, fork restart, telomere maintenance and cellular survival during replicative stress. USP50 supports WRN:FEN1 at stalled replication forks, suppresses MUS81-dependent fork collapse and restricts double-strand DNA breaks at GC-rich sequences. Surprisingly we find that cells depleted for USP50 and recovering from a replication block exhibit increased DNA2 and RECQL4 foci and that the defects in ongoing replication, poor fork restart and increased fork collapse seen in these cells are mediated by DNA2, RECQL4 and RECQL5. These data define a novel ubiquitin-dependent pathway that promotes the balance of helicase: nuclease use at ongoing and stalled replication forks.

The Importance of Dosimetry Standardization in Radiobiology.

Radiation dose is central to much of radiobiological research. Precision and accuracy of dose measurements and reporting of the measurement details should be sufficient to allow the work to be interpreted and repeated and to allow valid comparisons to be made, both in the same laboratory and by other laboratories. Despite this, a careful reading of published manuscripts suggests that measurement and reporting of radiation dosimetry and setup for radiobiology research is frequently inadequate, thus undermining the reliability and reproducibility of the findings. To address these problems and propose a course of action, the National Cancer Institute (NCI), the National Institute of Allergy and Infectious Diseases (NIAID), and the National Institute of Standards and Technology (NIST) brought together representatives of the radiobiology and radiation physics communities in a workshop in September, 2011. The workshop participants arrived at a number of specific recommendations as enumerated in this paper and they expressed the desirability of creating dosimetry standard operating procedures (SOPs) for cell culture and for small and large animal experiments. It was also felt that these SOPs would be most useful if they are made widely available through mechanism(s) such as the web, where they can provide guidance to both radiobiologists and radiation physicists, be cited in publications, and be updated as the field and needs evolve. Other broad areas covered were the need for continuing education through tutorials at national conferences, and for journals to establish standards for reporting dosimetry. This workshop did not address issues of dosimetry for studies involving radiation focused at the sub-cellular level, internally-administered radionuclides, biodosimetry based on biological markers of radiation exposure, or dose reconstruction for epidemiological studies.

Variability of sputum inflammatory mediators in COPD and α1-antitrypsin deficiency.

There is inherent daily variability of sputum inflammatory mediators in stable-state patients with usual chronic obstructive pulmonary disease (COPD). The variability of pulmonary inflammation in patients with α(1)-antitrypsin deficiency (A1ATD) is unknown. Our study aimed to quantify this variability, in comparison to patients with usual COPD, in order to facilitate power calculations for proof of concept trials of putative specific anti-inflammatory agents in both groups. Sputum interleukin (IL)-8, myeloperoxidase (MPO), leukotriene B(4) (LTB(4)) and differential cell counts were measured in 12 usual COPD patients and 12 A1ATD patients on nine occasions over a 1-month period. All samples were obtained in the stable clinical state. There was significant daily variability in all mediators in all patients. A1ATD patients had higher sputum concentrations of IL-8 and LTB(4) compared with usual COPD, but lower levels of MPO and absolute neutrophil counts. Patients with usual COPD had more intra-patient variability, A1ATD patients demonstrated greater inter-patient variability. There are increased concentrations of pulmonary inflammatory mediators but fewer sputum neutrophils in A1ATD compared with usual COPD. The daily variability of inflammatory mediators and cell counts was significantly reduced in both groups by averaging sequential samples. This can be utilised to perform power calculations for future proof of concept studies; averaging three sequential samples appears optimum.

Impact of the COVID-19 pandemic on a respiratory physiology department and the patient's perception of rapid service change.

BACKGROUND: Originating as a cluster of unexplained cases of pneumonia in Wuhan, China, a novel coronavirus disease, officially named as COVID-19 by WHO, has now reached a pandemic level. In the wake of this global health crisis, stringent public health measures were implemented to curtail the spread of COVID-19. At a local level, the University Hospitals of North Midlands National Health Service Trust suspended all elective and outpatient activity, primarily to address the current potential implications of the COVID-19 outbreak. Within respiratory physiology, all but urgent and emergency work was suspended. METHODS: In June 2020, the service commenced its restoration/recovery plan, which was based on national and international guidelines to ensure safe practice for patients and staff alike. The plan was a roadmap developed to upscale the respiratory physiology service to deliver urgent and routine care and to assist the service to undertake the essential task of managing the patient backlog as a consequence of the interruption of service. Patient concerns and anxieties due to the pandemic was a key aspect of the restoration/recovery plan. The service developed numerous initiatives along with a questionnaire to assess patient experience following attendance for investigations or assessment. RESULTS: The questionnaire confirmed that the initiatives put in place as part of the restoration/recovery plan achieve high levels of satisfaction in terms of communication, interaction within the service, professionalism and importantly patient safety. CONCLUSION: COVID-19 had a significant impact on routine clinical care and out-patient activity. This brought about significant change in service delivery that required a strict regimen to ensure COVID-19 free status and minimise cross-contamination of service users. The systems and processes introduced demonstrated positive responses and confirmed the objective of patient safety, which translated to the service users.

The dedicated iron deficiency anaemia clinic: a 15-year experience.

OBJECTIVE: To report our cumulative experience from a dedicated iron deficiency anaemia (IDA) clinic over the last 15 years-with particular emphasis on referral rate, uptake of investigation, impact on endoscopy services, diagnostic yield of gastrointestinal (GI) investigation and the issue of recurrent IDA. METHOD: A series of analyses of a register of 2808 referrals to the Poole IDA clinic between 2004 and 2018. RESULTS: The study population of 2808 had a sex ratio of 1.9 (female/male ratio) and a median age of 72 years (IQR: 60-79). A rising referral rate over the study period appears to be plateauing at around 2 cases per 1000 population per annum. On the basis of a snapshot audit, investigation of IDA may now account for over 20% of all diagnostic endoscopies.Overall, 86% of cases underwent examination of the upper and lower GI tract. Significant GI pathology was identified in 27% of the investigated cohort. Adenocarcinoma of the upper or lower GI tract was found in 8.3%, the majority in the right colon. The prevalence of recurrent IDA was estimated at 12.4%, and the results of investigation of this subgroup are reported. CONCLUSION: Unexplained IDA is common, particularly in those over 60 years, and may be the first indication of underlying GI malignancy in over 8% of cases. Unresolved challenges include accommodating the resulting endoscopy workload, establishing a risk/benefit ratio for investigating those with major comorbidities and the management of recurrent IDA.

In vivo imaging of retrovirus infection reveals a role for Siglec-1/CD169 in multiple routes of transmission.

Early events in retrovirus transmission are determined by interactions between incoming viruses and frontline cells near entry sites. Despite their importance for retroviral pathogenesis, very little is known about these events. We developed a bioluminescence imaging (BLI)-guided multiscale imaging approach to study these events in vivo. Engineered murine leukemia reporter viruses allowed us to monitor individual stages of retrovirus life cycle including virus particle flow, virus entry into cells, infection and spread for retroorbital, subcutaneous, and oral routes. BLI permitted temporal tracking of orally administered retroviruses along the gastrointestinal tract as they traversed the lumen through Peyer's patches to reach the draining mesenteric sac. Importantly, capture and acquisition of lymph-, blood-, and milk-borne retroviruses spanning three routes was promoted by a common host factor, the I-type lectin CD169, expressed on sentinel macrophages. These results highlight how retroviruses co-opt the immune surveillance function of tissue-resident sentinel macrophages for establishing infection.

Live Imaging of SARS-CoV-2 Infection in Mice Reveals Neutralizing Antibodies Require Fc Function for Optimal Efficacy.

Neutralizing antibodies (NAbs) are effective in treating COVID-19 but the mechanism of immune protection is not fully understood. Here, we applied live bioluminescence imaging (BLI) to monitor the real-time effects of NAb treatment in prophylaxis and therapy of K18-hACE2 mice intranasally infected with SARS-CoV-2-nanoluciferase. We could visualize virus spread sequentially from the nasal cavity to the lungs and thereafter systemically to various organs including the brain, which culminated in death. Highly potent NAbs from a COVID-19 convalescent subject prevented, and also effectively resolved, established infection when administered within three days. In addition to direct Fab-mediated neutralization, Fc effector interactions of NAbs with monocytes, neutrophils and natural killer cells were required to effectively dampen inflammatory responses and limit immunopathology. Our study highlights that both Fab and Fc effector functions of NAbs are essential for optimal in vivo efficacy against SARS-CoV-2.

3-Chlorotyrosine in sputum of COPD patients: relationship with airway inflammation.

The airways of patients with chronic obstructive pulmonary disease (COPD) are associated with increased numbers of inflammatory cells, in particular neutrophils and macrophages. Contained within the primary granules of neutrophils is the heme enzyme called myeloperoxidase (MPO) that has been used as a biomarker of neutrophilic inflammation in COPD and other inflammatory diseases. MPO is the only enzyme in the body that produces hypochlorous acid (HOCl), which effectively chlorinates tyrosine residues in proteins. The presence of 3-chlorotyrosine (3Cl-Tyr) in sputum of COPD patients has yet to be established. Spontaneously produced sputum was collected from 14 stable COPD patients, and ultra-centrifuged to prepare sol phase samples for analysis. Sputum 3Cl-Tyr levels were measured using Mass Spectrometry (LC-MSMS). Sputum MPO activity was measured using a standard chromogenic substrate assay. The Spearman rank correlation was used to analyse the data. We report for the first time the measurement of 3Cl-Tyr in sputum from stable COPD patients. The sputum levels of 3Cl-Tyr correlated well with sputum MPO activity (r = 0.88; p < 0.0001). The presence of 3Cl-Tyr in the sputum of stable COPD patients suggests an active process related to MPO that may play a role in the pathophysiology of this disease.

Phenotypic plasticity, but not adaptive tracking, underlies seasonal variation in post-cold hardening freeze tolerance of Drosophila melanogaster.

In temperate regions, an organism's ability to rapidly adapt to seasonally varying environments is essential for its survival. In response to seasonal changes in selection pressure caused by variation in temperature, humidity, and food availability, some organisms exhibit plastic changes in phenotype. In other cases, seasonal variation in selection pressure can rapidly increase the frequency of genotypes that offer survival or reproductive advantages under the current conditions. Little is known about the relative influences of plastic and genetic changes in short-lived organisms experiencing seasonal environmental fluctuations. Cold hardening is a seasonally relevant plastic response in which exposure to cool, but nonlethal, temperatures significantly increases the organism's ability to later survive at freezing temperatures. In the present study, we demonstrate seasonal variation in cold hardening in Drosophila melanogaster and test the extent to which plasticity and adaptive tracking underlie that seasonal variation. We measured the post-cold hardening freeze tolerance of flies from outdoor mesocosms over the summer, fall, and winter. We bred outdoor mesocosm-caught flies for two generations in the laboratory and matched each outdoor cohort to an indoor control cohort of similar genetic background. We cold hardened all flies under controlled laboratory conditions and then measured their post-cold hardening freeze tolerance. Comparing indoor and field-caught flies and their laboratory-reared G1 and G2 progeny allowed us to determine the roles of seasonal environmental plasticity, parental effects, and genetic changes on cold hardening. We also tested the relationship between cold hardening and other factors, including age, developmental density, food substrate, presence of antimicrobials, and supplementation with live yeast. We found strong plastic responses to a variety of field- and laboratory-based environmental effects, but no evidence of seasonally varying parental or genetic effects on cold hardening. We therefore conclude that seasonal variation in post-cold hardening freeze tolerance results from environmental influences and not genetic changes.