Gene conversion in human rearranged immunoglobulin genes

Over the past 20 years, many DNA sequences have been published suggesting that all or part of the V<sub>H</sub> segment of a rearranged immunoglobulin gene may be replaced in vivo. Two different mechanisms appear to be operating. One of these is very similar to primary V(D)J recombination, involving the RAG proteins acting upon recombination signal sequences, and this has recently been proven to occur. Other sequences, many of which show partial V<sub>H</sub> replacements with no addition of untemplated nucleotides at the V<sub>H</sub>–V<sub>H</sub> joint, have been proposed to occur by an unusual RAG-mediated recombination with the formation of hybrid (coding-to-signal) joints. These appear to occur in cells already undergoing somatic hypermutation in which, some authors are convinced, RAG genes are silenced. We recently proposed that the latter type of V<sub>H</sub> replacement might occur by homologous recombination initiated by the activity of AID (activation-induced cytidine deaminase), which is essential for somatic hypermutation and gene conversion. The latter has been observed in other species, but not in human Ig genes, so far. In this paper, we present a new analysis of sequences published as examples of the second type of rearrangement. This not only shows that AID recognition motifs occur in recombination regions but also that some sequences show replacement of central sections by a sequence from another gene, similar to gene conversion in the immunoglobulin genes of other species. These observations support the proposal that this type of rearrangement is likely to be AID-mediated rather than RAG-mediated and is consistent with gene conversion

Eukaryotic cell biology is temporally coordinated to support the energetic demands of protein homeostasis

Yeast physiology is temporally regulated, this becomes apparent under nutrient-limited conditions and results in respiratory oscillations (YROs). YROs share features with circadian rhythms and interact with, but are independent of, the cell division cycle. Here, we show that YROs minimise energy expenditure by restricting protein synthesis until sufficient resources are stored, while maintaining osmotic homeostasis and protein quality control. Although nutrient supply is constant, cells sequester and store metabolic resources via increased transport, autophagy and biomolecular condensation. Replete stores trigger increased H+ export which stimulates TORC1 and liberates proteasomes, ribosomes, chaperones and metabolic enzymes from non-membrane bound compartments. This facilitates translational bursting, liquidation of storage carbohydrates, increased ATP turnover, and the export of osmolytes. We propose that dynamic regulation of ion transport and metabolic plasticity are required to maintain osmotic and protein homeostasis during remodelling of eukaryotic proteomes, and that bioenergetic constraints selected for temporal organisation that promotes oscillatory behaviour

Mechanisms and physiological function of daily haemoglobin oxidation rhythms in red blood cells

Cellular circadian rhythms confer temporal organisation upon physiology that is fundamental to human health. Rhythms are present in red blood cells (RBCs), the most abundant cell type in the body, but their physiological function is poorly understood. Here, we present a novel biochemical assay for haemoglobin (Hb) oxidation status which relies on a redox-sensitive covalent haem-Hb linkage that forms during SDS-mediated cell lysis. Formation of this linkage is lowest when ferrous Hb is oxidised, in the form of ferric metHb. Daily haemoglobin oxidation rhythms are observed in mouse and human RBCs cultured in vitro, or taken from humans in vivo, and are unaffected by mutations that affect circadian rhythms in nucleated cells. These rhythms correlate with daily rhythms in core body temperature, with temperature lowest when metHb levels are highest. Raising metHb levels with dietary sodium nitrite can further decrease daytime core body temperature in mice via nitric oxide (NO) signalling. These results extend our molecular understanding of RBC circadian rhythms and suggest they contribute to the regulation of body temperature

Reduced antibody cross-reactivity following infection with B.1.1.7 than with parental SARS-CoV-2 strains

Background: The degree of heterotypic immunity induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains is a major determinant of the spread of emerging variants and the success of vaccination campaigns, but remains incompletely understood. Methods: We examined the immunogenicity of SARS-CoV-2 variant B.1.1.7 (Alpha) that arose in the United Kingdom and spread globally. We determined titres of spike glycoprotein-binding antibodies and authentic virus neutralising antibodies induced by B.1.1.7 infection to infer homotypic and heterotypic immunity. Results: Antibodies elicited by B.1.1.7 infection exhibited significantly reduced recognition and neutralisation of parental strains or of the South Africa variant B.1.351 (Beta) than of the infecting variant. The drop in cross-reactivity was significantly more pronounced following B.1.1.7 than parental strain infection. Conclusions: The results indicate that heterotypic immunity induced by SARS-CoV-2 variants is asymmetric

Consensus on circulatory shock and hemodynamic monitoring. Task force of the European Society of Intensive Care Medicine.

OBJECTIVE: Circulatory shock is a life-threatening syndrome resulting in multiorgan failure and a high mortality rate. The aim of this consensus is to provide support to the bedside clinician regarding the diagnosis, management and monitoring of shock. METHODS: The European Society of Intensive Care Medicine invited 12 experts to form a Task Force to update a previous consensus (Antonelli et al.: Intensive Care Med 33:575-590, 2007). The same five questions addressed in the earlier consensus were used as the outline for the literature search and review, with the aim of the Task Force to produce statements based on the available literature and evidence. These questions were: (1) What are the epidemiologic and pathophysiologic features of shock in the intensive care unit ? (2) Should we monitor preload and fluid responsiveness in shock ? (3) How and when should we monitor stroke volume or cardiac output in shock ? (4) What markers of the regional and microcirculation can be monitored, and how can cellular function be assessed in shock ? (5) What is the evidence for using hemodynamic monitoring to direct therapy in shock ? Four types of statements were used: definition, recommendation, best practice and statement of fact. RESULTS: Forty-four statements were made. The main new statements include: (1) statements on individualizing blood pressure targets; (2) statements on the assessment and prediction of fluid responsiveness; (3) statements on the use of echocardiography and hemodynamic monitoring. CONCLUSIONS: This consensus provides 44 statements that can be used at the bedside to diagnose, treat and monitor patients with shock

Exploratory outcome analyses according to stage and/or residual disease in the ICON7 trial of carboplatin and paclitaxel with or without bevacizumab for newly diagnosed ovarian cancer

Objective: In the randomized phase 3 ICON7 trial (ISRCTN91273375), adding bevacizumab to chemotherapy for newly diagnosed ovarian cancer significantly improved progression-free survival (PFS; primary endpoint) but not overall survival (OS; secondary endpoint) in the intent-to-treat (ITT) population. We explored treatment effect according to stage and extent of residual disease. Methods: Patients with stage IIB–IV or high-risk (grade 3/clear-cell) stage I–IIA ovarian cancer were randomized to receive six cycles of carboplatin and paclitaxel either alone or with bevacizumab 7.5 mg/kg every 3 weeks followed by single-agent bevacizumab for 12 further cycles (total duration 12 months). Post hoc exploratory analyses of subgroups defined by stage and extent of residual disease at diagnosis within the stage IIIB–IV population (European indication) was performed. Results: The PFS benefit from bevacizumab was seen consistently in all subgroups explored. The PFS hazard ratio was 0.77 (95% confidence interval [CI], 0.59–0.99) in 411 patients with stage IIIB–IV ovarian cancer with no visible residuum and 0.81 (95% CI, 0.69–0.95) in 749 patients with stage IIIB–IV disease and visible residuum. As in the ITT population, no OS difference was detected in any subgroup except the previously described ‘high-risk’ subgroup. Safety results in analyzed subgroups were consistent with the overall population. Conclusions: Adding bevacizumab to front-line chemotherapy improves PFS irrespective of stage/residual disease. In patients with stage III with >1 cm residuum, stage IV or inoperable disease, this translates into an OS benefit. No OS benefit or detriment was seen in other subgroups explored

Deaminase-Independent Inhibition of Parvoviruses by the APOBEC3A Cytidine Deaminase

The APOBEC3 proteins form a multigene family of cytidine deaminases with inhibitory activity against viruses and retrotransposons. In contrast to APOBEC3G (A3G), APOBEC3A (A3A) has no effect on lentiviruses but dramatically inhibits replication of the parvovirus adeno-associated virus (AAV). To study the contribution of deaminase activity to the antiviral activity of A3A, we performed a comprehensive mutational analysis of A3A. By mutation of non-conserved residues, we found that regions outside of the catalytic active site contribute to both deaminase and antiviral activities. Using A3A point mutants and A3A/A3G chimeras, we show that deaminase activity is not required for inhibition of recombinant AAV production. We also found that deaminase-deficient A3A mutants block replication of both wild-type AAV and the autonomous parvovirus minute virus of mice (MVM). In addition, we identify specific residues of A3A that confer activity against AAV when substituted into A3G. In summary, our results demonstrate that deaminase activity is not necessary for the antiviral activity of A3A against parvoviruses

Different Modes of Retrovirus Restriction by Human APOBEC3A and APOBEC3G In Vivo

The apolipoprotein B editing complex 3 (A3) cytidine deaminases are among the most highly evolutionarily selected retroviral restriction factors, both in terms of gene copy number and sequence diversity. Primate genomes encode seven A3 genes, and while A3F and 3G are widely recognized as important in the restriction of HIV, the role of the other genes, particularly A3A, is not as clear. Indeed, since human cells can express multiple A3 genes, and because of the lack of an experimentally tractable model, it is difficult to dissect the individual contribution of each gene to virus restriction in vivo. To overcome this problem, we generated human A3A and A3G transgenic mice on a mouse A3 knockout background. Using these mice, we demonstrate that both A3A and A3G restrict infection by murine retroviruses but by different mechanisms: A3G was packaged into virions and caused extensive deamination of the retrovirus genomes while A3A was not packaged and instead restricted infection when expressed in target cells. Additionally, we show that a murine leukemia virus engineered to express HIV Vif overcame the A3G-mediated restriction, thereby creating a novel model for studying the interaction between these proteins. We have thus developed an in vivo system for understanding how human A3 proteins use different modes of restriction, as well as a means for testing therapies that disrupt HIV Vif-A3G interactions.United States. Public Health Service (Grant R01-AI-085015)United States. Public Health Service (Grant T32-CA115299 )United States. Public Health Service (Grant F32-AI100512

SARS-CoV-2 can recruit a haem metabolite to evade antibody immunity.

The coronaviral spike is the dominant viral antigen and the target of neutralizing antibodies. We show that SARS-CoV-2 spike binds biliverdin and bilirubin, the tetrapyrrole products of haem metabolism, with nanomolar affinity. Using cryo-electron microscopy and X-ray crystallography, we mapped the tetrapyrrole interaction pocket to a deep cleft on the spike N-terminal domain (NTD). At physiological concentrations, biliverdin significantly dampened the reactivity of SARS-CoV-2 spike with immune sera and inhibited a subset of neutralizing antibodies. Access to the tetrapyrrole-sensitive epitope is gated by a flexible loop on the distal face of the NTD. Accompanied by profound conformational changes in the NTD, antibody binding requires relocation of the gating loop, which folds into the cleft vacated by the metabolite. Our results indicate that SARS-CoV-2 spike NTD harbors a dominant epitope, access to which can be controlled by an allosteric mechanism that is regulated through the recruitment of a metabolite