Slow evolution toward "Super-Aggregation" of the oligomers formed through the swapping of RNase A N-Termini: a wish for amyloidosis?

Natively monomeric RNase A can oligomerize upon lyophilization from 40% acetic acid solutions or when it is heated at high concentrations in various solvents. In this way, it produces many dimeric or oligomeric conformers through the three-dimensional domain swapping (3D-DS) mechanism involving both RNase A N- or/and C-termini. Here, we found many of these oligomers evolving toward not negligible amounts of large derivatives after being stored for up to 15 months at 4 degrees C in phosphate buffer. We call these species super-aggregates (SAs). Notably, SAs do not originate from native RNase A monomer or from oligomers characterized by the exclusive presence of the C-terminus swapping of the enzyme subunits as well. Instead, the swapping of at least two subunits' N-termini is mandatory to produce them. Through immunoblotting, SAs are confirmed to derive from RNase A even if they retain only low ribonucleolytic activity. Then, their interaction registered with Thioflavin-T (ThT), in addition to TEM analyses, indicate SAs are large and circular but not "amyloid-like" derivatives. This confirms that RNase A acts as an "auto-chaperone", although it displays many amyloid-prone short segments, including the 16-22 loop included in its N-terminus. Therefore, we hypothesize the opening of RNase A N-terminus, and hence its oligomerization through 3D-DS, may represent a preliminary step favoring massive RNase A aggregation. Interestingly, this process is slow and requires low temperatures to limit the concomitant oligomers' dissociation to the native monomer. These data and the hypothesis proposed are discussed in the light of protein aggregation in general, and of possible future applications to contrast amyloidosis

Burnout among surgeons before and during the SARS-CoV-2 pandemic: an international survey

Background: SARS-CoV-2 pandemic has had many significant impacts within the surgical realm, and surgeons have been obligated to reconsider almost every aspect of daily clinical practice. Methods: This is a cross-sectional study reported in compliance with the CHERRIES guidelines and conducted through an online platform from June 14th to July 15th, 2020. The primary outcome was the burden of burnout during the pandemic indicated by the validated Shirom-Melamed Burnout Measure. Results: Nine hundred fifty-four surgeons completed the survey. The median length of practice was 10 years; 78.2% included were male with a median age of 37 years old, 39.5% were consultants, 68.9% were general surgeons, and 55.7% were affiliated with an academic institution. Overall, there was a significant increase in the mean burnout score during the pandemic; longer years of practice and older age were significantly associated with less burnout. There were significant reductions in the median number of outpatient visits, operated cases, on-call hours, emergency visits, and research work, so, 48.2% of respondents felt that the training resources were insufficient. The majority (81.3%) of respondents reported that their hospitals were included in the management of COVID-19, 66.5% felt their roles had been minimized; 41% were asked to assist in non-surgical medical practices, and 37.6% of respondents were included in COVID-19 management. Conclusions: There was a significant burnout among trainees. Almost all aspects of clinical and research activities were affected with a significant reduction in the volume of research, outpatient clinic visits, surgical procedures, on-call hours, and emergency cases hindering the training. Trial registration: The study was registered on clicaltrials.gov "NCT04433286" on 16/06/2020

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance

INTRODUCTION Investment in Africa over the past year with regard to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing has led to a massive increase in the number of sequences, which, to date, exceeds 100,000 sequences generated to track the pandemic on the continent. These sequences have profoundly affected how public health officials in Africa have navigated the COVID-19 pandemic. RATIONALE We demonstrate how the first 100,000 SARS-CoV-2 sequences from Africa have helped monitor the epidemic on the continent, how genomic surveillance expanded over the course of the pandemic, and how we adapted our sequencing methods to deal with an evolving virus. Finally, we also examine how viral lineages have spread across the continent in a phylogeographic framework to gain insights into the underlying temporal and spatial transmission dynamics for several variants of concern (VOCs). RESULTS Our results indicate that the number of countries in Africa that can sequence the virus within their own borders is growing and that this is coupled with a shorter turnaround time from the time of sampling to sequence submission. Ongoing evolution necessitated the continual updating of primer sets, and, as a result, eight primer sets were designed in tandem with viral evolution and used to ensure effective sequencing of the virus. The pandemic unfolded through multiple waves of infection that were each driven by distinct genetic lineages, with B.1-like ancestral strains associated with the first pandemic wave of infections in 2020. Successive waves on the continent were fueled by different VOCs, with Alpha and Beta cocirculating in distinct spatial patterns during the second wave and Delta and Omicron affecting the whole continent during the third and fourth waves, respectively. Phylogeographic reconstruction points toward distinct differences in viral importation and exportation patterns associated with the Alpha, Beta, Delta, and Omicron variants and subvariants, when considering both Africa versus the rest of the world and viral dissemination within the continent. Our epidemiological and phylogenetic inferences therefore underscore the heterogeneous nature of the pandemic on the continent and highlight key insights and challenges, for instance, recognizing the limitations of low testing proportions. We also highlight the early warning capacity that genomic surveillance in Africa has had for the rest of the world with the detection of new lineages and variants, the most recent being the characterization of various Omicron subvariants. CONCLUSION Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve. This is important not only to help combat SARS-CoV-2 on the continent but also because it can be used as a platform to help address the many emerging and reemerging infectious disease threats in Africa. In particular, capacity building for local sequencing within countries or within the continent should be prioritized because this is generally associated with shorter turnaround times, providing the most benefit to local public health authorities tasked with pandemic response and mitigation and allowing for the fastest reaction to localized outbreaks. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century

Structural versatility of BS-RNase: Different oligomeric isomers formed through 3D domain swapping of N- and C-termini

Bovine seminal RNase (BS RNase) is the only naturally dimeric member of the pancreatic-type RNase superfamily. The enzyme is a mixture of two isoforms: (i) M=M, dimeric through two antiparallel disulfides occurring between the two subunits; (ii) MxM (70% of the total), characterized by the swapping of the N-termini besides the mentioned disulfides (1). When dissolved in 40% acetic acid and subjected to lyophilisation, BS RNase forms a mixture of oligomeric aggregates (2), as does RNase A (3), the proto-type of the super-family. However, while the oligomers of the pancreatic variant have been extensively characterized (3\u20135), the multimers of BS RNase (6) are presently less known. To deepen their characterization we induced BS RNase self-association by using the same conditions used with RNase A, i.e. lyophilisation of enzyme solutions at low pH, or thermally-induced aggregation of concentrated protein solutions in various media. The multimers obtained were analyzed by SEC, cation-exchange chromatography, cross-linking, electrophoresis, proteolysis, and enzymatic assays. The main results obtained are: (a) BS-RNase forms several 3D domain-swapped conformers, in particular at least two tetramers; one is probably a totally N terminal-swapped isoform (4), while the other contains a C terminal swapping. (b) The C-swapped tetramer is less stable than the N-swapped one. (c) BS RNase multimers larger than tetramers appear to be a mixture of various isoforms, similarly to what occurs with RNase A (3). These multimers seem to contain, again, a Cterminus swapping. (4) The validity of the results reported have been strengthened by the studies performed with a K113N-BS RNase mutant. References: 1. Piccoli R., et al. Proc Natl Acad Sci USA 1992; 89: 1870\u20131874. 2. Libonati M. Int J Biochem 1969; 18: 407\u2013417. 3. Libonati M. & Gotte G. Biochem J 2004; 380: 311\u2013327. 4. Liu Y., et al. Proc Natl Acad Sci USA 1998; 95: 3437\u20133442. 5. Liu Y., et al. Nat Str Biol 2001; 8: 211\u2013214. 6. Adinolfi S., et al. FEBS Lett 1996; 398: 326\u2013332

Structural versatility of BS-RNase: Different oligomeric isomers formed through 3D domain swapping of N- and C-termini

Bovine seminal RNase (BS RNase) is the only naturally dimeric member of the pancreatic-type RNase superfamily. The enzyme is a mixture of two isoforms: (i) M=M, dimeric through two antiparallel disulfides occurring between the two subunits; (ii) MxM (70% of the total), characterized by the swapping of the N-termini besides the mentioned disulfides (1). When dissolved in 40% acetic acid and subjected to lyophilisation, BS RNase forms a mixture of oligomeric aggregates (2), as does RNase A (3), the proto-type of the super-family. However, while the oligomers of the pancreatic variant have been extensively characterized (3–5), the multimers of BS RNase (6) are presently less known. To deepen their characterization we induced BS RNase self-association by using the same conditions used with RNase A, i.e. lyophilisation of enzyme solutions at low pH, or thermally-induced aggregation of concentrated protein solutions in various media. The multimers obtained were analyzed by SEC, cation-exchange chromatography, cross-linking, electrophoresis, proteolysis, and enzymatic assays. The main results obtained are: (a) BS-RNase forms several 3D domain-swapped conformers, in particular at least two tetramers; one is probably a totally N terminal-swapped isoform (4), while the other contains a C terminal swapping. (b) The C-swapped tetramer is less stable than the N-swapped one. (c) BS RNase multimers larger than tetramers appear to be a mixture of various isoforms, similarly to what occurs with RNase A (3). These multimers seem to contain, again, a Cterminus swapping. (4) The validity of the results reported have been strengthened by the studies performed with a K113N-BS RNase mutant. References: 1. Piccoli R., et al. Proc Natl Acad Sci USA 1992; 89: 1870–1874. 2. Libonati M. Int J Biochem 1969; 18: 407–417. 3. Libonati M. & Gotte G. Biochem J 2004; 380: 311–327. 4. Liu Y., et al. Proc Natl Acad Sci USA 1998; 95: 3437–3442. 5. Liu Y., et al. Nat Str Biol 2001; 8: 211–214. 6. Adinolfi S., et al. FEBS Lett 1996; 398: 326–332

Double domain swapping in bovine seminal RNase: formation of distinct N- and C-swapped tetramers and multimers with increasing biological activities

Bovine seminal (BS) RNase, the unique natively dimeric member of the RNase super-family, represents a special case not only for its additional biological actions but also for the singular features of 3D domain swapping. The native enzyme is indeed a mixture of two isoforms: M = M, a dimer held together by two inter-subunit disulfide bonds, and MxM, 70% of the total, which, besides the two mentioned disulfides, is additionally stabilized by the swapping of its N-termini. When lyophilized from 40% acetic acid, BS-RNase oligomerizes as the super-family proto-type RNase A does. In this paper, we induced BS-RNase self-association and analyzed the multimers by size-exclusion chromatography, cross-linking, electrophoresis, mutagenesis, dynamic light scattering, molecular modelling. Finally, we evaluated their enzymatic and cytotoxic activities. Several BS-RNase domain-swapped oligomers were detected, including two tetramers, one exchanging only the N-termini, the other being either N- or C-swapped. The C-swapping event, confirmed by results on a BS-K113N mutant, has been firstly seen in BS-RNase here, and probably stabilizes also multimers larger than tetramers. Interestingly, all BS-RNase oligomers are more enzymatically active than the native dimer and, above all, they display a cytotoxic activity that definitely increases with the molecular weight of the multimers. This latter feature, to date unknown for BS-RNase, suggests again that the self-association of RNases strongly modulates their biological and potentially therapeutic properties. © 2012 Gotte et al.This work has been supported by the grants ‘‘Gottex60%-08’’ and ‘‘FR-GOTTEG-09’’, funded by the Italian Ministero Istruzione Università e Ricerca (MIUR), and by the University of Verona, Italy. ‘‘CTQ2010-21567-C02-02’’ from the Spanish Ministerio de Ciencia e Innovación (MICINN)

Double Domain Swapping in Bovine Seminal RNase: Formation of Distinct N- and C-swapped Tetramers and Multimers with Increasing Biological Activities

<div><p>Bovine seminal (BS) RNase, the unique natively dimeric member of the RNase super-family, represents a special case not only for its additional biological actions but also for the singular features of 3D domain swapping. The native enzyme is indeed a mixture of two isoforms: M = M, a dimer held together by two inter-subunit disulfide bonds, and MxM, 70% of the total, which, besides the two mentioned disulfides, is additionally stabilized by the swapping of its N-termini.</p> <p>When lyophilized from 40% acetic acid, BS-RNase oligomerizes as the super-family proto-type RNase A does. In this paper, we induced BS-RNase self-association and analyzed the multimers by size-exclusion chromatography, cross-linking, electrophoresis, mutagenesis, dynamic light scattering, molecular modelling. Finally, we evaluated their enzymatic and cytotoxic activities.</p> <p>Several BS-RNase domain-swapped oligomers were detected, including two tetramers, one exchanging only the N-termini, the other being either N- or C-swapped. The C-swapping event, confirmed by results on a BS-K113N mutant, has been firstly seen in BS-RNase here, and probably stabilizes also multimers larger than tetramers.</p> <p>Interestingly, all BS-RNase oligomers are more enzymatically active than the native dimer and, above all, they display a cytotoxic activity that definitely increases with the molecular weight of the multimers. This latter feature, to date unknown for BS-RNase, suggests again that the self-association of RNases strongly modulates their biological and potentially therapeutic properties.</p> </div

SEC chromatograms and PAGE under non denaturing conditions of BS-RNase aggregates obtained by lyophilising the protein from a 40% (v/v) acetic acid solution.

<p>(<b>A</b>) SEC pattern obtained with a Sephadex G100 column. Elution with ammonium acetate 0.1 M, pH 5.65, flow rate of 0.4 ml/min. (<b>B</b>) SEC chromatogram of BS-RNase multimers superimposed with that of RNase A oligomers: both patterns were obtained with a Superdex 75 10/300 GL column. Elution with 0.2 M NaPi, pH 6.7, flow rate 0.1 ml/min. (<b>C</b>) Enlarged Superdex 75 SEC pattern of BS-RNase aggregates; in the inset, 7.5% non denaturing PAGE of the two BS-tetramers, run-time 110 min. (<b>D</b>) Additional purification of the two BS-RNase tetramers: their mixture was concentrated to 25 µl in 0.4 M NaPi, and re-chromatographed in the Superdex 75 column equilibrated with the same buffer (dashed+dotted line). Then, TT₁ and TT₂ fractions were further purified: once for TT₁, continuous line; twice for TT₂, dotted and dashed lines, respectively. In the right part of the panel are reported the models of two N-swapped BS-RNase tetramers proposed by Adinolfi <i>et al. </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046804#pone.0046804-Adinolfi1" target="_blank">[13]</a>: they cannot be associated to both tetramers. The various BS-RNase species are: D, native dimer; TT₁ and TT₂, two tetrameric conformers, H (1 and 2), hexamers; L.O., larger oligomers. Concerning RNase A, grey italics labels: <i>M</i>, native monomer, <i>N_D</i>, N-terminal-swapped dimer, <i>C_D</i>, C-terminal-swapped dimer; <i>T</i>, trimers; <i>NCN_TT</i>: double N+C-swapped tetramer; <i>CNC_TT</i>: double C+N-swapped tetramer; <i>P*</i>: pentamers; <i>H*</i>: hexamers. The asterisk* is present to mention that P and H positions are derived from data obtained in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046804#pone.0046804-Gotte1" target="_blank">[21]</a>.</p