The seesaw between normal function and protein aggregation: How functional interactions may increase protein solubility

AbstractProtein aggregation has been studied for at least 3 decades, and many of the principles that regulate this event are relatively well understood. Here, however, we present a different perspective to explain why proteins aggregate: we argue that aggregation may occur as a side‐effect of the lack of one or more natural partners that, under physiologic conditions, would act as chaperones. This would explain why the same surfaces that have evolved for functional purposes are also those that favour aggregation. In the course of reviewing this field, we substantiate our hypothesis with three paradigmatic examples that argue for the generality of our proposal. An obvious corollary of this hypothesis is, of course, that targeting the physiological partners of a protein could be the most direct and specific approach to designing anti‐aggregation molecules. Our analysis may thus inform a different strategy for combating diseases of protein aggregation and misfolding

The structural properties of full-length annexin A11

Annexin A11 (ANXA11) is a calcium-dependent phospholipid-binding protein belonging to the annexin protein family and implicated in the neurodegenerative amyotrophic lateral sclerosis. Structurally, ANXA11 contains a conserved calcium-binding C-terminal domain common to all annexins and a putative intrinsically unfolded N-terminus specific for ANXA11. Little is known about the structure and functions of this region of the protein. By analogy with annexin A1, it was suggested that residues 38 to 59 within the ANXA11 N-terminus could form a helical region that would be involved in interactions. Interestingly, this region contains residues that, when mutated, may lead to clinical manifestations. In the present study, we have studied the structural features of the full-length protein with special attention to the N-terminal region using a combination of biophysical techniques which include nuclear magnetic resonance and small angle X-ray scattering. We show that the N-terminus is intrinsically disordered and that the overall features of the protein are not markedly affected by the presence of calcium. We also analyzed the 38–59 helix hypothesis using synthetic peptides spanning both the wild-type sequence and clinically relevant mutations. We show that the peptides have a remarkable character typical of a native helix and that mutations do not alter the behaviour suggesting that they are required for interactions rather than being structurally important. Our work paves the way to a more thorough understanding of the ANXA11 functions

RNA aptamer reveals nuclear TDP-43 pathology is an early aggregation event that coincides with STMN-2 cryptic splicing and precedes clinical manifestation in ALS

Open Access via the Springer Agreement The research leading to this manuscript has been supported by (i) a Target ALS foundation grant to JMG, MHH, GGT, EZ and NS and employing MG and FMW BB-2022-C4-L2; (ii) an NIH grant to JG and MHH, employing HS and FR R01NS127186; (iii) the European Research Council (RIBOMYLOME_309545 and ASTRA_855923) to GGT; and (iv) an MND Association Lady Edith Wolfson Junior Non-Clinical Fellowship to RS Saleeb/Oct22/980-799 (RSS). The authors would also like to thank the University of Aberdeen Microscopy and Histology Core Facility in the Institute of Medical Sciences.Peer reviewe

RNA aptamer reveals nuclear TDP-43 pathology is an early aggregation event that coincides with STMN-2 cryptic splicing and precedes clinical manifestation in ALS

TDP-43 is an aggregation-prone protein which accumulates in the hallmark pathological inclusions of amyotrophic lateral sclerosis (ALS). However, the analysis of deeply phenotyped human post-mortem samples has shown that TDP-43 aggregation, revealed by standard antibody methods, correlates poorly with symptom manifestation. Recent identification of cryptic-splicing events, such as the detection of Stathmin-2 (STMN-2) cryptic exons, are providing evidence implicating TDP-43 loss-of-function as a potential driving pathomechanism but the temporal nature of TDP-43 loss and its relation to the disease process and clinical phenotype is not known. To address these outstanding questions, we used a novel RNA aptamer, TDP-43APT, to detect TDP-43 pathology and used single molecule in situ hybridization to sensitively reveal TDP-43 loss-of-function and applied these in a deeply phenotyped human post-mortem tissue cohort. We demonstrate that TDP-43APT identifies pathological TDP-43, detecting aggregation events that cannot be detected by classical antibody stains. We show that nuclear TDP-43 pathology is an early event, occurring prior to cytoplasmic accumulation and is associated with loss-of-function measured by coincident STMN-2 cryptic splicing pathology. Crucially, we show that these pathological features of TDP-43 loss-of-function precede the clinical inflection point and are not required for region specific clinical manifestation. Furthermore, we demonstrate that gain-of-function in the form of extensive cytoplasmic accumulation, but not loss-of-function, is the primary molecular correlate of clinical manifestation. Taken together, our findings demonstrate implications for early diagnostics as the presence of STMN-2 cryptic exons and early TDP-43 aggregation events could be detected prior to symptom onset, holding promise for early intervention in ALS

Immunogenicity and reactogenicity of modified vaccinia Ankara pre-exposure vaccination against mpox according to previous smallpox vaccine exposure and HIV infection. Prospective cohort study

Background: Pre-exposure vaccination with MVA-BN has been widely used against mpox to contain the 2022 outbreak. Many countries have defined prioritized strategies, administering a single dose to those historically vaccinated for smallpox, to achieve quickly adequate coverage in front of low supplies. Using epidemiological models, real-life effectiveness was estimated at approximately 36%-86%, but no clinical trials were performed. Few data on MVA-BN immunogenicity are currently available, and there are no established correlates of protection. Immunological response in PLWH in the context of the 2022 outbreak was also poorly described. Methods: Blood samples were collected from participants eligible for pre-exposure MVA-BN vaccination before (T1) receiving a full course of vaccine (single-dose for vaccine-experienced or smallpox-primed and two-dose for smallpox vaccine-naïve or smallpox non-primed) and one month after the last dose (T2 and T3, respectively). MPXV-specific IgGs were measured by in-house immunofluorescence assay, using 1:20 as screening dilution, MPXV-specific nAbs by 50% plaque reduction neutralization test (PRNT50, starting dilution 1:10), and IFN-γ-producing specific T cells to MVA-BN vaccine, by ELISpot assay. Paired or unpaired t-test and Wilcoxon or Mann-Whitney test were used to analyse IgG and nAbs, and T-cell response, as appropriate. The probability of IgG and nAb response in vaccine-experienced vs. vaccine-naïve was estimated in participants not reactive at T1. The McNemar test was used to evaluate vaccination's effect on humoral response both overall and by smallpox vaccination history. In participants who were not reactive at T1, the proportion of becoming responders one month after full-cycle completion by exposure groups was compared by logistic regression and then analysed by HIV status strata (interaction test). The response was also examined in continuous, and the Average Treatment Effect (ATE) of the difference from baseline to schedule completion according to previous smallpox vaccination was estimated after weighting for HIV using a linear regression model. Self-reports of adverse effects following immunization (AEFIs) were prospectively collected after the first MVA-BN dose (T1). Systemic (S-AEFIs: fatigue, myalgia, headache, GI effects, chills) and local (L-AEFIs: redness, swelling, pain) AEFIs were graded as absent (grade 0), mild (1), moderate (2), or severe (3). The maximum level of severity for S-AEFIs and L-AEFIs ever experienced over the 30 days post-dose by vaccination exposure groups were analysed using a univariable multinomial logistic regression model and after adjusting for HIV status; for each of the symptoms, we also compared the mean duration by exposure group using an unpaired t-test. Findings: Among the 164 participants included, 90 (54.8%) were smallpox vaccine-experienced. Median age was 49 years (IQR 41-55). Among the 76 (46%) PLWH, 76% had a CD4 count >500 cells/μL. There was evidence that both the IgG and nAbs titers increased after administration of the MVA-BN vaccine. However, there was no evidence for a difference in the potential mean change in humoral response from baseline to the completion of a full cycle when comparing primed vs. non-primed participants. Similarly, there was no evidence for a difference in the seroconversion rate after full cycle vaccination in the subset of participants not reactive for nAbs at T1 (p = 1.00 by Fisher's exact test). In this same analysis and for the nAbs outcome, there was some evidence of negative effect modification by HIV (interaction p-value = 0.17) as primed people living with HIV (PLWH) showed a lower probability of seroconversion vs. non-primed, and the opposite was seen in PLWoH. When evaluating the response in continuous, we observed an increase in T-cell response after MVA-BN vaccination in both primed and non-primed. There was evidence for a larger increase when using the 2-dose vs. one-dose strategy with a mean difference of -2.01 log2 (p ≤ 0.0001), after controlling for HIV. No evidence for a difference in the risk of developing any AEFIs of any grade were observed by exposure group, except for the lower risk of grade 2 (moderate) fatigue, induration and local pain which was lower in primed vs. non-primed [OR 0.26 (0.08-0.92), p = 0.037; OR 0.30 (0.10-0.88), p = 0.029 and OR 0.19 (0.05-0.73), p = 0.015, respectively]. No evidence for a difference in symptom duration was also detected between the groups. Interpretation: The evaluation of the humoral and cellular response one month after the completion of the vaccination cycle suggested that MVA-BN is immunogenic and that the administration of a two-dose schedule is preferable regardless of the previous smallpox vaccination history, especially in PLWH, to maximize nAbs response. MVA-BN was safe as well tolerated, with grade 2 reactogenicity higher after the first administration in vaccine-naïve than in vaccine-experienced individuals, but with no evidence for a difference in the duration of these adverse effects. Further studies are needed to evaluate the long-term duration of immunity and to establish specific correlates of protection. Funding: The study was supported by the National Institute for Infectious Disease Lazzaro Spallanzani IRCCS "Advanced grant 5 × 1000, 2021" and by the Italian Ministry of Health "Ricerca Corrente Linea 2"

Predicting needlestick and sharps injuries in nursing students: Development of the SNNIP scale

© 2020 The Authors. Nursing Open published by John Wiley & Sons Ltd. Aim: To develop an instrument to investigate knowledge and predictive factors of needlestick and sharps injuries (NSIs) in nursing students during clinical placements. Design: Instrument development and cross-sectional study for psychometric testing. Methods: A self-administered instrument including demographic data, injury epidemiology and predictive factors of NSIs was developed between October 2018–January 2019. Content validity was assessed by a panel of experts. The instrument's factor structure and discriminant validity were explored using principal components analysis. The STROBE guidelines were followed. Results: Evidence of content validity was found (S-CVI 0.75; I-CVI 0.50–1.00). A three-factor structure was shown by exploratory factor analysis. Of the 238 participants, 39% had been injured at least once, of which 67.3% in the second year. Higher perceptions of “personal exposure” (4.06, SD 3.78) were reported by third-year students. Higher scores for “perceived benefits” of preventive behaviours (13.6, SD 1.46) were reported by second-year students

Immunogenicity and reactogenicity of modified vaccinia Ankara pre-exposure vaccination against mpox according to previous smallpox vaccine exposure and HIV infection: prospective cohort study

BACKGROUND: Pre-exposure vaccination with MVA-BN has been widely used against mpox to contain the 2022 outbreak. Many countries have defined prioritized strategies, administering a single dose to those historically vaccinated for smallpox, to achieve quickly adequate coverage in front of low supplies. Using epidemiological models, real-life effectiveness was estimated at approximately 36%–86%, but no clinical trials were performed. Few data on MVA-BN immunogenicity are currently available, and there are no established correlates of protection. Immunological response in PLWH in the context of the 2022 outbreak was also poorly described. METHODS: Blood samples were collected from participants eligible for pre-exposure MVA-BN vaccination before (T1) receiving a full course of vaccine (single-dose for vaccine-experienced or smallpox-primed and two-dose for smallpox vaccine-naïve or smallpox non-primed) and one month after the last dose (T2 and T3, respectively). MPXV-specific IgGs were measured by in-house immunofluorescence assay, using 1:20 as screening dilution, MPXV-specific nAbs by 50% plaque reduction neutralization test (PRNT50, starting dilution 1:10), and IFN-γ-producing specific T cells to MVA-BN vaccine, by ELISpot assay. Paired or unpaired t-test and Wilcoxon or Mann–Whitney test were used to analyse IgG and nAbs, and T-cell response, as appropriate. The probability of IgG and nAb response in vaccine-experienced vs. vaccine-naïve was estimated in participants not reactive at T1. The McNemar test was used to evaluate vaccination's effect on humoral response both overall and by smallpox vaccination history. In participants who were not reactive at T1, the proportion of becoming responders one month after full-cycle completion by exposure groups was compared by logistic regression and then analysed by HIV status strata (interaction test). The response was also examined in continuous, and the Average Treatment Effect (ATE) of the difference from baseline to schedule completion according to previous smallpox vaccination was estimated after weighting for HIV using a linear regression model. Self-reports of adverse effects following immunization (AEFIs) were prospectively collected after the first MVA-BN dose (T1). Systemic (S-AEFIs: fatigue, myalgia, headache, GI effects, chills) and local (L-AEFIs: redness, swelling, pain) AEFIs were graded as absent (grade 0), mild (1), moderate (2), or severe (3). The maximum level of severity for S-AEFIs and L-AEFIs ever experienced over the 30 days post-dose by vaccination exposure groups were analysed using a univariable multinomial logistic regression model and after adjusting for HIV status; for each of the symptoms, we also compared the mean duration by exposure group using an unpaired t-test. FINDING: Among the 164 participants included, 90 (54.8%) were smallpox vaccine-experienced. Median age was 49 years (IQR 41–55). Among the 76 (46%) PLWH, 76% had a CD4 count >500 cells/μL. There was evidence that both the IgG and nAbs titers increased after administration of the MVA-BN vaccine. However, there was no evidence for a difference in the potential mean change in humoral response from baseline to the completion of a full cycle when comparing primed vs. non-primed participants. Similarly, there was no evidence for a difference in the seroconversion rate after full cycle vaccination in the subset of participants not reactive for nAbs at T1 (p = 1.00 by Fisher's exact test). In this same analysis and for the nAbs outcome, there was some evidence of negative effect modification by HIV (interaction p-value = 0.17) as primed people living with HIV (PLWH) showed a lower probability of seroconversion vs. non-primed, and the opposite was seen in PLWoH. When evaluating the response in continuous, we observed an increase in T-cell response after MVA-BN vaccination in both primed and non-primed. There was evidence for a larger increase when using the 2-dose vs. one-dose strategy with a mean difference of −2.01 log2 (p ≤ 0.0001), after controlling for HIV. No evidence for a difference in the risk of developing any AEFIs of any grade were observed by exposure group, except for the lower risk of grade 2 (moderate) fatigue, induration and local pain which was lower in primed vs. non-primed [OR 0.26 (0.08–0.92), p = 0.037; OR 0.30 (0.10–0.88), p = 0.029 and OR 0.19 (0.05–0.73), p = 0.015, respectively]. No evidence for a difference in symptom duration was also detected between the groups. INTERPRETATION: The evaluation of the humoral and cellular response one month after the completion of the vaccination cycle suggested that MVA-BN is immunogenic and that the administration of a two-dose schedule is preferable regardless of the previous smallpox vaccination history, especially in PLWH, to maximize nAbs response. MVA-BN was safe as well tolerated, with grade 2 reactogenicity higher after the first administration in vaccine-naïve than in vaccine-experienced individuals, but with no evidence for a difference in the duration of these adverse effects. Further studies are needed to evaluate the long-term duration of immunity and to establish specific correlates of protection