Search

Abstract Alzheimer’s disease (AD) is characterized by genetic and multifactorial risk factors. Many studies correlate AD to sleep disorders. In this study, we performed and validated a mouse model of AD and sleep fragmentation, which properly mimics a real condition of intermittent awakening. We noticed that sleep fragmentation induces a general acceleration of AD progression in 5xFAD mice, while in wild type mice it affects cognitive behaviors in particular learning and memory. Both these events may be correlated to aquaporin-4 (AQP4) modulation, a crucial player of the glymphatic system activity. In particular, sleep fragmentation differentially affects aquaporin-4 channel (AQP4) expression according to the stage of the disease, with an up-regulation in younger animals, while such change cannot be detected in older ones. Moreover, in wild type mice sleep fragmentation affects cognitive behaviors, in particular learning and memory, by compromising the glymphatic system through the decrease of AQP4. Nevertheless, an in-depth study is needed to better understand the mechanism by which AQP4 is modulated and whether it could be considered a risk factor for the disease development in wild type mice. If our hypotheses are going to be confirmed, AQP4 modulation may represent the convergence point between AD and sleep disorder pathogenic mechanisms.

Abstract Background There are a growing number of evidence-based interventions (EBIs) for autistic individuals, but few are successfully implemented with fidelity in under-resourced communities and with families from traditionally disenfranchised groups. Implementation science offers tools to increase EBI use in communities, but most implementation strategies are designed specific to a single EBI. It is not feasible to develop a new implementation strategy each time a new EBI is introduced in the community. Therefore, to test the effectiveness and generalizability of implementation strategies we are developing and testing a multifaceted implementation strategy with three EBIs concurrently. The goal of this protocol paper is to describe the randomized field trial of an implementation strategy for use across autism EBIs, diverse settings and participants, with the goal of increasing rapid uptake of effective practices to reach our most vulnerable children. Methods We developed a multifaceted implementation strategy called Using Novel Implementation Tools for Evidence-based intervention Delivery (UNITED) to facilitate the implementation and sustainment of three EBIs in under-resourced settings. We will compare fidelity to, and effectiveness of, each intervention [Mind the Gap (MTG), Remaking Recess (RR), Self-Determined Learning Model of Instruction (SDLMI)] with and without UNITED in a randomized field trial. Randomization will be stratified using a minimization allocation method. We will train community practitioners using remote delivery of modules specific to the intervention, and active coaching via Zoom for at least 6 sessions and up to 12 as dictated by each EBI. Our primary outcome is fidelity to each EBI, and our secondary outcome is at the child or family level (family empowerment for MTG, child peer social engagement for RR, and adolescent self-determination for SDLMI, respectively). We will measure progress through the implementation phases using the Stages of Implementation Completion and cost-effectiveness of UNITED. Discussion The results of this study will provide rigorous data on the effectiveness and generalizability of one relatively light-touch implementation strategy in increasing use of autism EBIs and associated outcomes in diverse under resourced public service settings for underrepresented autistic youth. Trial registration Mind the Gap: Clinicaltrials.gov Identifier: NCT04972825 (Date registered July 22, 2021); Remaking Recess: Clinicaltrials.gov Identifier: NCT04972838 (Date registered July 22, 2021); Self-Determined Learning Model of Instruction: Clinicaltrials.gov Identifier: NCT04972851 (Date registered July 22, 2021).

Abstract Bile duct injury (BDI) is a dangerous complication of cholecystectomy, with significant postoperative sequelae for the patient in terms of morbidity, mortality, and long-term quality of life. BDIs have an estimated incidence of 0.4–1.5%, but considering the number of cholecystectomies performed worldwide, mostly by laparoscopy, surgeons must be prepared to manage this surgical challenge. Most BDIs are recognized either during the procedure or in the immediate postoperative period. However, some BDIs may be discovered later during the postoperative period, and this may translate to delayed or inappropriate treatments. Providing a specific diagnosis and a precise description of the BDI will expedite the decision-making process and increase the chance of treatment success. Subsequently, the choice and timing of the appropriate reconstructive strategy have a critical role in long-term prognosis. Currently, a wide spectrum of multidisciplinary interventions with different degrees of invasiveness is indicated for BDI management. These World Society of Emergency Surgery (WSES) guidelines have been produced following an exhaustive review of the current literature and an international expert panel discussion with the aim of providing evidence-based recommendations to facilitate and standardize the detection and management of BDIs during cholecystectomy. In particular, the 2020 WSES guidelines cover the following key aspects: (1) strategies to minimize the risk of BDI during cholecystectomy; (2) BDI rates in general surgery units and review of surgical practice; (3) how to classify, stage, and report BDI once detected; (4) how to manage an intraoperatively detected BDI; (5) indications for antibiotic treatment; (6) indications for clinical, biochemical, and imaging investigations for suspected BDI; and (7) how to manage a postoperatively detected BDI.

Abstract Introduction Children with autism spectrum disorder (ASD) benefit when their caregivers can effectively advocate for appropriate services. Barriers to caregiver engagement such as provider mistrust, cultural differences, stigma, and lack of knowledge can interfere with timely service access. We describe Mind the Gap (MTG), an intervention that provides education about ASD, service navigation, and other topics relevant to families whose children have a new ASD diagnosis. MTG was developed via community partnerships and is explicitly structured to reduce engagement barriers (e.g., through peer matching, meeting flexibility, culturally-informed practices). We also present on the results of a pilot of MTG, conducted in preparation for a randomized controlled trial. Methods MTG was evaluated using mixed methods that included qualitative analysis and pre/post-test without concurrent comparison group. Participants (n=9) were primary caregivers of children (ages 2-7 years) with a recent ASD diagnosis and whose annual income was at or below 185% of the federal poverty level. In order to facilitate trust and relationship building, peer coaches delivered MTG. The coaches were parents of children with ASD who we trained to deliver the intervention. MTG consisted of up to 12 meetings between coaches and caregivers over the course of 18 weeks. Coaches delivered the intervention in homes and other community locations. Coaches shared information about various “modules,” which were topics identified as important for families with a new ASD diagnosis. Coaches worked with families to answer questions, set weekly goals, assess progress, and offer guidance. For the pilot, we focused on three primary outcomes: feasibility, engagement, and satisfaction. Feasibility was measured via enrollment and retention data, as well as coach fidelity (i.e., implementation of MTG procedures). Engagement was measured via number of sessions attended and percentage completion of the selected outcome measures. For completers (n=7), satisfaction was measured via a questionnaire (completed by caregivers) and open-ended interviews (completed by caregivers and coaches). Results We enrolled 56% of referred caregivers and 100% of eligible families. Retention was high (78%). Coaches could deliver the intervention with fidelity, completing, on average, 83% of program components. Engagement also was high; caregivers attended an average of 85% of total possible sessions and completed 100% of their measures. Caregivers indicated moderately high satisfaction with MTG. Qualitative data indicated that caregivers and coaches were positive about intervention content, and the coach-caregiver relationship was important. They also had suggestions for changes. Conclusion Mind the Gap demonstrates evidence of feasibility, and data from the pilot suggest that it addresses intervention engagement barriers for a population that is under-represented in research. The results and suggestions from participants were used to inform a large-scale RCT, which is currently underway. Overall, MTG shows promise as an intervention that can be feasibly implemented with under-resourced and ethnic minority families of children with ASD Trial registration This study is registered with ClinicalTrials.gov: NCT03711799 .

Abstract Background Nannochloropsis gaditana is a photosynthetic unicellular microalgae considered one of the most interesting marine algae to produce biofuels and food additive due to its rapid growth rate and high lipid accumulation. Although microalgae are attractive platforms for solar energy bioconversion, the overall efficiency of photosynthesis is reduced due to the steep light gradient in photobioreactors. Moreover, accumulation of lipids in microalgae for biofuels production is usually induced in a two-phase cultivation process by nutrient starvation, with additional time and costs associated. In this work, a biotechnological approach was directed for the isolation of strains with improved light penetration in photobioreactor combined with increased lipids productivity. Results Mutants of Nannochloropsis gaditana were obtained by chemical mutagenesis and screened for having both a reduced chlorophyll content per cell and increased affinity for Nile red, a fluorescent dye which binds to cellular lipid fraction. Accordingly, one mutant, called e8, was selected and characterized for having a 30% reduction of chlorophyll content per cell and an almost 80% increase of lipid productivity compared to WT in nutrient-replete conditions, with C16:0 and C18:0 fatty acids being more than doubled in the mutant. Whole-genome sequencing revealed mutations in 234 genes in e8 mutant among which there is a non-conservative mutation in the dgd1 synthase gene. This gene encodes for an enzyme involved in the biosynthesis of DGDG, one of the major lipids found in the thylakoid membrane and it is thus involved in chloroplast biogenesis. Lipid biosynthesis is strongly influenced by light availability in several microalgae species, including Nannochloropsis gaditana: reduced chlorophyll content per cell and more homogenous irradiance in photobioreactor is at the base for the increased lipid productivity observed in the e8 mutant. Conclusions The results herein obtained presents a promising strategy to produce algal biomass enriched in lipid fraction to be used for biofuel and biodiesel production in a single cultivation process, without the additional complexity of the nutrient starvation phase. Genome sequencing and identification of the mutations introduced in e8 mutant suggest possible genes responsible for the observed phenotypes, identifying putative target for future complementation and biotechnological application.

Abstract Background Ligation-mediated PCR protocols have diverse uses including the identification of integration sites of insertional mutagens, integrating vectors and naturally occurring mobile genetic elements. For approaches that employ NGS sequencing, the relative abundance of integrations within a complex mixture is typically determined through the use of read counts or unique fragment lengths from a ligation of sheared DNA; however, these estimates may be skewed by PCR amplification biases and saturation of sequencing coverage. Results Here we describe a modification of our previous splinkerette based ligation-mediated PCR using a novel Illumina-compatible adapter design that prevents amplification of non-target DNA and incorporates unique molecular identifiers. This design reduces the number of PCR cycles required and improves relative quantitation of integration abundance for saturating sequencing coverage. By inverting the forked adapter strands from a standard orientation, the integration-genome junction can be sequenced without affecting the sequence diversity required for cluster generation on the flow cell. Replicate libraries of murine leukemia virus-infected spleen samples yielded highly reproducible quantitation of clonal integrations as well as a deep coverage of subclonal integrations. A dilution series of DNAs bearing integrations of MuLV or piggyBac transposon shows linearity of the quantitation over a range of concentrations. Conclusions Merging ligation and library generation steps can reduce total PCR amplification cycles without sacrificing coverage or fidelity. The protocol is robust enough for use in a 96 well format using an automated liquid handler and we include programs for use of a Beckman Biomek liquid handling workstation. We also include an informatics pipeline that maps reads, builds integration contigs and quantitates integration abundance using both fragment lengths and unique molecular identifiers. Suggestions for optimizing the protocol to other target DNA sequences are included. The reproducible distinction of clonal and subclonal integration sites from each other allows for analysis of populations of cells undergoing selection, such as those found in insertional mutagenesis screens.

Abstract Aim The aim of this study is to review the literature focusing on various treatments based on time of tracheal injury and on different surgeons’ personal experience. Methods We retrospectively reviewed all cases of total thyroidectomy performed at the University Surgical Department of Ospedali Riuniti of Foggia from 2006 to 2017. Only a single case of tracheal lesion due to traditional total thyroidectomy was found. An extensive search of the relevant literature was carried out using MEDLINE (PubMed). We included articles that reported article type, patient number, sex, age, reasons for surgery, time of tracheal perforation intraoperatively or delayed rupture, symptoms, diagnosis, type of surgical procedure, pathological report and follow-up. Results A total of 156 published studies were screened from the sources listed. Of these, 15 studies were included in the present study. We introduced our case in the analysis. A total of 16 patients were totally analysed. There were seven males (43.7%) and seven females (43.7%), and for two patients, gender was not available. The mean patient age was 41.6 years. Conclusions The literature review showed very few cases treated differently. However, it would be good to standardise treatments. Tracheal perforation, if encountered, needs to be managed appropriately in centres of expertise with a high volume of thyroidectomies.

Abstract Background Little is known about the induction of humoral responses directed against human autoantigens during acute inflammation. We utilized a highly sensitive antibody profiling technology to study autoantibodies in patients with acute respiratory distress syndrome (ARDS) and severe sepsis, conditions characterized by intensive immune activation leading to multiple organ dysfunction. Methods Using Luciferase Immunoprecipitation Systems (LIPS), a cohort of control, ARDS and sepsis patients were tested for antibodies to a panel of autoantigens. Autoantibody titers greater than the mean plus 3 SD of the 24 control samples were used to identify seropositive samples. Available longitudinal samples from different seropositive ARDS and sepsis patient samples, starting from within the first two days after admission to the intensive care, were then analyzed for changes in autoantibody over time. Results From screening patient plasma, 57% of ARDS and 46% of septic patients without ARDS demonstrated at least one statistically significant elevated autoantibody compared to the controls. Frequent high titer antibodies were detected against a spectrum of autoantigens including potassium channel regulator, gastric ATPase, glutamic decarboxylase-65 and several cytokines. Analysis of serial samples revealed that several seropositive patients had low autoantibodies at early time points that often rose precipitously and peaked between days 7-14. Further, the use of therapeutic doses of corticosteroids did not diminish the rise in autoantibody titers. In some cases, the patient autoantibody titers remained elevated through the last serum sample collected. Conclusion The rapid induction of autoantibodies in ARDS and severe sepsis suggests that ongoing systemic inflammation and associated tissue destruction mediate the break in tolerance against these self proteins.

Abstract Background Sjögren's syndrome (SjS) is a relatively common autoimmune disease characterized by oral and ocular dryness. There is an increasing need for simple, sensitive and rapid technologies for the diagnosis of SjS and other autoimmune diseases. Here we investigated whether a quick version of luciferase immunoprecipitation systems (QLIPS) could be used to produce a rapid, specific and quantitative test to detect autoantibodies associated with SjS. Methods Using QLIPS, which requires only ten minutes of incubation, a cohort of control and SjS sera were tested for antibodies to three SjS autoantigens (La, Ro60 and Ro52). Sensitivity and specificity of the QLIPS tests were compared with LIPS and existing ELISA data. The QLIPS test for Ro52 was then evaluated with a new validation cohort and its diagnostic performance determined. Results Using QLIPS, autoantibodies to three SjS antigens, La, Ro60, and Ro52 were detected in 49%, 56% and 70%, respectively, of the SjS patients and none of the controls (100% specificity). With antibody titers in the Ro52-seropositive SjS samples approximately 1,000 times higher than the healthy controls, not only was Ro52 the most informative, but detection of anti-Ro52 antibodies under these non-equilibrium conditions was improved compared to the standard 2 hour LIPS format. Validation of the anti-Ro52 QLIPS test in a new, independent cohort of SjS and control serum samples showed 66% sensitivity and 100% specificity. Conclusion Together these results suggest that the QLIPS format for Ro52 yields both a more rapid and more discriminating test for detecting Ro52 autoantibodies than existing immunoassays and has the potential to be adapted for point-of-care evaluation of patients with SjS and other rheumatologic diseases.

Abstract Background Human cytomegalovirus infection is associated with a variety of pathological conditions including retinitis, pneumonia, hepatitis and encephalitis that may be transmitted congenitally, horizontally and parenterally and occurs both as a primary infection and as reactivation in immunocompromised individuals. Currently, there is a need for improved quantitative serological tests to document seropositivity with high sensitivity and specificity. Methods Here we investigated whether luciferase immunoprecipitation systems (LIPS) would provide a more quantitative and sensitive method for detecting anti-CMV antibodies. Four protein fragments of immunodominant regions of CMV antigens pp150 and pp65 were generated as Renilla luciferase (Ruc) fusion proteins and used in LIPS with two cohorts of CMV positive and negative sera samples previously tested by ELISA. Results Analysis of the antibody responses to two of these antigen fragments, pp150-d1 and pp150-d2, revealed geometric mean antibody titers in the first cohort that were 100–1000 fold higher in the CMV positive sera compared to the CMV negative samples (p < 0.0001) and infection status exactly matched the ELISA results for the 46 samples of the first cohort (100% sensitivity and 100% specificity). Two additional antigen fragments, pp65-d1 and pp65-d2 also showed robust antibody titers in some CMV-infected sera and yielded 50% and 96% sensitivity, respectively. Analysis of a second cohort of 70 samples using a mixture of the 4 antigens, which simplifies data collection and analysis, yielded values which correlated well with the sum of the values from the 4 separate tests (rs = 0.93, p < 0.00001). While comparison of the LIPS results from this second cohort with ELISA showed 100% sensitivity, LIPS detected six additional CMV positive samples that were not detected by ELISA. Heat map analysis revealed that several of the LIPS positive/ELISA negative samples had positive LIPS immunoreactivity with 3–4 of the CMV antigens. Conclusion These results suggest that LIPS provides a highly robust and quantitative method for studying anti-CMV antibodies and has the potential to more accurately document CMV infection than standard ELISA.

Abstract Background HTLV-I is the causal agent of adult T cell leukemia (ATLL) and HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Biomarkers are needed to diagnose and/or predict patients who are at risk for HAM/TSP or ATLL. Therefore, we investigated using luciferase immunoprecipitation technology (LIPS) antibody responses to seven HTLV-I proteins in non-infected controls, asymptomatic HTLV-I-carriers, ATLL and HAM/TSP sera samples. Antibody profiles were correlated with viral load and examined in longitudinal samples. Results Anti-GAG antibody titers detected by LIPS differentiated HTLV-infected subjects from uninfected controls with 100% sensitivity and 100% specificity, but did not differ between HTLV-I infected subgroups. However, anti-Env antibody titers were over 4-fold higher in HAM/TSP compared to both asymptomatic HTLV-I (P < 0.0001) and ATLL patients (P < 0.0005). Anti-Env antibody titers above 100,000 LU had 75% positive predictive value and 79% negative predictive value for identifying the HAM/TSP sub-type. Anti-Tax antibody titers were also higher (P < 0.0005) in the HAM/TSP compared to the asymptomatic HTLV-I carriers. Proviral load correlated with anti-Env antibodies in asymptomatic carriers (R = 0.76), but not in HAM/TSP. Conclusion These studies indicate that anti-HTLV-I antibody responses detected by LIPS are useful for diagnosis and suggest that elevated anti-Env antibodies are a common feature found in HAM/TSP patients.

Abstract Background Clear communication about the possible outcomes of proposed medical interventions is an integral part of medical care. Despite its importance, there have been few studies comparing different formats for presenting probabilistic information to patients, especially when small probabilities are involved. The purpose of this study was to explore the potential usefulness of several new small-risk graphic communication formats. Methods Information about the likelihoods of cancer and cancer prevention associated with two hypothetical cancer screening programs were used to create an augmented bar chart, an augmented grouped icon display, a flow chart, and three paired combinations of these formats. In the study scenario, the baseline risk of cancer was 53 per 1,000 (5.3%). The risk associated with cancer screening option A was 38 per 1,000 (3.8%) and the risk associated with screening option B was 29 per 1,000 (2.9%). Both the augmented bar chart and the augmented grouped icon display contained magnified views of the differences in cancer risk and cancer prevention associated with the screening programs. A convenience sample of 29 subjects (mean age 56.4 years; 76% men) used the Analytic Hierarchy Process (AHP) to indicate their relative preferences for the six formats using 15 sequential paired comparisons. Results The most preferred format was the combined augmented bar chart + flow diagram (mean preference score 0.43) followed by the combined augmented icon + augmented bar chart format (mean preference score 0.22). The overall differences among the six formats were statistically significant: Kruskal-Wallis Chi Square = 141.4, p < 0.0001. The three combined formats all had statistically significant higher preferences scores than the single format displays (p < 0.05). Conclusion These findings suggest that patients may prefer combined, rather than single, graphic risk presentation formats and that augmented bar charts and icon displays may be useful for conveying comparative information about small risks to clinical decision makers. Further research to confirm and extend these findings is warranted.

Abstract After the publication of this work 1, we became aware of the fact that one author was missing on the author list. Dr Jo.H.M. Berden contributed the JM403 antibody and advised on the methodology for the ELISA for JM403. He also contributed to the text of the manuscript. Apart from this correction concerning the authorship, the methods and interpretation of the data, the results reported in our publication and the conclusions are absolutely correct. We apologize for the inconvenience that this inaccuracy may have caused.

Abstract Background α1-antitrypsin and surfactant protein-A (SP-A) are major lung defense proteins. With the hypothesis that SP-A could bind α1-antitrypsin, we designed a series of in vitro experiments aimed at investigating the nature and consequences of such an interaction. Methods and results At an α1-antitrypsin:SP-A molar ratio of 1:1, the interaction resulted in a calcium-dependent decrease of 84.6% in the association rate constant of α1-antitrypsin for neutrophil elastase. The findings were similar when SP-A was coupled with the Z variant of α1-antitrypsin. The carbohydrate recognition domain of SP-A appeared to be a major determinant of the interaction, by recognizing α1-antitrypsin carbohydrate chains. However, binding of SP-A carbohydrate chains to the α1-antitrypsin amino acid backbone and interaction between carbohydrates of both proteins are also possible. Gel filtration chromatography and turnover per inactivation experiments indicated that one part of SP-A binds several molar parts of α1-antitrypsin. Conclusion We conclude that the binding of SP-A to α1-antitrypsin results in a decrease of the inhibition of neutrophil elastase. This interaction could have potential implications in the physiologic regulation of α1-antitrypsin activity, in the pathogenesis of pulmonary emphysema, and in the defense against infectious agents.

Abstract Background The burden of proteinases from inflammatory cells in the lung of subjects with type Pi ZZ of alpha-1-antitrypsin deficiency is higher than in those without the deficiency. Cross-sectional studies have shown increased levels of biomarkers of extracellular matrix degradation in vivo. Longitudinal variability of these biomarkers is unknown but desirable for clinical studies with proteinase inhibitors. Methods We measured three different types of biomarkers, including desmosines, elastase-formed fibrinogen fragments and heparan sulfate epitope JM403, in plasma and urine for a period of 7 weeks in a group of 12 patients who participated in a placebo-controlled study to assess the safety of a single inhalation of hyaluronic acid. Results Effect of study medication on any of the biomarkers was not seen. Baseline desmosines in plasma and urine correlated with baseline CO diffusion capacity (R = 0.81, p = 0.01 and R = 0.65, p = 0.05). Mean coefficient of variation within patients (CVi) for plasma and urine desmosines was 18.7 to 13.5%, respectively. Change in urinary desmosine levels correlated significantly with change in plasma desmosine levels (R = 0.84, p < 0.01). Mean CVi for fibrinogen fragments in plasma was 20.5% and for JM403 in urine was 27.8%. No correlations were found between fibrinogen fragments or JM403 epitope and desmosines. Conclusion We found acceptable variability in our study parameters, indicating the feasibility of their use in an evaluation of biochemical efficacy of alpha-1-antitrypsin augmentation therapy in Pi Z subjects.