Knowledge-building in an environment mediated by digital technology: A case study in higher education.

The advancement of technology in recent years seems to be prompting a re-ontologising of the world. Digital technology is transforming the educational spaces we inhabit, as well as our way of processing information. Although there are already numerous studies that have addressed this technological reality, only a handful have done so from a theoretical perspective. That is why we present research that seeks to reinforce the latest theoretical contributions for understanding how modern technology may be affecting the way in which knowledge is built. Based on the latest research in social constructivism, this is a qualitative study designed to contribute to the creation of a specific theoretical framework for an onlife world. An ill-structured task and a semi-structured interview were used to observe the use of the thinking skills that enable us to build knowledge and the relationship between them. The results show that the ways of building knowledge are changing, as digital technology fosters the use of higher-order thinking skills that, furthermore, operate in a chaotic, complex, and unpredictable manner. In conclusion, this study upholds the notion that the ways of building knowledge are changing, but we still need more empirical contributions to create a generally accepted theoretical construct for explaining how we build knowledge through digital technology.

Nanostructures in Hydrogen Peroxide Sensing.

In recent years, several devices have been developed for the direct measurement of hydrogen peroxide (H2O2), a key compound in biological processes and an important chemical reagent in industrial applications. Classical enzymatic biosensors for H2O2 have been recently outclassed by electrochemical sensors that take advantage of material properties in the nano range. Electrodes with metal nanoparticles (NPs) such as Pt, Au, Pd and Ag have been widely used, often in combination with organic and inorganic molecules to improve the sensing capabilities. In this review, we present an overview of nanomaterials, molecules, polymers, and transduction methods used in the optimization of electrochemical sensors for H2O2 sensing. The different devices are compared on the basis of the sensitivity values, the limit of detection (LOD) and the linear range of application reported in the literature. The review aims to provide an overview of the advantages associated with different nanostructures to assess which one best suits a target application.

Recombinant pharmaceuticals from microbial cells: a 2015 update.

Diabetes, growth or clotting disorders are among the spectrum of human diseases related to protein absence or malfunction. Since these pathologies cannot be yet regularly treated by gene therapy, the administration of functional proteins produced ex vivo is required. As both protein extraction from natural producers and chemical synthesis undergo inherent constraints that limit regular large-scale production, recombinant DNA technologies have rapidly become a choice for therapeutic protein production. The spectrum of organisms exploited as recombinant cell factories has expanded from the early predominating Escherichia coli to alternative bacteria, yeasts, insect cells and especially mammalian cells, which benefit from metabolic and protein processing pathways similar to those in human cells. Up to date, around 650 protein drugs have been worldwide approved, among which about 400 are obtained by recombinant technologies. Other 1300 recombinant pharmaceuticals are under development, with a clear tendency towards engineered versions with improved performance and new functionalities regarding the conventional, plain protein species. This trend is exemplified by the examination of the contemporary protein-based drugs developed for cancer treatment.

Do intangible factors enhance sociocultural productivity and economy in world heritage sites?

Measuring the sociocultural productivity of heritage sites remains an ongoing issue for international organizations concerned with the conservation and promotion of traditional sites. The productivity of these locations is not only affected by tangible elements but also by intangible factors, such as the emotions generated by the experiences. For this purpose, 597 employees of hotels in these historical locations who had visited one of the 14 heritage sites in Spain assessed what role emotions play in this contribution. The methodology used was the application of structural equations. Several conclusions have been drawn utilizing the SmartPLS 4 software. The first is that the generation of positive emotions comes exclusively from cultural and historical dynamization and not from technological advances or an eagerness to learn. The second is that both the application of technological advances and cultural dynamization have a direct impact on productivity.

The α-helical content of the transmembrane domain of the British dementia protein-2 (Bri2) determines its processing by signal peptide peptidase-like 2b (SPPL2b).

Regulated intramembrane proteolysis is a widely accepted concept describing the processing of various transmembrane proteins via ectodomain shedding followed by an intramembrane cleavage. The resulting cleavage products can be involved in reverse signaling. Presenilins, which constitute the active center of the γ-secretase complex, signal peptide peptidase (SPP), and its homologues, the SPP-like (SPPL) proteases are members of the family of intramembrane-cleaving aspartyl proteases of the GXGD-type. We recently demonstrated that Bri2 (itm2b) is a substrate for regulated intramembrane proteolysis by SPPL2a and SPPL2b. Intramembrane cleavage of Bri2 is triggered by an initial shedding event catalyzed by A Disintegrin and Metalloprotease 10 (ADAM10). Additionally primary sequence determinants within the intracellular domain, the transmembrane domain and the luminal juxtamembrane domain are required for efficient cleavage of Bri2 by SPPL2b. Using mutagenesis and circular dichroism spectroscopy we now demonstrate that a high α-helical content of the Bri2 transmembrane domain (TMD) reduces cleavage efficiency of Bri2 by SPPL2b, while the presence of a GXXXG dimerization motif influences the intramembrane cleavage only to a minor extent. Surprisingly, only one of the four conserved intramembrane glycine residues significantly affects the secondary structure of the Bri2 TMD and thereby its intramembrane cleavage. Other glycine residues do not influence the α-helical content of the transmembrane domain nor its intramembrane processing.

A "Kidney-on-the-Chip" Model Composed of Primary Human Tubular, Endothelial, and White Blood Cells.

State-of-the-art cell culture systems may enlist a variety of features to push the significance of in vitro models beyond classical 2D single cell culture; among them are the 3D scaffolds of organic or artificial materials, multi-cell setups, and the use of primary cells as source materials. Obviously, operational complexity increases with each additional feature and feasibility, whereas reproducibility may suffer.We report a multicellular setup using primary human cells and the Mimetas scaffold that aims to increase pathophysiological significance of in vitro culture and simultaneously allows for relatively high-throughput and easy handling.

Health behavioral responses to parental myocardial infarction and impact on own risk of disease in the general population.

Aims: A family history of coronary heart disease increases one's own risk of experiencing cardiovascular disease and death. An implication of the hereditary nature of the disease is that individuals are provided information about their own risk when a parent is affected, potentially leading them to engage in behaviors that reduce their own risk. In this study, we assessed how a 10-year risk of a cardiovascular event, measured by SCORE, changes for the offspring in response to a parent experiencing a myocardial infarction. Methods: We analyzed 19,995 individuals from the general population in the Copenhagen City Heart Study of whom 2,071 had a parent, who suffered from a myocardial infarction during four decades of observation using fixed-effects regressions. Results: Following a parental myocardial infarction, individuals reduced their 10-year risk by 0.16 percentage points constituting a 7.1% reduction of baseline risk. Male participants had the largest change in the risk SCORE following an event of the mother, with a 12.4% reduction from the baseline risk. The degree of response contingent on their own level of risk was found to be the largest for individuals with a 10-year risk between 5% and 10%, who also showed a reduction in systolic blood pressure following paternal myocardial infarction. Parental myocardial infarction was associated with an increased smoking rate in individuals with a baseline risk above 10%, while reductions in risk were seen for individuals with a lower baseline risk. Conclusion: Following a parental event, individuals reduced their 10-year risk with the largest reductions in their own risk, as observed in men and individuals experiencing a maternal event. The response was the largest for individuals with a 10-year risk for myocardial infarction between 5 and 10%.

What role geoparks play improving the health and well-being of senior tourists?

In recent years geoparks, helped by governmental policies, have become tourist destinations especially among senior visitors. The paper aimed to analyse whether geoparks contribute to improving the health of tourists older than 65 years and what were their main motives to visit geoparks. The data were collected from 398 senior tourists who visited the Villuerca- Ibores-Jara Geopark (Spain) in 2023, presenting our results using SmartPLS version 4. The results showed that senior tourists are very interested in visiting this geopark for psychotherapeutic reasons, given its high environmental and geological interest. In addition, they consider geoparks as spaces where they can socialise, which is beneficial considering the isolation that many often experience during the year. These findings are highly relevant for public authorities to protect, maintain and promote geoparks among senior tourists.

Efficacy and Toxicity Evaluation of Bepotastine Besilate 1.5% Preservative-Free Eye Drops Vs Olopatadine Hydrochloride 0.2% Bak-Preserved Eye Drops in Patients with Allergic Conjunctivitis.

Purpose: To study the efficacy and toxic effects of bepotastine besilate 1.5% preservative-free (BB-PF) and olopatadine 0.2% BAK-preserved (OL-BAK) drops on the ocular surface of patients with allergic conjunctivitis. Patients and Methods: Ninety-seven patients with allergic conjunctivitis diagnosis participated in a prospective, multicenter, randomized, double-blind, controlled, parallel-group clinical trial. Patients received either BB-PF (n=48) or OL-BAK (n=49), both administered once daily in the morning. The patients were followed for 60 days. Ocular itching was the primary outcome measure. Secondary outcomes included ocular symptoms, signs, and non-ocular symptoms associated with rhinoconjunctivitis. Conjunctival impression cytology (CIC) was performed to evaluate histopathological changes related to the toxic effects of preservatives. Results: BB-PF treatment was associated with a 1.30 more probability of diminished ocular itching than OL-BAK (odds ratio (OR)=1.30; 95% CI=(0.96-1.7); p=0.086). No statistically significant differences were found between treatments in the resolution of other ocular symptoms or signs, except for tearing, which was superior in the BB-PF (OR=1.37; 95% (1.26-1.47); p<0.0001). BB-PF was superior in terms of the resolution of rhinorrhea (p=0.040) and nasal itching (p=0.037). After 60 days of treatment, the BB-PF group exhibited 2.0 times higher probability of having a lower Nelson scale score compared to the OL-BAK group (OR=2.00; 95% CI=(1.19-3.34); p=0.010). Conclusion: Both medications presented a similar efficacy in terms of the resolution of ocular signs and symptoms associated with ocular conjunctivitis. BB-PF is superior in the resolution of non-ocular symptoms and safer for the ocular surface than OL-BAK.

Tracking excess deaths associated with the COVID-19 epidemic as an epidemiological surveillance strategy-preliminary results of the evaluation of six Brazilian capitals.

INTRODUCTION: In March 2020, the World Health Organization declared the coronavirus disease (COVID-19) outbreak a pandemic. In Brazil, 110 thousand cases and 5,901 deaths were confirmed by the end of April 2020. The scarcity of laboratory resources, the overload on the service network, and the broad clinical spectrum of the disease make it difficult to document all the deaths due to COVID-19. The aim of this study was to assess the mortality rate in Brazilian capitals with a high incidence of COVID-19. METHODS: We assessed the weekly mortality between epidemiological week 1 and 16 in 2020 and the corresponding period in 2019. We estimated the expected mortality at 95% confidence interval by projecting the mortality in 2019 to the population in 2020, using data from the National Association of Civil Registrars (ARPEN-Brasil). RESULTS: In the five capitals with the highest incidence of COVID-19, we identified excess deaths during the pandemic. The age group above 60 years was severely affected, while 31% of the excess deaths occurred in the age group of 20-59 years. There was a strong correlation (r = 0.94) between excess deaths and the number of deaths confirmed by epidemiological monitoring. The epidemiological surveillance captured only 52% of all mortality associated with the COVID-19 pandemic in the cities examined. CONCLUSIONS: Considering the simplicity of the method and its low cost, we believe that the assessment of excess mortality associated with the COVID-19 pandemic should be used as a complementary tool for regular epidemiological surveillance.

Establishing a malaria diagnostics centre of excellence in Kisumu, Kenya.

BACKGROUND: Malaria microscopy, while the gold standard for malaria diagnosis, has limitations. Efficacy estimates in drug and vaccine malaria trials are very sensitive to small errors in microscopy endpoints. This fact led to the establishment of a Malaria Diagnostics Centre of Excellence in Kisumu, Kenya. The primary objective was to ensure valid clinical trial and diagnostic test evaluations. Key secondary objectives were technology transfer to host countries, establishment of partnerships, and training of clinical microscopists. CASE DESCRIPTION: A twelve-day "long" and a four-day "short" training course consisting of supervised laboratory practicals, lectures, group discussions, demonstrations, and take home assignments were developed. Well characterized slides were developed and training materials iteratively improved. Objective pre- and post-course evaluations consisted of 30 slides (19 negative, 11 positive) with a density range of 50-660 parasites/mul, a written examination (65 questions), a photographic image examination (30 images of artifacts and species specific characteristics), and a parasite counting examination. DISCUSSION AND EVALUATION: To date, 209 microscopists have participated from 11 countries. Seventy-seven experienced microscopists participated in the "long" courses, including 47 research microscopists. Sensitivity improved by a mean of 14% (CI 9-19%) from 77% baseline (CI 73-81 %), while specificity improved by a mean of 17% (CI 11-23%) from 76% (CI 70-82%) baseline. Twenty-three microscopists who had been selected for a four-day refresher course showed continued improvement with a mean final sensitivity of 95% (CI 91-98%) and specificity of 97% (CI 95-100%). Only 9% of those taking the pre-test in the "long" course achieved a 90% sensitivity and 95% specificity, which increased to 61% of those completing the "short" course. All measures of performance improved substantially across each of the five organization types and in each course offered. CONCLUSION: The data clearly illustrated that false positive and negative malaria smears are a serious problem, even with research microscopists. Training dramatically improved performance. Quality microscopy can be provided by the Centre of Excellence concept. This concept can be extended to other diagnostics of public health importance, and comprehensive disease control strategies.

Effects of long-term gait training using visual cues in an individual with Parkinson disease.

BACKGROUND AND PURPOSE: In an attempt to improve the gait of people with Parkinson disease (PD), researchers have examined the effect of visual cues placed on the floor. These studies typically have used a single session of training with such cues and have not examined the long-term carryover of such training. In the present study, therefore, gait was analyzed during uncued, cued, and retention phases, each lasting 1 month. SUBJECT: A 78-year-old woman who had been diagnosed with PD 12 years previously (Hoehn and Yahr classification of disability, stage III) volunteered for the study. METHODS: During the initial uncued gait phase, the subject was required to walk a distance of 10 m as many times as she could in 30 minutes, 3 times per week for 4 weeks. During the 4-week cued gait phase, visual cues were placed on the floor along the 10-m walkway. The cues were initially 110% of the uncued step length and were later increased to 120%. Following this cued gait phase, the subject's gait was recorded periodically for 1 month without cues available. Step length, gait speed, and 2-dimensional lower-limb kinematics were compared within and across the 3 experimental phases. Celeration lines were calculated for the initial uncued phase and then extrapolated across the cued training and uncued retention phases. Binomial tests were used to analyze the significance of changes from the initial phase of the experiment. RESULTS: Step length (0.53-0.56 m) and gait speed (0.77-0.82 m x s(-1)) were essentially unchanged during uncued gait training after the first day; however, during the cued gait phase, gait speed improved, from 0.87 m x s(-1) to 1.13 m x s(-1), as step length was increased with visual cues. Improvements in step length (0.68 m) and gait speed (1.08 m x s(-1)) were still evident 1 month following the removal of the cues. Analyses of angle-angle diagrams and phase-plane portraits revealed that training with visual cues increased hip and knee range of motion and engendered more stable motor control of the lower limb. DISCUSSION AND CONCLUSION: In contrast to previous studies in which the benefits of visual cueing were relatively short-lived, in this study, 1 month of gait training with visual cues was successful in establishing a lasting improvement in gait speed and step length while increasing the stability of the underlying motor control system.

Tracking excess deaths associated with the COVID-19 epidemic as an epidemiological surveillance strategy-preliminary results of the evaluation of six Brazilian capitals

Abstract INTRODUCTION: In March 2020, the World Health Organization declared the coronavirus disease (COVID-19) outbreak a pandemic. In Brazil, 110 thousand cases and 5,901 deaths were confirmed by the end of April 2020. The scarcity of laboratory resources, the overload on the service network, and the broad clinical spectrum of the disease make it difficult to document all the deaths due to COVID-19. The aim of this study was to assess the mortality rate in Brazilian capitals with a high incidence of COVID-19. METHODS: We assessed the weekly mortality between epidemiological week 1 and 16 in 2020 and the corresponding period in 2019. We estimated the expected mortality at 95% confidence interval by projecting the mortality in 2019 to the population in 2020, using data from the National Association of Civil Registrars (ARPEN-Brasil). RESULTS: In the five capitals with the highest incidence of COVID-19, we identified excess deaths during the pandemic. The age group above 60 years was severely affected, while 31% of the excess deaths occurred in the age group of 20-59 years. There was a strong correlation (r = 0.94) between excess deaths and the number of deaths confirmed by epidemiological monitoring. The epidemiological surveillance captured only 52% of all mortality associated with the COVID-19 pandemic in the cities examined. CONCLUSIONS: Considering the simplicity of the method and its low cost, we believe that the assessment of excess mortality associated with the COVID-19 pandemic should be used as a complementary tool for regular epidemiological surveillance.

Tissucol application in dermolipectomy and incisional hernia repair.

Biological adhesives have a lot of applications in surgical procedures. Here we present a prospective study with the aim of analyzing results of the application of Tissucol between the muscle layers and subcutaneous tissue after incisional hernia repair with polypropylene mesh and associated dermolipectomy. We assess clinical and technical parameters, local morbidity, and hospital stay. Fifty-six patients were divided into two groups. Patients with whom we used fibrin glue were older, with more obesity (P < 0.005) with associated diseases, and their incisional hernias were larger and more complicated to repair. Patients in the Tissucol group developed less local morbidity (hematomas or abscesses; P < 0.01), had a shorter mean hospital stay (P < 0.01), and required less wound care. The use of Tissucol improves the results of surgical repair of large abdominal incisional hernias repaired by mesh placement and dermolipectomy, and it decreases global morbidity and hospital stay are reduced.

[A study of the fragile X syndrome among the employees of the Spanish National Telephone Company]. / Estudio del síndrome X-frágil en la población de la compañía telefónica de España.

Our aim was to study the incidence rate of the X-fragile syndrome among the male employees throughout Spain and that of the physically handicapped dependants of those workers at the Spanish National Telephone Company (TESA), this last goal being to offer genetic counselling to their families. The method used was to check the census of the Telephone Company Handicapped Assistance Association (ATAM), which embraces all handicapped dependents of TESA employees. 386 mentally backward (IQ under 70) males were found. A cytogenic study was offered to all those cases of unknown etiology. A total of 49 cases (12.7%) without any data to lead us into thinking of other etiological factors refused to take part. Molecular genetic studies were carried out on families with male members diagnosed as having X-fragile syndrome. The results were as follows: the total number of patients suffering with this syndrome was eleven. The incidence rates of X-fragile syndrome in the group studied was 14.3/386 (3.7% of mentally backward males). Molecular genetics was more sensitive than cytogenetics in diagnosing the state of mother and sister carriers.

Gaseous emissions from a heavy-duty engine equipped with SCR aftertreatment system and fuelled with diesel and biodiesel: assessment of pollutant dispersion and health risk.

The changes in the composition of fuels in combination with selective catalytic reduction (SCR) emission control systems bring new insights into the emission of gaseous and particulate pollutants. The major goal of our study was to quantify NOx, NO, NO2, NH3 and N2O emissions from a four-cylinder diesel engine operated with diesel and a blend of 20% soybean biodiesel. Exhaust fume samples were collected from bench dynamometer tests using a heavy-duty diesel engine equipped with SCR. The target gases were quantified by means of Fourier transform infrared spectrometry (FTIR). The use of biodiesel blend presented lower concentrations in the exhaust fumes than using ultra-low sulfur diesel. NOx and NO concentrations were 68% to 93% lower in all experiments using SCR, when compared to no exhaust aftertreatment. All fuels increased NH3 and N2O emission due to SCR, a precursor secondary aerosol, and major greenhouse gas, respectively. An AERMOD dispersion model analysis was performed on each compound results for the City of Curitiba, assumed to have a bus fleet equipped with diesel engines and SCR system, in winter and summer seasons. The health risks of the target gases were assessed using the Risk Assessment Information System For 1-h exposure of NH3, considering the use of low sulfur diesel in buses equipped with SCR, the results indicated low risk to develop a chronic non-cancer disease. The NOx and NO emissions were the lowest when SCR was used; however, it yielded the highest NH3 concentration. The current results have paramount importance, mainly for countries that have not yet adopted the Euro V emission standards like China, India, Australia, or Russia, as well as those already adopting it. These findings are equally important for government agencies to alert the need of improvements in aftertreatment technologies to reduce pollutants emissions.

In vitro characterization of [18F]-florbetaben, an Aß imaging radiotracer.

PURPOSE: Amyloid-ß (Aß) plaques are a major pathological hallmark of Alzheimer's disease (AD). The noninvasive detection of Aß plaques may increase the accuracy of clinical diagnosis as well as monitor therapeutic interventions. While [(11)C]-PiB is the most widely used Aß positron emission tomography (PET) radiotracer, due to the short half-life of (11)C (20 min), its application is limited to centers with an on-site cyclotron and (11)C radiochemistry expertise. Therefore, novel [(18)F] (half-life 110 min)-labeled Aß PET tracers have been developed. We have demonstrated that [(18)F]-florbetaben-PET can differentiate individuals diagnosed with AD from healthy elderly, Parkinson's disease and frontotemporal lobe dementia (FTLD-tau) patients. While [(18)F]-florbetaben-PET retention matched the reported postmortem distribution of Aß plaques, the nature of [(18)F]-florbetaben binding to other pathological lesions comprising misfolded proteins needs further assessment. The objective of this study was to determine whether Florbetaben selectively binds to Aß plaques in postmortem tissue specimens containing mixed pathological hallmarks (i.e., tau and α-synuclein aggregates). METHOD: Human AD, FTLD-tau and dementia with Lewy bodies (DLB) brain sections were analyzed by [(18)F]-florbetaben autoradiography and [(3)H]-florbetaben high-resolution emulsion autoradiography and [(19)F]-florbetaben fluorescence microscopy. RESULTS: Both autoradiographical analyses demonstrated that Florbetaben exclusively bound Aß plaques in AD brain sections at low nanomolar concentrations. Furthermore, at concentrations thousand-folds higher than those during a PET scan, [(19)F]-florbetaben did not bind to α-synuclein or tau aggregates in DLB and FTLD-tau brain sections, respectively. Detection of [(19)F]-florbetaben staining by fluorescence microscopy in several AD brain regions demonstrated that Florbetaben identified Aß plaques in all brain regions examined. CONCLUSION: This study provides further evidence that [(18)F]-florbetaben-PET is a highly selective radiotracer to assess Aß plaque deposition in the brain.

Substrate requirements for SPPL2b-dependent regulated intramembrane proteolysis.

Intramembrane proteolysis is now widely recognized as an important physiological pathway required for reverse signaling and membrane protein degradation. Aspartyl intramembrane cleaving proteases of the GXGD-type play an important regulatory role in health and disease. Besides gamma-secretase/presenilin, signal peptide peptidase (SPP) and SPP-like (SPPL) peptidases also belong to the family of GXGD-type aspartyl proteases. Although recently the first SPPL2a/b substrates have been identified, very little is known about substrate requirements, which allow them to be efficiently processed within the membrane. We demonstrate that similar to gamma-secretase substrates, intramembrane proteolysis of Bri2 (Itm2b) is greatly facilitated by an initial shedding event mediated by ADAM-10. Serial deletions revealed that the length of the ectodomain negatively correlates with efficient intramembrane proteolysis. Bri3 (Itm2c), which is highly homologous to Bri2, fails to be shed. Failure of shedding of Bri3 is accompanied by a lack of intramembrane proteolysis by SPPL2b. Surprisingly, a low molecular weight membrane-retained stub of Bri3 also fails to be processed by SPPL2b, indicating that shedding per se is not sufficient for subsequent intramembrane proteolysis. Extensive domain swapping analysis reveals that primary sequence determinants within the intracellular domain and the transmembrane domain together with short luminal juxtamembrane sequences are required for efficient intramembrane proteolysis.

Regulated intramembrane proteolysis of Bri2 (Itm2b) by ADAM10 and SPPL2a/SPPL2b.

Presenilin, the catalytic component of the gamma-secretase complex, type IV prepilin peptidases, and signal peptide peptidase (SPP) are the founding members of the family of intramembrane-cleaving GXGD aspartyl proteases. SPP-like (SPPL) proteases, such as SPPL2a, SPPL2b, SPPL2c, and SPPL3, also belong to the GXGD family. In contrast to gamma-secretase, for which numerous substrates have been identified, very few in vivo substrates are known for SPP and SPPLs. Here we demonstrate that Bri2 (Itm2b), a type II-oriented transmembrane protein associated with familial British and Danish dementia, undergoes regulated intramembrane proteolysis. In addition to the previously described ectodomain processing by furin and related proteases, we now describe that the Bri2 protein, similar to gamma-secretase substrates, undergoes an additional cleavage by ADAM10 in its ectodomain. This cleavage releases a soluble variant of Bri2, the BRICHOS domain, which is secreted into the extracellular space. Upon this shedding event, a membrane-bound Bri2 N-terminal fragment remains, which undergoes intramembrane proteolysis to produce an intracellular domain as well as a secreted low molecular weight C-terminal peptide. By expressing all known SPP/SPPL family members as well as their loss of function variants, we demonstrate that selectively SPPL2a and SPPL2b mediate the intramembrane cleavage, whereas neither SPP nor SPPL3 is capable of processing the Bri2 N-terminal fragment.

Intramembrane proteolysis of GXGD-type aspartyl proteases is slowed by a familial Alzheimer disease-like mutation.

More than 150 familial Alzheimer disease (FAD)-associated missense mutations in presenilins (PS1 and PS2), the catalytic subunit of the gamma-secretase complex, cause aberrant amyloid beta-peptide (Abeta) production, by increasing the relative production of the highly amyloidogenic 42-amino acid variant. The molecular mechanism behind this pathological activity is unclear, and different possibilities ranging from a gain of function to a loss of function have been discussed. gamma-Secretase, signal peptide peptidase (SPP) and SPP-like proteases (SPPLs) belong to the same family of GXGD-type intramembrane cleaving aspartyl proteases and share several functional similarities. We have introduced the FAD-associated PS1 G384A mutation, which occurs within the highly conserved GXGD motif of PS1 right next to the catalytically critical aspartate residue, into the corresponding GXGD motif of the signal peptide peptidase-like 2b (SPPL2b). Compared with wild-type SPPL2b, mutant SPPL2b slowed intramembrane proteolysis of tumor necrosis factor alpha and caused a relative increase of longer intracellular cleavage products. Because the N termini of the secreted counterparts remain unchanged, the mutation selectively affects the liberation of the intracellular processing products. In vitro experiments demonstrate that the apparent accumulation of longer intracellular cleavage products is the result of slowed sequential intramembrane cleavage. The longer cleavage products are still converted to shorter peptides, however only after prolonged incubation time. This suggests that FAD-associated PS mutation may also result in reduced intramembrane cleavage of beta-amyloid precursor protein (betaAPP). Indeed, in vitro experiments demonstrate slowed intramembrane proteolysis by gamma-secretase containing PS1 with the G384A mutation. As compared with wild-type PS1, the mutation selectively slowed Abeta40 production, whereas Abeta42 generation remained unaffected. Thus, the PS1 G384A mutation causes a selective loss of function by slowing the processing pathway leading to the benign Abeta40.