Factors associated with post-hospitalization dialysis dependence in ECMO patients who required continuous renal replacement therapy.

OBJECTIVE: This single center retrospective study aimed to describe the variables associated with outpatient dialysis dependence in extracorporeal membrane oxygenation (ECMO) patients who needed continuous renal replacement therapy (CRRT) for acute kidney injury (AKI) during their hospitalization. METHODS: Retrospective study of patients who required ECMO-CRRT. RESULTS: Between the years of 2016 and 2022, 202 patients required ECMO-CRRT. One hundred and six patients (52.5%) survived their hospitalization and were followed up for a median of 391 [133, 1005] days. Eighty-one patients (76.5%) recovered kidney function and were dialysis-free before hospital discharge. Twenty-five patients (23.5%) were hemodialysis-dependent after hospitalization. On multivariate regression analysis, hyperlipidemia (odds ratio, OR 6.08 [1.67-22]) and CRRT duration (OR 1.09 [1.03-1.15]) were associated with the need for dialysis post-hospitalization. In this group, 16 patients eventually became dialysis-free, after a median of 49 [34.7, 78.5] days. These patients had a higher median baseline glomerular filtration rate (GFR) compared to those who never recovered renal function (93 mL/min/1.73 m2 [82.4, 104.3] vs. 63.8 mL/min/1.73 m2 [37.9, 83], p = .009). Their follow-up GFR was lower compared to those who recovered renal function before hospital discharge; (87 mL/min/1.73 m2 [68.2, 98.9] vs. 99 mL/min/1.73 m2 [79, 118], p = .07). CONCLUSIONS: AKI requiring CRRT was associated with high mortality in patients receiving ECMO. Nonetheless, most ECMO survivors became dialysis-free before hospital discharge. Variables associated with the need for outpatient dialysis included hyperlipidemia and prolonged need for CRRT during hospitalization.

Bacterial and parasite co-infection in Mexican golden trout (Oncorhynchus chrysogaster) by Aeromonas bestiarum, Aeromonas sobria, Plesiomonas shigelloides and Ichthyobodo necator.

BACKGROUND: Bacterial infections are responsible of high economic losses in aquaculture. Mexican golden trout (Oncorhynchus chrysogaster) is a threatened native trout species that has been introduced in aquaculture both for species conservation and breeding for production and for which no studies of bacterial infections have been reported. CASE PRESENTATION: Fish from juvenile stages of Mexican golden trout showed an infectious outbreak in a farm in co-culture with rainbow trout (Oncorhynchus mykiss), showing external puntiform red lesions around the mouth and caudal pedunculus resembling furuncles by Aeromonas spp. and causing an accumulated mortality of 91%. Isolation and molecular identification of bacteria from lesions and internal organs showed the presence of Aeromonas bestiarum, Aeromonas sobria, Plesiomonas shigelloides and Ichthyobodo necator isolated from a single individual. All bacterial isolates were resistant to amoxicillin-clavulanic acid and cefazoline. P. shigelloides was resistant to third generation ß-lactamics. CONCLUSIONS: This is the first report of coinfection by Aeromonas bestiarum, Aeromonas sobria, Plesiomonas shigelloides and Ichthyobodo necator in an individual of Mexican golden trout in co-culture with rainbow trout. Resistance to ß-lactams suggests the acquisition of genetic determinants from water contamination by human- or livestock-associated activities.

Effect of oils on the transmission properties of a terahertz photonic crystal.

The transmission properties of a photonic crystal immersed in several different oils have been characterized using terahertz time domain spectroscopy in the spectral range of 0.3-1.5 THz. As in previous works, oil samples can be distinguished using terahertz transmission measurements. When the same oils are introduced into a photonic crystal, we find that the effective refractive index of the photonic crystal is sensitive to the properties of the oils and shows differences not seen in bulk measurements. These effects are described in detail and have potential applications in both the sensing of very small volumes of oils and in the fine control of the refractive indices of photonic crystals.

Influence of Mediterranean climate and lunar calendar on milk production in Lacaune breed ewes.

Environmental factors affect daily milk production in dairy animals. The aim of this study was to quantify the effects of environmental factors, specifically mean temperature (°C), relative humidity (%), temperature-humidity index (THI), solar radiation (°), pluvial precipitation (mm) and lunar calendar (full moon, waxing quarter, waning quarter, new moon), on milk production (kg/d). The analysis was based on 96,195 morning and evening milking records documented on 109 consecutive days, from 869 Lacaune ewes. Ewes were housed in groups of 174 individuals. The analysis was performed in two independent procedures, a Pearson correlation analysis and a multivariate analysis of the ewe's interrelationships, which was based on the total variance estimate and a Varimax-rotated factorial analysis. Milk yield (kg/d) was significantly (p < 0.05) negatively correlated with mean temperature (-0.24), relative humidity (-0.16), THI (-0.24), and radiation (-0.18), which suggests that the higher these environmental factors, the lower the milk yield. Lunar calendar had a significant (p < 0.01) effect on milk production yield; specifically, yields were higher on the full moon and new moon (2.25 ± 0.05 kg/day) than they were on the crescent or waning moon (2.17 ± 0.05 kg/day). In conclusion, ewes that had been exposed to higher mean temperature, relative humidity, THI and solar radiation had the lowest milk yield, and milk yields are highest on full and new moons. The results of this work may be helpful in making predictions for milk production in Lacaune ewes in the Mediterranean region throughout the year.

Sub-wavelength continuous THz imaging system based on interferometric detection.

We have developed a continuous wave sub-wavelength terahertz (THz) imaging system that combines two prominent classical optical techniques: solid immersion microscopy and interferometric detection. This combination allows for resolution beyond the diffraction limit at 703 GHz. We experimentally demonstrate sub-wavelength spatial resolution working with a relatively low-cost pyroelectric detector and with both high and low contrast samples.

Molecular mechanism for bidirectional regulation of CD44 for lipid raft affiliation by palmitoylations and PIP2.

The co-localization of Cluster-of-Differentiation-44 protein (CD44) and cytoplasmic adaptors in specific membrane environments is crucial for cell adhesion and migration. The process is controlled by two different pathways: On the one hand palmitoylation keeps CD44 in lipid raft domains and disables the linking to the cytoplasmic adaptor, whereas on the other hand, the presence of phosphatidylinositol-4,5-biphosphate (PIP2) lipids accelerates the formation of the CD44-adaptor complex. The molecular mechanism explaining how CD44 is migrating into and out of the lipid raft domains and its dependence on both palmitoylations and the presence of PIP2 remains, however, elusive. In this study, we performed extensive molecular dynamics simulations to study the raft affinity and translocation of CD44 in phase separated model membranes as well as more realistic plasma membrane environments. We observe a delicate balance between the influence of the palmitoylations and the presence of PIP2 lipids: whereas the palmitoylations of CD44 increases the affinity for raft domains, PIP2 lipids have the opposite effect. Additionally, we studied the association between CD44 and the membrane adaptor FERM in dependence of these factors. We find that the presence of PIP2 lipids allows CD44 and FERM to associate in an experimentally observed binding mode whereas the highly palmitoylated species shows no binding affinity. Together, our results shed light on the sophisticated mechanism on how membrane translocation and peripheral protein association can be controlled by both protein modifications and membrane composition.

Computational Prediction of ω-Transaminase Specificity by a Combination of Docking and Molecular Dynamics Simulations.

ω-Transaminases (ω-TAs) catalyze the conversion of ketones to chiral amines, often with high enantioselectivity and specificity, which makes them attractive for industrial production of chiral amines. Tailoring ω-TAs to accept non-natural substrates is necessary because of their limited substrate range. We present a computational protocol for predicting the enantioselectivity and catalytic selectivity of an ω-TA from Vibrio fluvialis with different substrates and benchmark it against 62 compounds gathered from the literature. Rosetta-generated complexes containing an external aldimine intermediate of the transamination reaction are used as starting conformations for multiple short independent molecular dynamics (MD) simulations. The combination of molecular docking and MD simulations ensures sufficient and accurate sampling of the relevant conformational space. Based on the frequency of near-attack conformations observed during the MD trajectories, enantioselectivities can be quantitatively predicted. The predicted enantioselectivities are in agreement with a benchmark dataset of experimentally determined ee% values. The substrate-range predictions can be based on the docking score of the external aldimine intermediate. The low computational cost required to run the presented framework makes it feasible for use in enzyme design to screen thousands of enzyme variants.

Sperm characterization and cryopreservation of the endangered freshwater fish Chirostoma estor (Atheriniformes).

The knowledge of the physiology of sperm of an endangered species allows the implantation of reproductive biotechnologies that aim at conservation. The aim of this study was to characterize fresh sperm and evaluate different cryopreservation solutions for sperm in Chirostoma estor. The characterization of Chirostoma estor fresh sperm (n = 22 males) was performed through analyzes of sperm concentration, membrane integrity, sperm morphology, motility rate, motility quality score, and motility duration. For cryopreservation (n = 42 males), 3 extenders (BTS™, MIII™, or Androstar Plus™) in combination with 2 permeable cryoprotectants (dimethyl sulfoxide (DMSO) or methyl glycol (Methyl)) were used. Analyzes of post-thaw sperm were performed as described for fresh sperm and additionally the fertilization rate analysis was performed. Fresh sperm presented a sperm concentration of 29.2 × 109 spermatozoa/mL, membrane integrity of 82.4%, and morphologically normal cells of 53%. After glucose activation (150 mM) a motility rate of 87.5%, sperm quality score of 5.0, and a duration of motility of 285 s were observed. For post-thaw sperm, MIII + Methyl and Androstar + Methyl solutions resulted in the highest motility rates of 40-48%. No differences were observed for motility duration, membrane integrity, and sperm morphology. Samples cryopreserved in Methyl (12-20%) showed a higher fertilization rate than DMSO, independently of the extender. In conclusion, the fresh sperm collected artificially from Chirostoma estor presents a compatible quality to carry out fertilization and can be cryopreserved in the commercial extenders MIII™ and Androstar Plus™ together with the cryoprotectant Methyl glycol.

Chronic kidney disease recognition amongst physicians and advanced practice providers.

OBJECTIVE: Chronic kidney disease is a worldwide public health issue, with increasing prevalence resulting in high morbidity and mortality. As a result, recognizing and treating it early can lead to improved outcomes. We hypothesized that some providers might be more comfortable making this diagnosis than others. METHODS: Retrospective study of 380 patients with chronic kidney disease seen between 2012 and 2016 in an outpatient setting. RESULTS: Three hundred and sixteen patients were treated by physicians and sixty-four by advanced practice providers. Chronic kidney disease was identified by the primary care providers in 318 patients (83.6%). Patients recognized with chronic kidney disease were older, 76 ± 8.8 vs 72 ± 7.45 years, p = 0.001; had lower GFR, 37 [29, 46] vs 57 [37, 76] ml/min/1.73 m2, p < 0.0001 and were more likely to be seen by a physician compared to an advanced practice provider: 272/316 (86%) vs 46/64 (71.8%), p = 0.008. In multivariate analyses, care by a physician, OR = 2.27 (1.13-4.58), p = 0.02 was associated with increased recognition of chronic kidney disease. On the other hand, higher GFR was associated with decreased diagnosis of chronic kidney disease, OR = 0.95 (0.93-0.96), p < 0.0001. CONCLUSION: The odds of chronic kidney disease recognition were higher amongst physicians in comparison to non-physician providers.

Knowledge Discovery for Higher Education Student Retention Based on Data Mining: Machine Learning Algorithms and Case Study in Chile.

Data mining is employed to extract useful information and to detect patterns from often large data sets, closely related to knowledge discovery in databases and data science. In this investigation, we formulate models based on machine learning algorithms to extract relevant information predicting student retention at various levels, using higher education data and specifying the relevant variables involved in the modeling. Then, we utilize this information to help the process of knowledge discovery. We predict student retention at each of three levels during their first, second, and third years of study, obtaining models with an accuracy that exceeds 80% in all scenarios. These models allow us to adequately predict the level when dropout occurs. Among the machine learning algorithms used in this work are: decision trees, k-nearest neighbors, logistic regression, naive Bayes, random forest, and support vector machines, of which the random forest technique performs the best. We detect that secondary educational score and the community poverty index are important predictive variables, which have not been previously reported in educational studies of this type. The dropout assessment at various levels reported here is valid for higher education institutions around the world with similar conditions to the Chilean case, where dropout rates affect the efficiency of such institutions. Having the ability to predict dropout based on student's data enables these institutions to take preventative measures, avoiding the dropouts. In the case study, balancing the majority and minority classes improves the performance of the algorithms.

Information retrieval using overlapping holograms with partial complementarity.

An information retrieval technique from superimposed holograms representing 2D and 3D objects using complementary fringes is presented. By adding two different computer generated holograms with quasi-complementarity information is possible to retrieve information at a specific depth.

Experimental evidence of masculinization by continuous illumination in a temperature sex determination teleost (Atherinopsidae) model: is oxidative stress involved?

The present study evaluates the influence of continuous light on phenotypic sex ratios in Chirostoma estor, a temperature sex determination animal model. Relative gene expression levels of 5 day old larvae were performed on two early gonad differentiation genes (sox9 and foxl2), two stress axis activation genes (gcr1 and crf) and four reactive oxygen species (ROS) antagonist effector genes (sod2, ucp2, gsr and cat). Two light treatments were applied from fertilization; control (12L:12D) simulated natural photoperiod and a continuous illumination photoperiod. By the end of the trial (12 weeks after hatching), differentiated and normal gonads were clearly identifiable in both treatments by histological observations. Regarding sex ratio, 73% of phenotypic males were found in continuous illumination compared with 40% in controls. Consistently, the sox9 gene (involved in early testis differentiation) showed an over expression in 64% of the individual larvae analysed compared with foxl2 (ovarian differentiation) suggesting a masculinization tendency in continuous illumination. On the other hand, only 36% of individuals showed the same tendency in the control treatment consistent with phenotypic sex ratios found under normal culture conditions. Relative gene expression results did not show significant difference in sod2, ucp2 and gcr1 levels, but cat, gsr and crf showed significantly higher expression levels in the continuous illumination treatment suggesting that both, the stress axis and ROS response mechanisms were activated at this time. This study suggests, a link between continuous light, oxidative stress and environmental sex determination in vertebrates. However, further research is necessary to describe this possible upstream mechanism that may drive some aspects of sexual plasticity in vertebrates.

Clinical outcomes with a systolic blood pressure lower than 120 mmHg in older patients with high disease burden.

BACKGROUND: The target blood pressure in older patients is controversial. Recent studies provided clinical evidence supporting a target systolic blood pressure <120 mmHg in patients >50 years at high risk of cardiovascular events. METHODS: Retrospective study of 380 consecutive patients ≥60 years with stages 1-5 pre-dialysis chronic kidney disease seen between January 2013 and November 2015. The outcomes of a systolic blood pressure <120 mmHg in older patients with chronic kidney disease and multiple comorbidities were analyzed. RESULTS: Sixty-eight patients had a systolic blood pressure <120 mmHg, 312 patients had a systolic blood pressure ≥120 mmHg. Forty-three patients died during the follow up (11.3%). Patients with a systolic blood pressure <120 mmHg had a higher risk of death: 21 (30.9%) vs 22 (7%). Primary cause of death: Cardiovascular: 11 (25.6%), infectious 9 (20.9%), cancer 5 (11.6%), renal failure 6 (13.9%), COPD/pulmonary fibrosis 2 (4.6%), end stage liver disease 3 (6.9%), traumatic brain injury 1 (2.3%), gastrointestinal hemorrhage 4 (9.3%), complications of diabetes 1 (2.3%), unknown 1 (2.3%). After adjusting for confounding factors, a systolic blood pressure <120 mmHg remained associated with increased mortality. There was a trend to more cardiovascular outcomes in those with a lower blood pressure. CONCLUSIONS: A systolic blood pressure below 120 mmHg in older patients with high disease burden was associated with adverse outcomes. Individualization of blood pressure therapy to each specific patient is warranted.

Identification, molecular and phylogenetic analysis of poxvirus in skin lesions of southern right whale.

Poxvirus skin disease has been reported in several species of cetaceans, principally in odontocetes, and a single report in mysticetes. Southern right whales Eubalaena australis in Peninsula Valdes, Argentina, show a variety of skin lesions of unknown etiology, and the number of these lesions has increased in recent years. Samples from dead whales were taken in order to establish the etiology of these lesions. One calf and one adult presented ring-type lesions, characterized by a circumscribed and slightly raised area of skin. Lesions were histologically characterized by the presence of microvesicles and vacuolated cells in the stratum spinosum, along with hyperplasia of the stratum corneum and eosinophilic inclusion bodies in the cytoplasm of the epithelial cells. Transmission electron microscopy showed aggregations of virions with typical poxvirus morphology. PCR of cetacean poxvirus (CPV) DNA polymerase, DNA topoisomerase I and parapoxvirus DNA polymerase gene fragments was done, and confirmed the presence of poxvirus in one sample. Phylogenetic analysis showed that the detected poxvirus belongs to the CPV-2 group. This is the first confirmed report of poxvirus in southern right whales in Argentina.

Avoidance of polypharmacy and excessive blood pressure control is associated with improved renal function in older patients.

BACKGROUND: Elderly patients are particularly susceptible to polypharmacy. The present study evaluated the renal effects of optimizing potentially nephrotoxic medications in an older population. METHODS: Retrospective study of patients' ≥ 60 years treated between January of 2013 and February of 2015 in a Nephrology Clinic. The renal effect of avoiding polypharmacy was studied. RESULTS: Sixty-one patients were studied. Median age was 81 years (range 60-94). Twenty-five patients (41%) were male. NSAIDs alone were stopped in seven patients (11.4%), a dose reduction in antihypertensives was done in 11 patients (18%), one or more antihypertensives were discontinued in 20 patients (32.7%) and discontinuation and dose reduction of multiple medications was carried out in 23 patients (37.7%). The number of antihypertensives was reduced from a median of 3 (range of 0-8) at baseline to a median of 2 (range 0-7), p < 0.001 after intervention. After intervention, the glomerular filtration rate (GFR) improved significantly, from a baseline of 32 ± 15.5 cc/min/1.73 m(2) to 39.5 ± 17 cc/min/1.73 m(2) at t1 (p < 0.001) and 44.5 ± 18.7 cc/min/1.73 m(2) at t2 (p < 0.001 vs. baseline). In a multivariate model, after adjusting for ACEIs/ARBs discontinuation/dose reduction, NSAIDs use and change in DBP, an increase in SBP at time 1 remained significantly associated with increments in GFR on follow-up (estimate = 0.20, p = 0.01). CONCLUSIONS: Avoidance of polypharmacy was associated with an improvement in renal function.

Diversification of substrate specificities in teleostei Fads2: characterization of Δ4 and Δ6Δ5 desaturases of Chirostoma estor.

Currently existing data show that the capability for long-chain PUFA (LC-PUFA) biosynthesis in teleost fish is more diverse than in other vertebrates. Such diversity has been primarily linked to the subfunctionalization that teleostei fatty acyl desaturase (Fads)2 desaturases have undergone during evolution. We previously showed that Chirostoma estor, one of the few representatives of freshwater atherinopsids, had the ability for LC-PUFA biosynthesis from C18 PUFA precursors, in agreement with this species having unusually high contents of DHA. The particular ancestry and pattern of LC-PUFA biosynthesis activity of C. estor make this species an excellent model for study to gain further insight into LC-PUFA biosynthetic abilities among teleosts. The present study aimed to characterize cDNA sequences encoding fatty acyl elongases and desaturases, key genes involved in the LC-PUFA biosynthesis. Results show that C. estor expresses an elongase of very long-chain FA (Elovl)5 elongase and two Fads2 desaturases displaying Δ4 and Δ6/Δ5 specificities, thus allowing us to conclude that these three genes cover all the enzymatic abilities required for LC-PUFA biosynthesis from C18 PUFA. In addition, the specificities of the C. estor Fads2 enabled us to propose potential evolutionary patterns and mechanisms for subfunctionalization of Fads2 among fish lineages.

Carcass traits and meat quality assessment of two slow-growing chicks strains fed Acheta domesticus larval meal.

Presently, there has been a noticeable rise in the consumption of poultry meat within the general population, particularly focusing on poultry sourced from alternative rearing systems as opposed to intensive ones. This study evaluated the impact of incorporating house cricket (Acheta domesticus,AD) larvae meal into the diet of 2 slow-growing chicken strains on their carcass traits and meat quality. A total of 256 one-day-old male chicks were utilized, with 128 belonging to the Red (R) strain and 128 to the Naked Neck (NN) strain. Chicks from each strain were randomly assigned to 16 replicates of 8 chicks each. Eight replicates were allocated to 2 distinct treatments (n=64 chicks/treatment) based on the diet employed: the control treatments for the Red (R-C) and Naked Neck (NN-C) strains were fed soybean meal, while the other 2 treatments were fed AD meal (R-AD and NN-AD). Three different rations were used throughout the growth cycle of the animals, tailored to meet the nutritional needs of the birds. All chickens were slaughtered at 95 d of age, and eight chickens per treatment were randomly chosen to assess carcass traits and meat quality. Near-infrared reflectance spectroscopy (NIRS) was employed to classify meat based on the feed and the chick strain, achieving a correct discrimination of 100% of the samples. The carcass traits exhibited significant alterations due to the inclusion of insect larval meal in the diet, resulting in reduced values across all parameters for chickens consuming AD meal, irrespective of genotype. The pH and cooking losses were generally higher for chicks fed cricket meal, as occurred with moisture. However, protein content was lower in the meat of chicks consuming cricket meal. Fat content showed lower values for NN chickens. There were minimal differences in the fatty acid profile, with lower C18:3n-3 values for chickens fed cricket meal. Therefore, including Acheta domesticus meal in the diets of slow-growing chick strains is a viable alternative to replace soybean meal. It provides meat quality characteristics comparable to those obtained in conventional systems.

Combined Metabolomic and NIRS Analyses Reveal Biochemical and Metabolite Changes in Goat Milk Kefir under Different Heat Treatments and Fermentation Times.

Dairy products are an important source of protein and other nutrients in the Mediterranean diet. In these countries, the most common sources of milk for producing dairy products are cow, goat, sheep, and buffalo. Andalusia is traditionally the largest producer of goat milk in Spain. Kefir is a fermented product made from bacteria and yeasts and has health benefits beyond its nutritional properties. There is a lack of knowledge about the molecular mechanisms and metabolites that bring about these benefits. In this work, the combination of analytical techniques (GC-FID, UHPLC-MS-QToF, GC-QqQ-MS, and GC-ToF-MS) resulted in the detection of 105 metabolites in kefir produced with goat milk from two different thermal treatments (raw and pasteurized) fermented at four time points (12, 24, 36, and 48 h, using 0 h as the control). Of these, 27 metabolites differed between kefir produced with raw and pasteurized milk. These changes could possibly be caused by the effect of pasteurization on the microbial population in the starting milk. Some interesting molecules were identified, such as shikimic acid, dehydroabietic acid, GABA, and tyramine, which could be related to antibacterial properties, strengthening of the immune system, and arterial pressure. Moreover, a viability assay of the NIRS technique was performed to evaluate its use in monitoring the fermentation and classification of samples, which resulted in a 90% accuracy in comparison to correctly classified samples according to their fermentation time. This study represents the most comprehensive metabolomic analysis of goat milk kefir so far, revealing the intricate changes in metabolites during fermentation and the impact of milk treatment.

Effect of pH and temperature on tropane alkaloids within a processing strategy to provide safe infant cereal-based food.

Tropane alkaloids (TAs) are secondary metabolites from weeds that can contaminate cereals and vegetables during harvest. Due to their toxicity, the Regulation (EC) 2023/915 sets maximum levels for atropine and scopolamine in cereal-based foods for infants containing millet, sorghum, buckwheat or their derived products. The aim of this study was to evaluate the effect of pH and temperature on the stability of TAs, as possible parameters in thermal processing to mitigate this chemical hazard in cereal-based infant food. The effect of pH (4 and 7) and temperature (80 °C and 100 °C) was assessed in buffer solutions. Also, treatment at 180 °C was performed in spiked and naturally incurred millet flour to assess the effect of high temperature, simulating cooking or drying, on the stability of TAs in the cereal matrix. The fate of 24 TAs was assessed by UHPLC-MS/MS. TAs showed high thermostability, although it was variable depending on the specific compound, pH, temperature and treatment time. In buffer solutions, higher degradation was found at 100 °C and pH 7. In spiked millet flour at 180 °C for 10 min, scopolamine and atropine contents decreased by 25 % and 22 %, similarly to other TAs which also showed a slow thermal degradation. Atropine, scopolamine, anisodamine, norscopolamine, scopine and scopoline were found in naturally contaminated millet flour. Interestingly, naturally incurred atropine was more thermostable than when spiked, showing a protective effect of the cereal matrix on TAs degradation. The present results highlight the need for an accurate monitorization of TAs in raw materials, as this chemical hazard may remain in infant cereal-based food even after intense thermal processing.

Structural and time-resolved mechanistic investigations of protein hydrolysis by the acidic proline-specific endoprotease from Aspergillus niger.

Proline-specific endoproteases have been successfully used in, for example, the in-situ degradation of gluten, the hydrolysis of bitter peptides, the reduction of haze during beer production, and the generation of peptides for mass spectroscopy and proteomics applications. Here we present the crystal structure of the extracellular proline-specific endoprotease from Aspergillus niger (AnPEP), a member of the S28 peptidase family with rarely observed true proline-specific endoprotease activity. Family S28 proteases have a conventional Ser-Asp-His catalytic triad, but their oxyanion-stabilizing hole shows a glutamic acid, an amino acid not previously observed in this role. Since these enzymes have an acidic pH optimum, the presence of a glutamic acid in the oxyanion hole may confine their activity to an acidic pH. Yet, considering the presence of the conventional catalytic triad, it is remarkable that the A. niger enzyme remains active down to pH 1.5. The determination of the primary cleavage site of cytochrome c along with molecular dynamics-assisted docking studies indicate that the active site pocket of AnPEP can accommodate a reverse turn of approximately 12 amino acids with proline at the S1 specificity pocket. Comparison with the structures of two S28-proline-specific exopeptidases reveals not only a more spacious active site cavity but also the absence of any putative binding sites for amino- and carboxyl-terminal residues as observed in the exopeptidases, explaining AnPEP's observed endoprotease activity.