Epileptic Encephalopathy with Variants in the PHACTR1 and AFF2 Genes: A Case Report.

Developmental and epileptic encephalopathy 70 (DEE70) is an epileptic encephalopathy associated with multiple neurological abnormalities and global developmental delay, among other characteristics. It has recently been established that it is caused by a heterozygous variant of the PHACTR1 gene, with currently four cases reported in the literature. This article presents a case report of a patient with DEE70 with a heterozygous variant in the PHACTR1 gene, who also presents a hemizygous variant in the AFF2 gene, associated with FRAXE syndrome. A phenotypic comparison is made between this case and the four other previously reported cases with variants in the PHACTR1 gene. In addition, the possible participation of the PHACTR1 and AFF2 genes in the clinical characteristics of the individual is discussed.

Predicting Perceived Exhaustion in Rehabilitation Exercises Using Facial Action Units.

Physical exercise has become an essential tool for treating various non-communicable diseases (also known as chronic diseases). Due to this, physical exercise allows to counter different symptoms and reduce some risk of death factors without medication. A solution to support people in doing exercises is to use artificial systems that monitor their exercise progress. While one crucial aspect is to monitor the correct physical motions for rehabilitative exercise, another essential element is to give encouraging feedback during workouts. A coaching system can track a user's exhaustion and give motivating feedback accordingly to boost exercise adherence. For this purpose, this research investigates whether it is possible to predict the subjective exhaustion level based on non-invasive and non-wearable technology. A novel data set was recorded with the facial record as the primary predictor and individual exhaustion levels as the predicted variable. 60 participants (30 male, 30 female) took part in the data recording. 17 facial action units (AU) were extracted as predictor variables for the perceived subjective exhaustion measured using the BORG scale. Using the predictor and the target variables, several regression and classification methods were evaluated aiming to predict exhaustion. The results showed that the decision tree and support vector methods provide reasonable prediction results. The limitation of the results, depending on participants being in the training data set and subjective variables (e.g., participants smiling during the exercises) were further discussed.

Machine Learning Approach for Fatigue Estimation in Sit-to-Stand Exercise.

Physical exercise (PE) has become an essential tool for different rehabilitation programs. High-intensity exercises (HIEs) have been demonstrated to provide better results in general health conditions, compared with low and moderate-intensity exercises. In this context, monitoring of a patients' condition is essential to avoid extreme fatigue conditions, which may cause physical and physiological complications. Different methods have been proposed for fatigue estimation, such as: monitoring the subject's physiological parameters and subjective scales. However, there is still a need for practical procedures that provide an objective estimation, especially for HIEs. In this work, considering that the sit-to-stand (STS) exercise is one of the most implemented in physical rehabilitation, a computational model for estimating fatigue during this exercise is proposed. A study with 60 healthy volunteers was carried out to obtain a data set to develop and evaluate the proposed model. According to the literature, this model estimates three fatigue conditions (low, moderate, and high) by monitoring 32 STS kinematic features and the heart rate from a set of ambulatory sensors (Kinect and Zephyr sensors). Results show that a random forest model composed of 60 sub-classifiers presented an accuracy of 82.5% in the classification task. Moreover, results suggest that the movement of the upper body part is the most relevant feature for fatigue estimation. Movements of the lower body and the heart rate also contribute to essential information for identifying the fatigue condition. This work presents a promising tool for physical rehabilitation.

A novel lecithin:cholesterol acyltransferase for soybean oil refining provides higher yields and extra nutritional value with a cleaner process.

The growing demand for food and biofuels urges the vegetable oil processing industry to adopt cleaner technologies to mitigate the environmental pollution caused by chemical refining processes. Over the past decade, several enzymatic methods have proven to be efficient at reducing the generated waste, but improving the benefit-cost ratio is still necessary for the widespread adoption of this technology. In this work, we show that lecithin:cholesterol acyltransferase from Aeromonas enteropelogenes (LCATAE) provides a higher extra-yield of soybean oil than a type A1 phospholipase (PLA) enzyme currently commercialized for soybean oil deep degumming. Our model indicates that crude soybean oil treated with the new enzyme generates 87% more neutral oil from phospholipids than the widely used PLA, with the corresponding reduction in waste and byproducts generation. The refined oil retains the phytosterols naturally present in crude oil, enriching its nutritional value. The results presented here position LCATAE as a promising candidate to provide the green solutions needed by the industrial oil processing sector. Key points • Selected LCAT gene candidates were expressed in E. coli. • Aeromonas enteropelogenes LCAT hydrolyzes all the phospholipids present in crude soybean oil. • The LCAT enzyme provides a higher yield of neutral oil than commercial PLA enzymes and generates less waste. • The degummed oil retains sterols with high nutritional value.

Industrial uses of phospholipases: current state and future applications.

Phospholipids play a central role in all living organisms. Phospholipases, the enzymes aimed at modifying phospholipids, are consequently widespread in nature and play diverse roles, from lipid metabolism and cellular signaling in eukaryotes to virulence and nutrient acquisition in microbes. Phospholipases catalyze the hydrolysis of one or more ester or phosphodiester bonds of glycerophospholipids. The use of phospholipases with industrial purposes has constantly increased over the last 30 years. This demand is rapidly growing given the ongoing improvements in protein engineering and the reduction of enzymes manufacturing costs, making them suitable for industrial use. Here, a general overview of phopholipases A, B, C, and D and their industrial application is presented along with potential new uses for these enzymes. We draw attention to commercial phospholipases used to improve the emulsifying properties of products in the baking, egg, and dairy industries. On the other hand, the improvement of oil degumming by phospholipases is thoroughly analyzed. Moreover, recent developments in enzymatic biodiesel production and the use of phospholipases for the synthesis of phospholipids with pharmaceutical or nutritional value are reviewed.

Pilot-scale process development for low-cost production of a thermostable biodiesel refining enzyme in Escherichia coli.

Biodiesels produced from vegetable oils have a major quality problem due to the presence of steryl glucosides (SGs), which form precipitates that clog filters and cause engine failures. Recently, we described an enzymatic process for removing SGs from biodiesel. However, industrial adoption of this technology was hindered by the cost of the steryl glucosidase (SGase) enzyme used. Here we report the development and validation at the pilot scale of a cost-efficient process for manufacturing the SGase. First, we tested various low-cost carbon sources for the Escherichia coli producing strain, ultimately developing a fed-batch fermentation process that utilizes crude glycerol as a feedstock. Next, we designed an efficient process for isolating the SGase. That process uses a novel thermolysis approach in the presence of a non-ionic detergent, centrifugation to separate the solids, and ultrafiltration to concentrate and formulate the final product. Our cost analysis indicates that on a large scale, the dose of enzyme required to eliminate SGs from each ton of biodiesel will have a manufacturing cost below $1. The new process for manufacturing the SGase, which will lead to biodiesels of a higher quality, should contribute to facilitate the global adoption of this renewable fuel. Our technology could also be used to manufacture other thermostable proteins in E. coli.

The production, properties, and applications of thermostable steryl glucosidases.

Extremophilic microorganisms are a rich source of enzymes, the enzymes which can serve as industrial catalysts that can withstand harsh processing conditions. An example is thermostable ß-glucosidases that are addressing a challenging problem in the biodiesel industry: removing steryl glucosides (SGs) from biodiesel. Steryl glucosidases (SGases) must be tolerant to heat and solvents in order to function efficiently in biodiesel. The amphipathic nature of SGs also requires enzymes with an affinity for water/solvent interfaces in order to achieve efficient hydrolysis. Additionally, the development of an enzymatic process involving a commodity such as soybean biodiesel must be cost-effective, necessitating an efficient manufacturing process for SGases. This review summarizes the identification of microbial SGases and their applications, discusses biodiesel refining processes and the development of analytical methods for identifying and quantifying SGs in foods and biodiesel, and considers technologies for strain engineering and process optimization for the heterologous production of a SGase from Thermococcus litoralis. All of these technologies might be used for the production of other thermostable enzymes. Structural features of SGases and the feasibility of protein engineering for novel applications are explored.

Strain engineering and process optimization for enhancing the production of a thermostable steryl glucosidase in Escherichia coli.

Biodiesels produced from transesterification of vegetable oils have a major problem in quality due to the presence of precipitates, which are mostly composed of steryl glucosides (SGs). We have recently described an enzymatic method for the efficient removal of SGs from biodiesel, based on the activity of a thermostable ß-glycosidase from Thermococcus litoralis. In the present work, we describe the development of an Escherichia coli-based expression system and a high cell density fermentation process. Strain and process engineering include the assessment of different promoters to drive the expression of a codon-optimized gene, the co-expression of molecular chaperones and the development of a high cell density fermentation process. A 200-fold increase in the production titers was achieved, which directly impacts on the costs of the industrial process for treating biodiesel.

Enzymatic hydrolysis of steryl glucosides, major contaminants of vegetable oil-derived biodiesel.

Biodiesels are mostly produced from lipid transesterification of vegetable oils, including those from soybean, jatropha, palm, rapeseed, sunflower, and others. Unfortunately, transesterification of oil produces various unwanted side products, including steryl glucosides (SG), which precipitate and need to be removed to avoid clogging of filters and engine failures. So far, efficient and cost-effective methods to remove SGs from biodiesel are not available. Here we describe for the first time the identification, characterization and heterologous production of an enzyme capable of hydrolyzing SGs. A synthetic codon-optimized version of the lacS gene from Sulfolobus solfataricus was efficiently expressed and purified from Escherichia coli, and used to treat soybean derived biodiesel containing 100 ppm of SGs. After optimizing different variables, we found that at pH 5.5 and 87 °C, and in the presence of 0.9 % of the emulsifier polyglycerol polyricinoleate, 81 % of the total amount of SGs present in biodiesel were hydrolyzed by the enzyme. This remarkable reduction in SGs suggests a path for the removal of these contaminants from biodiesel on industrial scale using an environmentally friendly enzymatic process.

Three cases of kyphoplasty performed in the lateral position due to significant comorbidities.

Background: More than 700,000 people suffer from vertebral compression fractures attributed to osteoporosis, metastatic disease, or trauma each year in the United States, and undergo kyphoplasty. They are typical. These often undergo kyphoplasty to treat resultant pain or new neurological deficits. Here, we present three patients who, due to significant comorbidities, underwent kyphoplasty performed in the lateral decubitus rather than the prone position. Case Description: Three females, two with metastatic cancer and one with osteoporosis, presented with lumbar compression fractures and new accompanying pain and/or neurological deficits. Due to significant accompanying comorbidities, kyphoplasty was safely and effectively performed in all three patients utilizing the lateral decubitus rather than the prone position. Conclusion: Although vertebral kyphoplasties are typically performed in the prone position, here, we present three patients who, due to significant comorbidities, safely and effectively underwent kyphoplasties performed in the lateral decubitus position.

The evaluation of NIR-absorbing porphyrin derivatives as contrast agents in photoacoustic imaging.

Six free base tetrapyrrolic chromophores, three quinoline-annulated porphyrins and three morpholinobacteriochlorins, that absorb light in the near-IR range and possess, in comparison to regular porphyrins, unusually low fluorescence emission and (1)O2 quantum yields were tested with respect to their efficacy as novel molecular photo-acoustic imaging contrast agents in a tissue phantom, providing an up to ∼2.5-fold contrast enhancement over that of the benchmark contrast agent ICG. The testing protocol compares the photoacoustic signal output strength upon absorption of approximately the same light energy. Some relationships between photophysical parameters of the dyes and the resulting photoacoustic signal strength could be derived.

Intraocular Lens Unfurling Time Exponentially Decays with Increased Solution Temperature.

Purpose: Intraocular lens (IOL) unfurling can be a rate-limiting step in cataract surgery, limiting operative efficiency. Furthermore, inefficient unfurling has important implications for clinical outcomes. We examine the effects of solution temperature on IOL unfurling time using three in vitro models of the ocular environment. Methods: IOLs were injected into a 6-well plate filled with balanced salt solution (BSS), dispersive ophthalmic viscoelastic device (OVD), or cohesive OVD. Experiments were also performed in a plastic eye filled with dispersive or cohesive OVD. IOL unfurling time was recorded against the temperature of the respective solution. Results: IOL unfurling time decayed exponentially as solution temperature increased in all experiments, including the BSS-filled 6-well plate, the OVD-filled 6-well plate, and the OVD-filled plastic eye. IOLs failed to unfurl within 10 min at 10°C, below the glass transition temperature of the tested IOLs. Increasing solution temperature from 20°C to 30°C decreases IOL unfurling by greater than 2 min. Further heating to 40°C did not significantly decrease IOL unfurling time. Conclusion: Increased solution temperature rapidly decreases IOL unfurling time in vitro. IOLs do not unfurl within a clinically acceptable timeframe at or below their glass transition temperature. Increased BSS and/or OVD temperature may be a potential method to decrease IOL unfurling time in cataract surgery. However, future research is needed to elucidate potential consequences of warmed BSS and/or OVD on post-operative outcomes. This study demonstrates the potential for temperature regulation to decrease cataract surgery operative time and provides preliminary evidence to justify future clinical validation of this relationship.

During cataract surgery, a prosthetic intraocular lens (IOL) is inserted into the eye once the clouded lens is removed. The IOL must then unfurl before the procedure can proceed. When IOLs fail to unfurl or unfurl slowly, this can delay the operation and may even cause post-operative complications. Thus, we studied the effect temperature may have on IOL unfurling time to optimize this segment of the operation. We injected IOLs into solutions of saline (balanced salt solution) or ophthalmic viscoelastic device (OVD), two fluids injected into the eye during surgery. In both a well plate and a plastic eye, we found that increasing the temperature of the solution significantly affected IOL unfurling time. Specifically, heating the solution from refrigeration to room temperature decreased unfurling time from over 10 min to less than four. Heating to physiological temperature further decreased unfurling time to less than a minute. Our results show promise for potentially utilizing heated BSS and/or OVD to accelerate IOL unfurling and decrease cataract surgery operative time.

Sustainable Refining of Vegetable Oil Made Easy with a Designer Phospholipase C Enzyme.

The increasing demand pressures the vegetable oil industry to develop novel refining methods. Degumming with type C phospholipases (PLCs) is a green technology and provides extra oil. However, natural PLCs are not active under the harsh conditions used in oil refining plants, requiring additional unit operations. These upfront capital expenditures and the associated operational costs hinder the adoption of this method. Here, we present a process based on ChPLC, a synthetic PLC obtained by consensus sequence design, possessing superior thermal stability and catalytic properties. Using ChPLC, crude soybean oil degumming was completed at 80 °C in 30 min, the temperature and residence time imposed by the design of existing oil refining plants. Remarkably, an extra yield of oil of 2% was obtained using 60% of the dose recommended for PLCs marketed today, saving upfront investments and reducing the operational cost of degumming. A techno-economic analysis indicates that, for medium size plants, ChPLC reduces the overall cost of soybean oil enzymatic degumming by 58%. The process presented here facilitates the implementation of enzymatic technologies to oil producers, regardless of their processing capacity, bringing potential annual benefits in the billion-dollar range for the global economy.

Single-domain llama antibodies as specific intracellular inhibitors of SpvB, the actin ADP-ribosylating toxin of Salmonella typhimurium.

ADP-ribosylation of host cell proteins is a common mode of cell intoxication by pathogenic bacterial toxins. Antibodies induced by immunization with inactivated ADP-ribosylating toxins provide efficient protection in case of some secreted toxins, e.g., diphtheria and pertussis toxins. However, other ADP-ribosylating toxins, such as Salmonella SpvB toxin, are secreted directly from the Salmonella-containing vacuole into the cytosol of target cells via the SPI-2 encoded bacterial type III secretion system, and thus are inaccessible to conventional antibodies. Small-molecule ADP-ribosylation inhibitors are fraught with potential side effects caused by inhibition of endogenous ADP-ribosyltransferases. Here, we report the development of a single-domain antibody from an immunized llama that blocks the capacity of SpvB to ADP-ribosylate actin at a molar ratio of 1:1. The single-domain antibody, when expressed as an intrabody, effectively protected cells from the cytotoxic activity of a translocation-competent chimeric C2IN-C/SpvB toxin. Transfected cells were also protected against cytoskeletal alterations induced by wild-type SpvB-expressing strains of Salmonella. This proof of principle paves the way for developing new antidotes against intracellular toxins.

Consistency and validity of the inventory of callous- unemotional traits in a multi-centric community sample.

The purpose of this research was to validate the Inventory of Callous Unemotional Traits in a multi-centric community sample of Colombian children and adolescents aged between 9 and 18 years. An adapted version to the Colombian Spanish was applied to 903 school students without significant medical background (neurotypical behavior), and 118 with a clinical history of internalizing or externalizing conditions. A group of specialized judges approved the content validity of the instrument in terms of relevance and intelligibility, but concept factorial validity was low for the uncaring and callousness factors. Exploratory factor analysis confirmed the existence of three dimensions (uncaring, unemotional, and callousness), but only 17 out of 24 items demonstrated adequate psychometric statistics. The consistency for the 17-item Colombian adaptation was acceptable (α = .78). Goodness-of-fit calculated through confirmatory analysis was satisfactory for a bifactor structure (model C). Neurotypical participants showed lower total scores in comparison to the other groups. Participants with internalizing conditions had higher unemotional traits, while those with externalizing behaviors more commonly presented uncaring behaviors. This study is important for psychopathy research in Colombia as provides a validated adaption of the most used instrument to assess callous-unemotional traits in children and adolescents.

Fast, limited-data photoacoustic imaging for multiplexed systems using a frequency-domain estimation technique.

PURPOSE: A new frequency-domain estimation algorithm has been developed that uses a priori information to simultaneously improve imaging quality and time resolution in photoacoustic tomography with incomplete data sets. METHODS: The method involves application of a single-stage Wiener optimal filter to augment data sets by interpolation between measurement locations using relationships determined in a reference scan. The filter can be applied in real-time using FFT methods using either fixed or dynamic references and used with any imaging algorithm. The performance of the method is compared to a modified version of constrained backprojection algorithms using simulations and experimental investigations. RESULTS: Simulations demonstrate the effectiveness of the approach for tracking dynamic photoacoustic activity for data sets with limited views (90 degrees) or tomographic views with a reduced number of acquisition angles at any given time (< or = 32). Experimental data of contrast uptake and washout using a 512-element curved transducer with 8:1 electronic multiplexing with the algorithm demonstrate full two-dimensional tomographic imaging with a temporal resolution better than 130 ms. CONCLUSIONS: The estimation algorithm enables high spatial resolution, real-time imaging of dynamic physiological events or volumetric regions for photoacoustic systems employing multiplexing or scanning.

Cloning and Production of Thermostable Enzymes for the Hydrolysis of Steryl Glucosides in Biodiesel.

Vegetable oil-derived biodiesels have a major quality problem due to the presence of precipitates formed by steryl glucosides, which clog filters and injectors of diesel engines. An efficient, scalable, and cost-effective method to hydrolyze steryl glucosides using thermostable enzymes has been developed. Here, methods to discover, express in recombinant microorganisms and manufacture enzymes with SGase activity, as well as methods to treat biodiesel with such enzymes, and to measure the content of steryl glucosides in biodiesel samples are presented.

Early-stage invasive breast cancers: potential role of optical tomography with US localization in assisting diagnosis.

PURPOSE: To investigate the potential role of optical tomography in the near-infrared (NIR) spectrum with ultrasonographic (US) localization as a means of differentiating early-stage cancers from benign lesions of the breast. MATERIALS AND METHODS: The protocol was approved by the institutional review boards and was HIPAA compliant; all participants signed an informed consent. One hundred seventy-eight consecutive women (mean age, 52 years; range, 21-89 years) who underwent US-guided biopsy were imaged with a hand-held probe consisting of a coregistered US transducer and an NIR imager. The lesion location provided by coregistered US was used to guide optical imaging. Light absorption was measured at two optical wavelengths. From this measurement, tumor angiogenesis was assessed on the basis of calculated total hemoglobin concentration (tHb) and was correlated with core biopsy results. For patients diagnosed with carcinomas and followed up with subsequent excision, the tHb was correlated with pathologic parameters. RESULTS: There were two in situ carcinomas (Tis), 35 T1 carcinomas, 24 T2-T4 carcinomas, and 114 benign lesions. The mean maximum and mean average tHb of the Tis-T1 group were 102.0 micromol/L +/- 28.5 (standard deviation) and 71.9 micromol/L +/- 18.8, and those of the T2-T4 group were 100.3 micromol/L +/- 26.4 and 67.0 micromol/L +/- 18.3, respectively. The mean maximum and mean average tHb of the benign group were 55.1 micromol/L +/- 22.7 and 39.1 micromol/L +/- 14.9, respectively. Both mean maximum and mean average tHb levels were significantly higher in the malignant groups than they were in the benign group (P < .001). The sensitivity, specificity, positive predictive value, and negative predictive value for Tis-T1 cancers were 92%, 93%, 81%, and 97%. The corresponding values for T2-T4 tumors were 75%, 93%, 69%, and 95%. CONCLUSION: The angiogenesis (tHb) contrast imaged by using the NIR technique with US holds promise as an adjunct to mammography and US for distinguishing early-stage invasive breast cancers from benign lesions.

A real-time photoacoustic tomography system for small animals.

A real-time 512-element photoacoustic tomography system for small animal imaging using a ring ultrasound array has been developed. The system, based upon a 5 MHz transducer array formed along a 50 mm circular aperture, achieves sub-200 micron lateral resolution over a 2 cm disk-shaped region. Corresponding elevation resolutions of 0.6 to 2.5 mm over the central volume enable depth-resolved 3D tomographic imaging with linear translation. Using 8:1 electronic multiplexing, imaging at up to 8 frame/sec is demonstrated for both dynamic phantoms and in vivo mouse and brain samples. The real-time, full 2D tomographic capability of the system paves the way for functional photoacoustic tomographic imaging studies in small animals with sub-second time frame.

Feasibility study: Towards Estimation of Fatigue Level in Robot-Assisted Exercise for Cardiac Rehabilitation.

Socially Assistive Robotics (SAR) has shown to be an important tool to assist patients in physical rehabilitation. SAR is used to provide feedback about patient's state and performance to users and health professionals, therefore, patients are monitored by means of sensor interfaces. In this context, aiming to avoid over-training conditions, one of the most important parameter to monitor is the fatigue level. However, it is usually measured by subjective scales such as Borg scale, thus, there is a need to develop systems that are able to estimate fatigue with greater accuracy. It has been demonstrated that fatigue can be associated to the decreasing performance of the exercise. Hence, this work carried out a study to determinate which temporal and kinematic features are related to the fatigue level during a sit-to-stand test. The procedure consisted of sitting down and standing up from a chair while kinematic data were measured by a Kinect sensor, in order to relate kinematic data and fatigue. Results show that temporal features (time stand-to-stand and time sit-to-stand) and 3 kinematic features (max vertical-velocity of the spin base, max knee-flexo-extension velocity, and max hip-flexo-extension velocity), have a significant correlation with the fatigue level $(p \lt 0.05)$.