Exploring Fracture Patterns: Assessing Representation Methods for Bone Fracture Simulation.

Fracture pattern acquisition and representation in human bones play a crucial role in medical simulation, diagnostics, and treatment planning. This article presents a comprehensive review of methodologies employed in acquiring and representing bone fracture patterns. Several techniques, including segmentation algorithms, curvature analysis, and deep learning-based approaches, are reviewed to determine their effectiveness in accurately identifying fracture zones. Additionally, diverse methods for representing fracture patterns are evaluated. The challenges inherent in detecting accurate fracture zones from medical images, the complexities arising from multifragmentary fractures, and the need to automate fracture reduction processes are elucidated. A detailed analysis of the suitability of each representation method for specific medical applications, such as simulation systems, surgical interventions, and educational purposes, is provided. The study explores insights from a broad spectrum of research articles, encompassing diverse methodologies and perspectives. This review elucidates potential directions for future research and contributes to advancements in comprehending the acquisition and representation of fracture patterns in human bone.

The neurovascular unit of capillary blood vessels in the rat nervous system. A rapid-Golgi electron microscopy study.

We describe a pericapillary organ in the rat forebrain and cerebellar cortex. It consists of a series of tripartite synapses with synaptic extensions enveloped by astrocytic endfeet that are linked to the capillary wall by synaptic extensions. Reciprocal specializations of the pericyte-capillary blood vessel (CBV) with such specialized synapses suggest a mechanoreceptor role. In Golgi-impregnated and 3D reconstructions of the cerebral cortex and thalamus, a series of TSs appear to be sequentially ordered in a common dendrite, paralleled by synaptic outgrowths termed golf club synaptic extensions (GCE) opposed to a longitudinal crest (LC) from the capillary basal lamina (BL). Our results show that, in the cerebellar cortex, afferent fibers and interneurons display microanatomical structures that strongly suggest an interaction with the capillary wall. Afferent mossy fiber (MF) rosettes and ascending granule cell axons and their dendrites define the pericapillary passage interactions that are entangled by endfeet. The presence of mRNA of the mechanosensitive channel Piezo1 in the MF rosettes, together with the surrounding end-feet and the capillary wall form mechanosensory units. The ubiquity of such units to modulate synaptic transmission is also supported by Piezo1 mRNA expressing pyramidal isocortical and thalamic neurons. This scenario suggests that ascending impulses to the cerebellar and cortical targets are presynaptically modulated by the reciprocal interaction with the mechanosensory pericapillary organ that ultimately modulates the vasomotor response.

Automatable downstream purification of the benzohydroxamic acid D-DIBOA from a biocatalytic synthesis.

Herbicides play a vital role in agriculture, contributing to increased crop productivity by minimizing weed growth, but their low degradability presents a threat to the environment and human health. Allelochemicals, such as DIBOA (2,4-dihydroxy-(2H)-1,4-benzoxazin-3(4 H)-one), are secondary metabolites released by certain plants that affect the survival or growth of other organisms. Although these metabolites have an attractive potential for use as herbicides, their low natural production is a critical hurdle. Previously, the synthesis of the biologically active analog D-DIBOA (4-hydroxy-(2H)-1,4-benzoxazin-3(4H)-one) was achieved, using an engineered E. coli strain as a whole-cell biocatalyst, capable of transforming a precursor compound into D-DIBOA and exporting it into the culture medium, although it cannot be directly applied to crops. Here a chromatographic method to purify D-DIBOA from this cell culture medium without producing organic solvent wastes is described. The purification of D-DIBOA from a filtered culture medium to the pure compound could also be automated. Biological tests with the purified compound on weed models showed that it has virtually the same activity than the chemically synthesized D-DIBOA.

Metabolomics for the design of new metabolic engineering strategies for improving aerobic succinic acid production in Escherichia coli.

INTRODUCTION: Glycerol is a byproduct from the biodiesel industry that can be biotransformed by Escherichia coli to high added-value products such as succinate under aerobic conditions. The main genetic engineering strategies to achieve this aim involve the mutation of succinate dehydrogenase (sdhA) gene and also those responsible for acetate synthesis including acetate kinase, phosphate acetyl transferase and pyruvate oxidase encoded by ackA, pta and pox genes respectively in the ΔsdhAΔack-ptaΔpox (M4) mutant. Other genetic manipulations to rewire the metabolism toward succinate consist on the activation of the glyoxylate shunt or blockage the pentose phosphate pathway (PPP) by deletion of isocitrate lyase repressor (iclR) or gluconate dehydrogenase (gnd) genes on M4-ΔiclR and M4-Δgnd mutants respectively. OBJECTIVE: To deeply understand the effect of the blocking of the pentose phosphate pathway (PPP) or the activation of the glyoxylate shunt, metabolite profiles were analyzed on M4-Δgnd, M4-ΔiclR and M4 mutants. METHODS: Metabolomics was performed by FT-IR and GC-MS for metabolite fingerprinting and HPLC for quantification of succinate and glycerol. RESULTS: Most of the 65 identified metabolites showed lower relative levels in the M4-ΔiclR and M4-Δgnd mutants than those of the M4. However, fructose 1,6-biphosphate, trehalose, isovaleric acid and mannitol relative concentrations were increased in M4-ΔiclR and M4-Δgnd mutants. To further improve succinate production, the synthesis of mannitol was suppressed by deletion of mannitol dehydrogenase (mtlD) on M4-ΔgndΔmtlD mutant that increase ~ 20% respect to M4-Δgnd. CONCLUSION: Metabolomics can serve as a holistic tool to identify bottlenecks in metabolic pathways by a non-rational design. Genetic manipulation to release these restrictions could increase the production of succinate.

Fracture pattern projection on 3D bone models as support for bone fracture simulations.

BACKGROUND AND OBJECTIVE: Obtaining bone models that represent certain types of fractures is limited by the need for such fractures to occur in real life and to be processed from medical images. This work aims to propose a method that starts from the design of specific fracture patterns in order to be projected on 3D geometric bone models, being prepared for their subsequent geometric fracturing. METHODS: The process of projecting expert-generated fracture patterns has been approached in such a way that they contain geometrical and topological information for the subsequent fracture of the triangle mesh representing the bone model, giving information about the validity of the fracture pattern due to the design process, the validation performed, and the relationships between the fracture lines. RESULTS: Different 3D models of long bones have been used (femur, humerus, ulna and fibula). Also, different types of fracture patterns have been created. These patterns have been used to obtain their projection on three-dimensional bones. In this study, an expert validation of the fracture patterns projected on the bone models is performed. A forensic validation of the fracture patterns used as starting point for the projection is also performed for cases in which this fracture is produced by impact, for which there is scientific evidence based on forensic analysis. This validation also supports the experts, giving them the necessary feedback to complete or modify their fracture patterns according to criteria analyzed from a forensic point of view. CONCLUSIONS: The patterns fit the bone models correctly, despite the irregularities of the bone models, and correspond to the expected projection. In addition, it provides us with a clear line of work, by using the topological information of the fracture pattern and the bone model, which allows us to establish a consistent basis for future guided fractures.

Identification of Enzymatic Bottlenecks for the Aerobic Production of Malate from Glycerol by the Systematic Gene Overexpression of Anaplerotic Enzymes in Escherichia coli.

The biotechnological production of dicarboxylic acids (C4) from renewable carbon sources represents an attractive approach for the provision of these valuable compounds by green chemistry means. Glycerol has become a waste product of the biodiesel industry that serves as a highly reduced carbon source for some microorganisms. Escherichia coli is capable of consuming glycerol to produce succinate under anaerobic fermentation, but with the deletion of some tricarboxylic acid (TCA) cycle genes, it is also able to produce succinate and malate in aerobiosis. In this study, we investigate possible rate-limiting enzymes by overexpressing the C-feeding anaplerotic enzymes Ppc, MaeA, MaeB, and Pck in a mutant that lacks the succinate dehydrogenase (Sdh) enzyme. The overexpression of the TCA enzyme Mdh and the activation of the glyoxylate shunt was also examined. Using this unbiased approach, we found that phosphoenol pyruvate carboxylase (Ppc) overexpression enhances an oxidative pathway that leads to increasing succinate, while phosphoenol pyruvate carboxykinase (Pck) favors a more efficient reductive branch that produces mainly malate, at 57.5% of the theoretical maximum molar yield. The optimization of the culture medium revealed the importance of bicarbonate and pH in the production of malate. An additional mutation of the ppc gene highlights its central role in growth and C4 production.

Optimization of the Biocatalysis for D-DIBOA Synthesis Using a Quick and Sensitive New Spectrophotometric Quantification Method.

D-DIBOA (4-hydroxy-(2H)-1,4-benzoxazin-3-(4H)-one) is an allelopathic-derived compound with interesting herbicidal, fungicidal, and insecticide properties whose production has been successfully achieved by biocatalysis using a genetically engineered Escherichia coli strain. However, improvement and scaling-up of this process are hampered by the current methodology for D-DIBOA quantification, which is based on high-performance liquid chromatographic (HPLC), a time-consuming technique that requires expensive equipment and the use of environmentally unsafe solvents. In this work, we established and validated a rapid, simple, and sensitive spectrophotometric method for the quantification of the D-DIBOA produced by whole-cell biocatalysis, with limits of detection and quantification of 0.0165 and 0.0501 µmol·mL-1 respectively. This analysis takes place in only a few seconds and can be carried out using 100 µL of the sample in a microtiter plate reader. We performed several whole-cell biocatalysis strategies to optimize the process by monitoring D-DIBOA production every hour to keep control of both precursor and D-DIBOA concentrations in the bioreactor. These experiments allowed increasing the D-DIBOA production from the previously reported 5.01 mM up to 7.17 mM (43% increase). This methodology will facilitate processes such as the optimization of the biocatalyst, the scaling up, and the downstream purification.

Immobilization of Cells on Polyurethane Foam.

In this chapter, protocols and details for the immobilization of a model cell onto polyurethane foam carriers are provided in order to facilitate the use of such systems in laboratory or industrial reactors. Polyurethane foam has recently acquired great relevance as a carrier for its good mechanical properties, high porosity, and large adsorption surface. In addition, it has a very low commercial cost. Two different immobilization protocols have been described, differing in the flow regime or the possibilities for the reactor: immobilization in a stirred tank reactor working in a discontinuous regime (by cycles) and immobilization in a packed column working in continuous operation mode. Protocols for carrier sterilization, analytical methodology, and immobilization are described.

A genetically engineered Escherichia coli strain overexpressing the nitroreductase NfsB is capable of producing the herbicide D-DIBOA with 100% molar yield.

BACKGROUND: The use of chemical herbicides has helped to improve agricultural production, although its intensive use has led to environmental damages. Plant allelochemicals are interesting alternatives due to their diversity and degradability in the environment. However, the main drawback of this option is their low natural production, which could be overcome by its chemical synthesis. In the case of the allelochemical DIBOA ((2,4-dihydroxy-2H)-1,4-benzoxazin-3(4H)-one), the synthesis of the analogous compound D-DIBOA (2-deoxy-DIBOA) has been achieved in two steps. However, the scale up of this synthesis is hindered by the second step, which uses an expensive catalyst and is an exothermic reaction, with hydrogen release and a relatively low molar yield (70%). We have previously explored the "Green Chemistry" alternative of using E. coli strains overexpressing the nitroreductase NfsB as a whole-cell-biocatalyst to replace this second step, although the molar yield in this case was lower than that of the chemical synthesis. RESULTS: In this work, we engineered an E. coli strain capable of carrying out this reaction with 100% molar yield and reaching a D-DIBOA concentration up to 379% respect to the highest biotransformation yield previously reported. This was achieved by a screening of 34 E. coli mutant strains in order to improve D-DIBOA production that led to the construction of the ΔlapAΔfliQ double mutant as an optimum genetic background for overexpression of the NfsB enzyme and D-DIBOA synthesis. Also, the use of a defined medium instead of a complex one, the optimization of the culture conditions and the development of processes with several substrate loads allowed obtaining maxima yields and concentrations. CONCLUSIONS: The high yields and concentrations of D-DIBOA reached by the microbial-cell-factory approach developed in this work will facilitate its application to industrial scale. Also, the use of an optimized defined medium with only an organic molecule (glucose as carbon and energy source) in its composition will also facilitate the downstream processes.

Overexpression of the nitroreductase NfsB in an E. coli strain as a whole-cell biocatalyst for the production of chlorinated analogues of the natural herbicide DIBOA.

Benzohydroxamic acids, such as DIBOA (2,4-dihydroxy-2 H)-1,4-benzoxazin-3(4 H)-one), are plant products that exhibit interesting herbicidal, fungicidal and bactericidal properties. A feasible alternative to their purification from natural sources is the synthesis of analogous compounds such as D-DIBOA (2-deoxy-DIBOA) and their chlorinated derivatives. Their chemical synthesis has been simplified into two steps. However, the second step is an exothermic reaction and involves hydrogen release, which makes this methodology expensive and difficult to scale up. The study reported here concerns the possibility of producing chlorobenzoxazinones by a whole-cell biocatalytic process using the ability of the engineered E. coli nfsB-/pBAD-NfsB to catalyse the synthesis of 6-Cl-D-DIBOA and 8-Cl-D-DIBOA from their respective precursors (PCs). The results show that this strain is able to grow in media that contain these compounds and to produce the target molecules with 59.3% and 46.7% biotransformation yields, respectively. Moreover, the strain is capable of processing non-purified PCs from the first chemical step to give similar yields to those obtained from the purified PCs. The kinetics of the reaction in vitro with purified recombinant NfsB nitroreductase were studied to characterise the catalysis further and evaluate the effects that several components of the non-purified PCs have on the process. The results revealed that the kinetics are that of an allosteric enzyme. The inhibitory effect of the substrate of the first step of the chemical synthesis, which is present in some non-purified PCs, was also demonstrated.

Heterologous expression of the human Phosphoenol Pyruvate Carboxykinase (hPEPCK-M) improves hydrogen and ethanol synthesis in the Escherichia coli dcuD mutant when grown in a glycerol-based medium.

The production of biodiesel has emerged as an alternative to fossil fuels. However, this industry generates glycerol as a by-product in such large quantities that it has become an environmental problem. The biotransformation of this excess glycerol into other renewable bio-energy sources, like H2 and ethanol, by microorganisms such as Escherichia coli is an interesting possibility that warrants investigation. In this work we hypothesized that the conversion of oxaloacetate (OAA) to phosphoenolpyruvate (PEP) could be improved by a controlled expression of the human mitochondrial GTP-dependent PEP carboxykinase. This heterologous expression was tested in several E. coli mutant backgrounds with increased availability of C4 intermediates. It was found that this metabolic rewiring improved the synthesis of the target products in several mutants, with the dcuD mutant being the most suitable background for hydrogen and ethanol specific productions and glycerol consumption. These factors increased by 2.46, 1.73 and 1.95 times, respectively, when compared to those obtained for the wild-type strain.

Identification of enhanced hydrogen and ethanol Escherichia coli producer strains in a glycerol-based medium by screening in single-knock out mutant collections.

BACKGROUND: Earth's climate is warming as a result of anthropogenic emissions of greenhouse gases from fossil fuel combustion. Bioenergy, which includes biodiesel, biohydrogen and bioethanol, has emerged as a sustainable alternative fuel source. For this reason, in recent years biodiesel production has become widespread but this industry currently generates a huge amount of glycerol as a by-product, which has become an environmental problem in its own right. A feasible possibility to solve this problem is the use of waste glycerol as a carbon source for microbial transformation into biofuels such as hydrogen and ethanol. For instance, Escherichia coli is a microorganism that can synthesize these compounds under anaerobic conditions. RESULTS: In this work an experimental procedure was established for screening E. coli single mutants to identify strains with enhanced ethanol and/or H2 productions compared to the wild type strain. In an initial screening of 150 single mutants, 12 novel strains (gnd, tdcE, rpiA nanE, tdcB, deoB, sucB, cpsG, frmA, glgC, fumA and gadB) were found to provide enhanced yields for at least one of the target products. The mutations, that improve most significantly the parameters evaluated (gnd and tdcE genes), were combined with other mutations in three engineered E. coli mutant strains in order to further redirect carbon flux towards the desired products. CONCLUSIONS: This methodology can be a useful tool to disclose the metabolic pathways that are more susceptible to manipulation in order to obtain higher molar yields of hydrogen and ethanol using glycerol as main carbon source in multiple E. coli mutants.

A systematic analysis of TCA Escherichia coli mutants reveals suitable genetic backgrounds for enhanced hydrogen and ethanol production using glycerol as main carbon source.

Biodiesel has emerged as an environmentally friendly alternative to fossil fuels; however, the low price of glycerol feed-stocks generated from the biodiesel industry has become a burden to this industry. A feasible alternative is the microbial biotransformation of waste glycerol to hydrogen and ethanol. Escherichia coli, a microorganism commonly used for metabolic engineering, is able to biotransform glycerol into these products. Nevertheless, the wild type strain yields can be improved by rewiring the carbon flux to the desired products by genetic engineering. Due to the importance of the central carbon metabolism in hydrogen and ethanol synthesis, E. coli single null mutant strains for enzymes of the TCA cycle and other related reactions were studied in this work. These strains were grown anaerobically in a glycerol-based medium and the concentrations of ethanol, glycerol, succinate and hydrogen were analysed by HPLC and GC. It was found that the reductive branch is the more relevant pathway for the aim of this work, with malate playing a central role. It was also found that the putative C4-transporter dcuD mutant improved the target product yields. These results will contribute to reveal novel metabolic engineering strategies for improving hydrogen and ethanol production by E. coli.

Study of the role played by NfsA, NfsB nitroreductase and NemA flavin reductase from Escherichia coli in the conversion of ethyl 2-(2'-nitrophenoxy)acetate to 4-hydroxy-(2H)-1,4-benzoxazin-3(4H)-one (D-DIBOA), a benzohydroxamic acid with interesting biological properties.

Benzohydroxamic acids, such as 4-hydroxy-(2H)-1,4-benzoxazin-3(4H)-one (D-DIBOA), exhibit interesting herbicidal, fungicidal and bactericidal properties. Recently, the chemical synthesis of D-DIBOA has been simplified to only two steps. In a previous paper, we demonstrated that the second step could be replaced by a biotransformation using Escherichia coli to reduce the nitro group of the precursor, ethyl 2-(2'-nitrophenoxy)acetate and obtain D-DIBOA. The NfsA and NfsB nitroreductases and the NemA xenobiotic reductase of E. coli have the capacity to reduce one or two nitro groups from a wide variety of nitroaromatic compounds, which are similar to the precursor. By this reason, we hypothesised that these three enzymes could be involved in this biotransformation. We have analysed the biotransformation yield (BY) of mutant strains in which one, two or three of these genes were knocked out, showing that only in the double nfsA/nfsB and in the triple nfsA/nfsB/nemA mutants, the BY was 0%. These results suggested that NfsA and NfsB are responsible for the biotransformation in the tested conditions. To confirm this, the nfsA and nfsB open reading frames were cloned into the pBAD expression vector and transformed into the nfsA and nfsB single mutants, respectively. In both cases, the biotransformation capacity of the strains was recovered (6.09 ± 0.06% as in the wild-type strain) and incremented considerably when NfsA and NfsB were overexpressed (40.33% ± 9.42% and 59.68% ± 2.0% respectively).

Daytime food restriction alters liver glycogen, triacylglycerols, and cell size. A histochemical, morphometric, and ultrastructural study.

BACKGROUND: Temporal restriction of food availability entrains circadian behavioral and physiological rhythms in mammals by resetting peripheral oscillators. This entrainment underlies the activity of a timing system, different from the suprachiasmatic nuclei (SCN), known as the food entrainable oscillator (FEO). So far, the precise anatomical location of the FEO is unknown. The expression of this oscillator is associated with an enhanced arousal prior to the food presentation that is called food anticipatory activity (FAA). We have focused on the study of the role played by the liver as a probable component of the FEO. The aim of this work was to identify metabolic and structural adaptations in the liver during the expression of the FEO, as revealed by histochemical assessment of hepatic glycogen and triacylglycerol contents, morphometry, and ultrastructure in rats under restricted feeding schedules (RFS). RESULTS: RFS promoted a decrease in the liver/body weight ratio prior to food access, a reduction of hepatic water content, an increase in cross-sectional area of the hepatocytes, a moderate reduction in glycogen content, and a striking decrease in triacylglyceride levels. Although these adaptation effects were also observed when the animal displayed FAA, they were reversed upon feeding. Mitochondria observed by electron microscopy showed a notorious opacity in the hepatocytes from rats during FAA (11:00 h). Twenty four hour fasting rats did not show any of the modifications observed in the animals expressing the FEO. CONCLUSIONS: Our results demonstrate that FEO expression is associated with modified liver handling of glycogen and triacylglycerides accompanied by morphometric and ultrastructural adaptations in the hepatocytes. Because the cellular changes detected in the liver cannot be attributed to a simple alternation between feeding and fasting conditions, they also strengthen the notion that RFS promotes a rheostatic adjustment in liver physiology during FEO expression.

Bacterial removal of chromium (VI) and (III) in a continuous system.

The capacity of Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans to reduce different concentrations of hexavalent chromium in shake flask cultures has been investigated. A. ferrooxidans reduces 100% of chromium (VI) at concentrations of 1, 2.5 and 5 ppm, but in the presence of 10 ppm only 42.9% of chromium (VI) was reduced after 11 days of incubation. A. thiooxidans showed a lower capacity to reduce this ion and total reduction of chromium (VI) was only obtained for concentrations of 1 and 2.5 ppm, whereas 64.7% and 30.5% was reached for 5 and 10 ppm, respectively, after 11 days. A continuous flow mode system was subsequently investigated, in which A. thiooxidans was immobilized on elemental sulphur and the acidic medium obtained was employed to solubilize chromium (III) and to reduce chromium (VI) present in a real electroplating waste [30% of chromium (III) and 0.1% of chromium (VI)]. The system enabled the reduction of 92.7% of hexavalent chromium and represents a promising way to treat this type of waste in the industry.

Electrophysiological evidence that a set of interfascicular cells of the rat anterior commissure are neurons.

In mammals, the anterior commissure (AC) provides a route that interconnects homonymous areas of the basal forebrain. Recently, we reported the presence of short-axon and projection neurons among the axonal fascicles of the rat AC (i.e. interfascicular neurons; IFNs). This, coupled with the commissural inputs to these neurons, suggests that in addition to conveying nerve impulses, the AC may be a site of neural processing. To test this hypothesis, the electrophysiological activity of IFNs was recorded in adult albino rats. From extracellular recordings performed in 11 IFNs, it was found that these cells: (1), have a spontaneous discharge of a relatively low frequency (i.e. 0.04 +/- 0.1 to 5.9 +/- 3.2 spikes per second); (2), application of anodic current in the adjacent commissural fibers decreased this frequency; and (3), application of cathodic current increased the number of action potentials. Since observations made in Golgi-impregnated sections suggest that the main input to IFNs arises from their commissural collaterals, it is concluded that these cells may participate in the integration of interhemispheric nerve impulses.

Histological and ultrastructural characterization of interfascicular neurons in the rat anterior commissure.

The histological, connectional, and ultrastructural characteristics of a peculiar neuron type in the rat anterior commissure (AC) are described. Since these cells are located among the axonal fascicles of the rostral and caudal parts of the AC, they are termed interfascicular neurons (IFN). In rapid-Golgi sections IFNs appeared in two forms: internuncial (i.e., short axon) and projection neurons (i.e., long axon). The axon of the internuncial neurons terminates upon neighboring IFNs. The projection neurons give rise to an axon which is either incorporated into commissural fibers or ramifies into 12-26 collaterals running laterally in opposite directions along commissural axons. Immunohistochemistry to microtubule-associated protein 2 combined with confocal microscopy showed that IFNs display short varicose dendrites which remain confined to the domain of the AC. The neuronal nature of IFNs was confirmed with the electron microscope on the basis of distinctive organelles and the presence of synaptic inputs. Small areas of neuropil surround some IFNs. These areas are composed of proximal dendrites, terminal axons, axo-shaft and axo-spinous synapses. Because IFNs with their afferents and efferents constitute sufficient elements to integrate neural inputs, it is proposed that they may be involved in processing nerve impulses proceeding from the bilateral cerebral structures connected by the AC.