PCDH19-clustering epilepsy, pathophysiology and clinical significance.

PCDH19 clustering epilepsy (PCDH19-CE) is an X-linked epilepsy disorder associated with intellectual disability (ID) and behavioral disturbances, which is caused by PCDH19 gene variants. PCDH19 pathogenic variant leads to epilepsy in heterozygous females, not in hemizygous males and the inheritance pattern is unusual. The hypothesis of cellular interference was described as a key pathogenic mechanism. According to that, males do not develop the disease because of the uniform expression of PCDH19 (variant or wild type) unless they have a somatic variation. We conducted a literature review on PCDH19-CE pathophysiology and concluded that other significant mechanisms could contribute to pathogenesis including: asymmetric cell division and heterochrony, female-related allopregnanolone deficiency, altered steroid gene expression, decreased Gamma-aminobutyric acid receptor A (GABAA) function, and blood-brain barrier (BBB) dysfunction. Being aware of these mechanisms helps us when we should decide which therapeutic option is more suitable for which patient.

The Value of Long-term Video EEG Monitoring to Diagnose and Track Childhood Epilepsy.

Objectives: Long-term video-EEG monitoring (LTM) is a new technique to assess and track fluctuations, classify seizures, identify epileptic syndromes, and determine the number of seizures and epilepsy-simulating disorders. The present study aims to evaluate the concordance of traditional EEG and LTM in assessing childhood epilepsy. Materials & Methods: This cross-sectional before-after study was performed on 120 children with epilepsy who were referred to the Epilepsy Monitoring Unit (EMU) at the Children's Medical Center between September 2021 and September 2022 and were monitored for at least eight hours in this unit. The source of the study information collection was the patients' recorded files. A neurologist reviewed the primary EEGs, and two experts blindly reviewed and interpreted the patients' LTMs under a clinical neurophysiologist's supervision. Results: The diagnoses changed after employing LTM in most children with epilepsy. Based on the diagnostic agreement analysis between EEG and LTM, the coefficient value for LTM was calculated at -0.37 (p= 0.229), showing that LTM has significantly expanded patients' diagnoses and care plans. Conclusion: The use of LTM improves the diagnosis, classification, and monitoring of epilepsy in affected children and can be a reliable supplement to EEG in some instances.

Temporal subcortical T2 hypointensity on MRI in epilepsia partialis continua, a non ketotic hyperglycemia rather than herpes encephalitis.

PURPOSE: Hyperglycemia can present as many neurological problems, one of them is seizure. Different brain MRI features can be seen in focal seizures associated with nonketotic hyperglycemia that subcortical T2 hypointensity is the only characteristic one. Finding this MRI feature is highly valuable in early diagnosis and treatment. METHODS: Our patient was a 60-year-old female, a case of type 2 diabetes mellitus. She was brought to Emergency Room (ER) with focal colonic status epilepticus of right face and arm associated with confusion and drowsiness progressed over 2 weeks prior to admission. At first, acyclovir was started alongside anti-seizure medication with doubt of herpes encephalitis but antiviral was discontinued after normal LP result and characteristic MRI features. RESULTS: Subcortical T2 hypointensity in left temporal and insular lobe was seen on first MRI that was resolved on follow up MRI after she was treated. CONCLUSION: Epilepsia partialis continua in the setting of non ketotic hyperglycemia should be differentiated from that in herpes encephalitis in a diabetic patient presenting with subacute confusional state and focal status epilepticus considering characteristic MRI finding of subcortical T2 hypointensity.

Patterns of bacterial motility in microfluidics-confining environments.

Understanding the motility behavior of bacteria in confining microenvironments, in which they search for available physical space and move in response to stimuli, is important for environmental, food industry, and biomedical applications. We studied the motility of five bacterial species with various sizes and flagellar architectures (Vibrio natriegens, Magnetococcus marinus, Pseudomonas putida, Vibrio fischeri, and Escherichia coli) in microfluidic environments presenting various levels of confinement and geometrical complexity, in the absence of external flow and concentration gradients. When the confinement is moderate, such as in quasi-open spaces with only one limiting wall, and in wide channels, the motility behavior of bacteria with complex flagellar architectures approximately follows the hydrodynamics-based predictions developed for simple monotrichous bacteria. Specifically, V. natriegens and V. fischeri moved parallel to the wall and P. putida and E. coli presented a stable movement parallel to the wall but with incidental wall escape events, while M. marinus exhibited frequent flipping between wall accumulator and wall escaper regimes. Conversely, in tighter confining environments, the motility is governed by the steric interactions between bacteria and the surrounding walls. In mesoscale regions, where the impacts of hydrodynamics and steric interactions overlap, these mechanisms can either push bacteria in the same directions in linear channels, leading to smooth bacterial movement, or they could be oppositional (e.g., in mesoscale-sized meandered channels), leading to chaotic movement and subsequent bacterial trapping. The study provides a methodological template for the design of microfluidic devices for single-cell genomic screening, bacterial entrapment for diagnostics, or biocomputation.

How to change the ratio of unsaturated (omega 3, 6, 7 and 9) to saturated fatty acids in Oenothera biennis L. oil under water deficit stress, fertilizers and geographical zones.

The 2015-2020 dietary guidelines for Americans advise substituting total unsaturated fatty acids (∑UFA) for total saturated fatty acids (∑SFA). Thus, field experiments were carried out to verify the influence of irrigation regime (well-irrigated and water deficit) and fertilizers (chemical and biological) on the ratio of ∑UFA to ∑SFA of evening primrose seed oil. Therefore, two experiments were conducted at the experimental stations (arid and semi-arid) of Iran in 2014 and 2015. Experiments were conducted in a split factorial layout within a randomized complete block design with three replications. Water deficit significantly reduced UFA (omega 3, 6, 7 and 9), ∑UFA and ratio of ∑UFA to ∑SFA (especially in the arid region), but it increased SFA and ∑SFA (especially in the arid region). In fact, fatty acid quality (increased ratio of ∑UFA to ∑SFA) of evening primrose seed oil was significantly increased in well-irrigated compared to water deficit stress (especially in the semi-arid region). Bio-fertilizers (Azospirillum lipoferum and Glomus mosseae) and chemical fertilizers (urea + triple superphosphate) increased the ratio of ∑UFA to ∑SFA of evening primrose seed oil (especially in the semi-arid region), but fatty acid quality of evening primrose oil was significantly increased in bio-fertilizers compared to the chemical fertilizers (especially in the arid region).

Magneto-aerotactic bacteria deliver drug-containing nanoliposomes to tumour hypoxic regions.

Oxygen-depleted hypoxic regions in the tumour are generally resistant to therapies. Although nanocarriers have been used to deliver drugs, the targeting ratios have been very low. Here, we show that the magneto-aerotactic migration behaviour of magnetotactic bacteria, Magnetococcus marinus strain MC-1 (ref. 4), can be used to transport drug-loaded nanoliposomes into hypoxic regions of the tumour. In their natural environment, MC-1 cells, each containing a chain of magnetic iron-oxide nanocrystals, tend to swim along local magnetic field lines and towards low oxygen concentrations based on a two-state aerotactic sensing system. We show that when MC-1 cells bearing covalently bound drug-containing nanoliposomes were injected near the tumour in severe combined immunodeficient beige mice and magnetically guided, up to 55% of MC-1 cells penetrated into hypoxic regions of HCT116 colorectal xenografts. Approximately 70 drug-loaded nanoliposomes were attached to each MC-1 cell. Our results suggest that harnessing swarms of microorganisms exhibiting magneto-aerotactic behaviour can significantly improve the therapeutic index of various nanocarriers in tumour hypoxic regions.

Switching between Magnetotactic and Aerotactic Displacement Controls to Enhance the Efficacy of MC-1 Magneto-Aerotactic Bacteria as Cancer-Fighting Nanorobots.

The delivery of drug molecules to tumor hypoxic areas could yield optimal therapeutic outcomes. This suggests that effective cancer-fighting micro- or nanorobots would require more integrated functionalities than just the development of directional propelling constructs which have so far been the main general emphasis in medical micro- and nanorobotic research. Development of artificial agents that would be most effective in targeting hypoxic regions may prove to be a very challenging task considering present technological constraints. Self-propelled, sensory-based and directionally-controlled agents in the form of Magnetotactic Bacteria (MTB) of the MC-1 strain have been investigated as effective therapeutic nanorobots in cancer therapy. Following computer-based magnetotactic guidance to reach the tumor area, the microaerophilic response of drug-loaded MC-1 cells could be exploited in the tumoral interstitial fluid microenvironments. Accordingly, their swimming paths would be guided by a decreasing oxygen concentration towards the hypoxic regions. However, the implementation of such a targeting strategy calls for a method to switch from a computer-assisted magnetotactic displacement control to an autonomous aerotactic displacement control. In this way, the MC-1 cells will navigate to tumoral regions and, once there, target hypoxic areas through their microaerophilic behavior. Here we show not only how the magnitude of the magnetic field can be used for this purpose but how the findings could help determine the specifications of a future compatible interventional platform within known technological and medical constraints.

Covalent binding of nanoliposomes to the surface of magnetotactic bacteria for the synthesis of self-propelled therapeutic agents.

The targeted and effective delivery of therapeutic agents remains an unmet goal in the field of controlled release systems. Magnetococcus marinus MC-1 magnetotactic bacteria (MTB) are investigated as potential therapeutic carriers. By combining directional magnetotaxis-microaerophilic control of these self-propelled agents, a larger amount of therapeutics can be delivered surpassing the diffusion limits of large drug molecules toward hard-to-treat hypoxic regions in solid tumors. The potential benefits of these carriers emphasize the need to develop an adequate method to attach therapeutic cargos, such as drug-loaded nanoliposomes, without substantially affecting the cell's ability to act as delivery agents. In this study, we report on a strategy for the attachment of liposomes to MTB (MTB-LP) through carbodiimide chemistry. The attachment efficacy, motility, and magnetic response of the MTB-LP were investigated. Results confirm that a substantial number of nanoliposomes (∼70) are efficiently linked with MTB without compromising functionality and motility. Cytotoxicity assays using three different cell types (J774, NIH/3T3, and Colo205) reveal that liposomal attachments to MTB formulation improve the biocompatibility of MTB, whereas attachment does not interfere with liposomal uptake.

Structural insights into the metabolism of 2-chlorodibenzofuran by an evolved biphenyl dioxygenase.

The biphenyl dioxygenase of Burkholderia xenovorans LB400 (BphAE(LB400)) is a Rieske-type oxygenase that catalyzes the stereospecific oxygenation of many heterocyclic aromatics including dibenzofuran. In a previous work, we evolved BphAE(LB400) and obtained BphAE(RR41). This variant metabolizes dibenzofuran and 2-chlorodibenzofuran more efficiently than BphAE(LB400). However, the regiospecificity of BphAE(RR41) toward these substrates differs. Dibenzofuran is metabolized principally through a lateral dioxygenation whereas 2-chlorodibenzofuran is metabolized principally through an angular dioxygenation. In order to explain this difference, we examined the crystal structures of both substrate-bound forms of BphAE(RR41) obtained under anaerobic conditions. This structure analysis, in combination with biochemical data for a Ser283Gly mutant provided evidences that the substrate is compelled to move after oxygen-binding in BphAE(RR41):dibenzofuran. In BphAE(RR41):2-chlorodibenzofuran, the chlorine atom is close to the side chain of Ser283. This contact is missing in the BphAE(RR41):dibenzofuran, and strong enough in the BphAE(RR41):2-chlorodibenzofuran to help prevent substrate movement during the catalytic reaction.

Metabolism of chlorobiphenyls by a variant biphenyl dioxygenase exhibiting enhanced activity toward dibenzofuran.

The biphenyl dioxygenase of Burkholderia xenovorans LB400 (BphAE(LB400)) catalyzes the dihydroxylation of biphenyl and of several polychlorinated biphenyls (PCBs) but it poorly oxidizes dibenzofuran. In this work we showed that BphAE(RR41), a variant which was previously found to metabolize dibenzofuran more efficiently than its parent BphAE(LB400), metabolized a broader range of PCBs than BphAE(LB400). Hence, BphAE(RR41) was able to metabolize 2,6,2',6'-, 3,4,3',5'- and 2,4,3',4'-tetrachlorobiphenyl that BphAE(LB400) is unable to metabolize. BphAE(RR41) was obtained by changing Thr335Phe336Asn338Ile341Leu409 of BphAE(LB400) to Ala335Met336Gln338Val341Phe409. Site-directed mutagenesis was used to create combinations of each substitution, in order to assess their individual contributions. Data show that the same Asn338Glu/Leu409Phe substitution that enhanced the ability to metabolize dibenzofuran resulted in a broadening of the PCB substrates range of the enzyme. The role of these substitutions on regiospecificities toward selected PCBs is also discussed.

Biochemical studies and ligand-bound structures of biphenyl dehydrogenase from Pandoraea pnomenusa strain B-356 reveal a basis for broad specificity of the enzyme.

Biphenyl dehydrogenase, a member of short-chain dehydrogenase/reductase enzymes, catalyzes the second step of the biphenyl/polychlorinated biphenyls catabolic pathway in bacteria. To understand the molecular basis for the broad substrate specificity of Pandoraea pnomenusa strain B-356 biphenyl dehydrogenase (BphB(B-356)), the crystal structures of the apo-enzyme, the binary complex with NAD(+), and the ternary complexes with NAD(+)-2,3-dihydroxybiphenyl and NAD(+)-4,4'-dihydroxybiphenyl were determined at 2.2-, 2.5-, 2.4-, and 2.1-Å resolutions, respectively. A crystal structure representing an intermediate state of the enzyme was also obtained in which the substrate binding loop was ordered as compared with the apo and binary forms but it was displaced significantly with respect to the ternary structures. These five structures reveal that the substrate binding loop is highly mobile and that its conformation changes during ligand binding, starting from a disorganized loop in the apo state to a well organized loop structure in the ligand-bound form. Conformational changes are induced during ligand binding; forming a well defined cavity to accommodate a wide variety of substrates. This explains the biochemical data that shows BphB(B-356) converts the dihydrodiol metabolites of 3,3'-dichlorobiphenyl, 2,4,4'-trichlorobiphenyl, and 2,6-dichlorobiphenyl to their respective dihydroxy metabolites. For the first time, a combination of structural, biochemical, and molecular docking studies of BphB(B-356) elucidate the unique ability of the enzyme to transform the cis-dihydrodiols of double meta-, para-, and ortho-substituted chlorobiphenyls.

Retuning Rieske-type oxygenases to expand substrate range.

Rieske-type oxygenases are promising biocatalysts for the destruction of persistent pollutants or for the synthesis of fine chemicals. In this work, we explored pathways through which Rieske-type oxygenases evolve to expand their substrate range. BphAE(p4), a variant biphenyl dioxygenase generated from Burkholderia xenovorans LB400 BphAE(LB400) by the double substitution T335A/F336M, and BphAE(RR41), obtained by changing Asn(338), Ile(341), and Leu(409) of BphAE(p4) to Gln(338), Val(341), and Phe(409), metabolize dibenzofuran two and three times faster than BphAE(LB400), respectively. Steady-state kinetic measurements of single- and multiple-substitution mutants of BphAE(LB400) showed that the single T335A and the double N338Q/L409F substitutions contribute significantly to enhanced catalytic activity toward dibenzofuran. Analysis of crystal structures showed that the T335A substitution relieves constraints on a segment lining the catalytic cavity, allowing a significant displacement in response to dibenzofuran binding. The combined N338Q/L409F substitutions alter substrate-induced conformational changes of protein groups involved in subunit assembly and in the chemical steps of the reaction. This suggests a responsive induced fit mechanism that retunes the alignment of protein atoms involved in the chemical steps of the reaction. These enzymes can thus expand their substrate range through mutations that alter the constraints or plasticity of the catalytic cavity to accommodate new substrates or that alter the induced fit mechanism required to achieve proper alignment of reaction-critical atoms or groups.

Anaerobic crystallization and initial X-ray diffraction data of biphenyl 2,3-dioxygenase from Burkholderia xenovorans LB400: addition of agarose improved the quality of the crystals.

Biphenyl 2,3-dioxygenase (BPDO; EC 1.14.12.18) catalyzes the initial step in the degradation of biphenyl and some polychlorinated biphenyls (PCBs). BPDOLB400, the terminal dioxygenase component from Burkholderia xenovorans LB400, a proteobacterial species that degrades a broad range of PCBs, has been crystallized under anaerobic conditions by sitting-drop vapour diffusion. Initial crystals obtained using various polyethylene glycols as precipitating agents diffracted to very low resolution (∼8 Å) and the recorded reflections were diffuse and poorly shaped. The quality of the crystals was significantly improved by the addition of 0.2% agarose to the crystallization cocktail. In the presence of agarose, wild-type BPDOLB400 crystals that diffracted to 2.4 Šresolution grew in space group P1. Crystals of the BPDOP4 and BPDORR41 variants of BPDOLB400 grew in space group P2(1).

Structural insight into the expanded PCB-degrading abilities of a biphenyl dioxygenase obtained by directed evolution.

The biphenyl dioxygenase of Burkholderia xenovorans LB400 is a multicomponent Rieske-type oxygenase that catalyzes the dihydroxylation of biphenyl and many polychlorinated biphenyls (PCBs). The structural bases for the substrate specificity of the enzyme's oxygenase component (BphAE(LB400)) are largely unknown. BphAE(p4), a variant previously obtained through directed evolution, transforms several chlorobiphenyls, including 2,6-dichlorobiphenyl, more efficiently than BphAE(LB400), yet differs from the parent oxygenase at only two positions: T335A/F336M. Here, we compare the structures of BphAE(LB400) and BphAE(p4) and examine the biochemical properties of two BphAE(LB400) variants with single substitutions, T335A or F336M. Our data show that residue 336 contacts the biphenyl and influences the regiospecificity of the reaction, but does not enhance the enzyme's reactivity toward 2,6-dichlorobiphenyl. By contrast, residue 335 does not contact biphenyl but contributes significantly to expansion of the enzyme's substrate range. Crystal structures indicate that Thr335 imposes constraints through hydrogen bonds and nonbonded contacts to the segment from Val320 to Gln322. These contacts are lost when Thr is replaced by Ala, relieving intramolecular constraints and allowing for significant movement of this segment during binding of 2,6-dichlorobiphenyl, which increases the space available to accommodate the doubly ortho-chlorinated congener 2,6-dichlorobiphenyl. This study provides important insight about how Rieske-type oxygenases can expand substrate range through mutations that increase the plasticity and/or mobility of protein segments lining the catalytic cavity.

Encapsulation of magnetotactic bacteria for targeted and controlled delivery of anticancer agents for tumor therapy.

We showed that magnetotactic bacteria (MTB) have great potentials to be used as microcarriers for targeted delivery of therapeutic agents. Indeed, magnetotaxis inherent in MTB can be exploited to direct them towards a tumor while being propelled by their own flagellated molecular motors. Nonetheless, although the thrust propelling force above 4 pN of the MC-1 MTB showed to be superior compared to other technologies for displacement in the microvasculature, MTB becomes much less efficient when travelling in larger blood vessels due to higher blood flow. In the latter case, a new technique developed by our group and referred to as Magnetic Resonance Navigation (MRN), has been successfully applied in larger vessels using synthetic microcarriers nut proved to be less efficient in the microvasculature due mainly to technological constraints. These findings called for the need to integrate both approaches by encapsulating MTB in special MRN-compatible microcarriers to be release in the vicinity of microvascular networks where they becomes more effective for targeting purposes in tumoral lesions. In this study Magnetococcus strain MC-1 were encapsulated in giant vesicles. The survival of the encapsulated bacteria was monitored. The release of bacteria from giant vesicles was also studied in different time intervals and conditions.

Effect of the chain of magnetosomes embedded in magnetotactic bacteria and their motility on magnetic resonance imaging.

This paper investigates the influence of the magnetosome's chain, the motility, and the bacterial cell of MC-1 magnetotactic bacteria (MTB) on the Magnetic Resonance imaging (MRI) contrast. Because of its embedded magnetic nanoparticles, that allow magnetic guidance and imaging contrast generation under MRI, magnetotactic bacteria are being considered for therapeutic drug delivery to tumors. In order to separately investigate the different potential sources of contrast in MRI, we used three samples of MC-1 MTB. The first sample was constituted of magnetic bacteria that successfully synthesize magnetic nanoparticles. MC-1 bacteria that do not synthesize magnetosomes form the second sample while the third sample is constituted from dead MC-1 magnetic bacteria containing magnetic nanoparticle. T(2)-weighted magnetic resonance images were obtained for multiple echo times. T(2) was then estimated by fitting the signal intensity data for different echo time values to a monoexponential decay curve. It is found that nanoparticles synthesized by MC-1 MTB are the predominant source of contrast in MRI over motion and the cell body.

Magnetotactic bacteria penetration into multicellular tumor spheroids for targeted therapy.

Preliminary experiments showed that MC-1 magnetotactic bacteria (MTB) could be used for the delivery of therapeutic agents to tumoral lesions. Each bacterium can provide a significant thrust propulsion force generated by two flagella bundles exceeding 4pN. Furthermore, a chain of single-domain magnetosomes embedded in the cell allows computer directional control and tracking using a magnetic resonance imaging (MRI) system. Although these embedded functionalities suggest that MTB when under the influence of an external computer could be considered as biological microrobots with the potential of targeting tumors, little is known about their level of penetration in tumoral tissues. In this paper, in vitro experiments were performed to assess the capability of these bacteria to penetrate tumor tissue for the delivery of therapeutic agents. Multicellular tumor spheroids were used since they reproduce many properties of solid tumors. The results show the ability of these MTB when submitted to a directional magnetic field to penetrate inside a 3D multicellular tumor spheroid through openings present in the tissue.

Flagellated Magnetotactic Bacteria as Controlled MRI-trackable Propulsion and Steering Systems for Medical Nanorobots Operating in the Human Microvasculature.

Although nanorobots may play critical roles for many applications in the human body such as targeting tumoral lesions for therapeutic purposes, miniaturization of the power source with an effective onboard controllable propulsion and steering system have prevented the implementation of such mobile robots. Here, we show that the flagellated nanomotors combined with the nanometer-sized magnetosomes of a single Magnetotactic Bacterium (MTB) can be used as an effective integrated propulsion and steering system for devices such as nanorobots designed for targeting locations only accessible through the smallest capillaries in humans while being visible for tracking and monitoring purposes using modern medical imaging modalities such as Magnetic Resonance Imaging (MRI). Through directional and magnetic field intensities, the displacement speeds, directions, and behaviors of swarms of these bacterial actuators can be controlled from an external computer.

MRI-based Medical Nanorobotic Platform for the Control of Magnetic Nanoparticles and Flagellated Bacteria for Target Interventions in Human Capillaries.

Medical nanorobotics exploits nanometer-scale components and phenomena with robotics to provide new medical diagnostic and interventional tools. Here, the architecture and main specifications of a novel medical interventional platform based on nanorobotics and nanomedicine, and suited to target regions inaccessible to catheterization are described. The robotic platform uses magnetic resonance imaging (MRI) for feeding back information to a controller responsible for the real-time control and navigation along pre-planned paths in the blood vessels of untethered magnetic carriers, nanorobots, and/or magnetotactic bacteria (MTB) loaded with sensory or therapeutic agents acting like a wireless robotic arm, manipulator, or other extensions necessary to perform specific remote tasks. Unlike known magnetic targeting methods, the present platform allows us to reach locations deep in the human body while enhancing targeting efficacy using real-time navigational or trajectory control. The paper describes several versions of the platform upgraded through additional software and hardware modules allowing enhanced targeting efficacy and operations in very difficult locations such as tumoral lesions only accessible through complex microvasculature networks.