Role of F-actin-mediated endocytosis and exercise in mitochondrial transplantation in an experimental Parkinson's disease mouse model.

Dopaminergic neurons gradually deteriorate in Parkinson's Disease (PD), which is characterized by the intracellular accumulation of Lewy bodies that are enriched with α-synuclein protein. Mitochondrial dysfunction is one of the primary contributors to this and is considered as the central player in the pathogenesis of PD. Recently, improving mitochondrial function has been extensively explored as a therapeutic strategy in various preclinical PD models. Mitochondrial transplantation is one such naïve yet highly efficient technique that has been well explored in diseases like diabetes, NAFLD, and cardiac ischemia but not in PD. Here, we compared the effects of transplanting normal allogenic mitochondria to those of transplanting exercise-induced allogenic mitochondria isolated from the liver into the PD mouse model. It is already known that normal Mitochondrial Transplant (MT) reduces the PD pathology, but our research found out that exercise-induced mitochondria were more effective in treating the PD pathology because they had higher respiratory capacities. Additionally, compared to a standard transplant, this therapy significantly boosted the rate of mitochondrial biogenesis and the quantity of mitochondrial subunits in PD mice. Further, we also explored the mechanism of mitochondrial uptake into the cells and found that F-actin plays a key role in the internalization of mitochondria. This study is the first to demonstrate the relevance of exercise-induced allogenic MT and the function of F-actin in the internalization of mitochondria in PD mice.

Fingolimod exerts neuroprotection by regulating S1PR1 mediated BNIP3-PINK1-Parkin dependent mitophagy in rotenone induced mouse model of Parkinson's disease.

The motor impairments brought on by the loss of dopaminergic neurons in the substantia nigra are the most well-known symptoms of Parkinson's disease (PD). It is believed that dopaminergic neurons are especially vulnerable to mitochondrial malfunction. For the maintenance of mitochondrial integrity, selective autophagic removal of dysfunctional mitochondria via mitophagy primarily regulated by PINK1/Parkin pathway is essential. Moreover, newer studies also implicate the role of phospholipid metabolism, such as that of Sphingosine-1-phosphate (S1P) as a contributor to PD. S1P receptors have been reported to influence mitochondrial function in neurodegenerative diseases. Fingolimod (FTY720), an S1P receptor-1 modulator has been proven effective in PD but its regulation of mitophagy in PD is still elusive. In this study, the neuroprotective effect of FTY720 by modulating mitophagy, has been explored against rotenone (ROT) induced neurotoxicity in in-vivo. The animals were randomly divided into 5 groups namely, Normal Control (NC); Disease control (DC): ROT (1.5 mg/kg); Low dose (LD): ROT + FTY720 (0.5 mg/kg); High dose (HD): ROT + FTY720 (1 mg/kg) and Vehicle control (VC): 1 % DMSO. ROT was administered through i.p. and FTY720 through p.o. for 21 days. At the end of the study, various neurobehavioral studies (rotarod test and actimeter), western blot techniques, and immunofluorescence studies were performed. FTY720 restored the neurobehavioural functions and protein expression of PINK1, Parkin and BNIP3 in ROT-induced PD mice. The results obtained in our study suggest that FTY720 has a neuroprotective effect in ROT-induced mice model of PD via PINK1-Parkin mediated mitophagy.

Inter and intracellular mitochondrial transfer: Future of mitochondrial transplant therapy in Parkinson's disease.

Parkinson's disease (PD) is marked by the gradual degeneration of dopaminergic neurons and the intracellular build-up of Lewy bodies rich in α-synuclein protein. This impairs various aspects of the mitochondria including the generation of ROS, biogenesis, dynamics, mitophagy etc. Mitochondrial dynamics are regulated through the inter and intracellular movement which impairs mitochondrial trafficking within and between cells. This inter and intracellular mitochondrial movement plays a significant role in maintaining neuronal dynamics in terms of energy and growth. Kinesin, dynein, myosin, Mitochondrial rho GTPase (Miro), and TRAK facilitate the retrograde and anterograde movement of mitochondria. Enzymes such as Kinases along with Calcium (Ca2+), Adenosine triphosphate (ATP) and the genes PINK1 and Parkin are also involved. Extracellular vesicles, gap junctions, and tunneling nanotubes control intercellular movement. The knowledge and understanding of these proteins, enzymes, molecules, and movements have led to the development of mitochondrial transplant as a therapeutic approach for various disorders involving mitochondrial dysfunction such as stroke, ischemia and PD. A better understanding of these pathways plays a crucial role in establishing extracellular mitochondrial transplant therapy for reverting the pathology of PD. Currently, techniques such as mitochondrial coculture, mitopunch and mitoception are being utilized in the pre-clinical stages and should be further explored for translational value. This review highlights how intercellular and intracellular mitochondrial dynamics are affected during mitochondrial dysfunction in PD. The field of mitochondrial transplant therapy in PD is underlined in particular due to recent developments and the potential that it holds in the near future.

Endodontic Management of Mandibular anterior teeth and premolars with Vertucci's Type VIII canal morphology: A Rare Case.

A thorough knowledge of variable and complex tooth morphology, detailed exploration of the internal anatomy and underlying pathology, proper interpretation of radiographic images, conservative access to explore all the canals, thorough debridement and disinfection of canal system, three-dimensional seal by obturation, and good coronal seal by final restoration are essential steps in the management for a successful endodontic treatment outcome. Clinical management of rare case with extra canals in the lower anterior teeth and premolars had to undergo root canal therapy has been described. Referring to the hard-tissue repository of the human dental internal morphology, carefully interpreting multiangled radiography/cone-beam computed tomography, using tools such as magnifying loupes with illumination and ultrasonics, thermoplasticized gutta-percha system to obturate, are very helpful to the clinician can achieve this goal. This article describes and illustrates the management of a rare case with Vertucci's Type VIII canal anatomy in lower anterior teeth and premolars.

Demonstration of a low-power, local-oscillator-less, and DSP-free coherent receiver for data center interconnects.

Performance limitations of currently employed four-level pulse amplitude modulation links and high power consumption of digital signal processing (DSP)-based coherent links for further increase in capacity create an urgent demand for low-power coherent solutions for short-reach data center interconnects. We propose a low-power coherent receiver with analog domain processing for a self-homodyne link. To validate the proposed scheme, a 10 GBd polarization multiplexed carrier-based self-homodyne quadrature phase-shift keying system with a constant modulus algorithm-based equalizer chip is experimentally demonstrated. Also, energy consumption per bit estimates show that the proposed approach results in significant power reduction in comparison with conventional DSP-based solutions.

Establishing a novel biosynthetic pathway for the production of 3,4-dihydroxybutyric acid from xylose in Escherichia coli.

3-Hydroxy-γ-butyrolactone (3HBL) is an attractive building block owing to its broad applications in pharmaceutical industry. Currently, 3HBL is commercially produced by chemical routes using petro-derived carbohydrates, which involves hazardous materials and harsh processing conditions. Only one biosynthetic pathway has been reported for synthesis of 3HBL and its hydrolyzed form 3,4-dihydroxybutyric acid (3,4-DHBA) using glucose and glycolic acid as the substrates and coenzyme A as the activator, which involves multiple steps (>10 steps) and suffers from low productivity and yield. Here we established a novel five-step biosynthetic pathway for 3,4-DHBA generation from D-xylose based on the non-phosphorylative D-xylose metabolism, which led to efficient production of 3,4-DHBA in Escherichia coli. Pathway optimization by incorporation of efficient enzymes for each step and host strain engineering by knocking out competing pathways enabled 1.27g/L 3,4-DHBA produced in shake flasks, which is the highest titer reported so far. The novel pathway established in engineered E. coli strain demonstrates a new route for 3,4-DHBA biosynthesis from xylose, and this engineered pathway has great potential for industrial biomanufacturing of 3,4-DHBA and 3HBL.

Rational engineering of diol dehydratase enables 1,4-butanediol biosynthesis from xylose.

Establishing novel synthetic routes for microbial production of chemicals often requires overcoming pathway bottlenecks by tailoring enzymes to enhance bio-catalysis or even achieve non-native catalysis. Diol dehydratases have been extensively studied for their interactions with C2 and C3 diols. However, attempts on utilizing these insights to enable catalysis on non-native substrates with more than two hydroxyl groups have been plagued with low efficiencies. Here, we rationally engineered the Klebsiella oxytoca diol dehydratase to enable and enhance catalytic activity toward a non-native C4 triol, 1,2,4-butanetriol. We analyzed dehydratase's interaction with 1,2-propanediol and glycerol, which led us to develop rationally conceived hypotheses. An in silico approach was then developed to identify and screen candidate mutants with desired activity. This led to an engineered diol dehydratase with nearly 5 fold higher catalytic activity toward 1,2,4-butanetriol than the wild type as determined by in vitro assays. Based on this result, we then expanded the 1,2,4-butanetriol pathway to establish a novel 1,4-butanediol production platform. We engineered Escherichia coli's xylose catabolism to enhance the biosynthesis of 1,2,4-butanetriol from 224mg/L to 1506mg/L. By introducing the complete pathway in the engineered strain we achieve de novo biosynthesis of 1,4-butanediol at 209mg/L from xylose. This work expands the repertoire of substrates catalyzed by diol dehydratases and serves as an elucidation to establish novel biosynthetic pathways involving dehydratase based biocatalysis.

Establishing a synergetic carbon utilization mechanism for non-catabolic use of glucose in microbial synthesis of trehalose.

In nature glucose is a common carbon and energy source for catabolic use and also a building unit of polysaccharides and glycosylated compounds. The presence of strong glucose catabolic pathways in microorganism rapidly decomposes glucose into smaller metabolites and challenges non-catabolic utilization of glucose as C6 building unit or precursor. To address this dilemma, we design a synergetic carbon utilization mechanism (SynCar), in which glucose catabolism is inactivated and a second carbon source (e.g. glycerol) is employed to maintain cell growth and rationally strengthen PEP driving force for glucose uptake and non-catabolic utilization. Remarkably, a trehalose biosynthesis model developed for proof-of-concept indicates that SynCar leads to 131% and 200% improvement in trehalose titer and yield, respectively. The conversion rate of glucose to trehalose reaches 91% of the theoretical maximum. This work demonstrates the broad applicability of SynCar in the biosynthesis of molecules derived from non-catabolic glucose.

Aerobic biosynthesis of hydrocinnamic acids in Escherichia coli with a strictly oxygen-sensitive enoate reductase.

3-Phenylpropionic acid (3PPA) and 3-(4-hydroxyphenyl) propionic acid (HPPA) are important commodity aromatic acids widely used in food, pharmaceutical and chemical industries. Currently, 3PPA and HPPA are mainly manufactured through chemical synthesis, which contains multiple steps involving toxic solvents and catalysts harmful to environment. Therefore, replacement of such existing petroleum-derived approaches with simple and environmentally friendly biological processes is highly desirable for manufacture of these chemicals. Here, for the first time we demonstrated the de novo biosynthesis of 3PPA and HPPA using simple carbon sources in E. coli by extending the cinnamic acids biosynthesis pathways through biological hydrogenation. We first screened 11 2-enoate reductases (ER) from nine microorganisms, leading to efficient conversion of cinnamic acid and p-coumaric acid to 3PPA and HPPA, respectively. Surprisingly, we found a strictly oxygen-sensitive Clostridia ER capable of functioning efficiently in E. coli even under aerobic conditions. On this basis, reconstitution of the full pathways led to the de novo production of 3PPA and HPPA and the accumulation of the intermediates (cinnamic acid and p-coumaric acid) with cell toxicity. To address this problem, different expression strategies were attempted to optimize individual enzyme׳s expression level and minimize intermediates accumulation. Finally, the titers of 3PPA and HPPA reached 366.77mg/L and 225.10mg/L in shake flasks, respectively. This study not only demonstrated the potential of microbial approach as an alternative to chemical process, but also proved the possibility of using oxygen-sensitive enzymes under aerobic conditions.

Engineering the Autotroph Methanococcus maripaludis for Geraniol Production.

The rapid autotrophic growth of the methanogenic archaeon Methanococcus maripaludis on H2 and CO2 makes it an attractive microbial chassis to inexpensively produce biochemicals. To explore this potential, a synthetic gene encoding geraniol synthase (GES) derived from Ocimum basilicum was cloned into a M. maripaludis expression vector under selection for puromycin resistance. Recombinant expression of GES in M. maripaludis during autotrophic growth on H2/CO2 or formate yielded geraniol at 2.8 and 4.0 mg g(-1) of dry weight, respectively. The yield of geraniol decreased 2-3-fold when organic carbon sources were added to stimulate heterotrophic growth. In the absence of puromycin, geraniol production during autotrophic growth on formate increased to 4.6 mg g(-1) of dry weight. A conceptual model centered on the autotrophic acetyl coenzyme A biosynthetic pathway identified strategies to divert more autotrophic carbon flux to geraniol production.

Enhanced photo-bioelectrochemical energy conversion by genetically engineered cyanobacteria.

Photosynthetic energy conversion using natural systems is increasingly being investigated in the recent years. Photosynthetic microorganisms, such as cyanobacteria, exhibit light-dependent electrogenic characteristics in photo-bioelectrochemical cells (PBEC) that generate substantial photocurrents, yet the current densities are lower than their photovoltaic counterparts. Recently, we demonstrated that a cyanobacterium named Nostoc sp. employed in PBEC could generate up to 35 mW m(-2) even in a non-engineered PBEC. With the insights obtained from our previous research, a novel and successful attempt has been made in the current study to genetically engineer the cyanobacteria to further enhance its extracellular electron transfer. The cyanobacterium Synechococcus elongatus PCC 7942 was genetically engineered to express a non-native redox protein called outer membrane cytochrome S (OmcS). OmcS is predominantly responsible for metal reducing abilities of exoelectrogens such as Geobacter sp. The engineered S. elongatus exhibited higher extracellular electron transfer ability resulting in approximately ninefold higher photocurrent generation on the anode of a PBEC than the corresponding wild-type cyanobacterium. This work highlights the scope for enhancing photocurrent generation in cyanobacteria, thereby benefiting faster advancement of the photosynthetic microbial fuel cell technology.

Resurrecting an unsalvageable lower incisor with a mono-block approach.

Contemporary techniques, as well as the availability of bioactive and adhesive materials in endodontics, have helped revivifying teeth that were deemed hopeless. These newer materials and techniques would enable the clinician: (a) to predictably stop microbial activity (b) to achieve a total corono-apical fluid tight seal and (c) to strengthen mutilated teeth by obtaining intra-radicular reinforcement through mono-block effect. This case report demonstrates the successful treatment of a mutilated anterior tooth with the use of bioactive and adhesive materials to obtain a total seal and mono-block effect. This article also shows the use of a simple method in the placement of root filling cement into the root canal.

Engineering microaerobic metabolism of E. coli for 1,2-propanediol production.

Establishment of novel metabolic pathways for biosynthesis of chemicals, fuels and pharmaceuticals has been demonstrated in Escherichia coli due to its ease of genetic manipulation and adaptability to varying oxygen levels. E. coli growing under microaerobic condition is known to exhibit features of both aerobic and anaerobic metabolism. In this work, we attempt to engineer this metabolism for production of 1,2-propanediol. We first redirect the carbon flux by disrupting carbon-competing pathways to increase the production of 1,2-propanediol microaerobically from 0.25 to 0.85 g/L. We then disrupt the first committed step of E. coli's ubiquinone biosynthesis pathway (ubiC) to prevent the oxidation of NADH in microaerobic conditions. Coupling this strategy with carbon flux redirection leads to enhanced production of 1,2-propanediol at 1.2 g/L. This work demonstrates the production of non-native reduced chemicals in E. coli by engineering its microaerobic metabolism.

Synthesis of chemicals by metabolic engineering of microbes.

Metabolic engineering is a powerful tool for the sustainable production of chemicals. Over the years, the exploration of microbial, animal and plant metabolism has generated a wealth of valuable genetic information. The prudent application of this knowledge on cellular metabolism and biochemistry has enabled the construction of novel metabolic pathways that do not exist in nature or enhance existing ones. The hand in hand development of computational technology, protein science and genetic manipulation tools has formed the basis of powerful emerging technologies that make the production of green chemicals and fuels a reality. Microbial production of chemicals is more feasible compared to plant and animal systems, due to simpler genetic make-up and amenable growth rates. Here, we summarize the recent progress in the synthesis of biofuels, value added chemicals, pharmaceuticals and nutraceuticals via metabolic engineering of microbes.

Systematically engineering Escherichia coli for enhanced production of 1,2-propanediol and 1-propanol.

The biological production of high value commodity 1,2-propanediol has been established by engineering the glycolysis pathway. However, the simultaneous achievement of high titer and high yield has not been reported yet, as all efforts in increasing the titer have resulted in low yields. In this work, we overcome this limitation by employing an optimal minimal set of enzymes, channeling the carbon flux into the 1,2-propanediol pathway, increasing NADH availability, and improving the anaerobic growth of the engineered Escherichia coli strain by developing a cell adaptation method. These efforts lead to 1,2-propanediol production at a titer of 5.13 g/L with a yield of 0.48 g/g glucose in 20 mL shake flask studies. On this basis, we pursue the enhancement of 1-propanol production from the 1,2-propanediol platform. By constructing a fusion diol dehydratase and developing a dual strain process, we achieve a 1-propanol titer of 2.91 g/L in 20 mL shake flask studies. To summarize, we report the production of 1,2-propanediol at enhanced titer and enhanced yield simultaneously in E. coli for the first time. Furthermore, we establish an efficient system for the production of biofuel 1-propanol biologically.

Microbial production of antioxidant food ingredients via metabolic engineering.

Antioxidants are biological molecules with the ability to protect vital metabolites from harmful oxidation. Due to this fascinating role, their beneficial effects on human health are of paramount importance. Traditional approaches using solvent-based extraction from food/non-food sources and chemical synthesis are often expensive, exhaustive, and detrimental to the environment. With the advent of metabolic engineering tools, the successful reconstitution of heterologous pathways in Escherichia coli and other microorganisms provides a more exciting and amenable alternative to meet the increasing demand of natural antioxidants. In this review, we elucidate the recent progress in metabolic engineering efforts for the microbial production of antioxidant food ingredients - polyphenols, carotenoids, and antioxidant vitamins.

Retrieval of a metallic obstruction from the root canal of a premolar using Masserann technique.

A metallic obstruction in the root canal blocks canal cleaning and shaping procedures and requires either bypassing or retrieval. Many methods have been recommended to retrieve a metallic obstruction from the root canal. This article describes the retrieval of a metallic obstruction from the root canal of a premolar using Masserann technique to facilitate endodontic retreatment. Masserann technique is said to have limited application in posteriors. However, in this case, the obstruction was successfully retrieved by employing Masserann technique which consisted of using a trephan to cut the dentine and extractor tube to retrieve the obstruction. The retrieved obstruction was found to be a separated H-file. Endodontic retreatment was completed following the detection and negotiation of an extra canal in the same tooth.

3D molecular descriptors important for clinical success.

The pharmacokinetic and safety profiles of clinical drug candidates are greatly influenced by their requisite physicochemical properties. In particular, it has been shown that 2D molecular descriptors such as fraction of Sp3 carbon atoms (Fsp3) and number of stereo centers correlate with clinical success. Using the proteomic off-target hit rate of nicotinic ligands, we found that shape-based 3D descriptors such as the radius of gyration and shadow indices discriminate off-target promiscuity better than do Fsp3 and the number of stereo centers. We have deduced the relevant descriptor values required for a ligand to be nonpromiscuous. Investigating the MDL Drug Data Report (MDDR) database as compounds move from the preclinical stage toward the market, we have found that these shape-based 3D descriptors predict clinical success of compounds at preclinical and phase1 stages vs compounds withdrawn from the market better than do Fsp3 and LogD. Further, these computed 3D molecular descriptors correlate well with experimentally observed solubility, which is among well-known physicochemical properties that drive clinical success. We also found that about 84% of launched drugs satisfy either Shadow index or Fsp3 criteria, whereas withdrawn and discontinued compounds fail to meet the same criteria. Our studies suggest that spherical compounds (rather than their elongated counterparts) with a minimal number of aromatic rings may exhibit a high propensity to advance from clinical trials to market.

Inhibition of acetate accumulation leads to enhanced production of (R,R)-2,3-butanediol from glycerol in Escherichia coli.

This work describes the production of (R,R)-2,3-butanediol in Escherichia coli using glycerol by metabolic engineering approaches. The introduction of a synthetic pathway converting pyruvate to (R,R)-2,3-butanediol into wild-type E. coli strain BW25113 led to the production of (R,R)-2,3-butanediol at a titer of 3.54 g/l and a yield of 0.131 g product/g glycerol (26.7 % of theoretical maximum) with acetate (around 3.00 g/l) as the dominant by-product. We therefore evaluated the impacts of deleting the genes ackA or/and poxB that are responsible for the major by-product, acetate. This increased production of (R,R)-2,3-butanediol to 9.54 g/l with a yield of 0.333 g product/g glycerol (68.0 % of theoretical maximum) in shake flask studies. The utilization of low-priced crude glycerol to produce value-added chemicals is of great significance to the economic viability of the biodiesel industry.

Dehydratase mediated 1-propanol production in metabolically engineered Escherichia coli.

BACKGROUND: With the increasing consumption of fossil fuels, the question of meeting the global energy demand is of great importance in the near future. As an effective solution, production of higher alcohols from renewable sources by microorganisms has been proposed to address both energy crisis and environmental concerns. Higher alcohols contain more than two carbon atoms and have better physiochemical properties than ethanol as fuel substitutes. RESULTS: We designed a novel 1-propanol metabolic pathway by expanding the well-known 1,2-propanediol pathway with two more enzymatic steps catalyzed by a 1,2-propanediol dehydratase and an alcohol dehydrogenase. In order to engineer the pathway into E. coli, we evaluated the activities of eight different methylglyoxal synthases which play crucial roles in shunting carbon flux from glycolysis towards 1-propanol biosynthesis, as well as two secondary alcohol dehydrogenases of different origins that reduce both methylglyoxal and hydroxyacetone. It is evident from our results that the most active enzymes are the methylglyoxal synthase from Bacillus subtilis and the secondary alcohol dehydrogenase from Klebsiella pneumoniae, encoded by mgsA and budC respectively. With the expression of these two genes and the E. coli ydjG encoding methylglyoxal reductase, we achieved the production of 1,2-propanediol at 0.8 g/L in shake flask experiments. We then characterized the catalytic efficiency of three different diol dehydratases on 1,2-propanediol and identified the optimal one as the 1,2-propanediol dehydratase from Klebsiella oxytoca, encoded by the operon ppdABC. Co-expressing this enzyme with the above 1,2-propanediol pathway in wild type E. coli resulted in the production of 1-propanol at a titer of 0.25 g/L. CONCLUSIONS: We have successfully established a new pathway for 1-propanol production by shunting the carbon flux from glycolysis. To our knowledge, it is the first time that this pathway has been utilized to produce 1-propanol in E. coli. The work presented here forms a basis for further improvement in production. We speculate that dragging more carbon flux towards methylglyoxal by manipulating glycolytic pathway and eliminating competing pathways such as lactate generation can further enhance the production of 1-propanol.