PLD3 and PLD4 synthesize S,S-BMP, a key phospholipid enabling lipid degradation in lysosomes.

Bis(monoacylglycero)phosphate (BMP) is an abundant lysosomal phospholipid required for degradation of lipids, particularly gangliosides. Alterations in BMP levels are associated with neurodegenerative diseases. Unlike typical glycerophospholipids, lysosomal BMP has two chiral glycerol carbons in the S (rather than the R) stereo-conformation, protecting it from lysosomal degradation. How this unusual and yet crucial S,S-stereochemistry is achieved is unknown. Here, we report that phospholipases D3 and D4 (PLD3 and PLD4) synthesize lysosomal S,S-BMP, with either enzyme catalyzing the critical glycerol stereo-inversion reaction in vitro. Deletion of PLD3 or PLD4 markedly reduced BMP levels in cells or in murine tissues where either enzyme is highly expressed (brain for PLD3; spleen for PLD4), leading to gangliosidosis and lysosomal abnormalities. PLD3 mutants associated with neurodegenerative diseases, including risk of Alzheimer's disease, diminished PLD3 catalytic activity. We conclude that PLD3/4 enzymes synthesize lysosomal S,S-BMP, a crucial lipid for maintaining brain health.

Kupyaphores are zinc homeostatic metallophores required for colonization of Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) endures a combination of metal scarcity and toxicity throughout the human infection cycle, contributing to complex clinical manifestations. Pathogens counteract this paradoxical dysmetallostasis by producing specialized metal trafficking systems. Capture of extracellular metal by siderophores is a widely accepted mode of iron acquisition, and Mtb iron-chelating siderophores, mycobactin, have been known since 1965. Currently, it is not known whether Mtb produces zinc scavenging molecules. Here, we characterize low-molecular-weight zinc-binding compounds secreted and imported by Mtb for zinc acquisition. These molecules, termed kupyaphores, are produced by a 10.8 kbp biosynthetic cluster and consists of a dipeptide core of ornithine and phenylalaninol, where amino groups are acylated with isonitrile-containing fatty acyl chains. Kupyaphores are stringently regulated and support Mtb survival under both nutritional deprivation and intoxication conditions. A kupyaphore-deficient Mtb strain is unable to mobilize sufficient zinc and shows reduced fitness upon infection. We observed early induction of kupyaphores in Mtb-infected mice lungs after infection, and these metabolites disappeared after 2 wk. Furthermore, we identify an Mtb-encoded isonitrile hydratase, which can possibly mediate intracellular zinc release through covalent modification of the isonitrile group of kupyaphores. Mtb clinical strains also produce kupyaphores during early passages. Our study thus uncovers a previously unknown zinc acquisition strategy of Mtb that could modulate host-pathogen interactions and disease outcome.

Iso-Pentadienyl Carbonate as a Five Carbon Synthon in Manganese(I)-Catalyzed Selective Linear 1,3-Dienylation.

Selective linear 1,3-dienylations are essential transformations, and numerous synthetic efforts have been documented. However, a general method enabling access to electron-rich, -poor, and biologically relevant dienyl molecules is in high demand. Hence, we report a straightforward method of manganese(I)-catalyzed C-H dienylation of arenes by using iso-pentadienyl carbonate as a five carbon synthon. This is a highly unprecedented report for selective linear 1,3-dienylation using manganese C-H activation catalysis. Our method facilitates the synthesis of varieties of dienes, including those suitable for normal or inverse electron demand Diels-Alder reactions, dienyl glycoconjugates, and unnatural amino acids. Extensive mechanistic studies, including isolation of C-H activated organo-manganese complex and isotopic analyses, have supported the proposed mechanism of this dienylation. The synthetic applicability of this method eased to deliver a 6/6/5-fused tricyclic nagilactone scaffold.

Robust Synthesis of Terpenoid Scaffolds under Mn(I)-Catalysis.

The 6/6/5-fused tricyclic scaffold is a central feature of structurally complex terpenoid natural products. A step-economical cascade transformation that leads to a complex molecular skeleton is regarded as a sustainable methodology. Therefore, we report the first Mn(I)-catalyzed C(sp2)-H chemoselective in situ dienylation and diastereoselective intramolecular Diels-Alder reaction using iso-pentadienyl carbonate to access 6/6/5-fused tricyclic scaffolds. To the best of our knowledge, there is no such report thus far to utilize iso-pentadienyl carbonate as a substrate in C-H activation catalysis. Extensive mechanistic studies, such as the isolation of catalytically active organo-manganese(I) complexes, 1,3-dienyl-intermediates, and isotopic labeling experiments have supported the proposed mechanism of this cascade reaction.

Discovery of novel microRNAs and their pathogenic responsive target genes in mild traumatic brain injury.

MicroRNAs (miRNAs) are non-coding RNA molecules that function in RNA silencing and post-transcriptional regulation of gene expression. They are profound mediators of molecular and cellular changes in several pathophysiological conditions. Since miRNAs play major roles in regulating gene expression after traumatic brain injury (TBI), their possible role in diagnosis, prognosis, and therapy is not much explored. In this study, we aimed to identify specific miRNAs that are involved in the pathophysiological conditions in the first 24 h after mild TBI (mTBI). The genome-wide expression of miRNAs was evaluated by applying RNA sequence in the injury area of the cerebral cortex 24 after inflicting the injury using a mouse model of mild fluid percussion injury (FPI; 10 psi). Here, we identified different annotated, conserved, and novel miRNAs. A total of 978 miRNAs after 24 h of TBI were identified, and among these, 906 miRNAs were differentially expressed between control and mTBI groups. In this study, 146 miRNAs were identified as novel to mTBI and among them, 21 miRNAs were significant (p < 0.05). Using q-RT-PCR, we validated 10 differentially and significantly expressed novel miRNAs. Further, we filtered the differentially expressed miRNAs that were linked with proinflammatory cytokines, apoptosis, matrix metalloproteinases (MMPs), and tight junction and junctional adhesion molecule genes. Overall, this work shows that mTBI induces widespread changes in the expression of miRNAs that may underlie the progression of the TBI pathophysiology. The detection of several novel TBI-responsive miRNAs and their solid link with pathophysiological genes may help in identifying novel therapeutic targets.

A Review on Physicochemical Properties of Polymers Used as Filaments in 3D-Printed Tablets.

Three-dimensional (3D) printing technology has presently been explored widely in the field of pharmaceutical research to produce various conventional as well as novel dosage forms such as tablets, capsules, oral films, pellets, subcutaneous implants, scaffolds, and vaginal rings. The use of this innovative method is a good choice for its advanced technologies and the ability to make tailored medicine specifically for individual patient. There are many 3D printing systems that are used to print tablets, implants, and vaginal rings. Among the available systems, the fused deposition modeling (FDM) is widely utilized. The FDM has been regarded as the best choice of printer as it shows high potential in the production of tablets as a unit dose in 3D printing medicine manufacturing. In order to design a 3D-printed tablet or other dosage forms, the physicochemical properties of polymers play a vital role. One should have proper knowledge about the polymer's properties so that one can select appropriate polymers in order to design 3D-printed dosage form. This review highlighted the various physicochemical properties of polymers that are currently used as filaments in 3D printing. In this manuscript, the authors also discussed various systems that are currently adopted in the 3D printing.

An approach to quantify heavy metals and their source apportionment in coal mine soil: a study through PMF model.

Mining activities in the opencast coal mines contaminate the surrounding soil by releasing coal dust containing heavy metals (HMs). The objective of the present study was to quantify the concentration of HMs like Fe, Cu, Mn, Ni, Cr, Zn, and Co in soil on profile and distance basis in the vicinity of the coal mines. This research also proposed the synthesis application of positive matrix factorization (PMF) model for the quantitative assessment of pollution sources. The results showed that the soil was more affected due to the presence of Cr in mining areas., and the contamination factor (Cf) of Cr was high at the edge of coal mine. It was observed from the study that Cf of the HMs was decreased with the increase in distance from the mine edge. The application of PMF model demonstrated that the contributions of Zn (4.2%), Ni (16.8%), and Mn (100%) were maximum in the pollution. The study concluded that soil contamination is inexorable due to opencast coal mining activities, and it can be mitigated by developing a green belt or through the process of ecological restoration and phytoremediation.

The loss of enzymatic activity of the PHARC-associated lipase ABHD12 results in increased phagocytosis that causes neuroinflammation.

Phagocytosis is an important evolutionary conserved process, essential for clearing pathogens and cellular debris in higher organisms, including humans. This well-orchestrated innate immunological response is intricately regulated by numerous cellular factors, important amongst which are the immunomodulatory lysophosphatidylserines (lyso-PSs) and the pro-apoptotic oxidized phosphatidylserines (PSs) signalling lipids. Interestingly, in mammals, both these signalling lipids are physiologically regulated by the lipase ABHD12, mutations of which cause the human neurological disorder PHARC. Despite the biomedical significance of this lipase, detailed mechanistic studies and the specific contribution of ABHD12 to innate processes like phagocytosis remain poorly understood. Here, by immunohistochemical and immunofluorescence approaches, using the murine model of PHARC, we show, that upon an inflammatory stimulus, activated microglial cells in the cerebellum of mice deficient in ABHD12 have an amoeboid morphology, increased soma size and display heightened phagocytosis activity. We also report that upon an inflammatory stimulus, cerebellar levels of ABHD12 increase to possibly metabolize the heightened oxidized PS levels, temper phagocytosis and, in turn, control neuroinflammation during oxidative stress. Next, to complement these findings, with the use of biochemical approaches in cultured microglial cells, we show that the pharmacological inhibition and/or genetic deletion of ABHD12 results in increased phagocytic uptake in a fluorescent bead uptake assay. Together, our studies provide compelling evidence that ABHD12 plays an important role in regulating phagocytosis in cerebellar microglial cells and provides a possible explanation, as to why human PHARC subjects display neuroinflammation and atrophy in the cerebellum.

A chemical-genetic screen identifies ABHD12 as an oxidized-phosphatidylserine lipase.

Reactive oxygen species (ROS) are transient, highly reactive intermediates or byproducts produced during oxygen metabolism. However, when innate mechanisms are unable to cope with sequestration of surplus ROS, oxidative stress results, in which excess ROS damage biomolecules. Oxidized phosphatidylserine (PS), a proapoptotic 'eat me' signal, is produced in response to elevated ROS, yet little is known regarding its chemical composition and metabolism. Here, we report a small molecule that generates ROS in different mammalian cells. We used this molecule to detect, characterize and study oxidized PS in mammalian cells. We developed a chemical-genetic screen to identify enzymes that regulate oxidized PS in mammalian cells and found that the lipase ABHD12 hydrolyzes oxidized PS. We validated these findings in different physiological settings including primary peritoneal macrophages and brains from Abhd12-/- mice under inflammatory stress, and in the process, we functionally annotated an enzyme regulating oxidized PS in vivo.

Mapping the Neuroanatomy of ABHD16A, ABHD12, and Lysophosphatidylserines Provides New Insights into the Pathophysiology of the Human Neurological Disorder PHARC.

Lysophosphatidylserine (lyso-PS), a lysophospholipid derived from phosphatidylserine (PS), has emerged as a potent signaling lipid in mammalian physiology. In vivo, the metabolic serine hydrolases ABHD16A and ABHD12 are major lipases that biosynthesize and degrade lyso-PS, respectively. Of biomedical relevance, deleterious mutations to ABHD12 cause accumulation of lyso-PS in the brain, and this deregulated lyso-PS metabolism leads to the human genetic neurological disorder PHARC (polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, and cataract). While the roles of ABHD16A and ABHD12 in lyso-PS metabolism in the mammalian brain are well established, the anatomical and (sub)cellular localizations of both lipases and the functional cross-talk between them with respect to regulating lyso-PS lipids remain under investigated. Here, using subcellular organelle fractionation, biochemical assays, and immunofluorescence-based high-resolution microscopy, we show that the PS lipase ABHD16A is an endoplasmic reticulum-localized enzyme, an organelle intricately regulating cellular PS levels. In addition, leveraging immunohistochemical analysis using genetic ABHD16A and ABHD12 knockout mice as important controls, we map the anatomical distribution of both of these lipases in tandem in the murine brain and show for the first time the distinct localization of these lipases to different regions and cells of the cerebellum. We complement the aforementioned immunohistochemical studies by quantitatively measuring lyso-PS concentrations in various brain regions using mass spectrometry and find that the cerebellar lyso-PS levels are most affected by deletion of ABHD16A (decreased) or ABHD12 (increased). Taken together, our studies provide new insights into lyso-PS signaling in the cerebellum, the most atrophic brain region in human PHARC subjects.

Autosomal dominant polycystic kidney disease and pioglitazone for its therapy: a comprehensive review with an emphasis on the molecular pathogenesis and pharmacological aspects.

Autosomal dominant polycystic kidney disease (ADPKD) is an inherited chronic kidney disorder (CKD) that is characterized by the development of numerous fluid-filled cysts in kidneys. It is caused either due to the mutations in the PKD1 or PKD2 gene that encodes polycystin-1 and polycystin-2, respectively. This condition progresses into end-stage renal disorder if the renal or extra-renal clinical manifestations remain untreated. Several clinical trials with a variety of drugs have failed, and the only Food and Drugs Administration (FDA) approved drug to treat ADPKD to date is tolvaptan that works by antagonizing the vasopressin-2 receptor (V2R). The pathology of ADPKD is complex and involves the malfunction of different signaling pathways like cAMP, Hedgehog, and MAPK/ERK pathway owing to the mutated product that is polycystin-1 or 2. A measured yet substantial number of preclinical studies have found pioglitazone to decrease the cystic burden and improve the renal function in ADPKD. The peroxisome proliferator-activated receptor-gamma is found on the epithelial cells of renal collecting tubule and when it gets agonized by pioglitazone, confers efficacy in ADPKD treatment through multiple mechanisms. There is only one clinical trial (ongoing) wherein it is being assessed for its benefits and risk in patients with ADPKD, and is expected to get approval from the regulatory body owing to its promising therapeutic effects. This article would encompass the updated information on the epidemiology, pathophysiology of ADPKD, different mechanisms of action of pioglitazone in the treatment of ADPKD with preclinical and clinical shreds of evidence, and related safety updates.

Peri-natal growth retardation rate and fat mass accumulation in mice lacking Dip2A is dependent on the dietary composition.

Disco-interacting protein 2 is a highly conserved three-domain protein with two tandem Adenylate-forming domains. It is proposed to influence the processes involved in neuronal development by influencing lipid metabolism and remains to be characterized. In this study, we show that Disco-interacting protein 2a null mice do not exhibit overt phenotype defects. However, the body composition differences were observed in these mice under different dietary regimens. The neutral lipid composition of two different diets was characterized, and it was observed that the new-born mice grow relatively slower than the wild-type mice with delayed appearance of features such as dentition when fed with high-triacylglycerol NIN-formulation diet. The high-diacylglycerol Safe-formulation diet was found to accumulate more fat mass in mice than those fed with high-triacylglycerol NIN-formulation diet beyond 10 months. These findings point to a proposed relationship between dietary components (particularly the lipid composition) and body composition along with the growth of neonates in mice lacking the gene Disco-interacting protein 2a.

Competitive Electrohydrodynamic and Electrosolutal Advection Arrests Evaporation Kinetics of Droplets.

This article reports the hitherto unreported phenomenon of arrested evaporation dynamics in pendant droplets because of electric field stimulus. The evaporation kinetics of pendant droplets of electrically conducting saline solutions in the presence of a transverse, alternating electric field is investigated experimentally. While the increase of field strength reduces the evaporation rate, increment in field frequency has the opposite effect. The same has been explained on the solvation kinetics of ions in polar water. Theoretical analysis reveals that change in surface tension and the diffusion-driven evaporation model cannot predict the decelerated evaporation. With the aid of particle image velocimetry, suppression of internal circulation velocity within the droplet is observed under electric field stimulus, which directly affects the evaporation rate. A mathematical scaling model is proposed to quantify the effects of electrohydrodynamic circulation and electrothermal and electrosolutal advection on the evaporation kinetics. The analysis encompasses major governing parameters, namely, the thermal and solutal Marangoni numbers, the electrohydrodynamic number, the electro-Prandtl and electro-Schmidt numbers, and their respective contributions. It has been shown that the electrothermal Marangoni effect is suppressed by the electric field, leading to deteriorated evaporation rates. Additionally, the electrosolutal Marangoni effect further suppresses the internal advection, further reducing the evaporation rate by a larger proportion. Stability analysis reveals that the electric body force retards the stable internal advection. The stability mapping also illustrates that if the field strength is high enough for the electrosolutal advection to overshadow the solutal Marangoni effect completely, it can lead to improvement in evaporation rates.

Biochemical characterization of the PHARC-associated serine hydrolase ABHD12 reveals its preference for very-long-chain lipids.

Polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, and cataract (PHARC) is a rare genetic human neurological disorder caused by null mutations to the Abhd12 gene, which encodes the integral membrane serine hydrolase enzyme ABHD12. Although the role that ABHD12 plays in PHARC is understood, the thorough biochemical characterization of ABHD12 is lacking. Here, we report the facile synthesis of mono-1-(fatty)acyl-glycerol lipids of varying chain lengths and unsaturation and use this lipid substrate library to biochemically characterize recombinant mammalian ABHD12. The substrate profiling study for ABHD12 suggested that this enzyme requires glycosylation for optimal activity and that it has a strong preference for very-long-chain lipid substrates. We further validated this substrate profile against brain membrane lysates generated from WT and ABHD12 knockout mice. Finally, using cellular organelle fractionation and immunofluorescence assays, we show that mammalian ABHD12 is enriched on the endoplasmic reticulum membrane, where most of the very-long-chain fatty acids are biosynthesized in cells. Taken together, our findings provide a biochemical explanation for why very-long-chain lipids (such as lysophosphatidylserine lipids) accumulate in the brains of ABHD12 knockout mice, which is a murine model of PHARC.

Real-Time Conformational Changes and Controlled Orientation of Native Proteins Inside a Protein Nanoreactor.

Protein conformations play crucial roles in most, if not all, biological processes. Here we show that the current carried through a nanopore by ions allows monitoring conformational changes of single and native substrate-binding domains (SBD) of an ATP-Binding Cassette importer in real-time. Comparison with single-molecule Förster Resonance Energy Transfer and ensemble measurements revealed that proteins trapped inside the nanopore have bulk-like properties. Two ligand-free and two ligand-bound conformations of SBD proteins were inferred and their kinetic constants were determined. Remarkably, internalized proteins aligned with the applied voltage bias, and their orientation could be controlled by the addition of a single charge to the protein surface. Nanopores can thus be used to immobilize proteins on a surface with a specific orientation, and will be employed as nanoreactors for single-molecule studies of native proteins. Moreover, nanopores with internal protein adaptors might find further practical applications in multianalyte sensing devices.

Synthesis of dispiro-orthoester via an acetal oxo-carbenium ion.

The first dispiro orthoester via a spiroacetal oxo-carbenium ion is presented. Oxidative dearomatization of phloretic esters results in a bifunctional electrophilic spiroacetal oxo-carbenium ion, which undergoes a double nucleophilic addition by diol delivering a range of unusual dispiro-orthoesters with an excellent diversity.

Effect of Formulation Parameters on Enalapril Maleate Mucoadhesive Buccal Tablet Using Quality by Design (QbD) Approach.

The buccal route has great prospects and possible benefits for the administration of drugs systemically. The present study involves designing, developing and optimising the buccal tablet formulation of Enalapril Maleate (EM) by using the QbD approach. We prepared the EM buccal tablets using the dry granulation method. In the QTPP profile, the CQAs for EM buccal tablets are Mucoadhesive strength, swelling index and drug release (dependent variables); the CMAs identified for EM buccal tablets were Carbopol 934P, HPMC-K100M and chitosan (independent variables). Diluent quantity, blending time and compression force were selected as CPPs; the Box-Behnkentdesign was used to evaluate the relationship between the CMAs and CPPs. Based on the DoE, the composition of the optimised formulation of EM BT-18 consists of 20mg of EM, 15 mg of carbopol 934p, 17 mg of HPMC-K100M, 10mg of chitosan, 30 mg of PVP K-30, 1 mg of magnesium stearate, 16 mg of Mannitol, 1 mg of aspartame, and 50 mg of Ethyl cellulose. The optimised formulation of EM BT 18 was found to have a Mucoadhesive strength of 24.32±0.30g. The swelling index was 90.74±0.25% and drug release was sustained up to 10 hours 98.4±3.62% compared to the marketed product, whose release was up to 8 hours. We attempted to design a buccal tablet of Enalapril Maleate for sustained drug release in the treatment of hypertension. Patients who cannot take oral medication due to trauma or unconscious conditions could receive the formulation. Development of a newly P.ceutical product is very time-consuming, extremely costly and high-risk, with very little chance of a successful outcome. Hence, this study showed EM tablets are already available on the market but we have chosen a buccal drug delivery system using a novel approach using QbD tools to target the quality of the product accurately.

Comparative role of acetaminophen, carbon tetrachloride and thioacetamide in development of fibrosis in rats.

Background: Several hepatotoxicants such as acetaminophen, carbon tetrachloride, and thioacetamide are repeatedly used to develop hepatic fibrosis to mimic the histological and hemodynamic characteristics of human illness. It may be a good idea to establish a better model among these hepatotoxicants to develop hepatic fibrosis. Aim: The present study evaluated comparative toxic effects of three model hepatotoxicants for experimental progression of fibrosis or cirrhosis. Materials and methods: Acetaminophen (200 mg/kg), carbon tetrachloride (200 µl/kg) and thioacetamide (200 mg/kg) were administered orally, thrice in a week for 8 weeks in different groups. After 8 weeks of exposure, animals were euthanized, blood and tissues were collected for various hematological, serological, tissue biochemical analysis and histological observations for comparative assessment of toxic consequences. Results: Significant deviation was noted in liver function tests, lipid peroxidation, glutathione, activities of superoxide dismutase, catalase, and GSH cycle enzymes; aniline hydroxylase, amidopyrine-N-demethylase, DNA fragmentation and level of hydroxyproline when compared with control group. Histology also depicted damage in liver histoarchitecture with exposure to acetaminophen, carbon tetrachloride and thioacetamide. Tukey's HSD post hoc test confirmed that thioacetamide produced severe toxic effects in comparison to carbon tetrachloride and acetaminophen. Conclusion: In conclusion, toxic effects were noted in ascending order as acetaminophen.

Quality by Design Perspective for Designing Foam-based Formulation: Current State of Art.

Foam-based delivery systems contain one or more active ingredients and dispersed solid or liquid components that transform into gaseous form when the valve is actuated. Foams are an attractive and effective delivery approach for medical, cosmetic, and pharmaceutical uses. The foams-based delivery systems are gaining attention due to ease of application as they allow direct application onto the affected area of skin without using any applicator or finger, hence increasing the compliance and satisfaction of the patients. In order to develop foam-based delivery systems with desired qualities, it is vital to understand which type of material and process parameters impact the quality features of foams and which methodologies may be utilized to investigate foams. For this purpose, Quality-by-Design (QbD) approach is used. It aids in achieving quality-based development during the development process by employing the QbD concept. The critical material attributes (CMAs) and critical process parameters (CPPs) were discovered through the first risk assessment to ensure the requisite critical quality attributes (CQAs). During the initial risk assessment, the high-risk CQAs were identified, which affect the foam characteristics. In this review, the authors discussed the various CMAs, CPPs, CQAs, and risk factors associated in order to develop an ideal foam-based formulation with desired characteristics.