Defects of the translation apparatus in human mitochondria are known to cause disease, yet details of how protein synthesis is regulated in this organelle remain to be unveiled. Ribosome production ...in all organisms studied thus far entails a complex, multistep pathway involving a number of auxiliary factors. This includes several RNA processing and modification steps required for correct rRNA maturation. Little is known about the maturation of human mitochondrial 16S rRNA and its role in biogenesis of the mitoribosome. Here we investigate two methyltransferases, MRM2 (also known as RRMJ2, encoded by FTSJ2) and MRM3 (also known as RMTL1, encoded by RNMTL1), that are responsible for modification of nucleotides of the 16S rRNA A-loop, an essential component of the peptidyl transferase center. Our studies show that inactivation of MRM2 or MRM3 in human cells by RNA interference results in respiratory incompetence as a consequence of diminished mitochondrial translation. Ineffective translation in MRM2- and MRM3-depleted cells results from aberrant assembly of the large subunit of the mitochondrial ribosome (mt-LSU). Our findings show that MRM2 and MRM3 are human mitochondrial methyltransferases involved in the modification of 16S rRNA and are important factors for the biogenesis and function of the large subunit of the mitochondrial ribosome.
Adverse drug reactions (ADRs) are considered an inherent risk of medication use, and some ADRs have been associated with off-target drug interactions with mitochondria. Metabolites that reflect ...mitochondrial function may help identify patients at risk of mitochondrial toxicity. We employed a database strategy to identify candidate mitochondrial metabolites that could be clinically useful to identify individuals at increased risk of mitochondrial-related ADRs. This led to L-carnitine being identified as the candidate mitochondrial metabolite. L-carnitine, its acetylated metabolite, acetylcarnitine and other acylcarnitines are mitochondrial biomarkers used to detect inborn errors of metabolism. We hypothesized that changes in L-carnitine disposition, induced by a "challenge test" of intravenous L-carnitine, could identify mitochondrial-related ADRs by provoking variation in L-carnitine and/or acetylcarnitine blood levels. To test this hypothesis, we induced mitochondrial drug toxicity with clofazimine (CFZ) in a mouse model. Following CFZ treatment, mice received an L-carnitine "challenge test". CFZ-induced changes in weight were consistent with previous work and reflect CFZ-induced catabolism. L-carnitine induced differences in whole blood acetylcarnitine concentrations in a manner that was dependent on CFZ treatment. This supports the usefulness of a database strategy for the discovery of candidate metabolite biomarkers of drug toxicity and substantiates the potential of the L-carnitine "challenge test" as a "probe" to identify drug-related toxicological manifestations.
The approaches for maintaining hepatocytes in vitro are aimed at recapitulating aspects of the native liver microenvironment through the use of co-cultures, surface coatings and 3D spheroids. This ...study highlights the effects of spatial confinement-a less studied component of the in vivo microenvironment. We demonstrate that hepatocytes cultured in low-volume microfluidic channels (microchambers) retain differentiated hepatic phenotype for 21 days whereas cells cultured in regular culture plates under identical conditions de-differentiate after 7 days. Careful consideration of nutrient delivery and oxygen tension suggested that these factors could not solely account for enhanced cell function in microchambers. Through a series of experiments involving microfluidic chambers of various heights and inhibition of key molecular pathways, we confirmed that phenotype of hepatocytes in small volumes was shaped by endogenous signals, both hepato-inductive growth factors (GFs) such as hepatocyte growth factor (HGF) and hepato-disruptive GFs such as transforming growth factor (TGF)-β1. Hepatocytes are not generally thought of as significant producers of GFs-this role is typically assigned to nonparenchymal cells of the liver. Our study demonstrates that, in an appropriate microenvironment, hepatocytes produce hepato-inductive and pro-fibrogenic signals at the levels sufficient to shape their phenotype and function.
Mammalian mitochondria can be transferred between cells both in culture and in vivo. There is evidence that isolated mitochondria enter cells by endocytosis, but the mechanism has not been fully ...characterised. We investigated the entry mechanism of isolated mitochondria into human osteosarcoma (HOS) cells. Initially we confirmed that respiratory-competent cells can be produced following incubation of HOS cells lacking mitochondrial DNA (mtDNA) with functional exogenous mitochondria and selection in a restrictive medium. Treatment of HOS cells with inhibitors of different endocytic pathways suggest that uptake of EGFP-labelled mitochondria occurs via an actin-dependent endocytic pathway which is consistent with macropinocytosis. We later utilised time-lapse microscopy to show that internalised mitochondria were found in large, motile cellular vesicles. Finally, we used confocal imaging to show that EGFP-labelled mitochondria colocalise with a macropinocytic cargo molecule during internalisation, HOS cells produce membrane ruffles interacting with external mitochondria during uptake and EGFP-labelled mitochondria are found within early macropinosomes inside cells. In conclusion our results are consistent with isolated mitochondria being internalised by macropinocytosis in HOS cells.
Classic galactosemia is a potentially lethal disease caused by the dysfunction of galactose 1-phosphate uridylyltransferase (GALT). Over 300 disease-associated GALT mutations have been reported, with ...the majority being missense changes, although a better understanding of their underlying molecular effects has been hindered by the lack of structural information for the human enzyme. Here, we present the 1.9 Å resolution crystal structure of human GALT (hGALT) ternary complex, revealing a homodimer arrangement that contains a covalent uridylylated intermediate and glucose-1-phosphate in the active site, as well as a structural zinc-binding site, per monomer. hGALT reveals significant structural differences from bacterial GALT homologues in metal ligation and dimer interactions, and therefore is a zbetter model for understanding the molecular consequences of disease mutations. Both uridylylation and zinc binding influence the stability and aggregation tendency of hGALT. This has implications for disease-associated variants where p.Gln188Arg, the most commonly detected, increases the rate of aggregation in the absence of zinc likely due to its reduced ability to form the uridylylated intermediate. As such our structure serves as a template in the future design of pharmacological chaperone therapies and opens new concepts about the roles of metal binding and activity in protein misfolding by disease-associated mutants.
•Sulfur deficiency and utilization of available sulfur are enhanced by supplementation with sulfur-oxidizing bacteria (SOB).•Different SOBs have been isolated from the soil and shown to have higher ...sulfide oxidase activity and produce higher sulfate ions.•Bacteria shown to have properties that promote plant growth, namely IAA, ammonia, phosphate solution, and HCN.•Experiments were conducted on chickpea supplemented with elemental sulfur and granular sulfur in pots and in the field.•SOB amendment with elemental and granular sulfur significantly influenced shoot length, root length, number of leaves and branches, and fresh dry weight of shoots and roots.
Sulphur deficiency affects several nations, including India, and has become a limiting issue for food productivity and quality. Sulphur deficiency causes plants to shrink, build up yellow chlorosis in their younger leaves, necrosis in their last stages of growth, and become stiff with short, slender stalks. The aim of this study was to identify and characterize Sulphur-oxidizing bacteria (SOB) from the rhizosphere and evaluate their potential to enhance chickpea growth with the application of Sulphur fertilizer. Out of 18 isolates, five potential Sulphur oxidizing bacteria were identified using 16 s rDNA sequencing Viz., Enterobacter cloacae KDNC31 (AD31; OR083345.1), Klebsiella oxytoca KDNC1 (OR083341.1), Raoultella planticola KDNC3 (OR083342.1), Enterobacter cloacae KDNC9 (OR083344.1), and Klebsiella pasteurii KDNC8 (OR083343.1). A significant sulphate content (621.2 µg/ml) and sulphide oxidase activity (19.18 U/ml) were reported after the AD31 isolate was inoculated in thiosulphate broth, which caused a fast pH reduction to pH 4.0 within 3 days. The strain produced IAA (1600 µg/ml), ammonia (278.78 µg/ml), solubilized tricalcium phosphate (1800 µg/ml), and HCN, among other plant growth-promoting substances. The Sulphur oxidation potential of AD31 was assessed by supplementing it with elemental Sulphur and granular Sulphur in a pot. The results indicated that, When AD31 and elemental Sulphur were applied together, the length of the shoots, the length of the roots, the number of leaves and branches, and the fresh and dry weight of the shoots and roots were all significantly higher compared to the corresponding control. Elemental Sulphur has a smaller particle size and a larger surface area, making it more suitable for Sulphur oxidation by AD31 compare to granular Sulphur. Therefore, SOB inoculation with elemental Sulphur might be employed to increase chick pea growth-related indices.
In the liver, hepatocytes are exposed to a large array of stimuli that shape hepatic phenotype. This in vivo microenvironment is lost when hepatocytes are cultured in standard cell cultureware, ...making it challenging to maintain hepatocyte function in vitro. Our article focused on one of the least studied inducers of the hepatic phenotype-the mechanical properties of the underlying substrate. Gel layers comprised of thiolated heparin (Hep-SH) and diacrylated poly(ethylene glycol) (PEG-DA) were formed on glass substrates via a radical mediated thiol-ene coupling reaction. The substrate stiffness varied from 10 to 110 kPa by changing the concentration of the precursor solution. ELISA analysis revealed that after 5 days, hepatocytes cultured on a softer heparin gel were synthesizing five times higher levels of albumin compared to those on a stiffer heparin gel. Immunofluorescent staining for hepatic markers, albumin and E-cadherin, confirmed that softer gels promoted better maintenance of the hepatic phenotype. Our findings point to the importance of substrate mechanical properties on hepatocyte function.
Background: Ropivacaine is more selective for sensory fibers when compared to other local anesthetics, producing less motor block. This permits better maternal ambulation and also allows for normal ...progression of labor, which translates into lesser instrumental deliveries and higher vaginal deliveries. Objective was to evaluate 0.125% versus 0.2% ropivacaine, with 2 microg/ml of fentanyl in epidural labor analgesia, regarding their sensory and motor block characteristics. Methods: This prospective study was conducted among 40 patients, 20 in each group group A (0.125% ropivacaine with 2 microg/ml fentanyl), group B (0.2% ropivacaine with 2 microg/ml fentanyl), for epidural labor analgesia in obstetrics and gynecology department (labor room). The efficacy of the drugs was tested by comparing the onset of analgesia, duration of labor analgesia, dose requirement, pulse, BP, sensory effect, motor effect, FHR, APGAR score and side effects. Results: Total duration of labor analgesia was 230.25 minutes with 55.68 SD and 186.25 minutes with 57.7 SD in group A and group B respectively (p<0.05). The total dose of ropivacaine used was 81.00 mg and 68.50 mg in group A and B respectively (p<0.05). Total dose of fentanyl required was 94.50 pg and 73.50 pg in group A and group B respectively (p<0.05). There was no significant difference found in hemodynamic parameters in both groups. Conclusions: Both the concentrations are effective in producing epidural labor analgesia. However, onset of analgesia was significantly faster with 0.2% ropivacaine. The required dose of ropivacaine was significantly higher in 0.125% ropivacaine. 0.2% ropivacaine shorten the duration of labor compared to 0.125% ropivacaine. Keywords: Epidural, Fentanyl, Labor analgesia, Local anesthetics, Ropivacaine
Cancer, developmental biology and tissue injury present multiple examples where groups of cells residing in close proximity communicate via paracrine factors. It is nearly impossible to dissect such ...cellular interactions in vivo and is quite challenging in vitro. The goal of this study is to utilize a reconfigurable microfluidic device in order to study paracrine signal exchange between groups of primary hepatocytes in vitro. Previously, we demonstrated that hepatocytes residing on protein spots containing collagen and hepatocyte growth factor (HGF) spots expressed epithelial (hepatic) phenotypes and also rescued them in neighboring hepatocytes on collagen spots that did not receive direct HGF stimulus. Herein, we designed a microfluidic device with parallel fluidic channels separated by retractable (reconfigurable) walls and employed this device to investigate interactions between groups of HGF-stimulated and unstimulated hepatocytes. Using a novel reconfigurable microfluidic device, we demonstrate that cultivation of HGF-containing protein spots upregulates the production of endogenous HGF in hepatocytes and that these HGF molecules diffuse over, causing phenotype enhancement in the recipient cells. We also show that selective treatment of the recipient hepatocytes with a c-met inhibitor (SU11274) diminishes the rescue effect, as gauged by the down-regulation of albumin and HGF expression. Our study is one of the first to demonstrate paracrine signaling via HGF in primary hepatocytes. More broadly, tools and methods described here may be used to study paracrine signaling in other types of cells and will have relevance for various fields of biomedical research from cancer to immunology.
To report the accuracy of intraocular lens (IOL) power estimation in eyes having combined phacoemulsification and vitrectomy for macular holes and to compare the axial length (AL) in those eyes with ...that in the fellow eyes.
Calderdale Royal Hospital, Halifax, West Yorkshire, United Kingdom.
The mean and standard deviation of the refractive aim, achieved refraction, and postoperative prediction error (calculated as difference between achieved refraction and refractive aim) were determined in 40 patients who had phacovitrectomy with gas tamponade for the treatment of idiopathic macular holes. The percentage of patients with an achieved refraction within +/-0.50 diopter (D), +/-1.00 D, and more than 2.00 D of the refractive aim was recorded. The mean absolute error (MAE) of the postoperative prediction error was calculated. In addition, the AL in eyes with macular holes was compared with that in fellow eyes. Axial lengths were measured using applanation A-scan ultrasound.
Of eyes having phacovitrectomy, 45.0%, 67.5%, and 90.0% achieved a postoperative refraction within +/-0.50 D, +/-1.00 D, and +/-2.00 D, respectively, of the refractive aim; 10.0% of eyes were more than -2.00 D from the refractive aim. The overall postoperative prediction error ranged from +1.64 D to -2.51 D. The mean refractive aim was +0.30 +/- 0.72 D and the mean achieved refraction, -0.09 +/-1.25 D. There was no clinically significant difference between the means. The mean postoperative prediction error was -0.39 +/- 1.01 D, suggesting a myopic overcorrection occurred postoperatively. The MAE of the postoperative prediction error was 0.83 D. The mean AL was 23.40 mm in operated eyes and 23.46 mm in fellow eyes.
The achieved refraction after phacovitrectomy for macular holes was comparable to results after phacoemulsification alone. The myopic overcorrection after phacovitrectomy might be a result of the gas bubble causing forward displacement of the capsular bag and IOL or inaccuracies in AL and keratometry measurements. Aiming for residual hyperopia may counteract the overcorrection. There was no difference in AL between eyes with macular holes and fellow eyes.
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