Analisis refleksi pada pembelajaran: review reasearch

Translation fidelity is the limiting factor in the accuracy of gene expression. With an estimated frequency of 10−4, errors in mRNA decoding occur in a mostly stochastic manner. Little is known about the response of higher eukaryotes to chronic loss of ribosomal accuracy as per an increase in the random error rate of mRNA decoding. Here, we present a global and comprehensive picture of the cellular changes in response to translational accuracy in mammalian ribosomes impaired by genetic manipulation. In addition to affecting established protein quality control pathways, such as elevated transcript levels for cytosolic chaperones, activation of the ubiquitin-proteasome system, and translational slowdown, ribosomal mistranslation led to unexpected responses. In particular, we observed increased mitochondrial biogenesis associated with import of misfolded proteins into the mitochondria and silencing of the unfolded protein response in the endoplasmic reticulum.ISSN:2399-364

Nonmutational compensation of the fitness cost of antibiotic resistance in mycobacteria by overexpression of tlyA rRNA methylase

Several studies over the last few decades have shown that antibiotic resistance mechanisms frequently confer a fitness cost and that these costs can be genetically ameliorated by intra- or extragenic second-site mutations, often without loss of resistance. Another, much less studied potential mechanism by which the fitness cost of antibiotic resistance could be reduced is via a regulatory response where the deleterious effect of the resistance mechanism is lowered by a physiological alteration that buffers the mutational effect. In mycobacteria, resistance to the clinically used tuberactinomycin antibiotic capreomycin involves loss-of-function mutations in rRNA methylase TlyA or point mutations in 16S rRNA (in particular the A1408G mutation). Both of these alterations result in resistance by reducing drug binding to the ribosome. Here we show that alterations of tlyA gene expression affect both antibiotic drug susceptibility and fitness cost of drug resistance. In particular, we demonstrate that the common resistance mutation A1408G is accompanied by a physiological change that involves increased expression of the tlyA gene. This gene encodes an enzyme that methylates neighboring 16S rRNA position C1409, and as a result of increased TlyA expression the fitness cost of the A1408G mutation is significantly reduced. Our findings suggest that in mycobacteria, a nonmutational mechanism (i.e., gene regulatory) can restore fitness to genetically resistant bacteria. Our results also point to a new and clinically relevant treatment strategy to combat evolution of resistance in multidrug-resistant tuberculosis. Thus, by utilizing antagonistic antibiotic interactions, resistance evolution could be reduced

Influence of 4'-O-Glycoside Constitution and Configuration on Ribosomal Selectivity of Paromomycin

A series of 20 4′-O-glycosides of the aminoglycoside antibiotic paromomycin were synthesized and evaluated for their ability to inhibit protein synthesis by bacterial, mitochondrial and cytosolic ribosomes. Target selectivity, i.e., inhibition of the bacterial ribosome over eukaryotic mitochondrial and cytosolic ribosomes, which is predictive of antibacterial activity with reduced ototoxicity and systemic toxicity, was greater for the equatorial than for the axial pyranosides, and greater for the d-pentopyranosides than for the l-pentopyranosides and d-hexopyranosides. In particular, 4′-O-β-d-xylopyranosyl paromomycin shows antibacterioribosomal activity comparable to that of paromomycin, but is significantly more selective showing considerably reduced affinity for the cytosolic ribosome and for the A1555G mutant mitochondrial ribosome associated with hypersusceptibility to drug-induced ototoxicity. Compound antibacterioribosomal activity correlates with antibacterial activity, and the ribosomally more active compounds show activity against Escherichia coli, Klebsiella pneumonia, Enterobacter cloacae, Acinetobacter baumannii, and methicillin-resistant Staphylococcus aureus (MRSA). The paromomycin glycosides retain activity against clinical strains of MRSA that are resistant to paromomycin, which is demonstrated to be a consequence of 4′-O-glycosylation blocking the action of 4′-aminoglycoside nucleotidyl transferases by the use of recombinant E. coli carrying the specific resistance determinant

Ribosome synthesis and MAPK activity modulate ionizing radiation-induced germ cell apoptosis in Caenorhabditis elegans

Synthesis of ribosomal RNA by RNA polymerase I (RNA pol I) is an elemental biological process and is key for cellular homeostasis. In a forward genetic screen in C. elegans designed to identify DNA damage-response factors, we isolated a point mutation of RNA pol I, rpoa-2(op259), that leads to altered rRNA synthesis and a concomitant resistance to ionizing radiation (IR)-induced germ cell apoptosis. This weak apoptotic IR response could be phenocopied when interfering with other factors of ribosome synthesis. Surprisingly, despite their resistance to DNA damage, rpoa-2(op259) mutants present a normal CEP-1/p53 response to IR and increased basal CEP-1 activity under normal growth conditions. In parallel, rpoa-2(op259) leads to reduced Ras/MAPK pathway activity, which is required for germ cell progression and physiological germ cell death. Ras/MAPK gain-of-function conditions could rescue the IR response defect in rpoa-2(op259), pointing to a function for Ras/MAPK in modulating DNA damage-induced apoptosis downstream of CEP-1. Our data demonstrate that a single point mutation in an RNA pol I subunit can interfere with multiple key signalling pathways. Ribosome synthesis and growth-factor signalling are perturbed in many cancer cells; such an interplay between basic cellular processes and signalling might be critical for how tumours evolve or respond to treatment

Epistasis between antibiotic resistance mutations drives the evolution of extensively drug-resistant tuberculosis

Background and objectives: Multidrug resistant (MDR) bacteria are a growing threat to global health. Studies focusing on single antibiotics have shown that drug resistance is often associated with a fitness cost in the absence of drug. However, little is known about the fitness cost associated with resistance to multiple antibiotics. Methodology: We used Mycobacterium smegmatis as a model for human tuberculosis (TB) and an in vitro competitive fitness assay to explore the combined fitness effects and interaction between mutations conferring resistance to rifampicin (RIF) and ofloxacin (OFX); two of the most important first- and second-line anti-TB drugs, respectively. Results: We found that 4 out of 17 M. smegmatis mutants (24%) resistant to RIF and OFX showed a statistically significantly higher or lower competitive fitness than expected when assuming a multiplicative model of fitness effects of each individual mutation. Moreover, 6 out of the 17 double drug-resistant mutants (35%) had a significantly higher fitness than at least one of the corresponding single drug-resistant mutants. The particular combinations of resistance mutations associated with no fitness deficit in M. smegmatis were the most frequent among 151 clinical isolates of MDR and extensively drug-resistant (XDR) Mycobacterium tuberculosis from South Africa. Conclusions and implications: Our results suggest that epistasis between drug resistance mutations in mycobacteria can lead to MDR strains with no fitness deficit, and that these strains are positively selected in settings with a high burden of drug-resistant TB. Taken together, our findings support a role for epistasis in the evolution and epidemiology of MDR- and XDR-TB

The <i>rpoa-2(op259)</i> mutation reduces irradiation-induced germ cell apoptosis but not cell cycle arrest response or DNA damage repair.

<p>A) DIC images of adult worms 24 hours after irradiation. Basal (physiologic) as well as DNA damage-induced germ cell death is confined to the late meiotic pachytene region of the germ line tubes; corpses are visible as cellularised, refractile discs (arrowheads). Size bar, 15 µm. B) Apoptotic cell corpses in the germ lines of X-ray treated adult hermaphrodites. Dashed lines represent basal levels (0 Gy), straight lines the levels following irradiation (60 Gy). At least three independent experiments (with n = 20 animals per condition) were performed with wild-type and <i>rpoa-2(op259)</i> worms, and one with animals mutant for the 9-1-1 complex subunit HUS-1. Error bars, standard deviation (SD) of corpse number per gonad over all experiments. C, D) In contrast to the DNA damage response mutant <i>hus-1(op241)</i>, cell cycle arrest is induced normally in <i>rpoa-2(op259)</i>. C) Percentage of mitotic cells with nuclear enlargement following IR. Error bars, SD from at least 12 gonads (approx. 40–50 total mitotic cells per gonad). D) Gonads were dissected from <i>opIs257[P_rad-54::rad-54::yfp 3′UTR; unc-119(+)]; unc-119(ed3)</i> or <i>rpoa-2(op259)</i>; <i>opIs257; unc-119(ed3)</i> adults, stained with Hoechst, and scored for YFP::RAD-54 foci. Size bar, 10 µm. E, F) A subset of rRNA processing and translation initiation mutants have reduced IR-induced apoptosis. Apoptotic response to IR irradiation in the gonads of the rRNA processing mutants <i>pro-2(na27)</i> and <i>pro-3(ar226)</i> (E) and of the eIF4E homolog mutants <i>ife-1(ok1978)</i> and <i>ife-2(ok306)</i> (F). Dashed lines, 0 Gy, straight lines, 60 Gy; error bars, SD of the number of germ cell corpses per gonad over at least 3 experiments (n = 20 animals per data point and experiment).</p

Influence of 4′‑<i>O</i>‑Glycoside Constitution and Configuration on Ribosomal Selectivity of Paromomycin

A series of 20 4′-<i>O</i>-glycosides of the aminoglycoside antibiotic paromomycin were synthesized and evaluated for their ability to inhibit protein synthesis by bacterial, mitochondrial and cytosolic ribosomes. Target selectivity, i.e., inhibition of the bacterial ribosome over eukaryotic mitochondrial and cytosolic ribosomes, which is predictive of antibacterial activity with reduced ototoxicity and systemic toxicity, was greater for the equatorial than for the axial pyranosides, and greater for the d-pentopyranosides than for the l-pentopyranosides and d-hexopyranosides. In particular, 4′-<i>O</i>-β-d-xylopyranosyl paromomycin shows antibacterioribosomal activity comparable to that of paromomycin, but is significantly more selective showing considerably reduced affinity for the cytosolic ribosome and for the A1555G mutant mitochondrial ribosome associated with hypersusceptibility to drug-induced ototoxicity. Compound antibacterioribosomal activity correlates with antibacterial activity, and the ribosomally more active compounds show activity against <i>Escherichia coli</i>, <i>Klebsiella pneumonia</i>, <i>Enterobacter cloacae</i>, <i>Acinetobacter baumannii</i>, and methicillin-resistant <i>Staphylococcus aureus</i> (MRSA). The paromomycin glycosides retain activity against clinical strains of MRSA that are resistant to paromomycin, which is demonstrated to be a consequence of 4′-<i>O</i>-glycosylation blocking the action of 4′-aminoglycoside nucleotidyl transferases by the use of recombinant <i>E. coli</i> carrying the specific resistance determinant

Activated Ras/MAPK pathway enhances the apoptotic DNA damage response and restores irradiation-induced germ cell death in <i>rpoa-2(op259)</i> mutants.

<p>A) Apoptotic germ cell corpses at 24 hours post irradiation. <i>lip-1(lf)</i> and <i>let-60(gf)</i> increase the IR response of <i>rpoa-2(op259)</i> mutants. <i>lip-1</i> is epistatic to <i>rpoa-2(op259)</i> regarding the IR response; the response in <i>rpoa-2(op259); let-60(gf)</i> is intermediate between the single mutants. Error bars, SD; n≥40 for each condition. B) Simplified model of the EGFR/Ras/MAPK signal axis. Gain-of-function mutations in LET-60 or loss of the phosphatase LIP-1 increase MPK-1 activity. GAP-1 is a GTPase-activating protein acting as a negative regulator of LET-60; <i>gap-1(lf)</i> mutants show synthetic effects on vulval induction with other genes but have no strong phenotype on their own. C) Reduced kinase activity in the Ras/MAPK pathway mutant <i>mpk-1(ga111)</i> or in the p38 MAPK mutant <i>pmk-1(km25)</i> but not the Jnk mutant <i>jnk-1(gk7)</i> reduces germ cell apoptosis. Experiments performed at 20°C; error bars, SD; n = 20 animals per condition. D) <i>rpoa-2(op259)</i> suppresses excessive vulval induction of <i>let-60(n1046gf)</i>. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003943#s4" target="_blank">Methods</a> for assessment of vulval induction index (VI) (reference value is 3.0 for wild type, corresponding to normal induction of one vulva). Error bars, SD of at least 20 animals.</p

Baseline levels of activated MPK-1 in the gonad are reduced in <i>rpoa-2(op259)</i> and do not increase upon irradiation.

<p>A) Gonads were extruded from non-irradiated (0 Gy) and irradiated (60 Gy) adult worms 4 hours after treatment, fixed, and immunostained for the doubly phosphorylated (activated) MPK-1. MPK-1 activity is high in the late meiotic pachytene region (white dashed lines) and is further increased following IR in wild-type worms, but not in <i>rpoa-2(op259)</i> mutants. Control staining included total MPK-1 with anti-ERK, and anti-dsDNA to detect an epitope other than protein for normalisation of fluorescence intensity. Size bar, 40 µm. B) Quantification of the anti-ppMPK signal intensity along the central axis (white streak) of the late meiotic pachytene region, starting distally to the first oocyte (white dashed reference line) and extending into the mid-pachytene region. Profiles were plotted for at least 8 worms per condition (thin grey lines for individual wild-type worms, thin green lines for individual <i>rpoa-2(op259)</i> mutants) and a spline was calculated from their overlay. Size bar, 25 µm.</p