506 research outputs found
Gap misrepair mutagenesis: efficient site-directed induction of transition, transversion, and frameshift mutations in vitro.
Soluble oligomerization provides a beneficial fitness effect on destabilizing mutations
Mutations create the genetic diversity on which selective pressures can act,
yet also create structural instability in proteins. How, then, is it possible
for organisms to ameliorate mutation-induced perturbations of protein stability
while maintaining biological fitness and gaining a selective advantage? Here we
used a new technique of site-specific chromosomal mutagenesis to introduce a
selected set of mostly destabilizing mutations into folA - an essential
chromosomal gene of E. coli encoding dihydrofolate reductase (DHFR) - to
determine how changes in protein stability, activity and abundance affect
fitness. In total, 27 E.coli strains carrying mutant DHFR were created. We
found no significant correlation between protein stability and its catalytic
activity nor between catalytic activity and fitness in a limited range of
variation of catalytic activity observed in mutants. The stability of these
mutants is strongly correlated with their intracellular abundance; suggesting
that protein homeostatic machinery plays an active role in maintaining
intracellular concentrations of proteins. Fitness also shows a significant
correlation with intracellular abundance of soluble DHFR in cells growing at
30oC. At 42oC, on the other hand, the picture was mixed, yet remarkable: a few
strains carrying mutant DHFR proteins aggregated rendering them nonviable, but,
intriguingly, the majority exhibited fitness higher than wild type. We found
that mutational destabilization of DHFR proteins in E. coli is counterbalanced
at 42oC by their soluble oligomerization, thereby restoring structural
stability and protecting against aggregation
Targeting the Blood-Brain Barrier to Prevent Sepsis-Associated Cognitive Impairment
Sepsis is a systemic inflammatory disease resulting from an infection. This disorder affects 750 000 people annually in the United States and has a 62% rehospitalization rate. Septic symptoms range from typical flu-like symptoms (eg, headache, fever) to a multifactorial syndrome known as sepsis-associated encephalopathy (SAE). Patients with SAE exhibit an acute altered mental status and often have higher mortality and morbidity. In addition, many sepsis survivors are also burdened with long-term cognitive impairment. The mechanisms through which sepsis initiates SAE and promotes long-term cognitive impairment in septic survivors are poorly understood. Due to its unique role as an interface between the brain and the periphery, numerous studies support a regulatory role for the blood-brain barrier (BBB) in the progression of acute and chronic brain dysfunction. In this review, we discuss the current body of literature which supports the BBB as a nexus which integrates signals from the brain and the periphery in sepsis. We highlight key insights on the mechanisms that contribute to the BBB’s role in sepsis which include neuroinflammation, increased barrier permeability, immune cell infiltration, mitochondrial dysfunction, and a potential barrier role for tissue non-specific alkaline phosphatase (TNAP). Finally, we address current drug treatments (eg, antimicrobials and intravenous immunoglobulins) for sepsis and their potential outcomes on brain function. A comprehensive understanding of these mechanisms may enable clinicians to target specific aspects of BBB function as a therapeutic tool to limit long-term cognitive impairment in sepsis survivors
Dynamics of DNA replication loops reveal temporal control of lagging-strand synthesis
In all organisms, the protein machinery responsible for the replication of DNA, the replisome, is faced with a directionality problem. The antiparallel nature of duplex DNA permits the leading-strand polymerase to advance in a continuous fashion, but forces the lagging-strand polymerase to synthesize in the opposite direction. By extending RNA primers, the lagging-strand polymerase restarts at short intervals and produces Okazaki fragments. At least in prokaryotic systems, this directionality problem is solved by the formation of a loop in the lagging strand of the replication fork to reorient the lagging-strand DNA polymerase so that it advances in parallel with the leading-strand polymerase. The replication loop grows and shrinks during each cycle of Okazaki fragment synthesis. Here we use single-molecule techniques to visualize, in real time, the formation and release of replication loops by individual replisomes of bacteriophage T7 supporting coordinated DNA replication. Analysis of the distributions of loop sizes and lag times between loops reveals that initiation of primer synthesis and the completion of an Okazaki fragment each serve as a trigger for loop release. The presence of two triggers may represent a fail-safe mechanism ensuring the timely reset of the replisome after the synthesis of every Okazaki fragment.
Efficiency of the EmERGE Pathway of Care in Five European HIV Centres
Objective: We aimed to calculate the efficiency of the EmERGE Pathway of Care in five European HIV clinics, developed and implemented for medically stable people living with HIV.
Methods: Participants were followed up for 1 year before and after implementation of EmERGE, between April 2016 and October 2019. Micro-costing studies were performed in the outpatient services of the clinics. Unit costs for outpatient services were calculated in national currencies and converted to US purchasing parity prices. Primary and secondary outcome measures of participants did not change during the study.
Conclusions: EmERGE is acceptable and provided cost savings in different socio-economic settings. Antiretroviral drug costs remain the main cost drivers in medically stable people living with HIV. While antiretroviral drug prices in local currencies did not differ that much between countries, conversion to US$ purchasing parity prices revealed antiretroviral drugs were more expensive in the least wealthy countries. This needs to be taken into consideration when countries negotiate drug prices with pharmaceutical vendors. Greater efficiencies can be anticipated by extending the use of the EmERGE Pathway to people with complex HIV infection or other chronic diseases. Extending such use should be systematically monitored, implementation should be evaluated and funding should be provided to monitor and evaluate future changes in service provision.info:eu-repo/semantics/publishedVersio
Terahertz underdamped vibrational motion governs protein-ligand binding in solution
Low-frequency collective vibrational modes in proteins have been proposed as being responsible for efficiently directing biochemical reactions and biological energy transport. However, evidence of the existence of delocalized vibrational modes is scarce and proof of their involvement in biological function absent. Here we apply extremely sensitive femtosecond optical Kerr-effect spectroscopy to study the depolarized Raman spectra of lysozyme and its complex with the inhibitor triacetylchitotriose in solution. Underdamped delocalized vibrational modes in the terahertz frequency domain are identified and shown to blue-shift and strengthen upon inhibitor binding. This demonstrates that the ligand-binding coordinate in proteins is underdamped and not simply solvent-controlled as previously assumed. The presence of such underdamped delocalized modes in proteins may have significant implications for the understanding of the efficiency of ligand binding and protein–molecule interactions, and has wider implications for biochemical reactivity and biological function
<Abstract of annual report>Stereochemical Analysis of the Methyl Transfer Catalyzed by Cobalamin-Dependent Methionine Synthase from Escherichia coli B.
Coupled Motions Direct Electrons along Human Microsomal P450 Chains
Directional electron transfer through biological redox chains can be achieved by coupling reaction chemistry to conformational changes in individual redox enzymes
Predominant Functional Expression of Kv1.3 by Activated Microglia of the Hippocampus after Status epilepticus
BACKGROUND:Growing evidence indicates that the functional state of microglial cells differs according to the pathological conditions that trigger their activation. In particular, activated microglial cells can express sets of Kv subunits which sustain delayed rectifying potassium currents (Kdr) and modulate differently microglia proliferation and ability to release mediators. We recently reported that hippocampal microglia is in a particular activation state after a status epilepticus (SE) and the present study aimed at identifying which of the Kv channels are functionally expressed by microglia in this model. METHODOLOGY/PRINCIPAL FINDINGS:SE was induced by systemic injection of kainate in CX3CR1(eGFP/+) mice and whole cell recordings of fluorescent microglia were performed in acute hippocampal slices prepared 48 h after SE. Microglia expressed Kdr currents which were characterized by a potential of half-maximal activation near -25 mV, prominent steady-state and cumulative inactivations. Kdr currents were almost abolished by the broad spectrum antagonist 4-Aminopyridine (1 mM). In contrast, tetraethylammonium (TEA) at a concentration of 1 mM, known to block Kv3.1, Kv1.1 and 1.2 subunits, only weakly reduced Kdr currents. However, at a concentration of 5 mM which should also affect Kv1.3 and 1.6, TEA inhibited about 30% of the Kdr conductance. Alpha-dendrotoxin, which selectively inhibits Kv1.1, 1.2 and 1.6, reduced only weakly Kdr currents, indicating that channels formed by homomeric assemblies of these subunits are not important contributors of Kdr currents. Finally, agitoxin-2 and margatoxin strongly inhibited the current. CONCLUSIONS/SIGNIFICANCE:These results indicate that Kv1.3 containing channels predominantly determined Kdr currents in activated microglia after SE
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