HIV versus the Terminator: Drug resistance of HIV reverse transcriptase with mutations at the connection subdomain

Abstract only availableAntiretroviral drug therapy can prolong the life of an HIV-infected individual, but this treatment also promotes drug-resistance mutations. The replicative enzyme of HIV, reverse transcriptase (RT), is a primary target for anti-HIV drug therapy because it is responsible for converting the single stranded RNA genome of HIV into double stranded DNA for integration into the host genome. Many current anti-HIV drugs belong to two classes of inhibitors that target RT: nucleoside reverse transcriptase inhibitors (NRTIs) incorporate into and chain-terminate nascent transcription products of RT, whereas non-nucleoside reverse transcriptase inhibitors (NNRTIs) alter enzyme-nucleic acid interactions, thereby affecting the efficiency of DNA polymerization. Here, we focus on NRTI resistance mutations that are located at the connection subdomain of the enzyme in the presence and absence of thymidine analog associated mutations (TAMs). TAMs cause resistance to the commonly prescribed chain terminator 3'-azido-3'-deoxythymidine (AZT) through excision of the incorporated AZT-monophosphate. Mutations in the connection domain, such as N348I, confer resistance to NRTIs and NNRTIs and augment AZT resistance when present in combination with TAMs. Although the underlying mechanism of N348I resistance remains elusive, it has been suggested that the mutation compromises ribonuclease (RNase) H activity, which is responsible for cleaving the viral genomic RNA of the RNA/DNA heterodimeric intermediate. Changes in RNase H cleavage affect the availability of AZT-terminated primers to be excised, thereby increasing the unblocking of template/primer and NRTI resistance. Our investigation attempts to determine if AZT-resistance mutations affect resistance to other commonly prescribed NRTIs, as well as to competitive substrate inhibitors currently in development, through changes in template/primer processing. In addition, we are examining the effects of NRTI and NNRTI cocktails on the RNase H activity of RT possessing connection domain mutations. Our findings should provide insight for screening novel inhibitors for their efficacy against emergent strains of drug-resistant HIV.Life Sciences Undergraduate Research Opportunity Progra

Evolving the Physical Global Ocean Observing System for Research and Application Services Through International Coordination

Climate change and variability are major societal challenges, and the ocean is an integral part of this complex and variable system. Key to the understanding of the ocean's role in the Earth's climate system is the study of ocean and sea-ice physical processes, including its interactions with the atmosphere, cryosphere, land and biosphere. These processes include those linked to ocean circulation; the storage and redistribution of heat, carbon, salt and other water properties; and air-sea exchanges of heat, momentum, freshwater, carbon and other gasses. Measurements of ocean physics variables are fundamental to reliable earth prediction systems for a range of applications and users. In addition, knowledge of the physical environment is fundamental to growing understanding of the ocean's biogeochemistry and biological/ecosystem variability and function. Through the progress from OceanObs'99 to OceanObs'09, the ocean observing system has evolved from a platform centric perspective to an integrated observing system. The challenge now is for the observing system to evolve to respond to an increasingly diverse end user group. The Ocean Observations Physics and Climate panel (OOPC), formed in 1995, has undertaken many activities that led to observing system-related agreements. Here, OOPC will explore the opportunities and challenges for the development of a fit-for-purpose, sustained and prioritized ocean observing system, focusing on physical variables that maximize support for fundamental research, climate monitoring, forecasting on different timescales, and society. OOPC recommendations are guided by the Framework for Ocean Observing (Lindstrom et al. 2012) which emphasizes identifying user requirements by considering time and space scales of the Essential Ocean Variables. This approach provides a framework for reviewing the adequacy of the observing system, looking for synergies in delivering an integrated observing system for a range of applications and focusing innovation in areas where existing technologies do not meet these requirement

Measuring global ocean heat content to estimate the earth energy imbalance

The energy radiated by the Earth toward space does not compensate the incoming radiation from the Sun leading to a small positive energy imbalance at the top of the atmosphere (0.4–1 Wm–2). This imbalance is coined Earth’s Energy Imbalance (EEI). It is mostly caused by anthropogenic greenhouse gas emissions and is driving the current warming of the planet. Precise monitoring of EEI is critical to assess the current status of climate change and the future evolution of climate. But the monitoring of EEI is challenging as EEI is two orders of magnitude smaller than the radiation fluxes in and out of the Earth system. Over 93% of the excess energy that is gained by the Earth in response to the positive EEI accumulates into the ocean in the form of heat. This accumulation of heat can be tracked with the ocean observing system such that today, the monitoring of Ocean Heat Content (OHC) and its long-term change provide the most efficient approach to estimate EEI. In this community paper we review the current four state-of-the-art methods to estimate global OHC changes and evaluate their relevance to derive EEI estimates on different time scales. These four methods make use of: (1) direct observations of in situ temperature; (2) satellite-based measurements of the ocean surface net heat fluxes; (3) satellite-based estimates of the thermal expansion of the ocean and (4) ocean reanalyses that assimilate observations from both satellite and in situ instruments. For each method we review the potential and the uncertainty of the method to estimate global OHC changes. We also analyze gaps in the current capability of each method and identify ways of progress for the future to fulfill the requirements of EEI monitoring. Achieving the observation of EEI with sufficient accuracy will depend on merging the remote sensing techniques with in situ measurements of key variables as an integral part of the Ocean Observing System

K70Q Adds High-Level Tenofovir Resistance to “Q151M Complex” HIV Reverse Transcriptase through the Enhanced Discrimination Mechanism

HIV-1 carrying the “Q151M complex” reverse transcriptase (RT) mutations (A62V/V75I/F77L/F116Y/Q151M, or Q151Mc) is resistant to many FDA-approved nucleoside RT inhibitors (NRTIs), but has been considered susceptible to tenofovir disoproxil fumarate (TFV-DF or TDF). We have isolated from a TFV-DF-treated HIV patient a Q151Mc-containing clinical isolate with high phenotypic resistance to TFV-DF. Analysis of the genotypic and phenotypic testing over the course of this patient's therapy lead us to hypothesize that TFV-DF resistance emerged upon appearance of the previously unreported K70Q mutation in the Q151Mc background. Virological analysis showed that HIV with only K70Q was not significantly resistant to TFV-DF. However, addition of K70Q to the Q151Mc background significantly enhanced resistance to several approved NRTIs, and also resulted in high-level (10-fold) resistance to TFV-DF. Biochemical experiments established that the increased resistance to tenofovir is not the result of enhanced excision, as K70Q/Q151Mc RT exhibited diminished, rather than enhanced ATP-based primer unblocking activity. Pre-steady state kinetic analysis of the recombinant enzymes demonstrated that addition of the K70Q mutation selectively decreases the binding of tenofovir-diphosphate (TFV-DP), resulting in reduced incorporation of TFV into the nascent DNA chain. Molecular dynamics simulations suggest that changes in the hydrogen bonding pattern in the polymerase active site of K70Q/Q151Mc RT may contribute to the observed changes in binding and incorporation of TFV-DP. The novel pattern of TFV-resistance may help adjust therapeutic strategies for NRTI-experienced patients with multi-drug resistant (MDR) mutations

Characterization of HIV-1 reverse transcriptase drug resistance connection subdomain mutation N348I

Title from PDF of title page (University of Missouri--Columbia, viewed on August 23, 2012).The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file.Thesis advisor: Dr. Donald BurkeIncludes bibliographical references.M.S. University of Missouri-Columbia 2011."May 2011"[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT REQUEST OF AUTHOR.] Connection subdomain mutations are a recently discovered class of reverse transcriptase (RT) drug resistance mutations which are positioned at some distance from previously described resistance mutations. The work presented in this thesis investigates drug resistance mechanisms conferred by HIV-1 RT connection subdomain mutation N348I. N348I was chosen as a representative connection subdomain mutation due to its clinical relevance to resistance against drugs belonging to both classes of HIV-1 RT inhibitors: the NRTIs and NNRTIs. N348I is the first, and currently only, clinically relevant HIV-1 RT single amino acid substitution mutation known to confer cross-resistance to drugs from both classes of RT inhibitors. I describe here a mechanism of HIV-1 RT N348I in vitro resistance against inhibition by the NNRTI nevirapine (NVP), where I show the mutant enzyme exhibits a decreased affinity towards inhibitor binding. Using pre-steady state kinetics techniques, I further show in detail how the N348I mutation on either of the heterodimer enzyme's subunits affects enzymatic activities. Interestingly, the mutation on either subunit decreases the rate of catalytic turnover for nucleotide incorporation reactions. The N348I mutant enzyme also displays an altered RNAse H activity, and I demonstrate that the N348I mutation on the p51 subunit provides the major contribution towards this altered activity. I also investigated RT N348I in vitro susceptibility to ddATP, the active form of the NRTI prodrug didanosine (ddI). Multiple mechanisms of NRTI resistance were studied, but significant levels of in vitro resistance to ddATP were not detected despite the fact that the N34I mutant virus has been shown to be resistant to ddI. Here I found that the N348I mutation does not negatively impact the steady-state kinetics of single nucleotide incorporation reactions, or the affinity for nucleotide substrate

Amino Acid Mutation N348I in the Connection Subdomain of Human Immunodeficiency Virus Type 1 Reverse Transcriptase Confers Multiclass Resistance to Nucleoside and Nonnucleoside Reverse Transcriptase Inhibitors▿ †

We identified clinical isolates with phenotypic resistance to nevirapine (NVP) in the absence of known nonnucleoside reverse transcriptase inhibitor (NNRTI) mutations. This resistance is caused by N348I, a mutation at the connection subdomain of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). Virologic analysis showed that N348I conferred multiclass resistance to NNRTIs (NVP and delavirdine) and to nucleoside reverse transcriptase inhibitors (zidovudine [AZT] and didanosine [ddI]). N348I impaired HIV-1 replication in a cell-type-dependent manner. Acquisition of N348I was frequently observed in AZT- and/or ddI-containing therapy (12.5%; n = 48; P < 0.0001) and was accompanied with thymidine analogue-associated mutations, e.g., T215Y (n = 5/6) and the lamivudine resistance mutation M184V (n = 1/6) in a Japanese cohort. Molecular modeling analysis shows that residue 348 is proximal to the NNRTI-binding pocket and to a flexible hinge region at the base of the p66 thumb that may be affected by the N348I mutation. Our results further highlight the role of connection subdomain residues in drug resistance

Evolving the global ocean observing system for research and application services through international coordination

Climate change and variability are major societal challenges, and the ocean is an integral part of this complex and variable system. Key to the understanding of the ocean’s role in the Earth’s climate system is the study of ocean and sea-ice physical processes, including its interactions with the atmosphere, cryosphere, land, and biosphere. These processes include those linked to ocean circulation; the storage and redistribution of heat, carbon, salt and other water properties; and air-sea exchanges of heat, momentum, freshwater, carbon, and other gasses. Measurements of ocean physics variables are fundamental to reliable earth prediction systems for a range of applications and users. In addition, knowledge of the physical environment is fundamental to growing understanding of the ocean’s biogeochemistry and biological/ecosystem variability and function. Through the progress from OceanObs’99 to OceanObs’09, the ocean observing system has evolved from a platform centric perspective to an integrated observing system. The challenge now is for the observing system to evolve to respond to an increasingly diverse end user group. The Ocean Observations Physics and Climate panel (OOPC), formed in 1995, has undertaken many activities that led to observing system-related agreements. Here, OOPC will explore the opportunities and challenges for the development of a fit-for-purpose, sustained and prioritized ocean observing system, focusing on physical variables that maximize support for fundamental research, climate monitoring, forecasting on different timescales, and society. OOPC recommendations are guided by the Framework for Ocean Observing which emphasizes identifying user requirements by considering time and space scales of the Essential Ocean Variables. This approach provides a framework for reviewing the adequacy of the observing system, looking for synergies in delivering an integrated observing system for a range of applications and focusing innovation in areas where existing technologies do not meet these requirements

Indicators of Global Climate Change 2022: annual update of large-scale indicators of the state of the climate system and human influence

Intergovernmental Panel on Climate Change (IPCC) assessments are the trusted source of scientific evidence for climate negotiations taking place under the United Nations Framework Convention on Climate Change (UNFCCC), including the first global stocktake under the Paris Agreement that will conclude at COP28 in December 2023. Evidence-based decision-making needs to be informed by up-to-date and timely information on key indicators of the state of the climate system and of the human influence on the global climate system. However, successive IPCC reports are published at intervals of 5–10 years, creating potential for an information gap between report cycles. We follow methods as close as possible to those used in the IPCC Sixth Assessment Report (AR6) Working Group One (WGI) report. We compile monitoring datasets to produce estimates for key climate indicators related to forcing of the climate system: emissions of greenhouse gases and short-lived climate forcers, greenhouse gas concentrations, radiative forcing, surface temperature changes, the Earth's energy imbalance, warming attributed to human activities, the remaining carbon budget, and estimates of global temperature extremes. The purpose of this effort, grounded in an open data, open science approach, is to make annually updated reliable global climate indicators available in the public domain (https://doi.org/10.5281/zenodo.8000192, Smith et al., 2023a). As they are traceable to IPCC report methods, they can be trusted by all parties involved in UNFCCC negotiations and help convey wider understanding of the latest knowledge of the climate system and its direction of travel. The indicators show that human-induced warming reached 1.14 [0.9 to 1.4] ∘C averaged over the 2013–2022 decade and 1.26 [1.0 to 1.6] ∘C in 2022. Over the 2013–2022 period, human-induced warming has been increasing at an unprecedented rate of over 0.2 ∘C per decade. This high rate of warming is caused by a combination of greenhouse gas emissions being at an all-time high of 54 ± 5.3 GtCO2e over the last decade, as well as reductions in the strength of aerosol cooling. Despite this, there is evidence that increases in greenhouse gas emissions have slowed, and depending on societal choices, a continued series of these annual updates over the critical 2020s decade could track a change of direction for human influence on climate.ISSN:1866-3516ISSN:1866-350

Indicators of Global Climate Change 2022: annual update of large-scale indicators of the state of the climate system and human influence

Abstract. Intergovernmental Panel on Climate Change (IPCC) assessments are the trusted source of scientific evidence for climate negotiations taking place under the United Nations Framework Convention on Climate Change (UNFCCC), including the first global stocktake under the Paris Agreement that will conclude at COP28 in December 2023. Evidence-based decision-making needs to be informed by up-to-date and timely information on key indicators of the state of the climate system and of the human influence on the global climate system. However, successive IPCC reports are published at intervals of 5–10 years, creating potential for an information gap between report cycles. We follow methods as close as possible to those used in the IPCC Sixth Assessment Report (AR6) Working Group One (WGI) report. We compile monitoring datasets to produce estimates for key climate indicators related to forcing of the climate system: emissions of greenhouse gases and short-lived climate forcers, greenhouse gas concentrations, radiative forcing, surface temperature changes, the Earth's energy imbalance, warming attributed to human activities, the remaining carbon budget, and estimates of global temperature extremes. The purpose of this effort, grounded in an open data, open science approach, is to make annually updated reliable global climate indicators available in the public domain (https://doi.org/10.5281/zenodo.8000192, Smith et al., 2023a). As they are traceable to IPCC report methods, they can be trusted by all parties involved in UNFCCC negotiations and help convey wider understanding of the latest knowledge of the climate system and its direction of travel. The indicators show that human-induced warming reached 1.14 [0.9 to 1.4] ∘C averaged over the 2013–2022 decade and 1.26 [1.0 to 1.6] ∘C in 2022. Over the 2013–2022 period, human-induced warming has been increasing at an unprecedented rate of over 0.2 ∘C per decade. This high rate of warming is caused by a combination of greenhouse gas emissions being at an all-time high of 54 ± 5.3 GtCO2e over the last decade, as well as reductions in the strength of aerosol cooling. Despite this, there is evidence that increases in greenhouse gas emissions have slowed, and depending on societal choices, a continued series of these annual updates over the critical 2020s decade could track a change of direction for human influence on climate