Intrinsically disordered regions regulate RhlE RNA helicase functions in bacteria.

RNA helicases-central enzymes in RNA metabolism-often feature intrinsically disordered regions (IDRs) that enable phase separation and complex molecular interactions. In the bacterial pathogen Pseudomonas aeruginosa, the non-redundant RhlE1 and RhlE2 RNA helicases share a conserved REC catalytic core but differ in C-terminal IDRs. Here, we show how the IDR diversity defines RhlE RNA helicase specificity of function. Both IDRs facilitate RNA binding and phase separation, localizing proteins in cytoplasmic clusters. However, RhlE2 IDR is more efficient in enhancing REC core RNA unwinding, exhibits a greater tendency for phase separation, and interacts with the RNase E endonuclease, a crucial player in mRNA degradation. Swapping IDRs results in chimeric proteins that are biochemically active but functionally distinct as compared to their native counterparts. The RECRhlE1-IDRRhlE2 chimera improves cold growth of a rhlE1 mutant, gains interaction with RNase E and affects a subset of both RhlE1 and RhlE2 RNA targets. The RECRhlE2-IDRRhlE1 chimera instead hampers bacterial growth at low temperatures in the absence of RhlE1, with its detrimental effect linked to aberrant RNA droplets. By showing that IDRs modulate both protein core activities and subcellular localization, our study defines the impact of IDR diversity on the functional differentiation of RNA helicases.

Reprogramming neuroblastoma by diet-enhanced polyamine depletion.

Neuroblastoma is a highly lethal childhood tumor derived from differentiation-arrested neural crest cells1,2. Like all cancers, its growth is fueled by metabolites obtained from either circulation or local biosynthesis3,4. Neuroblastomas depend on local polyamine biosynthesis, with the inhibitor difluoromethylornithine showing clinical activity5. Here we show that such inhibition can be augmented by dietary restriction of upstream amino acid substrates, leading to disruption of oncogenic protein translation, tumor differentiation, and profound survival gains in the TH-MYCN mouse model. Specifically, an arginine/proline-free diet decreases the polyamine precursor ornithine and augments tumor polyamine depletion by difluoromethylornithine. This polyamine depletion causes ribosome stalling, unexpectedly specifically at adenosine-ending codons. Such codons are selectively enriched in cell cycle genes and low in neuronal differentiation genes. Thus, impaired translation of these codons, induced by the diet-drug combination, favors a pro-differentiation proteome. These results suggest that the genes of specific cellular programs have evolved hallmark codon usage preferences that enable coherent translational rewiring in response to metabolic stresses, and that this process can be targeted to activate differentiation of pediatric cancers.

Safety Aspects and Rational Use of Single Intramuscular Dose Ceftriaxone: Clinical Insights on the Management of Uncomplicated Gonococcal Infections.

Gonorrhea, a sexually transmitted infection caused by Neisseria gonorrhoeae, is a grave public health concern. Gonorrhea is the second most reported sexually transmitted infection worldwide. The treatment of uncomplicated gonococcal infections has evolved dramatically in response to the emergence of antimicrobial resistance. Multiple resistance mechanisms (for example, beta-lactamase production, antimicrobial efflux, and target site modification) exist, some of which may cause multidrug-resistance. Ceftriaxone was first recommended as an option for uncomplicated gonococcal infections in 1985, and it is now a mainstay of therapy in all clinical practice guidelines. Ceftriaxone has consistently shown high microbiologic cure rates in clinical trials, and it has demonstrated an excellent safety profile. Although its use may be limited in patients with hypersensitivity to penicillins, the risk of using ceftriaxone in such patients is overestimated. The emergence of reduced ceftriaxone susceptibility in N. gonorrhoeae, coupled with a lack of diverse treatment alternatives and the limited pipeline of new antimicrobials, is a significant threat to the treatment of gonorrhea.

Specialty pharmacy interventions benefit patients receiving HIV post-exposure prophylaxis.

BACKGROUND: Specialty pharmacies service many different complex disease states that require high-cost medication, including the treatment of patients prescribed HIV post-exposure prophylaxis (PEP). PEP requires time-sensitive initiation and patient counseling for therapeutic efficacy. OBJECTIVE: The objective of this study was to examine all PEP referrals received at a specialty pharmacy and demonstrate how they aided in interventions including assisting in obtaining financial assistance, making clinical interventions, and offering counseling to patients. METHODS: This is an observational retrospective chart review of patients who received PEP from one specialty pharmacy. All patients that filled PEP at the pharmacy between January 1st, 2017-July 1st, 2022, were included. Information was collected from documentation provided in the electronic medication record utilized by the pharmacy. The PEP regimen prescribed were raltegravir (RAL) + emtricitabine/tenofovir disoproxil fumarate (FTC/TDF) and dolutegravir (DTG) + emtricitabine/tenofovir disoproxil fumarate (FTC/TDF). RESULTS: A total of 52 patients were treated with PEP during the measurement period. Patients who received a PEP regimen of RAL + FTC/TDF experienced a total cost-savings of $1,692.60 and $218.40 for those who were fully insured and uninsured, respectively. Patients who received a PEP regimen of DTG + FTC/TDF experienced a total cost-savings of $676.20 and $2,725.50 for those who were fully insured and uninsured, respectively. Counseling by a pharmacist was offered to all patients and 74.5% of patients accepted. Pharmacists made clinical interventions on 29.4% of PEP referrals. CONCLUSION: PEP medications are expensive, time-sensitive, and can require clinical interventions and specific patient counseling. This study indicates that specialty pharmacies can provide and ensure access to care in the areas of financial assistance, patient counseling, and clinical interventions.

Diminishing storage returns of reservoir construction.

Surface water reservoirs are increasingly being relied upon to meet rising demands in the context of growing population and changing climate. However, the amount of water available in reservoirs (and the corresponding trends) have not been well quantified at the global scale. Here we use satellite observations to estimate the storage variations of 7245 global reservoirs from 1999 to 2018. Total global reservoir storage has increased at a rate of 27.82 ± 0.08 km3/yr, which is mainly attributed to the construction of new dams. However, the normalized reservoir storage (NS)-the ratio of the actual storage to the storage capacity-has declined by 0.82 ± 0.01%. The decline of NS values is especially pronounced in the global south, while the global north mainly exhibits an NS increase. With predicted decreasing runoff and increasing water demand, these observed diminishing storage returns of reservoir construction will likely persist into the future.

Not4-dependent targeting of MMF1 mRNA to mitochondria limits its expression via ribosome pausing, Egd1 ubiquitination, Caf130, no-go-decay and autophagy.

The Ccr4-Not complex is a conserved multi protein complex with diverse roles in the mRNA life cycle. Recently we determined that the Not1 and Not4 subunits of Ccr4-Not inversely regulate mRNA solubility and thereby impact dynamics of co-translation events. One mRNA whose solubility is limited by Not4 is MMF1 encoding a mitochondrial matrix protein. In this work we uncover a mechanism that limits MMF1 overexpression and depends upon its co-translational targeting to the mitochondria. We have named this mechanism Mito-ENCay. This mechanism relies on Not4 promoting ribosome pausing during MMF1 translation, and hence the co-translational docking of the MMF1 mRNA to mitochondria via the mitochondrial targeting sequence of the Mmf1 nascent chain, the Egd1 chaperone, the Om14 mitochondrial outer membrane protein and the co-translational import machinery. Besides co-translational Mitochondrial targeting, Mito-ENCay depends upon Egd1 ubiquitination by Not4, the Caf130 subunit of the Ccr4-Not complex, the mitochondrial outer membrane protein Cis1, autophagy and no-go-decay.

Satellites reveal hotspots of global river extent change.

Rivers are among the most diverse, dynamic, and productive ecosystems on Earth. River flow regimes are constantly changing, but characterizing and understanding such changes have been challenging from a long-term and global perspective. By analyzing water extent variations observed from four-decade Landsat imagery, we here provide a global attribution of the recent changes in river regime to morphological dynamics (e.g., channel shifting and anabranching), expansion induced by new dams, and hydrological signals of widening and narrowing. Morphological dynamics prevailed in ~20% of the global river area. Booming reservoir constructions, mostly skewed in Asia and South America, contributed to ~32% of the river widening. The remaining hydrological signals were characterized by contrasting hotspots, including prominent river widening in alpine and pan-Arctic regions and narrowing in the arid/semi-arid continental interiors, driven by varying trends in climate forcing, cryospheric response to warming, and human water management. Our findings suggest that the recent river extent dynamics diverge based on hydroclimate and socio-economic conditions, and besides reflecting ongoing morphodynamical processes, river extent changes show close connections with external forcings, including climate change and anthropogenic interference.

Not1 and Not4 inversely determine mRNA solubility that sets the dynamics of co-translational events.

BACKGROUND: The Ccr4-Not complex is mostly known as the major eukaryotic deadenylase. However, several studies have uncovered roles of the complex, in particular of the Not subunits, unrelated to deadenylation and relevant for translation. In particular, the existence of Not condensates that regulate translation elongation dynamics has been reported. Typical studies that evaluate translation efficiency rely on soluble extracts obtained after the disruption of cells and ribosome profiling. Yet cellular mRNAs in condensates can be actively translated and may not be present in such extracts. RESULTS: In this work, by analyzing soluble and insoluble mRNA decay intermediates in yeast, we determine that insoluble mRNAs are enriched for ribosomes dwelling at non-optimal codons compared to soluble mRNAs. mRNA decay is higher for soluble RNAs, but the proportion of co-translational degradation relative to the overall mRNA decay is higher for insoluble mRNAs. We show that depletion of Not1 and Not4 inversely impacts mRNA solubilities and, for soluble mRNAs, ribosome dwelling according to codon optimality. Depletion of Not4 solubilizes mRNAs with lower non-optimal codon content and higher expression that are rendered insoluble by Not1 depletion. By contrast, depletion of Not1 solubilizes mitochondrial mRNAs, which are rendered insoluble upon Not4 depletion. CONCLUSIONS: Our results reveal that mRNA solubility defines the dynamics of co-translation events and is oppositely regulated by Not1 and Not4, a mechanism that we additionally determine may already be set by Not1 promoter association in the nucleus.

How Have Global River Widths Changed Over Time?

Changes in a river's width reflect natural and anthropogenic impacts on local and upstream/downstream hydraulic and hydrologic processes. Temporal variation of river width also impacts biogeochemical exchange and reflects geomorphologic evolution. However, while global maps of mean river width and dynamic water surface extent exist, there is currently no standardized global assessment of river widths that documents changes over time. Therefore, we made repeated width measurements from Landsat images for all rivers wider than 90 m collected from 1984 to 2020 (named Global LOng-term river Width, GLOW), which consists of ∼1.2 billion cross-sectional river width measurements, with an average of 3,000 width measurements per 10-km reach. With GLOW, we investigated the temporal variations of global river width, quantified by the interquartile range (IQR) and temporal trend. We found that 85% of global rivers have a width IQR <150 m. We also found that 37% of global river segments show significant temporal trends in width over the past 37 years, and this number is higher (46%) for human-regulated rivers. Further, we leveraged machine learning to identify the most important factors explaining river width variations and revealed that these driving factors are significantly different between free-flowing and non-free-flowing rivers. Specifically, the most important factor driving temporal variations in river width is the climate for free-flowing rivers, and is soil condition for human-impacted rivers. Finally, we anticipate that this study and the public release of GLOW will improve the understanding of river dynamics and catalyze additional interdisciplinary studies.

Assessing placement bias of the global river gauge network.

Knowing where and when rivers flow is paramount to managing freshwater ecosystems. Yet stream gauging stations are distributed sparsely across rivers globally and may not capture the diversity of fluvial network properties and anthropogenic influences. Here we evaluate the placement bias of a global stream gauge dataset on its representation of socioecological, hydrologic, climatic and physiographic diversity of rivers. We find that gauges are located disproportionally in large, perennial rivers draining more human-occupied watersheds. Gauges are sparsely distributed in protected areas and rivers characterized by non-perennial flow regimes, both of which are critical to freshwater conservation and water security concerns. Disparities between the geography of the global gauging network and the broad diversity of streams and rivers weakens our ability to understand critical hydrologic processes and make informed water-management and policy decisions. Our findings underscore the need to address current gauge placement biases by investing in and prioritizing the installation of new gauging stations, embracing alternative water-monitoring strategies, advancing innovation in hydrologic modelling, and increasing accessibility of local and regional gauging data to support human responses to water challenges, both today and in the future.

Residential wood heating: An overview of U.S. impacts and regulations.

Air pollution from residential wood heating poses a significant public health risk and is a primary cause of PM nonattainment in some areas of the United States. Those emissions also play a role in regional haze and climate change. While regulatory programs have focused on emissions reductions from large facilities, the residential heating sector has received limited attention. The failure to develop effective programs to address this emission source hampers the ability of state and local air quality programs to meet clean air goals. An updated New Source Performance Standard (NSPS) for Residential Wood Heaters was promulgated in 2015, which includes more stringent emissions standards for wood stoves and broadens its scope to regulate additional types of wood heating appliances. However, weaknesses in the test methods and programs used to certify compliance with the NSPS limits hamper the efficacy of those requirements. Current emissions certification tests measure stove performance under defined laboratory conditions that (1) do not adequately reflect operation and performance of appliances in homes, (2) are not sufficiently repeatable to allow for comparison of emissions of different appliances, and (3) allow manufacturers leeway to modify critical test fueling and operating parameters which can significantly impact performance outcomes. These foundational regulatory issues present substantial challenges to promoting the cleanest and most efficient wood heating systems. This paper provides an overview of the air quality and public health impacts of residential wood heating and discusses the weaknesses in the current emission certification approaches and work by the Northeast States for Coordinated Air Use Management (NESCAUM) and the New York State Energy Research and Development Authority to develop improved testing methods. Other articles in this issue discuss the development and testing of those methods in detail.Implications: Air pollution from residential wood heating poses a significant public health risk and is a primary cause of PM nonattainment in some areas of the United States. Those emissions also play a role in regional haze and climate change. While regulatory programs have focused on emissions reductions from large facilities, the residential heating sector has received limited attention. The failure to develop effective programs to address this emission source hampers the ability of state and local air quality programs to meet clean air goals. This paper provides an overview of the issue.

Impact of fueling protocols on emission outcomes for residential wood-fired appliances.

Many believe that certification testing of residential wood heat appliances should provide data indicative of installed performance. Operationally, test methods typically only assess steady-state emissions and fail to include other typical conditions for batch appliances such as start-up. From a fueling perspective, protocols should ensure a consistent approach reflecting common use practices. Ensuring representative conditions and accurate quantification of emissions requires assessing the impact of different start-up conditions and whether or not start-up conditions affect appliance operation during start-up and beyond. This study evaluated the impact of modifying fuel piece sizes and configurations using a "smart" wood-fired hydronic heater (WHH) cordwood appliance. The appliance represents technologies using software and oxygen sensors to improve performance. Since the study used a "smart" appliance, the results likely reflect the least amount of variability found in a WHH cordwood appliance. The analysis consisted of a series of tests that involved changing one fuel variable per series, including: (1) kindling fuel arrangement in the firebox; (2) fuel piece size; and (3) the amount of kindling and starter fuel used. A goal of the study was to determine how each variable affects emissions performance during start-up and the following steady state load. Testing used a dual-stage combustion cordwood WHH equipped with external thermal storage. Particulate matter (PM), carbon monoxide (CO), and delivered heating efficiency were measured, and visible emissions from the stack and secondary combustion chamber were observed. Replicate tests were conducted for each protocol series to evaluate WHH performance reproducibility. These tests found that for a low-mass staged combustion WHH with external thermal storage, the use of different fueling protocols can substantially affect PM and CO emissions.Implications: As test methods move to incorporate measurements beyond steady-state emissions, fueling protocols must be assessed to determine (1) if they reflect typical field procedures and (2) the impact of start-up procedures on the complete test run. This paper assessed how changing start-up conditions affected run variability and PM emission impacts.

Impacts of wood species and moisture content on emissions from residential wood heaters.

Homeowners burn wood of a wide range of species and moisture content (MC) in residential cordwood and pellet stoves. An effective emission certification test protocol must account for and accurately measure the impact of those variables in order to ensure a reasonable correlation between laboratory results and in-use emissions and to promote the design and manufacture of cleaner burning appliances. This study explored the effect of wood species and MC on emissions and efficiency in four cordwood and four pellet stoves. PM emissions were consistently lower with pellets manufactured from softwood than for hardwood species and were highly correlated with ash content. Higher MC oak fuel substantially increased PM emissions in a non-catalytic cordwood stove; however, a hybrid cordwood stove was able to meet federal emissions limits even with the higher MC fuel. The results of this study, in combination with previous research, suggest that certification tests that use softwood fuel likely report lower emissions than tests that use hardwood. Requiring hardwood and higher MC cordwood fuel in certification tests would enable the assessment of an appliance's ability to operate well even when fuel conditions are not optimized.Implications: The emission testing results reported in this paper call into question the adequacy of the fuel moisture content and fuel species specifications in testing protocols approved for certifying compliance with EPA's New Source Performance Standards for cordwood and pellet stoves. We recommend changes in those specifications, including the prohibition of testing with Douglas fir and other low ash softwood species, requiring the use of cordwood test fuel with a higher moisture content, and requiring pellet stoves to be tested using hardwood pellets. Adoption of these measures would increase the replicability of tests. allow for the identification of stoves that are unlikely to perform well in the field when fuel conditions are not ideal, and, ultimately, result in the design of cleaner burning stoves.

Development of an integrated duty cycle test method to assess cordwood stove performance.

The US Environmental Protection Agency's (EPA's) New Source Performance Standards (NSPS) for Residential Wood Heaters (RWH) require certification emission testing of prototype appliances. In 2015, EPA revised those standards to further reduce particulate matter emissions from this critical source. However, to achieve that goal, lower emissions measured in certification tests must reflect lower emissions when the appliance is operated in homes. Woodstove certification tests have used either the Federal Reference Method (FRM), a crib wood method, or a cordwood testing method developed by ASTM International that was designated as a broadly applicable Alternative Test Method (ATM) by the EPA until December 2021, when that status was revoked. There is broad agreement that the FRM and ASTM procedures do not simulate typical fueling and operating of wood stoves in the field, raising questions about the efficacy of the current program. Effective emission reduction efforts require robust, accurate, and reproducible test methods. With input from a range of stakeholders, the Northeast States for Coordinated Air Use Management (NESCAUM) developed the Integrated Duty Cycle Test Method for Certification of Wood-Fired Stoves Using Cordwood (IDC), a cordwood testing protocol designed to improve the efficacy of residential wood heater certification testing. That method was approved by EPA as a broadly applicable ATM in 2021. IDC test runs assess appliance performance under a range of operating and fueling conditions representative of typical consumer use patterns. Unlike previous test methods, the IDC protocol requires three replicate runs to assess appliance performance variability. Including variable fueling and operating conditions, along with the requirement for replicates runs, will increase the effectiveness of certification testing and promote the development of improved wood stove technology. This paper reports on experiments conducted to develop and test the IDC method.Implications: Residential wood heating is one of the largest sources of primary particulate matter pollution nationwide. EPA's New Source Performance Standards (NSPS) establish emission limits for this source category and require certification testing of prototype wood appliances to demonstrate compliance with those limits. However, the operating and fueling requirements in NSPS compliance testing protocols do not represent typical conditions in the field. We developed a new testing approach, the Integrated-Duty Cycle (IDC) Test Method, to address the shortcomings of current certification test approaches. The IDC procedure for cordwood stoves, which was approved by EPA as a broadly applicable alternative test method in 2021, assesses appliance operations over various operating and fueling conditions representing typical consumer use patterns in an integrated run and requires three replicate runs to enable the assessment of variability in stove performance. Stoves certified with this method will be equipped to meet the NSPS limits consistently in field operation.

Online measurement of PM from residential wood heaters in a dilution tunnel.

The U.S. Environmental Protection Agency (US EPA) requires residential wood heaters (RWHs) to meet particulate matter (PM) emission limits in order to lower ambient concentrations and reduce public exposure. The current US EPA dilution tunnel PM measurement methods for RWHs were developed several decades ago and use manual filter samples to generate a single PM value for tests that can last more than 12 hours for stoves and 30 hours for central heating appliances. This approach results in averaging periods of high and low emissions together and provides limited data on emissions over the entire burn profile. Over the last decade, the U.S. ambient fine particulate monitoring network has transitioned to the routine use of online automated methods. However, stationary source measurement methods have not made this transition. There are no substantial technical issues in implementing real-time automated methods to measure PM for RWH emission certification purposes. The Thermo Scientific Tapered Element Oscillating Microbalance (TEOM™) has been widely used for ambient PM measurements. It is a true inertial mass measurement with high time resolution and sensitivity. This work compares measurements obtained using a Thermo 1400 or 1405 TEOM with ASTM E2515 manual filter samples, the current US EPA Federal Reference Method, for 172 test runs across a wide range of stoves and PM loading conditions. The TEOM measurements used the same filter media, similar filter face velocities, and filter temperatures as manual methods. PM measurements were well correlated (R2 > 0.9), with TEOM values typically lower by 5% to 10%. TEOM data capture was high, with filter changes resulting in ~5 minutes of lost data, usually once or twice during a multi-hour test. We discuss differences between the two methods, such as post-sampling equilibration and measurement of PM on sample train surfaces (probe "catch"). We also provide examples of substantial non-water semi-volatile mass loss during sampling.Implications: Measurement methods for continuous PM and our understanding of their performance has dramatically improved over the last thirty years. Highly time-resolved measurements of PM from residential wood heating appliances in an appliance certification testing context provide additional insight into both appliance performance and the suitability of the test method to assess that performance. This continuous measurement approach offers new opportunities to replace traditional US regulatory PM sampling integrated manual source methods like ASTM E2515 or EPA Method 5G testing. For measurement of combustion products that can have a wide range of physical and chemical characteristics, the TEOM's actual mass measurement principle has advantages over the sensitivity of surrogate methods to different aerosols for use in a regulatory program. Although the TEOM is commonly used to measure ambient PM, it can readily be configured to meet the needs of continuous emission testing.

Computational modeling of woodstove pollutants in dilution tunnels.

The computational modeling of the dilution tunnels used for experimental measurement of the woodstove pollution was presented. Two EPA-approved test labs for residential wood heat appliances, referred to as Lab-1 and Lab-2 dilution tunnels were simulated. The Ansys-Fluent software was enhanced with the addition of user-defined functions (UDF) and was used to simulate the airflow velocity, temperature, and particle concentration in the dilution tunnels. Particular attention was given to the variation of concentration profile at the test section and its uniformity. The simulation results suggested that roughly uniform or somewhat non-uniform particle concentrations entering from the woodstove stack into the dilution tunnel led to the uniform concentration at the outlet of the tunnel. This is particularly the case for the Lab-1 dilution tunnel. However, for the Lab-2 dilution tunnel, a highly non-uniform concentration at the woodstove stack outlet flowing at a high velocity into the dilution tunnel led to a non-uniform profile for the particle concentration at the test section. For this case, replacing the second elbow that is downstream from the mixing section with a tee reduced the nonuniformity of the concentration profile at the tunnel outlet.Implications: This study numerically investigated two dilution tunnels used in EPA-approved test labs. The dilution tunnel is used to dilute and cool the exhaust flow of the woodstove's stack. A properly working dilution tunnel provides a uniform concentration at the test section. Under different conditions, particulate matter (PM) laden turbulent flows in the tunnels are simulated to assess the dilution tunnel's performance. The goal is to understand the conditions that the dilution tunnels provide uniform concentration at their test section. The presented results suggest that using a tee instead of an elbow would enhance mixing and the chance for generating uniform concentration at the test section.