Enhanced prime editing systems by manipulating cellular determinants of editing outcomes.

While prime editing enables precise sequence changes in DNA, cellular determinants of prime editing remain poorly understood. Using pooled CRISPRi screens, we discovered that DNA mismatch repair (MMR) impedes prime editing and promotes undesired indel byproducts. We developed PE4 and PE5 prime editing systems in which transient expression of an engineered MMR-inhibiting protein enhances the efficiency of substitution, small insertion, and small deletion prime edits by an average 7.7-fold and 2.0-fold compared to PE2 and PE3 systems, respectively, while improving edit/indel ratios by 3.4-fold in MMR-proficient cell types. Strategic installation of silent mutations near the intended edit can enhance prime editing outcomes by evading MMR. Prime editor protein optimization resulted in a PEmax architecture that enhances editing efficacy by 2.8-fold on average in HeLa cells. These findings enrich our understanding of prime editing and establish prime editing systems that show substantial improvement across 191 edits in seven mammalian cell types.

Metabolism of Free Guanidine in Bacteria Is Regulated by a Widespread Riboswitch Class.

The guanidyl moiety is a component of fundamental metabolites, including the amino acid arginine, the energy carrier creatine, and the nucleobase guanine. Curiously, reports regarding the importance of free guanidine in biology are sparse, and no biological receptors that specifically recognize this compound have been previously identified. We report that many members of the ykkC motif RNA, the longest unresolved riboswitch candidate, naturally sense and respond to guanidine. This RNA is found throughout much of the bacterial domain of life, where it commonly controls the expression of proteins annotated as urea carboxylases and multidrug efflux pumps. Our analyses reveal that these proteins likely function as guanidine carboxylases and guanidine transporters, respectively. Furthermore, we demonstrate that bacteria are capable of endogenously producing guanidine. These and related findings demonstrate that free guanidine is a biologically relevant compound, and several gene families that can alleviate guanidine toxicity exist.

Perceived stress and renewal: The effects of long-term stress on the renewal effect.

Two online experiments evaluated the relationship between long-term stress, as measured with the Perceived Stress Scale-10, and the Renewal Effect. In the first experiment renewal was assessed with a behavioral suppression task in a science-fiction based video game. Participants learned to suppress mouse clicking during a signal for an upcoming attack to avoid losing points. The signal was first paired with an attack in Context A and extinguished in Context B and tested back in Context A. The contexts were different space galaxies where the gameplay took place. Experiment 2 used a food/illness predictive-learning paradigm. Two food items were paired with stomachache in one restaurant (A) and extinguished in Context B prior to testing in both contexts without feedback. Positive correlations were obtained between renewal and stress in each experiment. Unlike acute stress (Drexler et al., 2017), long term stress was associated with greater renewal. The effects of stress, both chronic and punctual, on renewal are discussed.

Widespread habitat loss leads to ecosystem-scale decrease in trophic function.

Natural and anthropogenic disturbances have led to rapid declines in the amount and quality of available habitat in many ecosystems. Many studies have focused on how habitat loss has affected the composition and configuration of habitats, but there have been fewer studies that investigate how this loss affects ecosystem function. We investigated how a large-scale seagrass die-off altered the distribution of energetic resources of three seagrass-associated consumers with varied resource use patterns. Using long-term benthic habitat monitoring data and resource use data from Bayesian stable isotope mixing models, we generated energetic resource landscapes (E-scapes) annually between 2007 and 2019. E-scapes link the resources being used by a consumer to the habitats that produce those resources to calculate a habitat resource index as a measurement of energetic quality of the landscape. Overall, our results revealed that following the die-off there was a reduction in trophic function across all species in areas affected by the die-off event, but the response was species-specific and dependent on resource use and recovery patterns. This study highlights how habitat loss can lead to changes in ecosystem function. Incorporating changes in ecosystem function into models of habitat loss could improve understanding of how species will respond to future change.

Restructuring the Gut Microbiota by Intermittent Fasting Lowers Blood Pressure.

[Figure: see text].

Trophic ontogeny of a generalist predator is conserved across space.

Consumers can influence ecological patterns and processes through their trophic roles and contributions to the flow of energy through ecosystems. However, the diet and associated trophic roles of consumers commonly change during ontogeny. Despite the prevalence of ontogenetic variation in trophic roles of most animals, we lack an understanding of whether they change consistently across local populations and broad geographic gradients. We examined how the diet and trophic position of a generalist marine predator varied with ontogeny across seven broadly separated locations (~ 750 km). We observed a high degree of heterogeneity in prey consumed without evidence of spatial structuring in this variability. However, compound-specific isotope analysis of amino acids revealed remarkably consistent patterns of increasing trophic position through ontogeny across local populations, suggesting that the roles of this generalist predator scaled with its body size across space. Given the high degree of diet heterogeneity we observed, this finding suggests that even though the dietary patterns differed, the underlying food web architecture transcended variation in prey species across locations for this generalist consumer. Our research addresses a gap in empirical field work regarding the interplay between stage-structured populations and food webs, and suggests ontogenetic changes in trophic position can be consistent in generalist consumers.

An ancient riboswitch class in bacteria regulates purine biosynthesis and one-carbon metabolism.

Over 30 years ago, ZTP (5-aminoimidazole-4-carboxamide riboside 5'-triphosphate), a modified purine biosynthetic intermediate, was proposed to signal 10-formyl-tetrahydrofolate (10f-THF) deficiency in bacteria. However, the mechanisms by which this putative alarmone or its precursor ZMP (5-aminoimidazole-4-carboxamide ribonucleotide, also known as AICAR) brings about any metabolic changes remain unexplained. Herein, we report the existence of a widespread riboswitch class that is most commonly associated with genes related to de novo purine biosynthesis and one-carbon metabolism. Biochemical data confirm that members of this riboswitch class selectively bind ZMP and ZTP with nanomolar affinity while strongly rejecting numerous natural analogs. Indeed, increases in the ZMP/ZTP pool, caused by folate stress in bacterial cells, trigger changes in the expression of a reporter gene fused to representative ZTP riboswitches in vivo. The wide distribution of this riboswitch class suggests that ZMP/ZTP signaling is important for species in numerous bacterial lineages.

Renal Outcomes Following Transcatheter Mitral Valve Repair - Analysis of COAPT Trial Data.

The prevalence of valvular heart disease in the United States has been estimated at 4.2-to-5.6 million, with mitral regurgitation (MR) being the most common lesion. Significant MR is associated with heart failure (HF) and death if left untreated. When HF is present, renal dysfunction (RD) is common and is associated with worse outcomes (ie, it is a marker of HF disease progression). Additionally, a complex interplay exists in patients with HF who also have MR, as this combination further impairs renal function, and the presence of RD further worsens prognosis and often limits guideline-directed management and therapy (GDMT). This has important implications in secondary MR because GDMT is the standard of care. However, with the development of minimally invasive transcatheter mitral valve repair, mitral transcatheter edge-to-edge repair (TEER) has become a new treatment option for secondary MR that is now incorporated into current guidelines published in 2020 that listed mitral TEER as a class 2a recommendation (moderate recommendation with benefit >> risk) as an addition to GDMT in a subset of patients with left ventricular ejection fraction <50%. The Cardiovascular Outcomes Assessment of the MitraClip Percutaneous Therapy for Heart Failure Patients with Functional Mitral Regurgitation (COAPT) trial, which demonstrated favorable outcomes in secondary MR by adding mitral TEER to GDMT versus GDMT alone, was the evidence base for these guidelines. Considering these guidelines and the understanding that concomitant RD often limits GDMT in secondary MR, there is emerging research studying the renal outcomes from the COAPT trial. This review analyzes this evidence, which could further influence current decision-making and future guidelines.

The impacts of mangrove range expansion on wetland ecosystem services in the southeastern United States: Current understanding, knowledge gaps, and emerging research needs.

Climate change is transforming ecosystems and affecting ecosystem goods and services. Along the Gulf of Mexico and Atlantic coasts of the southeastern United States, the frequency and intensity of extreme freeze events greatly influence whether coastal wetlands are dominated by freeze-sensitive woody plants (mangrove forests) or freeze-tolerant grass-like plants (salt marshes). In response to warming winters, mangroves have been expanding and displacing salt marshes at varying degrees of severity in parts of north Florida, Louisiana, and Texas. As winter warming accelerates, mangrove range expansion is expected to increasingly modify wetland ecosystem structure and function. Because there are differences in the ecological and societal benefits that salt marshes and mangroves provide, coastal environmental managers are challenged to anticipate the effects of mangrove expansion on critical wetland ecosystem services, including those related to carbon sequestration, wildlife habitat, storm protection, erosion reduction, water purification, fisheries support, and recreation. Mangrove range expansion may also affect wetland stability in the face of extreme climatic events and rising sea levels. Here, we review the current understanding of the effects of mangrove range expansion and displacement of salt marshes on wetland ecosystem services in the southeastern United States. We also identify critical knowledge gaps and emerging research needs regarding the ecological and societal implications of salt marsh displacement by expanding mangrove forests. One consistent theme throughout our review is that there are ecological trade-offs for consideration by coastal managers. Mangrove expansion and marsh displacement can produce beneficial changes in some ecosystem services, while simultaneously producing detrimental changes in other services. Thus, there can be local-scale differences in perceptions of the impacts of mangrove expansion into salt marshes. For very specific local reasons, some individuals may see mangrove expansion as a positive change to be embraced, while others may see mangrove expansion as a negative change to be constrained.

The gut microbiome contributes to blood-brain barrier disruption in spontaneously hypertensive stroke prone rats.

In recent years, it has become apparent that the gut microbiome can influence the functioning and pathological states of organs and systems throughout the body. In this study, we tested the hypothesis that the gut microbiome has a major role in the disruption of the blood-brain barrier (BBB) in the spontaneously hypertensive stroke prone rats (SHRSP), an animal model for hypertensive cerebral small vessel disease (CSVD). Loss of BBB is thought to be an early and initiating component to the full expression of CSVD in animal models and humans. To test this hypothesis, newly born SHRSP pups were placed with foster dams of the SHRSP strain or dams of the WKY strain, the control strain that does not demonstrate BBB dysfunction or develop hypertensive CSVD. Similarly, WKY pups were placed with foster dams of the same or opposite strain. The rationale for cross fostering is that the gut microbiomes are shaped by environmental bacteria of the foster dam and the nesting surroundings. Analysis of the bacterial genera in feces, using 16S rRNA analysis, demonstrated that the gut microbiome in the rat pups was influenced by the foster dam. SHRSP offspring fostered on WKY dams had systolic blood pressures (SBPs) that were significantly decreased by 26 mmHg (P < .001) from 16-20 weeks, compared to SHRSP offspring fostered on SHRSP dams. Similarly WKY offspring fostered on SHRSP dams had significantly increased SBP compared to WKY offspring fostered on WKY dams, although the magnitude of SBP change was not as robust. At ~20 weeks of age, rats fostered on SHRSP dams showed enhanced inflammation in distal ileum regardless of the strain of the offspring. Disruption of BBB integrity, an early marker of CSVD onset, was improved in SHRSPs that were fostered on WKY dams when compared to the SHRSP rats fostered on SHRSP dams. Although SHRSP is a genetic model for CSVD, environmental factors such as the gut microbiota of the foster dam have a major influence in the loss of BBB integrity.

E-scape: Consumer-specific landscapes of energetic resources derived from stable isotope analysis and remote sensing.

Energetic resources and habitat distribution are inherently linked. Energetic resource availability is a major driver of the distribution of consumers, but estimating how much specific habitats contribute to the energetic resource needs of a consumer can be problematic. We present a new approach that combines remote sensing information and stable isotope ecology to produce maps of energetic resources (E-scapes). E-scapes project species-specific resource use information onto the landscape to classify areas based on energetic importance. Using our E-scapes, we investigated the relationship between energetic resource distribution and white shrimp distribution and how the scale used to generate the E-scape mediated this relationship. E-scapes successfully predicted the size, abundance, biomass, and total energy of a consumer in salt marsh habitats in coastal Louisiana, USA at scales relevant to the movement of the consumer. Our E-scape maps can be used alone or in combination with existing models to improve habitat management and restoration practices and have potential to be used to test fundamental movement theory.

Use of plasma late on cardiopulmonary bypass in patients undergoing left ventricular assist device implantation.

Coagulopathy is common during left ventricular assist device (LVAD) implantation, treatment of which can be challenging given the often-limited ability for the right ventricle to accommodate volume transfusion after device initiation with 20% to 40% of patients developing right ventricular failure (RVF). Transfusion of plasma late on cardiopulmonary bypass (CPB) combined with ultrafiltration may replace clotting factors while reducing volume administration. We compared outcomes in patients undergoing LVAD implantation receiving plasma on CPB and ultrafiltration with traditional transfusion practices. Co-primary outcomes needed for blood product transfusion in the first 6 and 24 hours after CPB. Secondary outcomes included metrics of morbidity and mortality. 396 patients were analyzed (59 plasma on CPB). Patients receiving plasma on CPB had a greater volume of blood products transfused (3764 vs. 2741 mL first 6 hours; 6059 vs. 4305 mL first 24 hours) in unadjusted analysis. In adjusted analysis, plasma transfusion on CPB with ultrafiltration had no significant effect on the primary outcomes of blood products given in the first 6 hours (estimated effect size 982 [-428, 2392] mL, P = .17) and 24 hours (estimated effect size 1076 [-904, 3057] mL, P = .29). Patients receiving plasma on CPB were more likely on either vasopressors or inotropes at 24 hours after ICU admission (P = .01), however, indices of coagulopathy and RVF were similar between groups. While prospective studies would be necessary to definitively evaluate the clinical utility of this strategy, no signal for benefit was observed suggesting plasma should not be used for this purpose.

Analysis of the ESC/EACTS 2020 Atrial Fibrillation Guidelines With Perioperative Implications.

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia worldwide, with an individual lifetime risk of approximately 37% in the United States. Broadly defined as a supraventricular tachyarrhythmia with disorganized atrial activation, AF results in an increased risk of stroke, heart failure, valvular heart disease, and impaired quality of life, and confers a significant burden on the health of individuals and society. AF in the perioperative setting is common and a significant source of perioperative morbidity and mortality worldwide. The latest iteration of the European Society of Cardiology AF guidelines published in 2020 provide the clinician a valuable road map for the management of this arrythmia. This expert review will comprehensively analyze the 2020 European Society of Cardiology guidelines and provide perioperative management tools for the clinician.

Impact of nutritional status on prevalence of left ventricular hypertrophy in children undergoing liver transplant.

OBJECTIVE: We sought to (1) determine the prevalence of cardiac changes in patients with ESLD awaiting OLT (2) determine relationship between nutritional indices and cardiac changes. METHODS: Retrospective review of transthoracic ECHO, clinical and nutritional information of pediatric patients evaluated for OLT. ECHO was analyzed for LVH, defined as LVMI > 95 g/m2.7 and/or RWT > 0.42. These findings were correlated with age, ESLD etiology, growth and nutritional parameters as well as pre- and post-OLT. RESULTS: Sixty-five patients were included, all had normal left ventricular systolic function. Nine patients (14%) had LVMI > 95 g/m2.7 , five patients (8%) had RWT > 0.42, none met both criteria. None had thickened interventricular septal wall. Fourteen patients (20%) had significant left ventricular dilation. Nutritional deprivation was modestly present-weight under third percentile in 22%, length under third percentile in 24%, and both weight and length under third percentile in 17%. There were 12 patients (17%) with MUAC below two standard deviations for age; of these one had an elevated LVMI and another had an RWT > 0.42. CONCLUSIONS: In this contemporary cross-sectional evaluation, a smaller proportion of patients with ESLD had LVH in contrast to prior studies. Despite a comparable disease burden, our cohort had better nutritional status. Though there was a trend between nutritional and LVH indices, this correlation may be better assessed prospectively in a larger cohort.

Acute Severe Functional Mitral Regurgitation After Non-Mitral Valve Cardiac Surgery-Left Ventricular Dyssynchrony as a Potential Mechanism.

Functional mitral regurgitation (MR) describes valve leakage in the absence of disease or damage to the mitral leaflets or subvalvular apparatus. Significant, new functional MR after cardiopulmonary bypass (CPB) may result from a number of intraoperative processes, including left ventricular (LV) ischemia and enlargement, left atrial enlargement secondary to increased filling pressure, and systolic anterior motion of the mitral valve after mitral repair. Assessment of new MR after CPB is important because it may direct hemodynamic maneuvers or prompt reinitiation of CPB if surgical intervention is deemed necessary. Described extensively in the electrophysiology literature but underreported as a cause of MR after CPB, LV dyssynchrony represents another possible mechanism of functional MR, in which resynchronization of conduction via pacing maneuvers may prove beneficial. Herein, a series of 4 patients in whom new MR was found after non-mitral valve cardiac surgery in the setting of normal LV systolic function is presented, and LV dyssynchrony is proposed as a major contributing factor. The findings suggested that the concomitant observation of new or worsened functional MR, together with normal global and regional LV systolic function, should lead the clinician to consider ventricular dyssynchrony as a possible cause. Attempts to improve or alter ventricular conduction should be considered before contemplating a return to CPB for mitral valve intervention.

Tricuspid Regurgitation in Congestive Heart Failure: Management Strategies and Analysis of Outcomes.

Tricuspid regurgitation is a notable aspect of congestive heart failure and is linked with worse outcomes if untreated. Functional tricuspid regurgitation commonly is seen in patients with heart failure, particularly in patients presenting for surgical management, such as those for mechanical cardiac assist device implantation. This review aims to study the published data related to the surgical management of tricuspid regurgitation in the cardiac surgical population comprehensively.

Repetitive binge-like consumption based on the Drinking-in-the-Dark model alters the microglial population in the mouse hippocampus.

Alcoholism causes various maladaptations in the central nervous system, including the neuroimmune system. Studies of alcohol-induced dysregulation of the neuroimmune system generally focus on alcohol dependence and brain damage, but our previous research indicates that repetitive binge-like consumption perturbs cytokines independent of cell death. This paper extends that research by examining the impact of binge-like consumption on microglia in the hippocampus and the amygdala. Microglia were assessed using immunohistochemistry following binge-like ethanol consumption based on Drinking-in-the-Dark model. Immunohistochemistry results showed that binge-like ethanol consumption caused an increase in Iba-1 immunoreactivity and the number of Iba-1+ cells after one Drinking-in-the-Dark cycle. However, after three Drinking-in-the-Dark cycles, the number of microglia decreased in the hippocampus. We showed that in the dentate gyrus, the average immunoreactivity/cell was increased following ethanol exposure despite the decrease in number after three cycles. Likewise, Ox-42, an indicator of microglia activation, was upregulated after ethanol consumption. No significant effects on microglia number or immunoreactivity (Iba-1 nor Ox-42) were observed in the amygdala. Finally, ethanol caused an increase in the expression of the microglial gene Aif-1 during intoxication and ten days into abstinence, suggesting persistence of ethanol-induced upregulation of microglial genes. Altogether, these findings indicate that repetitive binge-like ethanol is sufficient to elicit changes in microglial reactivity. This altered neuroimmune state may contribute to the development of alcohol use disorders.

Thrombolytic Enzymes of Microbial Origin: A Review.

Enzyme therapies are attracting significant attention as thrombolytic drugs during the current scenario owing to their great affinity, specificity, catalytic activity, and stability. Among various sources, the application of microbial-derived thrombolytic and fibrinolytic enzymes to prevent and treat vascular occlusion is promising due to their advantageous cost-benefit ratio and large-scale production. Thrombotic complications such as stroke, myocardial infarction, pulmonary embolism, deep venous thrombosis, and peripheral occlusive diseases resulting from blood vessel blockage are the major cause of poor prognosis and mortality. Given the ability of microbial thrombolytic enzymes to dissolve blood clots and prevent any adverse effects, their use as a potential thrombolytic therapy has attracted great interest. A better understanding of the hemostasis and fibrinolytic system may aid in improving the efficacy and safety of this treatment approach over classical thrombolytic agents. Here, we concisely discuss the physiological mechanism of thrombus formation, thrombo-, and fibrinolysis, thrombolytic and fibrinolytic agents isolated from bacteria, fungi, and algae along with their mode of action and the potential application of microbial enzymes in thrombosis therapy.

Monitoring Site-Specific Fermentation Outcomes via Oxidation Reduction Potential and UV-Vis Spectroscopy to Characterize "Hidden" Parameters of Pinot Noir Wine Fermentations.

Real-time process metrics are standard for the majority of fermentation-based industries but have not been widely adopted by the wine industry. In this study, replicate fermentations were conducted with temperature as the main process parameter and assessed via in-line Oxidation Reduction Potential (ORP) probes and at-line profiling of phenolics compounds by UV-Vis spectroscopy. The California and Oregon vineyards used in this study displayed consistent vinification outcomes over five vintages and are representative of sites producing faster- and slower-fermenting musts. The selected sites have been previously characterized by fermentation kinetics, elemental profile, phenolics, and sensory analysis. ORP probes were integrated into individual fermentors to record how ORP changed throughout the fermentation process. The ORP profiles generally followed expected trends with deviations revealing previously undetectable process differences between sites and replicates. Site-specific differences were also observed in phenolic and anthocyanin extraction. Elemental composition was also analyzed for each vineyard, revealing distinctive profiles that correlated with the fermentation kinetics and may influence the redox status of these wines. The rapid ORP responses observed related to winemaking decisions and yeast activity suggest ORP is a useful process parameter that should be tracked in addition to Brix, temperature, and phenolics extraction for monitoring fermentations.

Orthopaedists Versus Radiologists: A Prospective Comparison of Radiographic Interpretation Between Orthopaedists and Radiologists at a Level I Trauma Center.

The purpose of this investigation was to evaluate the difference in response time, accuracy of radiographic interpretation, and frequency of changes in clinical management necessary after inaccurate reads when comparing orthopaedic and radiology providers. Data including provider response time, accuracy of radiographic interpretation and the frequency of changes in clinical management necessary after imaging reads was collected over a continuous two-month period at a Level I Trauma center. A total of 188 orthopedic injuries involving imaging were included. Orthopedic providers responded 203.2 minutes sooner than radiology providers. Accuracy of radiographic interpretation of the orthopaedic and radiology providers was 100% and 91%, respectively. Frequency of changes in clinical management after inaccurate interpretation of imaging by the orthopaedic and radiology provider was 0% and 7.6%, respectively. Based on our study, orthopaedic providers are significantly faster, more accurate, and make fewer mistakes affecting patient care while interpreting images of orthopaedic injuries than our radiology colleagues. (Journal of Surgical Orthopaedic Advances 30(2):093-096, 2021).