Cognitive impairment in post-acute sequelae of COVID-19 and short duration myalgic encephalomyelitis patients is mediated by orthostatic hemodynamic changes.

Introduction: Cognitive impairment is experienced by people with myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) and post-acute sequelae of COVID-19 (PASC). Patients report difficulty remembering, concentrating, and making decisions. Our objective was to determine whether orthostatic hemodynamic changes were causally linked to cognitive impairment in these diseases. Methods: This prospective, observational cohort study enrolled PASC, ME/CFS, and healthy controls. All participants underwent clinical evaluation and assessment that included brief cognitive testing before and after an orthostatic challenge. Cognitive testing measured cognitive efficiency which is defined as the speed and accuracy of subject's total correct responses per minute. General linear mixed models were used to analyze hemodynamics and cognitive efficiency during the orthostatic challenge. Additionally, mediation analysis was used to determine if hemodynamic instability induced during the orthostatic challenge mediated the relationship between disease status and cognitive impairment. Results: Of the 276 participants enrolled, 256 were included in this study (34 PASC, 71 < 4 year duration ME/CFS, 69 > 10 year ME/CFS duration, and 82 healthy controls). Compared to healthy controls, the disease cohorts had significantly lower cognitive efficiency scores immediately following the orthostatic challenge. Cognitive efficiency remained low for the >10 year ME/CFS 2 and 7 days after orthostatic challenge. Narrow pulse pressure less than 25% of systolic pressure occurred at 4 and 5 min into the orthostatic challenge for the PASC and ME/CFS cohorts, respectively. Abnormally narrow pulse pressure was associated with slowed information processing in PASC patients compared to healthy controls (-1.5, p = 0.04). Furthermore, increased heart rate during the orthostatic challenge was associated with a decreased procedural reaction time in PASC and < 4 year ME/CFS patients who were 40 to 65 years of age. Discussion: For PASC patients, both their disease state and hemodynamic changes during orthostatic challenge were associated with slower reaction time and decreased response accuracy during cognitive testing. Reduced cognitive efficiency in <4 year ME/CFS patients was associated with higher heart rate in response to orthostatic stress. Hemodynamic changes did not correlate with cognitive impairment for >10 year ME/CFS patients, but cognitive impairment remained. These findings underscore the need for early diagnosis to mitigate direct hemodynamic and other physiological effects on symptoms of cognitive impairment.

Orthostatic Challenge Causes Distinctive Symptomatic, Hemodynamic and Cognitive Responses in Long COVID and Myalgic Encephalomyelitis/Chronic Fatigue Syndrome.

Background: Some patients with acute COVID-19 are left with persistent, debilitating fatigue, cognitive impairment ("brain fog"), orthostatic intolerance (OI) and other symptoms ("Long COVID"). Many of the symptoms are like those of other post-infectious fatigue syndromes and may meet criteria for myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). Common diagnostic laboratory tests are often unrevealing. Methods: We evaluated whether a simple, standardized, office-based test of OI, the 10-min NASA Lean Test (NLT), would aggravate symptoms and produce objective hemodynamic and cognitive abnormalities, the latter being evaluated by a simple smart phone-based app. Participants: People with Long COVID (N = 42), ME/CFS (N = 26) and healthy control subjects (N = 20) were studied just before, during, immediately after, 2 and 7 days following completion of the NLT. Results: The NLT provoked a worsening of symptoms in the two patient groups but not in healthy control subjects, and the severity of all symptoms was similar and significantly worse in the two patient groups than in the control subjects (p < 0.001). In the two patient groups, particularly those with Long COVID, the NLT provoked a marked and progressive narrowing in the pulse pressure. All three cognitive measures of reaction time worsened in the two patient groups immediately following the NLT, compared to the healthy control subjects, particularly in the Procedural Reaction Time (p < 0.01). Conclusions: A test of orthostatic stress easily performed in an office setting reveals different symptomatic, hemodynamic and cognitive abnormalities in people with Long COVID and ME/CFS, compared to healthy control subjects. Thus, an orthostatic challenge easily performed in an office setting, and the use of a smart phone app to assess cognition, can provide objective confirmation of the orthostatic intolerance and brain fog reported by patients with Long COVID and ME/CFS.

Biosensor for branched-chain amino acid metabolism in yeast and applications in isobutanol and isopentanol production.

Branched-chain amino acid (BCAA) metabolism fulfills numerous physiological roles and can be harnessed to produce valuable chemicals. However, the lack of eukaryotic biosensors specific for BCAA-derived products has limited the ability to develop high-throughput screens for strain engineering and metabolic studies. Here, we harness the transcriptional regulator Leu3p from Saccharomyces cerevisiae to develop a genetically encoded biosensor for BCAA metabolism. In one configuration, we use the biosensor to monitor yeast production of isobutanol, an alcohol derived from valine degradation. Small modifications allow us to redeploy Leu3p in another biosensor configuration that monitors production of the leucine-derived alcohol, isopentanol. These biosensor configurations are effective at isolating high-producing strains and identifying enzymes with enhanced activity from screens for branched-chain higher alcohol (BCHA) biosynthesis in mitochondria as well as cytosol. Furthermore, this biosensor has the potential to assist in metabolic studies involving BCAA pathways, and offers a blueprint to develop biosensors for other products derived from BCAA metabolism.

Mitochondrial Compartmentalization Confers Specificity to the 2-Ketoacid Recursive Pathway: Increasing Isopentanol Production in Saccharomyces cerevisiae.

Recursive elongation pathways produce compounds of increasing carbon-chain length with each iterative cycle. Of particular interest are 2-ketoacids derived from recursive elongation, which serve as precursors to a valuable class of advanced biofuels known as branched-chain higher alcohols (BCHAs). Protein engineering has been used to increase the number of iterative elongation cycles completed, yet specific production of longer-chain 2-ketoacids remains difficult to achieve. Here, we show that mitochondrial compartmentalization is an effective strategy to increase specificity of recursive pathways to favor longer-chain products. Using 2-ketoacid elongation as a proof of concept, we show that overexpression of the three elongation enzymes-LEU4, LEU1, and LEU2-in mitochondria of an isobutanol production strain results in a 2.3-fold increase in the isopentanol to isobutanol product ratio relative to overexpressing the same elongation enzymes in the cytosol, and a 31-fold increase relative to wild-type enzyme expression. Reducing the loss of intermediates allows us to further boost isopentanol production to 1.24 ± 0.06 g/L of isopentanol. In this strain, isopentanol accounts for 86% of the total BCHAs produced, while achieving the highest isopentanol titer reported for Saccharomyces cerevisiae. Localizing the elongation enzymes in mitochondria  enables the development of strains in which isopentanol constitutes as much as 93% of BCHA production. This work establishes mitochondrial compartmentalization as a new approach to favor high titers and product specificities of larger products from recursive pathways.

Critical Roles of the Pentose Phosphate Pathway and GLN3 in Isobutanol-Specific Tolerance in Yeast.

Branched-chain alcohols are attractive advanced biofuels; however, their cellular toxicity is an obstacle to engineering microbes to produce them at high titers. We performed genome-wide screens on the Saccharomyces cerevisiae gene deletion library to identify cell systems involved in isobutanol-specific tolerance. Deletion of pentose phosphate pathway genes GND1 or ZWF1 causes hypersensitivity to isobutanol but not to ethanol. By contrast, deletion of GLN3 increases yeast tolerance specifically to branched-chain alcohols. Transcriptomic analyses revealed that isobutanol induces a nitrogen starvation response via GLN3 and GCN4, upregulating amino acid biosynthesis and nitrogen scavenging while downregulating glycolysis, cell wall biogenesis, and membrane lipid biosynthesis. Disruption of this response by deleting GLN3 is enough to enhance tolerance and boost isobutanol production 4.9-fold in engineered strains. This study illustrates how adaptive mechanisms to tolerate stress can lead to toxicity in microbial fermentations for chemical production and how genetic interventions can boost production by evading such mechanisms.

Xylose utilization stimulates mitochondrial production of isobutanol and 2-methyl-1-butanol in Saccharomyces cerevisiae.

BACKGROUND: Branched-chain higher alcohols (BCHAs), including isobutanol and 2-methyl-1-butanol, are promising advanced biofuels, superior to ethanol due to their higher energy density and better compatibility with existing gasoline infrastructure. Compartmentalizing the isobutanol biosynthetic pathway in yeast mitochondria is an effective way to produce BCHAs from glucose. However, to improve the sustainability of biofuel production, there is great interest in developing strains and processes to utilize lignocellulosic biomass, including its hemicellulose component, which is mostly composed of the pentose xylose. RESULTS: In this work, we rewired the xylose isomerase assimilation and mitochondrial isobutanol production pathways in the budding yeast Saccharomyces cerevisiae. We then increased the flux through these pathways by making gene deletions of BAT1, ALD6, and PHO13, to develop a strain (YZy197) that produces as much as 4 g/L of BCHAs (3.10 ± 0.18 g isobutanol/L and 0.91 ± 0.02 g 2-methyl-1-butanol/L) from xylose. This represents approximately a 28-fold improvement on the highest isobutanol titers obtained from xylose previously reported in yeast and the first report of 2-methyl-1-butanol produced from xylose. The yield of total BCHAs is 57.2 ± 5.2 mg/g xylose, corresponding to ~ 14% of the maximum theoretical yield. Respirometry experiments show that xylose increases mitochondrial activity by as much as 7.3-fold compared to glucose. CONCLUSIONS: The enhanced levels of mitochondrial BCHA production achieved, even without disrupting ethanol byproduct formation, arise mostly from xylose activation of mitochondrial activity and are correlated with slow rates of sugar consumption.

Uncovering the role of branched-chain amino acid transaminases in Saccharomyces cerevisiae isobutanol biosynthesis.

Isobutanol and other branched-chain higher alcohols (BCHAs) are promising advanced biofuels derived from the degradation of branched-chain amino acids (BCAAs). The yeast Saccharomyces cerevisiae is a particularly attractive host for the production of BCHAs due to its high tolerance to alcohols and prevalent use in the bioethanol industry. Degradation of BCAAs begins with transamination reactions, catalyzed by branched-chain amino acid transaminases (BCATs) located in the mitochondria (Bat1p) and cytosol (Bat2p). However, the roles that these transaminases play in isobutanol production remain poorly understood and obscured by conflicting reports in the literature. In this work, we elucidate the influence of BCATs on isobutanol production in two genetic backgrounds (CEN.PK2-1C and BY4741). In the process, we uncover and characterize two competing isobutanol pathways, which can be manipulated by overexpressing or deleting BAT1 or BAT2, and adding or removing valine from the fermentation media. We show that deletion of BAT1 alone increases isobutanol production by 14.2-fold over wild type strains in media lacking valine, and examine how interactions between valine and the regulatory protein Ilv6p affect isobutanol production. Compartmentalizing the five-gene isobutanol biosynthetic pathway in mitochondria of BAT1 deletion strains results in an additional 2.1-fold increase in isobutanol production in the absence of valine. While valine inhibits isobutanol production, it boosts 2-methyl-1-butanol production. This work clarifies the role of transamination activity in BCHA biosynthesis, and develops valuable strategies and strains for future optimization of isobutanol production.

Harnessing yeast organelles for metabolic engineering.

Each subcellular compartment in yeast offers a unique physiochemical environment and metabolite, enzyme, and cofactor composition. While yeast metabolic engineering has focused on assembling pathways in the cell cytosol, there is growing interest in embracing subcellular compartmentalization. Beyond harnessing distinct organelle properties, physical separation of organelles from the cytosol has the potential to eliminate metabolic crosstalk and enhance compartmentalized pathway efficiency. In this Perspective we review the state of the art in yeast subcellular engineering, highlighting the benefits of targeting biosynthetic pathways to subcellular compartments, including mitochondria, peroxisomes, the ER and/or Golgi, vacuoles, and the cell wall, in different yeast species. We compare the performances of strains developed with subcellular engineering to those of native producers or yeast strains previously engineered with cytosolic pathways. We also identify important challenges that lie ahead, which need to be addressed for organelle engineering to become as mainstream as cytosolic engineering in academia and industry.

Biological lignocellulose solubilization: comparative evaluation of biocatalysts and enhancement via cotreatment.

BACKGROUND: Feedstock recalcitrance is the most important barrier impeding cost-effective production of cellulosic biofuels. Pioneer commercial cellulosic ethanol facilities employ thermochemical pretreatment and addition of fungal cellulase, reflecting the main research emphasis in the field. However, it has been suggested that it may be possible to process cellulosic biomass without thermochemical pretreatment using thermophilic, cellulolytic bacteria. To further explore this idea, we examine the ability of various biocatalysts to solubilize autoclaved but otherwise unpretreated cellulosic biomass under controlled but not industrial conditions. RESULTS: Carbohydrate solubilization of mid-season harvested switchgrass after 5 days ranged from 24 % for Caldicellulosiruptor bescii to 65 % for Clostridium thermocellum, with intermediate values for a thermophilic horse manure enrichment, Clostridium clariflavum, Clostridium cellulolyticum, and simultaneous saccharification and fermentation (SSF) featuring a fungal cellulase cocktail and yeast. Under a variety of conditions, solubilization yields were about twice as high for C. thermocellum compared to fungal cellulase. Solubilization of mid-season harvested switchgrass was about twice that of senescent switchgrass. Lower yields and greater dependence on particle size were observed for Populus as compared to switchgrass. Trends observed from data drawn from six conversion systems and three substrates, including both time course and end-point data, were (1) equal fractional solubilization of glucan and xylan, (2) no biological solubilization of the non-carbohydrate fraction of biomass, and (3) higher solubilization for three of the four bacterial cultures tested as compared to the fungal cellulase system. Brief (5 min) ball milling of solids remaining after fermentation of senescent switchgrass by C. thermocellum nearly doubled carbohydrate solubilization upon reinnoculation as compared to a control without milling. Greater particle size reduction and solubilization were observed for milling of partially fermented solids than for unfermented solids. Physical disruption of cellulosic feedstocks after initiation of fermentation, termed cotreatment, warrants further study. CONCLUSIONS: While the ability to achieve significant solubilization of minimally pretreated switchgrass is widespread, a fivefold difference between the most and least effective biocatalyst-feedstock combinations was observed. Starting with nature's best biomass-solubilizing systems may enable a reduction in the amount of non-biological processing required, and in particular substitution of cotreatment for pretreatment.

Multiple Retinal Axons Converge onto Relay Cells in the Adult Mouse Thalamus.

Activity-dependent refinement of neural circuits is a fundamental principle of neural development. This process has been well studied at retinogeniculate synapses-synapses that form between retinal ganglion cells (RGCs) and relay cells within the dorsal lateral geniculate nucleus. Physiological studies suggest that shortly after birth, inputs from ∼20 RGCs converge onto relay cells. Subsequently, all but just one to two of these inputs are eliminated. Despite widespread acceptance, this notion is at odds with ultrastructural studies showing numerous retinal terminals clustering onto relay cell dendrites in the adult. Here, we explored this discrepancy using brainbow AAVs and serial block face scanning electron microscopy (SBFSEM). Results with both approaches demonstrate that terminals from numerous RGCs cluster onto relay cell dendrites, challenging the notion that only one to two RGCs innervate each relay cell. These findings force us to re-evaluate our understanding of subcortical visual circuitry.

Nuclei-specific differences in nerve terminal distribution, morphology, and development in mouse visual thalamus.

BACKGROUND: Mouse visual thalamus has emerged as a powerful model for understanding the mechanisms underlying neural circuit formation and function. Three distinct nuclei within mouse thalamus receive retinal input, the dorsal lateral geniculate nucleus (dLGN), the ventral lateral geniculate nucleus (vLGN), and the intergeniculate nucleus (IGL). However, in each of these nuclei, retinal inputs are vastly outnumbered by nonretinal inputs that arise from cortical and subcortical sources. Although retinal and nonretinal terminals associated within dLGN circuitry have been well characterized, we know little about nerve terminal organization, distribution and development in other nuclei of mouse visual thalamus. RESULTS: Immunolabeling specific subsets of synapses with antibodies against vesicle-associated neurotransmitter transporters or neurotransmitter synthesizing enzymes revealed significant differences in the composition, distribution and morphology of nonretinal terminals in dLGN, vLGN and IGL. For example, inhibitory terminals are more densely packed in vLGN, and cortical terminals are more densely distributed in dLGN. Overall, synaptic terminal density appears least dense in IGL. Similar nuclei-specific differences were observed for retinal terminals using immunolabeling, genetic labeling, axonal tracing and serial block face scanning electron microscopy: retinal terminals are smaller, less morphologically complex, and more densely distributed in vLGN than in dLGN. Since glutamatergic terminal size often correlates with synaptic function, we used in vitro whole cell recordings and optic tract stimulation in acutely prepared thalamic slices to reveal that excitatory postsynaptic currents (EPSCs) are considerably smaller in vLGN and show distinct responses following paired stimuli. Finally, anterograde labeling of retinal terminals throughout early postnatal development revealed that anatomical differences in retinal nerve terminal structure are not observable as synapses initially formed, but rather developed as retinogeniculate circuits mature. CONCLUSIONS: Taken together, these results reveal nuclei-specific differences in nerve terminal composition, distribution, and morphology in mouse visual thalamus. These results raise intriguing questions about the different functions of these nuclei in processing light-derived information, as well as differences in the mechanisms that underlie their unique, nuclei-specific development.

Assessing teen smoking patterns: the weekend phenomenon.

BACKGROUND: Adolescent cigarette smokers may have more daily variability in their smoking patterns than adults. A better understanding of teen smoking patterns can inform the development of more effective adolescent smoking cessation interventions. METHODS: Teen smokers seeking cessation treatment (N=366) reported the number of cigarettes smoked on each day of a typical week. A paired t-test was used to examine differences between weekday (Sunday-Thursday) and weekend (Friday-Saturday) smoking. Main effects and interactions for race/ethnicity and gender were assessed using a 2-way ANOVA for the following variables: typical weekly smoking, average weekday smoking, average weekend smoking, and difference between weekday and weekend smoking. Scheffé post hoc tests were used to analyze any statistically significant differences. RESULTS: There was significantly more weekend smoking compared to weekday smoking, p<0.001. The difference in weekday versus weekend smoking levels was larger for females than for males, p<0.05. Hispanics reported less typical weekly smoking, p<0.001, less weekday smoking, p<0.001, and less weekend day smoking, p<0.01, compared to Caucasians and multi-racial teens. There was no difference in weekend day versus weekday smoking by race/ethnic background. CONCLUSIONS: Using a more detailed assessment of smoking quantity captures patterns of adolescent smoking that may lead to more effective smoking cessation interventions.

Using treatment process data to predict maintained smoking abstinence.

OBJECTIVES: To identify distinct subgroups of treatment responders and nonresponders to aid in the development of tailored smoking-cessation interventions for long-term maintenance using signal detection analysis (SDA). METHODS: The secondary analyses (n = 301) are based on data obtained in our randomized clinical trial designed to assess the efficacy of extended cognitive behavior therapy for cigarette smoking cessation. Model 1 included only pretreatment factors, demographic characteristics, and treatment assignment. Model 2 included all Model 1 variables, as well as clinical data measured during treatment. RESULTS: SDA was successfully able to identify smokers with varying probabilities of maintaining abstinence from end-of-treatment to 52-week follow-up; however, the inclusion of clinical data obtained over the course of treatment in Model 2 yielded very different partitioning parameters. CONCLUSIONS: The findings from this study may enable researchers to target underlying factors that may interact to promote maintenance of long-term smoking behavior change.