Increased Cortical Thickness in Alzheimer's Disease.

OBJECTIVE: Patients with Alzheimer's disease (AD) have diffuse brain atrophy, but some regions, such as the anterior cingulate cortex (ACC), are spared and may even show increase in size compared to controls. The extent, clinical significance, and mechanisms associated with increased cortical thickness in AD remain unknown. Recent work suggested neural facilitation of regions anticorrelated to atrophied regions in frontotemporal dementia. Here, we aim to determine whether increased thickness occurs in sporadic AD, whether it relates to clinical symptoms, and whether it occur in brain regions functionally connected to-but anticorrelated with-locations of atrophy. METHODS: Cross-sectional clinical, neuropsychological, and neuroimaging data from the Alzheimer's Disease Neuroimaging Initiative were analyzed to investigate cortical thickness in AD subjects versus controls. Atrophy network mapping was used to identify brain regions functionally connected to locations of increased thickness and atrophy. RESULTS: AD patients showed increased thickness in the ACC in a region-of-interest analysis and the visual cortex in an exploratory analysis. Increased thickness in the left ACC was associated with preserved cognitive function, while increased thickness in the left visual cortex was associated with hallucinations. Finally, we found that locations of increased thickness were functionally connected to, but anticorrelated with, locations of brain atrophy (r = -0.81, p < 0.05). INTERPRETATION: Our results suggest that increased cortical thickness in Alzheimer's disease is relevant to AD symptoms and preferentially occur in brain regions functionally connected to, but anticorrelated with, areas of brain atrophy. Implications for models of compensatory neuroplasticity in response to neurodegeneration are discussed. ANN NEUROL 2024;95:929-940.

Screening of Oligomeric (Meth)acrylate Vaccine Adjuvants Synthesized via Catalytic Chain Transfer Polymerization.

This report details the first systematic screening of free-radical-produced methacrylate oligomer reaction mixtures as alternative vaccine adjuvant components to replace the current benchmark compound squalene, which is unsustainably sourced from shark livers. Homo-/co-oligomer mixtures of methyl, butyl, lauryl, and stearyl methacrylate were successfully synthesized using catalytic chain transfer control, where the use of microwave heating was shown to promote propagation over chain transfer. Controlling the mixture material properties allowed the correct viscosity to be achieved, enabling the mixtures to be effectively used in vaccine formulations. Emulsions of selected oligomers stimulated comparable cytokine levels to squalene emulsion when incubated with human whole blood and elicited an antigen-specific cellular immune response when administered with an inactivated influenza vaccine, indicating the potential utility of the compounds as vaccine adjuvant components. Furthermore, the oligomers' molecular sizes were demonstrated to be large enough to enable greater emulsion stability than squalene, especially at high temperatures, but are predicted to be small enough to allow for rapid clearance from the body.

Physicochemical and immunological effects of adjuvant formulations with snake venom antigens for immunization of horses for antivenom production.

Enhancement of antivenom immune responses in horses through adjuvant technology improves antivenom production efficiency, but substantial local reactogenicity associated with some traditional veterinary adjuvants limits their usability. To explore modern adjuvant systems suitable for generating antivenom responses in horses, we first assessed their physicochemical compatibility with Bothrops asper snake venom. Liposome and nanoparticle aluminum adjuvants exhibited changes in particle size and phospholipid content after mixing with venom, whereas squalene emulsion-based adjuvants remained stable. Next, we evaluated serum antibody response magnitude and neutralization capacity in horses immunized with adjuvant-containing Echis ocellatus, Bitis arietans, Naja nigricollis, and Dendroaspis polylepis venom preparations. Whereas all tested adjuvants elicited significant neutralization capacity against the viperid venoms, the greatest antibody responses were generated by a squalene-in-water emulsion, thus representing a promising novel alternative for antivenom production.

Measuring Antisocial Behaviors in Behavioral Variant Frontotemporal Dementia With a Novel Informant-Based Questionnaire.

OBJECTIVE: Antisocial behaviors are common and problematic among patients with behavioral variant frontotemporal dementia (bvFTD). In the present study, the investigators aimed to validate an informant-based questionnaire developed to measure the extent and severity of antisocial behaviors among patients with dementia. METHODS: The Social Behavior Questionnaire (SBQ) was developed to measure 26 antisocial behaviors on a scale from absent (0) to very severe (5). It was administered to 23 patients with bvFTD, 19 patients with Alzheimer's disease, and 14 patients with other frontotemporal lobar degeneration syndromes. Group-level differences in the presence and severity of antisocial behaviors were measured. Psychometric properties of the SBQ were assessed by using Cronbach's alpha, exploratory factor analysis, and comparisons with a psychopathy questionnaire. Cluster analysis was used to determine whether the SBQ identifies different subgroups of patients. RESULTS: Antisocial behaviors identified by using the SBQ were common and severe among patients with bvFTD, with at least one such behavior endorsed for 21 of 23 (91%) patients. Antisocial behaviors were more severe among patients with bvFTD, including the subsets of patients with milder cognitive impairment and milder disease severity, than among patients in the other groups. The SBQ was internally consistent (Cronbach's α=0.81). Exploratory factor analysis supported separate factors for aggressive and nonaggressive behaviors. Among the patients with bvFTD, the factor scores for aggressive behavior on the SBQ were correlated with those for antisocial behavior measured on the psychopathy scale, but the nonaggressive scores were not correlated with psychopathy scale measures. The k-means clustering analysis identified a subset of patients with severe antisocial behaviors. CONCLUSIONS: The SBQ is a useful tool to identify, characterize, and measure the severity of antisocial behaviors among patients with dementia.

Stressed stability and protective efficacy of lead lyophilized formulations of ID93+GLA-SE tuberculosis vaccine.

With the recent exception of coronavirus disease 2019 (COVID-19), tuberculosis (TB) causes more deaths globally than any other infectious disease, and approximately 1/3 of the world's population is infected with Mycobacterium tuberculosis (Mtb). However, encouraging progress in TB vaccine development has been reported, with approximately 50% efficacy achieved in Phase 2b clinical testing of an adjuvanted subunit TB vaccine candidate. Nevertheless, current lead vaccine candidates require cold-chain transportation and storage. In addition to temperature stress, vaccines may be subject to several other stresses during storage and transport, including mechanical, photochemical, and oxidative stresses. Optimal formulations should enable vaccine configurations with enhanced stability and decreased sensitivity to physical and chemical stresses, thus reducing reliance on the cold chain and facilitating easier worldwide distribution. In this report, we describe the physicochemical stability performance of three lead thermostable formulations of the ID93 + GLA-SE TB vaccine candidate under various stress conditions. Moreover, we evaluate the impact of thermal stress on the protective efficacy of the vaccine formulations. We find that formulation composition impacts stressed stability performance, and our comprehensive evaluation enables selection of a lead single-vial lyophilized candidate containing the excipient trehalose and Tris buffer for advanced development.

Semi-synthetic terpenoids with differential adjuvant properties as sustainable replacements for shark squalene in vaccine emulsions.

Synthetic biology has allowed for the industrial production of supply-limited sesquiterpenoids such as the antimalarial drug artemisinin and ß-farnesene. One of the only unmodified animal products used in medicine is squalene, a triterpenoid derived from shark liver oil, which when formulated into an emulsion is used as a vaccine adjuvant to enhance immune responses in licensed vaccines. However, overfishing is depleting deep-sea shark populations, leading to potential supply problems for squalene. We chemically generated over 20 squalene analogues from fermentation-derived ß-farnesene and evaluated adjuvant activity of the emulsified compounds compared to shark squalene emulsion. By employing a desirability function approach that incorporated multiple immune readouts, we identified analogues with enhanced, equivalent, or decreased adjuvant activity compared to shark squalene emulsion. Availability of a library of structurally related analogues allowed elucidation of structure-function relationships. Thus, combining industrial synthetic biology with chemistry and immunology enabled generation of sustainable terpenoid-based vaccine adjuvants comparable to current shark squalene-based adjuvants while illuminating structural properties important for adjuvant activity.

Formulated Phospholipids as Non-Canonical TLR4 Agonists.

Immunogenic agents known as adjuvants play a critical role in many vaccine formulations. Adjuvants often signal through Toll-like receptor (TLR) pathways, including formulations in licensed vaccines that target TLR4. While TLR4 is predominantly known for responding to lipopolysaccharide (LPS), a component of Gram-negative bacterial membranes, it has been shown to be a receptor for a number of molecular structures, including phospholipids. Therefore, phospholipid-based pharmaceutical formulations might have off-target effects by signaling through TLR4, confounding interpretation of pharmaceutical bioactivity. In this study we examined the individual components of a clinical stage oil-in-water vaccine adjuvant emulsion (referred to as a stable emulsion or SE) and their ability to signal through murine and human TLR4s. We found that the phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) activated TLR4 and elicited many of the same immune phenotypes as canonical TLR4 agonists. This pathway was dependent on the saturation, size, and headgroup of the phospholipid. Interestingly, DMPC effects on human cells were evident but overall appeared less impactful than emulsion oil composition. Considering the prevalence of DMPC and other phospholipids used across the pharmaceutical space, these findings may contextualize off-target innate immune responses that could impact preclinical and clinical development.

Lyophilization Process Engineering and Thermostability of ID93 + GLA-SE, a Single-Vial Adjuvanted Subunit Tuberculosis Vaccine Candidate for Use in Clinical Studies.

Promising clinical efficacy results have generated considerable enthusiasm for the potential impact of adjuvant-containing subunit tuberculosis vaccines. The development of a thermostable tuberculosis vaccine formulation could have significant benefits on both the cost and feasibility of global vaccine distribution. The tuberculosis vaccine candidate ID93 + GLA-SE has reached Phase 2 clinical testing, demonstrating safety and immunogenicity as a two-vial point-of-care mixture. Earlier publications have detailed efforts to develop a lead candidate single-vial lyophilized thermostable ID93 + GLA-SE vaccine formulation. The present report describes the lyophilization process development and scale-up of the lead candidate thermostable ID93 + GLA-SE composition. The manufacture of three full-scale engineering batches was followed by one batch made and released under current Good Manufacturing Practices (cGMP). Up to 4.5 years of stability data were collected. The cGMP lyophilized ID93 + GLA-SE passed all manufacturing release test criteria and maintained stability for at least 3 months when stored at 37°C and up to 24 months when stored at 5°C. This work represents the first advancement of a thermostable adjuvant-containing subunit tuberculosis vaccine to clinical testing readiness.

Controlled Covalent Conjugation of a Tuberculosis Subunit Antigen (ID93) to Liposome Improved In Vitro Th1-Type Cytokine Recall Responses in Human Whole Blood.

Tuberculosis (TB) remains a foremost poverty-related disease with a high rate of mortality despite global immunization with Bacille Calmette-Guérin (BCG). Several adjuvanted recombinant proteins are in clinical development for TB to protect against the disease in infants and adults. Nevertheless, simple mixing of adjuvants with antigens may not be optimal for enhancing the immune response due to poor association. Hence, co-delivery of adjuvants with antigens has been advocated for improved immune response. This report, therefore, presents a strategy of using chemical conjugation to co-deliver an adjuvanted recombinant protein TB vaccine (ID93 + GLA-LSQ). Chemical conjugation involving glutaraldehyde (GA) or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) was used to associate the antigen (ID93) to the modified liposome (mGLA-LSQ). The physicochemical stability of the formulations was evaluated using high-performance liquid chromatography (HPLC) (adjuvant content), dynamic light scattering (DLS, particle size analysis), and sodium dodecyl sulfate-polyacrylamide gel (SDS) electrophoresis (protein analysis). The bioactivity was assessed by cytokine stimulation using fresh whole blood from 10 healthy donors. The conjugates of ID93 + mGLA_LSQ maintained liposomal and protein integrity with the two protein chemistries. The GLA and QS21 content of the vaccine were also stable for 3 months. However, only the glutaraldehyde conjugates provoked significant secretion of interleukin-2 (210.4 ± 11.45 vs 166.7 ± 9.15; p = 0.0059), interferon-gamma (210.5 ± 14.79 vs 144.1 ± 4.997; p = 0.0011), and tumor necrosis factor alpha (2075 ± 46.8 vs 1456 ± 144.8; p = 0.0082) compared to simple mixing. Conjugation of recombinant protein (ID93) to the liposome (mGLA_LSQ) through chemical conjugation resulted in a stable vaccine formulation, which could facilitate co-delivery of the subunit vaccine to promote a robust immune response.

Network Localization of Alien Limb in Patients with Corticobasal Syndrome.

OBJECTIVE: Perirolandic atrophy occurs in corticobasal syndrome (CBS) but is not specific versus progressive supranuclear palsy (PSP). There is heterogeneity in the locations of atrophy outside the perirolandic cortex and it remains unknown why atrophy in different locations would cause the same CBS-specific symptoms. In prior work, we used a wiring diagram of the brain called the human connectome to localize lesion-induced disorders to symptom-specific brain networks. Here, we use a similar technique termed "atrophy network mapping" to localize single-subject atrophy maps to symptom-specific brain networks. METHODS: Single-subject atrophy maps were generated by comparing cortical thickness in patients with CBS versus controls. Next, we performed seed-based functional connectivity using a large normative connectome to determine brain regions functionally connected to each patient's atrophied locations. RESULTS: Patients with CBS had perirolandic atrophy versus controls at the group level, but locations of atrophy in CBS were heterogeneous outside of the perirolandic cortex at the single-subject level (mean spatial correlation = 0.04). In contrast, atrophy occurred in locations functionally connected to the perirolandic cortex in all patients with CBS (spatial correlation = 0.66). Compared with PSP, patients with CBS had atrophy connected to a network of higher-order sensorimotor regions beyond perirolandic cortex, matching a CBS atrophy network from a recent meta-analysis. Finally, atrophy network mapping identified a symptom-specific network for alien limb, matching a lesion-induced alien limb network and a network associated with agency in healthy subjects. INTERPRETATION: We identified a syndrome-specific network for CBS and symptom-specific network for alien limb using single-subject atrophy maps and the human connectome. ANN NEUROL 2020;88:1118-1131.

Squalene Emulsion Manufacturing Process Scale-Up for Enhanced Global Pandemic Response.

Squalene emulsions are among the most widely employed vaccine adjuvant formulations. Among the demonstrated benefits of squalene emulsions is the ability to enable vaccine antigen dose sparing, an important consideration for pandemic response. In order to increase pandemic response capabilities, it is desirable to scale up adjuvant manufacturing processes. We describe innovative process enhancements that enabled the scale-up of bulk stable squalene emulsion (SE) manufacturing capacity from a 3000- to 5,000,000-dose batch size. Manufacture of concentrated bulk along with the accompanying viscosity change in the continuous phase resulted in a ≥25-fold process efficiency enhancement. Process streamlining and implementation of single-use biocontainers resulted in reduced space requirements, fewer unit operations, and minimization of cleaning requirements. Emulsion physicochemical characteristics were measured by dynamic light scattering, laser diffraction, and HPLC with charged aerosol detection. The newly developed full-scale process was demonstrated by producing two 5,000,000-dose batches of bulk concentrated SE. A scale-up of adjuvant manufacturing capacity through process innovation enables more efficient production capabilities for pandemic response.

Preparedness against pandemic influenza: Production of an oil-in-water emulsion adjuvant in Brazil.

Increasing pandemic influenza vaccine manufacturing capacity is considered strategic by WHO. Adjuvant use is key in this strategy in order to spare the vaccine doses and by increasing immune protection. We describe here the production and stability studies of a squalene based oil-in-water emulsion, adjuvant IB160, and the immune response of the H7N9 vaccine combined with IB160. To qualify the production of IB160 we produced 10 consistency lots of IB160 and the average results were: pH 6.4±0.05; squalene 48.8±.0.03 mg/ml; osmolality 47.6±6.9 mmol/kg; Z-average 157±2 nm, with polydispersity index (PDI) of 0.085±0.024 and endotoxin levels <0.5 EU/mL. The emulsion particle size was stable for at least six months at 25°C and 24 months at 4-8°C. Two doses of H7N9 vaccine formulated at 7.5 µg/dose or 15 µg/dose with adjuvant IB160 showed a significant increase of hemagglutination inhibition (HAI) titers in sera of immunized BALB/c mice when compared to control sera from animals immunized with the H7N9 antigens without adjuvant. Thus the antigen-sparing capacity of IB160 can potentially increase the production of the H7N9 pandemic vaccine and represents an important achievement for preparedness against pandemic influenza and a successful North (IDRI) to South (Butantan Institute) technology transfer for the production of the adjuvant emulsion IB160.

Network localization of clinical, cognitive, and neuropsychiatric symptoms in Alzheimer's disease.

There is both clinical and neuroanatomical variability at the single-subject level in Alzheimer's disease, complicating our understanding of brain-behaviour relationships and making it challenging to develop neuroimaging biomarkers to track disease severity, progression, and response to treatment. Prior work has shown that both group-level atrophy in clinical dementia syndromes and complex neurological symptoms in patients with focal brain lesions localize to brain networks. Here, we use a new technique termed 'atrophy network mapping' to test the hypothesis that single-subject atrophy maps in patients with a clinical diagnosis of Alzheimer's disease will also localize to syndrome-specific and symptom-specific brain networks. First, we defined single-subject atrophy maps by comparing cortical thickness in each Alzheimer's disease patient versus a group of age-matched, cognitively normal subjects across two independent datasets (total Alzheimer's disease patients = 330). No more than 42% of Alzheimer's disease patients had atrophy at any given location across these datasets. Next, we determined the network of brain regions functionally connected to each Alzheimer's disease patient's location of atrophy using seed-based functional connectivity in a large (n = 1000) normative connectome. Despite the heterogeneity of atrophied regions at the single-subject level, we found that 100% of patients with a clinical diagnosis of Alzheimer's disease had atrophy functionally connected to the same brain regions in the mesial temporal lobe, precuneus cortex, and angular gyrus. Results were specific versus control subjects and replicated across two independent datasets. Finally, we used atrophy network mapping to define symptom-specific networks for impaired memory and delusions, finding that our results matched symptom networks derived from patients with focal brain lesions. Our study supports atrophy network mapping as a method to localize clinical, cognitive, and neuropsychiatric symptoms to brain networks, providing insight into brain-behaviour relationships in patients with dementia.

Continuous flow, large-scale, microbial fuel cell system for the sustained treatment of swine waste.

Microbial fuel cells (MFCs) have long held the promise of being a cost-effective technology for the energy-neutral treatment of wastewater. However, successful pilot-scale demonstrations for this technology are still limited to very few. Here, we present a large-scale MFC system, composed of 12 MFCs with a total volume of 110 L, successfully treating swine wastewater at a small educational farm. The system was operated for over 200 days in continuous mode with hydraulic residence time of 4 hr. Very stable electrochemical and waste treatment performance was observed with up to 65% of chemical oxygen demand (COD) removed and a maximum treatment rate of 5.0 kg COD/m3 .day. Robust microbial enrichment was performed and adapted to metabolize and transform a diversity of compounds present. The Net Energy Recovery (NER = 0.11 kWhr/kg COD) is not only competitive with conventional cogeneration processes, but is in fact sufficient to sustain the operational energy requirements of the system. PRACTITIONER POINTS: This study demonstrates the design and operation of a large-scale microbial fuel cells (MFC) system for continuous treatment of swine wastewater. The system achieved a high chemical oxygen demand removal rate within a short hydraulic residence time. This study moves one-step closer to applying MFC technology for real wastewater treatment.

Preparedness against pandemic influenza: production of an oil-in-water emulsion adjuvant in Brazil

Increasing pandemic influenza vaccine manufacturing capacity is considered strategic by WHO. Adjuvant use is key in this strategy in order to spare the vaccine doses and by increasing immune protection. We describe here the production and stability studies of a squalene based oil-in-water emulsion, adjuvant IB160, and the immune response of the H7N9 vaccine combined with IB160. To qualify the production of IB160 we produced 10 consistency lots of IB160 and the average results were: pH 6.4±0.05; squalene 48.8±.0.03 mg/ml; osmolality 47.6±6.9 mmol/kg; Z-average 157±2 nm, with polydispersity index (PDI) of 0.085±0.024 and endotoxin levels <0.5 EU/mL. The emulsion particle size was stable for at least six months at 25°C and 24 months at 4–8°C. Two doses of H7N9 vaccine formulated at 7.5 µg/dose or 15 µg/dose with adjuvant IB160 showed a significant increase of hemagglutination inhibition (HAI) titers in sera of immunized BALB/c mice when compared to control sera from animals immunized with the H7N9 antigens without adjuvant. Thus the antigen-sparing capacity of IB160 can potentially increase the production of the H7N9 pandemic vaccine and represents an important achievement for preparedness against pandemic influenza and a successful North (IDRI) to South (Butantan Institute) technology transfer for the production of the adjuvant emulsion IB160.

Preparedness against pandemic influenza: production of an oil-in-water emulsion adjuvant in Brazil

Increasing pandemic influenza vaccine manufacturing capacity is considered strategic by WHO. Adjuvant use is key in this strategy in order to spare the vaccine doses and by increasing immune protection. We describe here the production and stability studies of a squalene based oil-in-water emulsion, adjuvant IB160, and the immune response of the H7N9 vaccine combined with IB160. To qualify the production of IB160 we produced 10 consistency lots of IB160 and the average results were: pH 6.4±0.05; squalene 48.8±.0.03 mg/ml; osmolality 47.6±6.9 mmol/kg; Z-average 157±2 nm, with polydispersity index (PDI) of 0.085±0.024 and endotoxin levels <0.5 EU/mL. The emulsion particle size was stable for at least six months at 25°C and 24 months at 4–8°C. Two doses of H7N9 vaccine formulated at 7.5 µg/dose or 15 µg/dose with adjuvant IB160 showed a significant increase of hemagglutination inhibition (HAI) titers in sera of immunized BALB/c mice when compared to control sera from animals immunized with the H7N9 antigens without adjuvant. Thus the antigen-sparing capacity of IB160 can potentially increase the production of the H7N9 pandemic vaccine and represents an important achievement for preparedness against pandemic influenza and a successful North (IDRI) to South (Butantan Institute) technology transfer for the production of the adjuvant emulsion IB160.

Interactions Between Antigens and Nanoemulsion Adjuvants: Separation and Characterization Techniques.

Determining the association of vaccine components in a formulation is of interest for designing and optimizing well characterized vaccines. Three methods are described to assess interactions between protein antigens and oil-in-water nanoemulsion adjuvants. The methods include (1) ultracentrifugation to measure free versus adjuvant-associated protein, (2) size exclusion chromatography (SEC) to qualitatively assess existing interactions, and (3) Native PAGE as a means to visualize the formulation run in its native state on a polyacrylamide gel. As with many techniques, the methods alone are not definitive, but data from multiple orthogonal assays can provide a more complete picture of protein-adjuvant interactions.

Adsorption of a synthetic TLR7/8 ligand to aluminum oxyhydroxide for enhanced vaccine adjuvant activity: A formulation approach.

For nearly a century, aluminum salts have been the most widely used vaccine adjuvant formulation, and have thus established a history of safety and efficacy. Nevertheless, for extremely challenging disease targets such as tuberculosis or HIV, the adjuvant activity of aluminum salts may not be potent enough to achieve protective efficacy. Adsorption of TLR ligands to aluminum salts facilitates enhanced adjuvant activity, such as in the human papilloma virus vaccine Cervarix®. However, some TLR ligands such as TLR7/8 agonist imidazoquinolines do not efficiently adsorb to aluminum salts. The present report describes a formulation approach to solving this challenge by developing a lipid-based nanosuspension of a synthetic TLR7/8 ligand (3M-052) that facilitates adsorption to aluminum oxyhydroxide via the structural properties of the helper lipid employed. In immunized mice, the aluminum oxyhydroxide-adsorbed formulation of 3M-052 enhanced antibody and TH1-type cellular immune responses to vaccine antigens for tuberculosis and HIV.

Functional and taxonomic dynamics of an electricity-consuming methane-producing microbial community.

The functional and taxonomic microbial dynamics of duplicate electricity-consuming methanogenic communities were observed over a 6 months period to characterize the reproducibility, stability and recovery of electromethanogenic consortia. The highest rate of methanogenesis was 0.72 mg-CH4/L/day, which occurred during the third month of enrichment when multiple methanogenic phylotypes and associated Desulfovibrionaceae phylotypes were present in the electrode-associated microbial community. Results also suggest that electromethanogenic microbial communities are very sensitive to electron donor-limiting open-circuit conditions. A 45 min exposure to open-circuit conditions induced an 87% drop in volumetric methane production rates. Methanogenic performance recovered after 4 months to a maximum value of 0.30 mg-CH4/L/day under set potential operation (-700 mV vs Ag/AgCl); however, current consumption and biomass production was variable over time. Long-term functional and taxonomic analyses from experimental replicates provide new knowledge toward understanding how to enrich electromethanogenic communities and operate bioelectrochemical systems for stable and reproducible performance.

Akt phosphorylation and regulation of transketolase is a nodal point for amino acid control of purine synthesis.

The phosphatidylinositol 3-kinase (PI3K)/Akt pathway integrates environmental clues to regulate cell growth and survival. We showed previously that depriving cells of a single essential amino acid rapidly and reversibly arrests purine synthesis. Here we demonstrate that amino acids via mammalian target of rapamycin 2 and IκB kinase regulate Akt activity and Akt association and phosphorylation of transketolase (TKT), a key enzyme of the nonoxidative pentose phosphate pathway (PPP). Akt phosphorylates TKT on Thr382, markedly enhancing enzyme activity and increasing carbon flow through the nonoxidative PPP, thereby increasing purine synthesis. Mice fed a lysine-deficient diet for 2 days show decreased Akt activity, TKT activity, and purine synthesis in multiple organs. These results provide a mechanism whereby Akt coordinates amino acid availability with glucose utilization, purine synthesis, and RNA and DNA synthesis.