Exploiting anaerobic consortia as new tools for biomass breakdown and sustainable chemistry

Anaerobic microbes work together in complex communities that decompose and recycle carbon biomass throughout the Earth. Compared to microbes that thrive in the presence of oxygen, anaerobic consortia remain understudied and recalcitrant to culture. However, they are a vast, untapped resource for novel enzymes and strains that degrade woody biomass into sugars for value-added chemical production. Here, we performed several enrichment experiments to isolate biomass-degrading consortia from goat feces, and identify microbes that drive the activity and stability of these cultures. Fecal samples were challenged by four types of biomass (alfalfa, bagasse, xylan, and reed canary grass) and two types of antibiotic treatments (chloramphenicol, penicillin-streptomycin) during cultivation to identify important cross-domain partnerships; 10 billion metagenomic reads spread across 402 enrichment samples tracked biological diversity as the cultures converged to a minimal set of ~20 microorganisms that were stable after more than ten culture generations. Nearly 200,000 carbohydrate active enzymes (CAZyme) domains were identified from the fecal samples alone, constituting nearly 25% of the known CAZymes in existence. 724 genomes were assembled for previously uncultured novel microbes within the herbivore rumen. Surprisingly, consortia dominated by anaerobic fungi generated more than twice the amount of methane compared to prokaryotic consortia, suggesting that fungi play a key role in methane release in ruminant herbivores. The most active microbial consortia comprise cross-domain partnerships between anaerobic fungi from the genus Neocallimastix and Piromyces, methanogenic archaea from the genus Methanobrevibacter, and bacteria from the phylum Firmicutes, some of which were enriched nearly 20-fold from the fecal microbiome, produce high yields of methane off-gas, and are capable of cryopreservation and revival. New routes for metabolic cooperation between enriched consortia were also identified, suggesting that an array of bacteria support biomass-degrading microbes by providing essential amino acids while consuming deleterious byproducts. Overall, our analysis points to natural compartmentalization between anaerobes as a means to degrade crude biomass, which can be exploited to harness nature’s microbes for sustainable chemical production

Facile interrogation of high-order epistasis between distal sites using next-generation sequencing

Deep mutational scanning (DMS) combines next-generation sequencing and protein engineering to construct sequence-function landscapes and rapidly identify fitness optima. Practical use of these landscapes requires identification of all mutations in each protein variant due to the potential effects of epistasis, the interdependence between residues resulting in non-additive phenotypes. This phenomenon plays an important role in protein evolution and is often a necessary step along the path towards protein fitness optima. However, current methods to assign distal mutations to their corresponding gene are work-intensive, costly, and introduce potential sources of error. To overcome these limitations, we introduce a method compatible with DMS that matches distal mutations to their corresponding gene without additional experimental steps. Using this approach to screen ~2,000,000 unique protein variants, we engineer a human G protein-coupled receptor with a 15-fold improvement in ligand binding affinity and observe prevalent epistasis between distal residues within the ligand binding pocket. Compared to variants containing only proximal substitutions, those harboring missense mutations in distal sites demonstrate significantly greater functional activity in our screen. This method can be applied immediately to all experiments using Illumina next-generation sequencing and provides a facile approach to illuminate complex mechanisms underlying key protein functions. Please click Additional Files below to see the full abstract

Strategies to engineer G protein-coupled receptor ligand binding properties

G protein-coupled receptors (GPCRs) comprise a family of integral membrane proteins that mediate eukaryotic cells\u27 responses to a wide array of extracellular signals. As a result of their ligand specificity, sensitivity, and capacity for signal transduction, GPCRs have great potential as biosensors for a wide range of molecules (e.g. toxins, value-added products, biomarkers for disease). Despite their inherent advantages, many GPCRs are difficult to express functionally and in high numbers within heterologous hosts such as yeast. Thus, there is a need to engineer functionally expressed GPCRs to bind ligands of interest with high affinities and specificities. Towards this end, we have optimized a high-throughput screening methodology to engineer variants of the human adenosine A2A receptor (hA2AR) with improved binding affinity towards a target ligand. Specifically, a fluorescent ligand binding assay was used in concert with fluorescence-activated cell sorting (FACS) to isolate yeast cells expressing desirable hA2AR mutants. After four rounds of sorting, we observed convergence of mutated residues towards a consensus sequence and a 3.5-fold increase in cellular mean fluorescence intensity upon incubation with fluorescent ligand. Additionally, we demonstrate the importance of vector choice and concomitant mitotic stability in influencing hA2AR yield and cellular homogeneity in yeast. The use of a mitotically stable integrating vector results in increased GPCR yield compared to non-integrating (i.e. centromeric and episomal) vectors. Yields of hA2AR are improved further by increasing vector integration frequency, where gene copy number is shown to have a greater effect on protein yield at lower relative copy numbers. Further, the growth of cells in raffinose-containing media prior to gene induction is shown to improve cellular homogeneity of yeast expressing hA2AR under the control of an inducible galactose promoter. In all, these results are envisioned to benefit both GPCR expression and engineering in yeast. The use of this platform to further evolve and isolate improved hA2AR variants is expected to generate mutants with even greater binding affinity and specificity towards ligands of interest. Please click Additional Files below to see the full abstract

Functional production of transporters from biomass-degrading anaerobic fungi for metabolic engineering

Membrane-embedded transporters and receptors are increasingly becoming targets for the metabolic engineering community that aims to enhance the performance and stability of microbial production strains. Anaerobic gut fungi inhabit the digestive tract of herbivores such as cows and sheep, and excel at degrading raw plant biomass into fermentable sugars. Recently, a transcriptomic analysis of three strains of gut fungi suggested that they display a plethora of carbohydrate binding proteins on their surface, including G-protein coupled receptors with a novel architecture; and possess a multitude of small-solute transporters that are of chief biotechnological interest: transporters for sugars, amino acids, lipids, drugs, and metals. Here, we introduced genes encoding gut fungal fluoride transporters into Saccharomyces cerevisiae, and show that with codon optimization, the yeast produce large quantities of functional and correctly membrane-localized transporters capable of bolstering solvent tolerance. We are currently expanding our approach to putative drug- and sugar-transporters and receptors sourced from the anaerobic fungi. These results in part explain the physiology of these understudied fungi, and highlight the critical role that their membrane proteins play towards their existence in competitive, extreme environments. Notably, the work expands on the toolbox of receptor and transporter proteins that can be used to enhance the performance and stability of model microbial cell factory strains. Please click Additional Files below to see the full abstract

A Randomized, Phase III Trial to Evaluate Rucaparib Monotherapy as Maintenance Treatment in Patients With Newly Diagnosed Ovarian Cancer (ATHENA–MONO/GOG-3020/ENGOT-ov45)

Cáncer de ovarios; MonoterapiaCàncer d'ovaris; MonoteràpiaOvarian cancer; MonotherapyPURPOSE ATHENA (ClinicalTrials.gov identifier: NCT03522246) was designed to evaluate rucaparib first-line maintenance treatment in a broad patient population, including those without BRCA1 or BRCA2 (BRCA) mutations or other evidence of homologous recombination deficiency (HRD), or high-risk clinical characteristics such as residual disease. We report the results from the ATHENA–MONO comparison of rucaparib versus placebo. METHODS Patients with stage III-IV high-grade ovarian cancer undergoing surgical cytoreduction (R0/complete resection permitted) and responding to first-line platinum-doublet chemotherapy were randomly assigned 4:1 to oral rucaparib 600 mg twice a day or placebo. Stratification factors were HRD test status, residual disease after chemotherapy, and timing of surgery. The primary end point of investigator-assessed progression-free survival was assessed in a step-down procedure, first in the HRD population (BRCA-mutant or BRCA wild-type/loss of heterozygosity high tumor), and then in the intent-to-treat population. RESULTS As of March 23, 2022 (data cutoff), 427 and 111 patients were randomly assigned to rucaparib or placebo, respectively (HRD population: 185 v 49). Median progression-free survival (95% CI) was 28.7 months (23.0 to not reached) with rucaparib versus 11.3 months (9.1 to 22.1) with placebo in the HRD population (log-rank P = .0004; hazard ratio [HR], 0.47; 95% CI, 0.31 to 0.72); 20.2 months (15.2 to 24.7) versus 9.2 months (8.3 to 12.2) in the intent-to-treat population (log-rank P < .0001; HR, 0.52; 95% CI, 0.40 to 0.68); and 12.1 months (11.1 to 17.7) versus 9.1 months (4.0 to 12.2) in the HRD-negative population (HR, 0.65; 95% CI, 0.45 to 0.95). The most common grade ≥ 3 treatment-emergent adverse events were anemia (rucaparib, 28.7% v placebo, 0%) and neutropenia (14.6% v 0.9%). CONCLUSION Rucaparib monotherapy is effective as first-line maintenance, conferring significant benefit versus placebo in patients with advanced ovarian cancer with and without HRD

Sympathy for the Devil: A Conservation Strategy for Devil and Manta Rays

Background&nbsp; International trade for luxury products, medicines, and tonics poses a threat to both terrestrial and marine wildlife. The demand for and consumption of gill plates (known as Peng Yu Sai, “Fish Gill of Mobulid Ray”) from devil and manta rays (subfamily Mobulinae, collectively referred to as mobulids) poses a significant threat to these marine fishes because of their extremely low productivity. The demand for these gill plates has driven an international trade supplied by largely unmonitored and unregulated catches from target and incidental fisheries around the world. Scientific research, conservation campaigns, and legal protections for devil rays have lagged behind those for manta rays despite similar threats across all mobulids. Methods&nbsp; To investigate the difference in attention given to devil rays and manta rays, we examined trends in the scientific literature and updated species distribution maps for all mobulids. Using available information on target and incidental fisheries, and gathering information on fishing and trade regulations (at international, national, and territorial levels), we examined how threats and protective measures overlap with species distribution. We then used a species conservation planning approach to develop the Global Devil and Manta Ray Conservation Strategy, specifying a vision, goals, objectives, and actions to advance the knowledge and protection of both devil and manta rays. Results and Discussion&nbsp; Our literature review revealed that there had been nearly 2.5-times more “manta”-titled publications, than “mobula” or “devil ray”-titled publications over the past 4.5 years (January 2012–June 2016). The majority of these recent publications were reports on occurrence of mobulid species. These publications contributed to updated Area of Occupancy and Extent of Occurrence maps which showed expanded distributions for most mobulid species and overlap between the two genera. While several international protections have recently expanded to include all mobulids, there remains a greater number of national, state, and territory-level protections for manta rays compared to devil rays. We hypothesize that there are fewer scientific publications and regulatory protections for devil rays due primarily to perceptions of charisma that favour manta rays. We suggest that the well-established species conservation framework used here offers an objective solution to close this gap. To advance the goals of the conservation strategy we highlight opportunities for parity in protection and suggest solutions to help reduce target and bycatch fisheries

Small business in Ukraine as the engine of national economic development

У статті розглянуто стан малого підприємництва в Україні, охарактеризовано слабкі сторони його діяльності та чинники, що впливають на даний сектор економіки. Для переконливішого пояснення зроблених висновків, наведено статистичну інформацію щодо частки малих підприємств України в загальній кількості підприємств і їх розподіл за регіонами.This article examines the state of small business in Ukraine, gives a description of the weaknesses of its activities; describes factors that affect this sector of the economy. For a more convincing explanation of the findings, statistics on the share of small business in Ukraine, the total number of companies and their distribution by region are presented

Health and wellbeing amongst older people research in Northamptonshire

The Ageing Research Centre of the University of Northampton (2014-current), in collaboration with the East Midlands Research into Ageing Network (EMRAN) is pleased to compile this brochure on research activity associated with older people across the county of Northamptonshire. This provides a comprehensive overview of activity that is relevant and of value to practice, identifying research outcomes that have real significance to age-related health and wellbeing. The brochure provides a summary of research activity over the last five years from academic, clinical and professional colleagues and demonstrates cross sector networks of collaboration around the common agenda of aging. Such collaboration will enhance the capacity of research understanding across the county and provide information and support for the needs of older people, their families and carers. The translation of research outcomes into practice is essential if we are to promote wellness, independence and healthy aging within the county and beyond and I would like to thank all contributors for their commitment and hard work in the production of this brochure

Health and wellbeing amongst older people research in Northamptonshire

The Ageing Research Centre of the University of Northampton (2014-current), in collaboration with the East Midlands Research into Ageing Network (EMRAN) is pleased to compile this brochure on research activity associated with older people across the county of Northamptonshire. This provides a comprehensive overview of activity that is relevant and of value to practice, identifying research outcomes that have real significance to age-related health and wellbeing. The brochure provides a summary of research activity over the last five years from academic, clinical and professional colleagues and demonstrates cross sector networks of collaboration around the common agenda of aging. Such collaboration will enhance the capacity of research understanding across the county and provide information and support for the needs of older people, their families and carers. The translation of research outcomes into practice is essential if we are to promote wellness, independence and healthy aging within the county and beyond and I would like to thank all contributors for their commitment and hard work in the production of this brochure

Racial differences in breast cancer survival in the Detroit Metropolitan area

African American (AA) women have poorer breast cancer survival compared to Caucasian American (CA) women. The purpose of this analysis was to determine whether socioeconomic status (SES) and treatment differences influence racial differences in breast cancer survival . The study population included 9,321 women (82% CA, 18% AA) diagnosed with local (63%) or regional (37%) stage disease between 1988 and 1992, identified through the Metropolitan Detroit SEER registry. Data on SES were obtained through linkage with the 1990 Census of Population and Housing Summary Tape and cases were geocoded to census block groups. Pathology, treatment and survival data were obtained through SEER. Cox proportional hazards models were used to compare survival for AA versus CA women after adjusting for age, SES, tumor size, number of involved lymph nodes, and treatment. AA␣women were more likely to live in a geographic area classified as working poor than were CA women ( p <0.001). AA women were less likely to have lumpectomy and radiation and more likely to have mastectomy with radiation ( p <0.001). After multivariable adjusted analysis, there were no significant racial differences in survival among women with local stage disease, although AA women with regional stage disease had persistent but attenuated poorer survival compared to CA women. After adjusting for known clinical and SES predictors of survival, AA and CA women who are diagnosed with local disease demonstrate similar overall and breast cancer-specific survival, while race continues to have an independent effect among women presenting at a later stage of disease.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44238/1/10549_2005_Article_9103.pd