Methods for genomic characterization and maintenance of anaerobic fungi

The rapid development of molecular biology and bioinformatics has fueled renewed interests in anaerobic fungi from the phylum Neocallimastigomycota. This chapter presents well-established methods for isolation, routine cultivation, and cryopreservation of anaerobic fungi. Moreover, detailed nucleic acid extraction protocols are provided, which should enable readers to isolate high-quality DNA and RNA from a variety of anaerobic fungal culture media for downstream applications such as next-generation sequencing

Metabolic characterization of anaerobic fungi provides a path forward for bioprocessing of crude lignocellulose

The conversion of lignocellulose-rich biomass to bio-based chemicals and higher order fuels remains a grand challenge, as single-microbe approaches often cannot drive both deconstruction and chemical production steps. In contrast, consortia based bioprocessing leverages the strengths of different microbes to distribute metabolic loads and achieve process synergy, product diversity, and bolster yields. Here, we describe a biphasic fermentation scheme that combines the lignocellulolytic action of anaerobic fungi isolated from large herbivores with domesticated microbes for bioproduction. When grown in batch culture, anaerobic fungi release excess sugars from both cellulose and crude biomass due to a wealth of highly expressed carbohydrate active enzymes (CAZymes), converting as much as 49% of cellulose to free glucose. This sugar-rich hydrolysate readily supports growth of Saccharomyces cerevisiae, which can be engineered to produce a range of value-added chemicals. Further, construction of metabolic pathways from transcriptomic data reveals that anaerobic fungi do not catabolize all sugars that their enzymes hydrolyze from biomass, leaving other carbohydrates such as galactose, arabinose, and mannose available as nutritional links to other microbes in their consortium. Although basal expression of CAZymes in anaerobic fungi is high, it is drastically amplified by cellobiose breakout products encountered during biomass hydrolysis. Overall, these results suggest that anaerobic fungi provide a nutritional benefit to the rumen microbiome, which can be harnessed to design synthetic microbial communities that compartmentalize biomass degradation and bioproduct formation

Early-branching gut fungi possess a large, comprehensive array of biomass-degrading enzymes

The fungal kingdom is the source of almost all industrial enzymes in use for lignocellulose bioprocessing. We developed a systems-level approach that integrates transcriptomic sequencing, proteomics, phenotype, and biochemical studies of relatively unexplored basal fungi. Anaerobic gut fungi isolated from herbivores produce a large array of biomass-degrading enzymes that synergistically degrade crude, untreated plant biomass and are competitive with optimized commercial preparations from Aspergillus and Trichoderma. Compared to these model platforms, gut fungal enzymes are unbiased in substrate preference due to a wealth of xylan-degrading enzymes. These enzymes are universally catabolite-repressed and are further regulated by a rich landscape of noncoding regulatory RNAs. Additionally, we identified several promising sequence-divergent enzyme candidates for lignocellulosic bioprocessing

Novel genotype-phenotype and MRI correlations in a large cohort of patients with SPG7 mutations

Objective: To clinically, genetically, and radiologically characterize a large cohort of SPG7 patients. Methods: We used data from next-generation sequencing panels for ataxias and hereditary spastic paraplegia to identify a characteristic phenotype that helped direct genetic testing for variations in SPG7. We analyzed MRI. We reviewed all published SPG7 mutations for correlations. Results: We identified 42 cases with biallelic SPG7 mutations, including 7 novel mutations, including a large multi-exon deletion, representing one of the largest cohorts so far described. We identified a characteristic phenotype comprising cerebellar ataxia with prominent cerebellar dysarthria, mild lower limb spasticity, and a waddling gait, predominantly from a cohort of idiopathic ataxia. We report a rare brain MRI finding of dentate nucleus hyperintensity on T2 sequences with SPG7 mutations. We confirm that the c.1529C>T allele is frequently present in patients with long-standing British ancestry. Based on the findings of the present study and existing literature, we confirm that patients with homozygous mutations involving the M41 peptidase domain of SPG7 have a younger age at onset compared to individuals with mutations elsewhere in the gene (14 years difference, p T compound heterozygous mutations are associated with a younger age at onset compared to homozygous cases (5.4 years difference, p < 0.022). Conclusions: Mutant SPG7 is common in sporadic ataxia. In patients with British ancestry, c.1529C>T allele represents the most frequent mutation. SPG7 mutations can be clinically predicted by the characteristic hybrid spastic-ataxic phenotype described above, along with T2 hyperintensity of the dentate nucleus on MRI

Analiza FPGA implementacije bilateralnih algoritama upravljanja za dodirnu teleoperaciju

This paper presents the FPGA implementation of sliding mode control algorithm for bilateral teleoperation, such that, the problem of haptic teleoperation is addressed. The presented study improves haptic fidelity by widening the control bandwidth. For wide control bandwidth, short control periods as well as short sampling periods are required that was achieved by the FPGA. The presented FPGA design methodology applies basic optimization methods in order to meet the required control period as well as the required hardware resource consumption. The circuit specification was performed by the high-level programing language LabVIEW using the fixed-point data type. Hence, short design times for producing the FPGA logic circuit can be achieved. The proposed FPGA-based bilateral teleoperation was validated by master-slave experimental device.Ovaj rad opisuje FPGA implementaciju algoritama upravljanja kliznim režimima za bilateralnu teleoperaciju, pri čemu je opisan problem haptičke teleoperacije. Prikazano istraživanje poboljšava dodirnu pouzdanost proširenjem upravljačkog propusnog pojasa. Za široki propusni pojas, potrebni su kratki upravljački periodi i brzo vrijeme uzorkovanja, što je postignuto primjenom FPGA sklopovlja. Prikazana metodologija za projektiranje FPGA sklopovlja koristi osnovne optimizacijske metode s ciljem postizanja potrebnih upravljačkih perioda i zahtijevane fizičke iskorištenosti sklopovlja. Specifikacije sklopovlja su provedene programskim jezikom visoke razine LabVIEW uz korištenje podataka s nepomičnim decimalnim zarezom. Stoga je moguće implementirati traženu logiku na FPGA sklopovlje u kratkom vremenu. Opisana bilateralna teleoperacija temeljena na FPGA slopovlju je testirana na eksperimentalnom postavu s nadre.enim i podre.enim čvorom

Functional mechanisms underlying pleiotropic risk alleles at the 19p13.1 breast-ovarian cancer susceptibility locus

A locus at 19p13 is associated with breast cancer (BC) and ovarian cancer (OC) risk. Here we analyse 438 SNPs in this region in 46,451 BC and 15,438 OC cases, 15,252 BRCA1 mutation carriers and 73,444 controls and identify 13 candidate causal SNPs associated with serous OC (P=9.2 × 10-20), ER-negative BC (P=1.1 × 10-13), BRCA1-associated BC (P=7.7 × 10-16) and triple negative BC (P-diff=2 × 10-5). Genotype-gene expression associations are identified for candidate target genes ANKLE1 (P=2 × 10-3) and ABHD8 (P<2 × 10-3). Chromosome conformation capture identifies interactions between four candidate SNPs and ABHD8, and luciferase assays indicate six risk alleles increased transactivation of the ADHD8 promoter. Targeted deletion of a region containing risk SNP rs56069439 in a putative enhancer induces ANKLE1 downregulation; and mRNA stability assays indicate functional effects for an ANKLE1 3′-UTR SNP. Altogether, these data suggest that multiple SNPs at 19p13 regulate ABHD8 and perhaps ANKLE1 expression, and indicate common mechanisms underlying breast and ovarian cancer risk

Robust and effective methodologies for cryopreservation and DNA extraction from anaerobic gut fungi

Cell storage and DNA isolation are essential to developing an expanded suite of microorganisms for biotechnology. However, many features of non-model microbes, such as an anaerobic lifestyle and rigid cell wall, present formidable challenges to creating strain repositories and extracting high quality genomic DNA. Here, we establish accessible, high efficiency, and robust techniques to store lignocellulolytic anaerobic gut fungi long term without specialized equipment. Using glycerol as a cryoprotectant, gut fungal isolates were preserved for a minimum of 23 months at −80 °C. Unlike previously reported approaches, this improved protocol is non-toxic and rapid, with samples surviving twice as long with negligible growth impact. Genomic DNA extraction for these isolates was optimized to yield samples compatible with next generation sequencing platforms (e.g. Illumina, PacBio). Popular DNA isolation kits and precipitation protocols yielded preps that were unsuitable for sequencing due to carbohydrate contaminants from the chitin-rich cell wall and extensive energy reserves of gut fungi. To address this, we identified a proprietary method optimized for hardy plant samples that rapidly yielded DNA fragments in excess of 10 kb with minimal RNA, protein or carbohydrate contamination. Collectively, these techniques serve as fundamental tools to manipulate powerful biomass-degrading gut fungi and improve their accessibility among researchers

Design, Control and Performance of RiceWrist: A Force Feedback Wrist Exoskeleton for Rehabilitation and Training

This paper presents the design, control, and performance of a high fidelity four degree-of-freedom wrist exoskeleton robot, RiceWrist, for training and rehabilitation. The RiceWrist is intended to provide kinesthetic feedback during the training of motor skills or rehabilitation of reaching movements. Motivation for such applications is based on findings that show robot-assisted physical therapy aids in the rehabilitation process following neurological injuries. The exoskeleton device accommodates forearm supination and pronation, wrist flexion and extension, and radial and ulnar deviation in a compact parallel mechanism design with low friction, zero backlash, and high stiffness. As compared to other exoskeleton devices, the RiceWrist allows easy measurement of human joint angles and independent kinesthetic feedback to individual human joints. In this paper, joint-space as well as task-space position controllers and an impedance-based force controller for the device are presented. The kinematic performance of the device is characterized in terms of its workspace, singularities, manipulability, backlash, and backdrivability. The dynamic performance of RiceWrist is characterized in terms of motor torque output, joint friction, step responses, behavior under closed loop set-point and trajectory tracking control, and display of virtual walls. The device is singularity-free, encompasses most of the natural workspace of the human joints, and exhibits low friction, zero-backlash, and high manipulability, which are kinematic properties that characterize a high-quality impedance display device. In addition, the device displays fast, accurate response under position control that matches human actuation bandwidth, and the capability to display sufficiently hard contact with little coupling between controlled degrees-of-freedom