Novel Therapeutic Targets in Acute Myeloid Leukemia (AML).

PURPOSE OF REVIEW: This review seeks to identify and describe novel genetic and protein targets and their associated therapeutics currently being used or studied in the treatment of acute myeloid leukemia (AML). RECENT FINDINGS: Over the course of the last 5-6 years, several targeted therapies have been approved by the FDA, for the treatment of both newly diagnosed as well as relapsed/refractory AML. These novel therapeutics, as well as several others currently under investigation, have demonstrated activity in AML and have improved outcomes for many patients. Patient outcomes in AML have slowly improved over time, though for many patients, particularly elderly patients or those with relapsed/refractory disease, mortality remains very high. With the identification of several molecular/genetic drivers and protein targets and development of therapeutics which leverage those mechanisms to target leukemic cells, outcomes for patients with AML have improved and continue to improve significantly.

Effects of Sugarcane-Derived Polyphenol Supplementation on Methane Production and Rumen Microbial Diversity of Second-Cross Lambs.

The objective of this study was to evaluate the effects of feeding sugarcane-derived polyphenolic supplement (Polygain, The Product Makers Australia, Keysborough, VIC, Australia) on enteric methane (CH4) emission, rumen microbiota, and performance of second-cross lambs. For this purpose, 24 Poll Dorset × (Border Leicester × Merino) lambs were allocated to 3 different treatments: Control (C), 0.25% Polygain (0.25 PG), and 1% Polygain (1 PG) diets with a uniform basal feed (25% cracked wheat grain, 25% cracked barley grain, 25% oaten chaff, 25% lucerne chaff). Both doses of Polygain reduced the total CH4 production (g/day; p = 0.006), CH4 yield (CH4, g/kg of dry matter intake; p = 0.003) and CH4 intensity (CH4, g/kg of BW; p = 0.003). Dry matter intake tended to be greater (p = 0.08) in sheep fed 1 PG compared to the C group, with the 0.25 PG group being intermediate. The average daily gain of the lambs was improved (p = 0.03) with 1% Polygain supplementation. The relative abundance of genera Methanobrevibacter_unidentified, Methanomethylophilaceae_uncultured, Methanogenic archaeon mixed culture ISO4-G1, Methanosphaera uncultured rumen methanogen, Methanogenic archaeon ISO4-H5, and Methanobrevibacter boviskoreani JH1 were reduced with Polygain supplementation. In conclusion, feeding Polygain reduced lambs' enteric CH4 emissions, altered the rumen microbiome, and improved the growth performance of lambs.

Identification of PS1/gamma-secretase and glutamate transporter GLT-1 interaction sites.

The recently discovered interaction between Presenilin 1 (PS1), a catalytic subunit of γ-secretase responsible for generating amyloid-ß peptides, and GLT-1, a major glutamate transporter in the brain (EAAT2), provides a mechanistic link between these two key factors involved in Alzheimer's disease (AD) pathology. Modulating this interaction can be crucial to understand the consequence of such crosstalk in AD context and beyond. However, the interaction sites between these two proteins are unknown. Herein, we utilized an alanine scanning approach coupled with FRET-based fluorescence lifetime imaging microscopy to identify the interaction sites between PS1 and GLT-1 in their native environment within intact cells. We found that GLT-1 residues at position 276 to 279 (TM5) and PS1 residues at position 249 to 252 (TM6) are crucial for GLT-1-PS1 interaction. These results have been cross validated using AlphaFold Multimer prediction. To further investigate whether this interaction of endogenously expressed GLT-1 and PS1 can be prevented in primary neurons, we designed PS1/GLT-1 cell-permeable peptides (CPPs) targeting the PS1 or GLT-1 binding site. We used HIV TAT domain to allow for cell penetration which was assayed in neurons. First, we assessed the toxicity and penetration of CPPs by confocal microscopy. Next, to ensure the efficiency of CPPs, we monitored the modulation of GLT-1-PS1 interaction in intact neurons by fluorescence lifetime imaging microscopy. We saw significantly less interaction between PS1 and GLT-1 with both CPPs. Our study establishes a new tool to study the functional aspect of GLT-1-PS1 interaction and its relevance in normal physiology and AD models.

PS1/gamma-secretase acts as rogue chaperone of glutamate transporter EAAT2/GLT-1 in Alzheimer's disease.

The recently discovered interaction between presenilin 1 (PS1), a catalytic subunit of γ-secretase responsible for the generation of amyloid-ß(Aß) peptides, and GLT-1, the major glutamate transporter in the brain (EAAT2 in the human) may provide a mechanistic link between two important pathological aspects of Alzheimer's disease (AD): abnormal Aßoccurrence and neuronal network hyperactivity. In the current study, we employed a FRET-based approach, fluorescence lifetime imaging microscopy (FLIM), to characterize the PS1/GLT-1 interaction in its native environment in the brain tissue of sporadic AD (sAD) patients. There was significantly less interaction between PS1 and GLT-1 in sAD brains, compared to tissue from patients with frontotemporal lobar degeneration (FTLD), or non-demented age-matched controls. Since PS1 has been shown to adopt pathogenic "closed" conformation in sAD but not in FTLD, we assessed the impact of changes in PS1 conformation on the interaction. Familial AD (fAD) PS1 mutations which induce a "closed" PS1 conformation similar to that in sAD brain and gamma-secretase modulators (GSMs) which induce a "relaxed" conformation, reduced and increased the interaction, respectively. This indicates that PS1 conformation seems to have a direct effect on the interaction with GLT-1. Furthermore, using biotinylation/streptavidin pull-down, western blotting, and cycloheximide chase assays, we determined that the presence of PS1 increased GLT-1 cell surface expression and GLT-1 homomultimer formation, but did not impact GLT-1 protein stability. Together, the current findings suggest that the newly described PS1/GLT-1 interaction endows PS1 with chaperone activity, modulating GLT-1 transport to the cell surface and stabilizing the dimeric-trimeric states of the protein. The diminished PS1/GLT-1 interaction suggests that these functions of the interaction may not work properly in AD.

Increased Milk Yield and Reduced Enteric Methane Concentration on a Commercial Dairy Farm Associated with Dietary Inclusion of Sugarcane Extract (Saccharum officinarum).

(1) Background: The purpose of this study was to assess the influence of a natural sugarcane extract (Polygain™) on milk production, milk composition and methane emissions on a commercial dairy farm. (2) Methods: A three-week baseline was established for lactating Holstein × Friesian animals. Following this baseline period, these animals were fed Polygain™ at 0.25% of their estimated dry matter intake for 3 weeks. Methane concentration in the feed bin was determined at each milking using the Gascard NG Infrared Sensor (Edinburgh Sensors LTD). (3) Results: During the intervention phase milk yield increased significantly from 26.43 kg to 28.54 kg per cow per day, whilst methane emissions and bulk tank somatic cell counts decreased significantly in the intervention phase. For methane concentration, an average of 246 ppm during the baseline periods reduced to an average of 161.09 ppm during the intervention phase. For the bulk tank somatic cell counts, the average was observed at 283,200 during the baseline and reduced to an average value of 151,100 during the intervention phase. (4) Conclusions: The natural sugarcane extract was shown to have the potential to mitigate enteric methane emissions while also increasing production and animal wellbeing outcomes in a commercial dairy setting.

Identification of PS1/gamma-secretase and glutamate transporter GLT-1 interaction sites.

The recently discovered interaction between Presenilin 1 (PS1), a catalytic subunit of γ-secretase responsible for generating amyloid-ß (Aß) peptides, and GLT-1, a major glutamate transporter in the brain (EAAT2) provides a mechanistic link between these two key factors involved in Alzheimer's disease (AD) pathology. Modulating this interaction can be crucial to understand the consequence of such crosstalk in AD context and beyond. However, the interaction sites between these two proteins are unknown. Herein, we utilized an alanine scanning approach coupled with FRET-based fluorescence lifetime imaging microscopy (FLIM) to identify the interaction sites between PS1 and GLT-1 in their native environment within intact cells. We found that GLT-1 residues at position 276 to 279 (TM5) and PS1 residues at position 249 to 252 (TM6) are crucial for GLT-1/PS1 interaction. These results have been cross validated using AlphaFold Multimer prediction. To further investigate whether this interaction of endogenously expressed GLT-1 and PS1 can be prevented in primary neurons, we designed PS1/GLT-1 cell-permeable peptides (CPPs) targeting the PS1 or GLT-1 binding site. We used HIV TAT domain to allow for cell penetration which was assayed in neurons. First, we assessed the toxicity and penetration of CPPs by confocal microscopy. Next, to ensure the efficiency of CPPs, we monitored the modulation of GLT-1/PS1 interaction in intact neurons by FLIM. We saw significantly less interaction between PS1 and GLT-1 with both CPPs. Our study establishes a new tool to study the functional aspect of GLT-1/PS1 interaction and its relevance in normal physiology and AD models.

Endosome and Lysosome Membrane Properties Functionally Link to γ-Secretase in Live/Intact Cells.

Our unique multiplexed imaging assays employing FRET biosensors have previously detected that γ-secretase processes APP C99 primarily in late endosomes and lysosomes in live/intact neurons. Moreover we have shown that Aß peptides are enriched in the same subcellular loci. Given that γ-secretase is integrated into the membrane bilayer and functionally links to lipid membrane properties in vitro, it is presumable that γ-secretase function correlates with endosome and lysosome membrane properties in live/intact cells. In the present study, we show using unique live-cell imaging and biochemical assays that the endo-lysosomal membrane in primary neurons is more disordered and, as a result, more permeable than in CHO cells. Interestingly, γ-secretase processivity is decreased in primary neurons, resulting in the predominant production of long Aß42 instead of short Aß38. In contrast, CHO cells favor Aß38 over the Aß42 generation. Our findings are consistent with the previous in vitro studies, demonstrating the functional interaction between lipid membrane properties and γ-secretase and provide further evidence that γ-secretase acts in late endosomes and lysosomes in live/intact cells.

RNA polymerase II transcription attenuation at the yeast DNA repair gene DEF1 is biologically significant and dependent on the Hrp1 RNA-recognition motif.

Premature transcription termination (i.e. attenuation) is a potent gene regulatory mechanism that represses mRNA synthesis. Attenuation of RNA polymerase II is more prevalent than once appreciated, targeting 10-15% of mRNA genes in yeast through higher eukaryotes, but its significance and mechanism remain obscure. In the yeast Saccharomyces cerevisiae, polymerase II attenuation was initially shown to rely on Nrd1-Nab3-Sen1 termination, but more recently our laboratory characterized a hybrid termination pathway involving Hrp1, an RNA-binding protein in the 3'-end cleavage factor. One of the hybrid attenuation gene targets is DEF1, which encodes a repair protein that promotes degradation of polymerase II stalled at DNA lesions. In this study, we characterized the chromosomal DEF1 attenuator and the functional role of Hrp1. DEF1 attenuator mutants overexpressed Def1 mRNA and protein, exacerbated polymerase II degradation, and hindered cell growth, supporting a biologically significant DEF1 attenuator function. Using an auxin-induced Hrp1 depletion system, we identified new Hrp1-dependent attenuators in MNR2, SNG1, and RAD3 genes. An hrp1-5 mutant (L205S) known to impair binding to cleavage factor protein Rna14 also disrupted attenuation, but surprisingly no widespread defect was observed for an hrp1-1 mutant (K160E) located in the RNA-recognition motif. We designed a new RNA recognition motif mutant (hrp1-F162W) that altered a highly conserved residue and was lethal in single copy. In a heterozygous strain, hrp1-F162W exhibited dominant-negative readthrough defects at several gene attenuators. Overall, our results expand the hybrid RNA polymerase II termination pathway, confirming that Hrp1-dependent attenuation controls multiple yeast genes and may function through binding cleavage factor proteins and/or RNA.

In-Depth Characterization of Endo-Lysosomal Aß in Intact Neurons.

Amyloid-beta (Aß) peptides are produced within neurons. Some peptides are released into the brain parenchyma, while others are retained inside the neurons. However, the detection of intracellular Aß remains a challenge since antibodies against Aß capture Aß and its precursor proteins (i.e., APP and C99). To overcome this drawback, we recently developed 1) the C99 720-670 biosensor for recording γ-secretase activity and 2) a unique multiplexed immunostaining platform that enables the selective detection of intracellular Aß with subcellular resolution. Using these new assays, we showed that C99 is predominantly processed by γ-secretase in late endosomes and lysosomes, and intracellular Aß is enriched in the same subcellular loci in intact neurons. However, the detailed properties of Aß in the acidic compartments remain unclear. Here, we report using fluorescent lifetime imaging microscopy (FLIM) that intracellular Aß includes both long Aß intermediates bound to γ-secretase and short peptides dissociated from the protease complex. Surprisingly, our results also suggest that the dissociated Aß is bound to the glycoproteins on the inner membrane of lysosomes. Furthermore, we show striking cell-to-cell heterogeneity in intracellular Aß levels in primary neurons and APP transgenic mouse brains. These findings provide a basis for the further investigation of the role(s) of intracellular Aß and its relevance to Alzheimer's disease (AD).

SARS-CoV-2 Delta variant isolates from vaccinated individuals.

BACKGROUND: The SARS-CoV-2 Delta variant was first identified in the U.S. in March 2021 and has rapidly become the predominant lineage across the U.S. due to increased transmissibility, immune evasion and vaccine breakthrough. The aim of this study was to better understand the genetic diversity and the potential impact of mutations observed in SARS-CoV-2 viruses circulating in the U.S. in vaccinated individuals. RESULTS: Whole genome sequencing was performed on thirty-four SARS-CoV-2 positive samples using the Oxford Nanopore MinION. Evolutionary genomic analysis revealed two novel mutations, ORF1b:V2354F and a premature stop codon, ORF7a:Q94*, identified in a cluster of SARS-CoV-2 Delta isolates collected from vaccinated individuals in Colorado. The ORF1b:V2354F mutation, corresponding to NSP15:V303F, may induce a conformational change and result in a disruption to a flanking beta-sheet structure. The premature stop codon, ORF7a:Q94*, truncates the transmembrane protein and cytosolic tail used to mediate protein transport. This may affect protein localization to the ER-Golgi. In addition to these novel mutations, the cluster of vaccinated isolates contain an additional mutation in the spike protein, at position 112, compared to the Delta variant defining mutations. This mutation, S112L, exists in isolates previously obtained in the U.S. The S112L mutation substitutes a bulky hydrophobic side chain for a polar side chain, which results in a non-conservative substitution within the protein that may affect antibody-binding affinity. Additionally, the vaccinated cluster of isolates contains non-synonymous mutations within ORF8 and NSPs which further distinguish this cluster from the respective ancestral Delta variant. CONCLUSIONS: These results show there is an emerging sub-lineage of the ancestral Delta variant circulating in the U.S. As mutations emerge in constellations, those with a potentially beneficial advantage to the virus may continue to circulate while others will cease.

Impact of SARS-CoV-2 Mutations on PCR Assay Sequence Alignment.

Real-time reverse transcription polymerase chain reaction (RT-PCR) assays are the most widely used molecular tests for the detection of SARS-CoV-2 and diagnosis of COVID-19 in clinical samples. PCR assays target unique genomic RNA regions to identify SARS-CoV-2 with high sensitivity and specificity. In general, assay development incorporates the whole genome sequences available at design time to be inclusive of all target species and exclusive of near neighbors. However, rapid accumulation of mutations in viral genomes during sustained growth in the population can result in signature erosion and assay failures, creating situational blind spots during a pandemic. In this study, we analyzed the signatures of 43 PCR assays distributed across the genome against over 1.6 million SARS-CoV-2 sequences. We present evidence of significant signature erosion emerging in just two assays due to mutations, while adequate sequence identity was preserved in the other 41 assays. Failure of more than one assay against a given variant sequence was rare and mostly occurred in the two assays noted to have signature erosion. Assays tended to be designed in regions with statistically higher mutations rates. in silico analyses over time can provide insights into mutation trends and alert users to the emergence of novel variants that are present in the population at low proportions before they become dominant. Such routine assessment can also potentially highlight false negatives in test samples that may be indicative of mutations having functional consequences in the form of vaccine and therapeutic failures. This study highlights the importance of whole genome sequencing and expanded real-time monitoring of diagnostic PCR assays during a pandemic.

Spider-Inspired Electrohydraulic Actuators for Fast, Soft-Actuated Joints.

The impressive locomotion and manipulation capabilities of spiders have led to a host of bioinspired robotic designs aiming to reproduce their functionalities; however, current actuation mechanisms are deficient in either speed, force output, displacement, or efficiency. Here-using inspiration from the hydraulic mechanism used in spider legs-soft-actuated joints are developed that use electrostatic forces to locally pressurize a hydraulic fluid, and cause flexion of a segmented structure. The result is a lightweight, low-profile articulating mechanism capable of fast operation, high forces, and large displacement; these devices are termed spider-inspired electrohydraulic soft-actuated (SES) joints. SES joints with rotation angles up to 70°, blocked torques up to 70 mN m, and specific torques up to 21 N m kg-1 are demonstrated. SES joints demonstrate high speed operation, with measured roll-off frequencies up to 24 Hz and specific power as high as 230 W kg-1 -similar to human muscle. The versatility of these devices is illustrated by combining SES joints to create a bidirectional joint, an artificial limb with independently addressable joints, and a compliant gripper. The lightweight, low-profile design, and high performance of these devices, makes them well-suited toward the development of articulating robotic systems that can rapidly maneuver.

HASEL Artificial Muscles for a New Generation of Lifelike Robots-Recent Progress and Future Opportunities.

Future robots and intelligent systems will autonomously navigate in unstructured environments and closely collaborate with humans; integrated with our bodies and minds, they will allow us to surpass our physical limitations. Traditional robots are mostly built from rigid, metallic components and electromagnetic motors, which make them heavy, expensive, unsafe near people, and ill-suited for unpredictable environments. By contrast, biological organisms make extensive use of soft materials and radically outperform robots in terms of dexterity, agility, and adaptability. Particularly, natural muscle-a masterpiece of evolution-has long inspired researchers to create "artificial muscles" in an attempt to replicate its versatility, seamless integration with sensing, and ability to self-heal. To date, natural muscle remains unmatched in all-round performance, but rapid advancements in soft robotics have brought viable alternatives closer than ever. Herein, the recent development of hydraulically amplified self-healing electrostatic (HASEL) actuators, a new class of high-performance, self-sensing artificial muscles that couple electrostatic and hydraulic forces to achieve diverse modes of actuation, is discussed; current designs match or exceed natural muscle in many metrics. Research on materials, designs, fabrication, modeling, and control systems for HASEL actuators is detailed. In each area, research opportunities are identified, which together lays out a roadmap for actuators with drastically improved performance. With their unique versatility and wide potential for further improvement, HASEL actuators are poised to play an important role in a paradigm shift that fundamentally challenges the current limitations of robotic hardware toward future intelligent systems that replicate the vast capabilities of biological organisms.

Design of a High-Speed Prosthetic Finger Driven by Peano-HASEL Actuators.

Current designs of powered prosthetic limbs are limited by the nearly exclusive use of DC motor technology. Soft actuators promise new design freedom to create prosthetic limbs which more closely mimic intact neuromuscular systems and improve the capabilities of prosthetic users. This work evaluates the performance of a hydraulically amplified self-healing electrostatic (HASEL) soft actuator for use in a prosthetic hand. We compare a linearly-contracting HASEL actuator, termed a Peano-HASEL, to an existing actuator (DC motor) when driving a prosthetic finger like those utilized in multi-functional prosthetic hands. A kinematic model of the prosthetic finger is developed and validated, and is used to customize a prosthetic finger that is tuned to complement the force-strain characteristics of the Peano-HASEL actuators. An analytical model is used to inform the design of an improved Peano-HASEL actuator with the goal of increasing the fingertip pinch force of the prosthetic finger. When compared to a weight-matched DC motor actuator, the Peano-HASEL and custom finger is 10.6 times faster, has 11.1 times higher bandwidth, and consumes 8.7 times less electrical energy to grasp. It reaches 91% of the maximum range of motion of the original finger. However, the DC motor actuator produces 10 times the fingertip force at a relevant grip position. In this body of work, we present ways to further increase the force output of the Peano-HASEL driven prosthetic finger system, and discuss the significance of the unique properties of Peano-HASELs when applied to the field of upper-limb prosthetic design. This approach toward clinically-relevant actuator performance paired with a substantially different form-factor compared to DC motors presents new opportunities to advance the field of prosthetic limb design.

An Easy-to-Implement Toolkit to Create Versatile and High-Performance HASEL Actuators for Untethered Soft Robots.

For soft robots to have ubiquitous adoption in practical applications they require soft actuators that provide well-rounded actuation performance that parallels natural muscle while being inexpensive and easily fabricated. This manuscript introduces a toolkit to rapidly prototype, manufacture, test, and power various designs of hydraulically amplified self-healing electrostatic (HASEL) actuators with muscle-like performance that achieve all three basic modes of actuation (expansion, contraction, and rotation). This toolkit utilizes easy-to-implement methods, inexpensive fabrication tools, commodity materials, and off-the-shelf high-voltage electronics thereby enabling a wide audience to explore HASEL technology. Remarkably, the actuators created from this easy-to-implement toolkit achieve linear strains exceeding 100%, a specific power greater than 150 W kg-1, and ≈20% strain at frequencies above 100 Hz. This combination of large strain, extreme speed, and high specific power yields soft actuators that jump without power-amplifying mechanisms. Additionally, an efficient fabrication technique is introduced for modular designs of HASEL actuators, which is used to develop soft robotic devices driven by portable electronics. Inspired by the versatility of elephant trunks, the above capabilities are combined to create an untethered continuum robot for grasping and manipulating delicate objects, highlighting the wide potential of the introduced methods for soft robots with increasing sophistication.

Peano-HASEL actuators: Muscle-mimetic, electrohydraulic transducers that linearly contract on activation.

Soft robotic systems are well suited to unstructured, dynamic tasks and environments, owing to their ability to adapt and conform without damaging themselves or their surroundings. These abilities are crucial in areas such as human-robot interaction. Soft robotic systems are currently limited by the soft actuators that power them. To date, most soft actuators are based on pneumatics or shape-memory alloys, which have issues with efficiency, response speed, and portability. Dielectric elastomer actuators (DEAs) are controlled and powered electrically and excel with muscle-like actuation, but they typically require a rigid frame and prestretch to perform effectively. In addition, DEAs require complex stacks or structures to achieve linear contraction modes. We present a class of soft electrohydraulic transducers, termed Peano-HASEL (hydraulically amplified self-healing electrostatic) actuators, that combine the strengths of fluidic actuators and electrostatic actuators, while addressing many of their issues. These actuators use both electrostatic and hydraulic principles to linearly contract on application of voltage in a muscle-like fashion, without rigid frames, prestretch, or stacked configurations. We fabricated these actuators using a facile heat-sealing method with inexpensive commercially available materials. These prototypical devices demonstrated controllable linear contraction up to 10%, a strain rate of 900% per second, actuation at 50 hertz, and the ability to lift more than 200 times their weight. In addition, these actuators featured characteristics such as high optical transparency and the ability to self-sense their deformation state. Hence, this class of actuators demonstrates promise for applications such as active prostheses, medical and industrial automation, and autonomous robotic devices.

Impact of maternal aging on the molecular signature of human cumulus cells.

OBJECTIVE: To investigate the impact of maternal aging on the molecular signature of cumulus cells. DESIGN: Experimental study. SETTING: Research laboratory. PATIENT(S): Patients, young fertile oocyte donors (n = 40) and infertile women of advanced maternal age (40-45 years; n = 48), donated, with Institutional Review Board consent, cumulus cells during routine infertility treatment. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Proteomic and gene expression profiles of cumulus cells. RESULT(S): Proteomic analysis identified a total of 1,423 cumulus cell proteins. Statistical analysis revealed 110 (7.7%) proteins to be differentially expressed in relation to female aging (>1.5-fold change). Pathway annotation revealed significant involvement in metabolism (ACAT2, HSD17B4, ALDH9A1, MVK, CYP11A1, and FDFT1), oxidative phosphorylation (OP; NDUFA1, UQCRC1, MT-ATP6, ATP5I, and MT-ATP8), and post-transcriptional mechanisms (KHSRP, SFPQ, DDX46, SNRPF, ADAR, NHPL1, and U2AF2) relative to advanced maternal age. Gene expression analysis also revealed altered profiles in cumulus cells from women in their early to mid-40s. CONCLUSION(S): This novel study reveals that the cumulus cell molecular signature, at both the gene and protein level, is impacted by advanced maternal aging. A compromised follicular environment is evident with altered energy metabolism and post-transcriptional processes.