•The most common exosome isolation methods are not selective, isolating total exosomes.•Ultracentrifugation is sensitive to factors that cannot be controlled or standardized.•Ultracentrifugation is ...associated with reduced protein and RNA recovery.•Size exclusion chromatography yields no diminished recovery of exosomal cargoes.•Immunoaffinity capture can maximize recovery of pathology-specific exosome cargo.
Viable tumor cells actively release vesicles into the peripheral circulation and other biologic fluids, which exhibit proteins and RNAs characteristic of that cell. Our group demonstrated the presence of these extracellular vesicles of tumor origin within the peripheral circulation of cancer patients and proposed their utility for diagnosing the presence of tumors and monitoring their response to therapy in the 1970s. However, it has only been in the past 10years that these vesicles have garnered interest based on the recognition that they serve as essential vehicles for intercellular communication, are key determinants of the immunosuppressive microenvironment observed in cancer and provide stability to tumor-derived components that can serve as diagnostic biomarkers. To date, the clinical utility of extracellular vesicles has been hampered by issues with nomenclature and methods of isolation. The term “exosomes” was introduced in 1981 to denote any nanometer-sized vesicles released outside the cell and to differentiate them from intracellular vesicles. Based on this original definition, we use “exosomes” as synonymous with “extracellular vesicles.” While our original studies used ultracentrifugation to isolate these vesicles, we immediately became aware of the significant impact of the isolation method on the number, type, content and integrity of the vesicles isolated. In this review, we discuss and compare the most commonly utilized methods for purifying exosomes for post-isolation analyses. The exosomes derived from these approaches have been assessed for quantity and quality of specific RNA populations and specific marker proteins. These results suggest that, while each method purifies exosomal material, there are pros and cons of each and there are critical issues linked with centrifugation-based methods, including co-isolation of non-exosomal materials, damage to the vesicle’s membrane structure and non-standardized parameters leading to qualitative and quantitative variability. The down-stream analyses of these resulting varying exosomes can yield misleading results and conclusions.
With the proliferation of online data collection in human-subjects research, concerns have been raised over the presence of inattentive survey participants and non-human respondents (bots). We ...compared the quality of the data collected through five commonly used platforms. Data quality was indicated by the percentage of participants who meaningfully respond to the researcher's question (high quality) versus those who only contribute noise (low quality). We found that compared to MTurk, Qualtrics, or an undergraduate student sample (i.e., SONA), participants on Prolific and CloudResearch were more likely to pass various attention checks, provide meaningful answers, follow instructions, remember previously presented information, have a unique IP address and geolocation, and work slowly enough to be able to read all the items. We divided the samples into high- and low-quality respondents and computed the cost we paid per high-quality respondent. Prolific ($1.90) and CloudResearch ($2.00) were cheaper than MTurk ($4.36) and Qualtrics ($8.17). SONA cost $0.00, yet took the longest to collect the data.
Autophagy constitutes a major cell-protective mechanism that eliminates damaged components and maintains energy homeostasis via recycling nutrients under normal/stressed conditions. Although the ...core components of autophagy have been well studied, regulation of autophagy at the transcriptional level is poorly understood. Herein, we establish ZKSCAN3, a zinc finger family DNA-binding protein, as a transcriptional repressor of autophagy. Silencing of ZKSCAN3 induced autophagy and increased lysosome biogenesis. Importantly, we show that ZKSCAN3 represses transcription of a large gene set (>60) integral to, or regulatory for, autophagy and lysosome biogenesis/function and that a subset of these genes, including Map1lC3b and Wipi2, represent direct targets. Interestingly, ZKSCAN3 and TFEB are oppositely regulated by starvation and in turn oppositely regulate lysosomal biogenesis and autophagy, suggesting that they act in conjunction. Altogether, our study uncovers an autophagy master switch regulating the expression of a transcriptional network of genes integral to autophagy and lysosome biogenesis/function.
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► Silencing of ZKSCAN3 induces autophagy and lysosome biogenesis ► ZKSCAN3 transcriptionally regulates an extensive set of genes involved in autophagy ► ZKSCAN3 nuclear localization is regulated by nutrient availability ► ZKSCAN3 and TFEB regulate autophagy in opposite directions
Computer science advances and ultra-fast computing speeds find artificial intelligence (AI) broadly benefitting modern society—forecasting weather, recognizing faces, detecting fraud, and deciphering ...genomics. AI's future role in medical practice remains an unanswered question. Machines (computers) learn to detect patterns not decipherable using biostatistics by processing massive datasets (big data) through layered mathematical models (algorithms). Correcting algorithm mistakes (training) adds to AI predictive model confidence. AI is being successfully applied for image analysis in radiology, pathology, and dermatology, with diagnostic speed exceeding, and accuracy paralleling, medical experts. While diagnostic confidence never reaches 100%, combining machines plus physicians reliably enhances system performance. Cognitive programs are impacting medical practice by applying natural language processing to read the rapidly expanding scientific literature and collate years of diverse electronic medical records. In this and other ways, AI may optimize the care trajectory of chronic disease patients, suggest precision therapies for complex illnesses, reduce medical errors, and improve subject enrollment into clinical trials.
Human and non-human animal behavior is highly malleable and adapts successfully to internal and external demands. Such behavioral success stands in striking contrast to the apparent instability in ...neural activity (i.e., variability) from which it arises. Here, we summon the considerable evidence across scales, species, and imaging modalities that neural variability represents a key, undervalued dimension for understanding brain-behavior relationships at inter- and intra-individual levels. We believe that only by incorporating a specific focus on variability will the neural foundation of behavior be comprehensively understood.
Successful behavior arises from brain activity exhibiting remarkable variability. Summoning evidence across species, scales, and techniques, Waschke et al. argue that neural variability represents a key, undervalued dimension essential for understanding inter- and intra-individual associations between brain and behavior.
Spreen (1992:41) noted that "the historical purpose that Coleman had in mind ... of using a snowball design to study social structure changed into a total sic different purpose of using some kind of ...snowball design, namely as an expethent for locating members of a special population." ... the transition from snowball sampling for studying network structure to snowball sampling as a convenience sampling method fit the needs of scholars whose exclusive concern was accessing hidden populations. 2.
Nitric oxide ((•)NO, nitrogen monoxide) is one of the most unique biological signaling molecules associated with a multitude of physiologic and pathological conditions. In order to fully appreciate ...its numerous roles, it is essential to understand its basic biochemical properties. Most signaling effector molecules such as steroids or proteins have a significant life-span and function through classical receptor-ligand interactions. (•)NO, however, is a short-lived free-radical gas that only reacts with two types of molecules under biological conditions; metals and other free radicals. These simple interactions can lead to a myriad of complex intermediates which in turn have their own phenotypic effects. For these reasons, responses to (•)NO often appear to be random or contradictory when outcomes are compared across various experimental settings. This article will serve as a brief overview of the chemical, biological, and microenvironmental factors that dictate (•)NO signaling with an emphasis on (•)NO metabolism. The prominent role that oxygen (dioxygen, O2) plays in (•)NO metabolism and how it influences the biological effects of (•)NO will be highlighted. This information and these concepts are intended to help students and investigators think about the interpretation of data from experiments where biological effects of (•)NO are being elucidated.
Hippocampal interactions with the motor system are often assumed to reflect the role of memory in motor learning. Here, we examine hippocampal connectivity with sensorimotor cortex during two tasks ...requiring paced movements, one with a mnemonic component (sequence learning) and one without (repetitive tapping). Functional magnetic resonance imaging activity was recorded from thirteen right-handed subjects; connectivity was identified from sensorimotor cortex correlations with psychophysiological interactions in hippocampal activity between motor and passive visual tasks. Finger movements in both motor tasks anticipated the timing of the metronome, reflecting cognitive control, yet evidence of motor learning was limited to the sequence learning task; nonetheless, hippocampal connectivity was observed during both tasks. Connectivity from corresponding regions in the left and right hippocampus overlapped extensively, with improved sensitivity resulting from their conjunctive (global) analysis. Positive and negative connectivity were both evident, with positive connectivity in sensorimotor cortex ipsilateral to the moving hand during unilateral movements, whereas negative connectivity was prominent in whichever hemisphere was most active during movements. Results implicate the hippocampus in volitional finger movements even in the absence of motor learning or recall.
Filamentous cyanobacteria exhibit some of the greatest developmental complexity observed in the prokaryotic domain. This includes the ability to differentiate nitrogen-fixing cells known as ...heterocysts, spore-like akinetes, and hormogonia, which are specialized motile filaments capable of gliding on solid surfaces. Hormogonia and motility play critical roles in several aspects of the biology of filamentous cyanobacteria, including dispersal, phototaxis, the formation of supracellular structures, and the establishment of nitrogen-fixing symbioses with plants. While heterocyst development has been investigated extensively at the molecular level, much less is known about akinete or hormogonium development and motility. This is due, in part, to the loss of developmental complexity during prolonged laboratory culture in commonly employed model filamentous cyanobacteria. In this review, recent progress in understanding the molecular level regulation of hormogonium development and motility in filamentous cyanobacteria is discussed, with a focus on experiments performed using the genetically tractable model filamentous cyanobacterium Nostoc punctiforme, which retains the developmental complexity of field isolates.
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