Cellular Biochemistry Research Articles

Tryptophan and indole metabolism in immune regulation.

L-tryptophan is an essential amino acid that undergoes complex metabolic routes, resulting in production of many types of signaling molecules that fall into two types: retaining the indole ring such as serotonin, melatonin and indole-pyruvate or breaking the indole ring to form kynurenine. Kynurenines are the precursor of signaling molecules and are the first step in de novo NAD+ synthesis. In mammalian cells, the kynurenine pathway is initiated by the rate-limiting enzymes tryptophan-2,3-dioxygenase (TDO) and interferon responsive indoleamine 2,3-dioxygenase (IDO1) and is the major route for tryptophan catabolism. IDO1 regulates immune cell function through the kynurenine pathway but also by depleting tryptophan in microenvironments, and especially in tumors, which led to the development of IDO1 inhibitors for cancer therapy. However, the connections between tryptophan depletion versus product supply remain an ongoing challenge in cellular biochemistry and metabolism. Here, we highlight current knowledge about the physiological and pathological roles of tryptophan signaling network with a focus on the immune system.

Ethanol affects fibroblast behavior differentially at low and high doses: A comprehensive, dose-response evaluation.

This study aims to develop a comprehensive understanding of effects of low and high doses of ethanol on cellular biochemistry and morphology. Here, fibroblast cells are exposed to ethanol of varied concentrations [0.005-10 % (v/v)] to investigate cellular activity, cytoskeletal organization, cellular stiffness, mitochondrial structure, and real-time behavior. Our results indicate a sharp difference in cellular behavior above and below 1 % ethanol concentration. A two-fold increase in MTT activity at low doses is observed, whereas at high doses it decreases. This increased activity at low doses does not involve cell proliferation changes or mitochondrial impairment, as seen at higher doses. Moreover, the study identifies different types of mitochondrial structure impairment at high doses. Morphologically, cells demonstrate a gradual change in cytoskeletal organization and an increase in cell stiffness with increase in doses. Cells exhibit adaptation to sub-toxic doses of ethanol, wherein recovery from ethanol-induced stress is a dose-dependent phenomenon. Cell survival at low doses and toxicity at higher doses are attributed to mild and strong oxidative stress, respectively. Overall, the study provides a comprehensive understanding of dose-dependent effects of ethanol, manifesting its biphasic or hormetic response, biochemically, at low doses and illustrating its toxicological effects at higher doses.

Evaluation of Early Knee Osteoarthritis Using Biomechanical and Biochemical Markers.

Altered cellular mechano-transduction and biochemistry lead to degeneration of articular cartilage in people with knee osteoarthritis. However, the influence of low-moderate exposure to weight-bearing activity such as squatting on cartilage metabolism has not been adequately studied. The current study explored associations between knee adduction moment (KAM) during walking, biochemical markers and daily squat exposure. 3D gait analysis was used to determine external loads acting on the knee as indicators of joint compressive forces whereas biomarkers-Urine type-II-collagen-telopeptide (uCTxII), antioxidant and phospholipase A2 (PLA2) activity reflected on articular cartilage status. Following ethical approval, 66 participants with varying daily squat exposure (non-squatters [n = 21, exposure = 0 min]; activity of daily living [ADL] squatters [n = 16, exposure = 34 min]; occupational squatters [n = 13, exposure = 102 min]) and people with grade 2-3 knee osteoarthritis (n = 16, exposure = 28 min) were evaluated using 3D gait and biomarker analysis. The PLA2 activity was lowest in ADL squatters while occupational squatters demonstrated highest activity (p < 0.05). KAM and urine biomarker were similar among the groups. Moderate-strong positive association was observed between sweat PLA2 activity and age (r = 0.819, p = 0.004), daily squat exposure and biomarker uCTxII (r = 0.604, p = 0.013), antioxidant activity and Right-KAM (r = -0.917, p = 0.001), and Left-KAM (r = -0.767, p = 0.016), in people with knee OA. Healthy people demonstrated weak positive associations between KAM, uCTxII, and BMI. Associations between non-invasive biomechanical and biochemical markers indicate their potential use to identify early knee osteoarthritis. Studies with larger sample size are necessary to support prescription of body weight joint loading activities such as squatting in moderation, to delay functional decline caused by knee OA.

Introductory Chapter: The Importance of Heat Shock Proteins in Survival and Pathogenesis of the Malaria Parasite Plasmodium falciparum.

Malaria did not die with the end of the age of western colonization but is still a major public health issue in large parts of the world. Despite repeated and concerted efforts to eradicate this disease, it has proved remarkably resilient, and constant vigilance and continuous research are required to discover new chinks in the parasite's armor and alleviate the suffering at both the individual and societal levels. A deeper understanding of the fundamental processes underlying parasite survival, propagation, virulence, and ability to cause disease is the key to the development of desperately needed new therapies and prophylactic drugs. Malaria parasites, by the nature of their lifecycle, are subject to a number of environmental and cellular stresses which they must overcome to survive. To this end, they express a number of heat shock proteins (HSPs), molecules specialized on buffering the effects of external stimuli, but which are also essential for normal cellular biochemistry. In this introductory chapter, I give a brief overview of the diversity of structure, function, and importance of these HSPs, and highlight some of the current and future research questions in this field. Additionally, this chapter acts as a bridge to the other chapters in this book. These chapters, I think you will agree, demonstrate that with regard to HSPs malaria parasites, as in so many things, obey the adage "Same same, but different."

Study of lead and nickel resistance mechanism in Enterobacter species

The aim of this study was to understand the mechanism of resistance towards the heavy metal Lead and Nickel using resistant and sensitive Enterobacter spp. The cellular biochemistry of this microorganism was checked with the help of estimations of dehydrogenases activity, and at the molecular biology level determination of Lead and Nickel resistant plasmid through Agarose gel Electrophoresis. The Minimum inhibitory concentration (MIC) determined was 100 ppm (Lead) and 50 ppm (Nickel) in sensitive Enterobacter spp. that completely inhibited the growth, and activity of dehydrogenases of the TCA (Tricarboxylic Acid) cycle. The tolerance level of Lead and Nickel in the resistant Enterobacter spp., were found to be 300 ppm and 200 ppm respectively. Mechanism of resistance was found to be the presence of the resistance plasmid. Keywords: Lead, Nickel, Resistant, Sensitive, Enterobacter, Mechanism

Quantifying the roles of space and stochasticity in computer simulations for cell biology and cellular biochemistry.

Most of the fascinating phenomena studied in cell biology emerge from interactions among highly organized multimolecular structures embedded into complex and frequently dynamic cellular morphologies. For the exploration of such systems, computer simulation has proved to be an invaluable tool, and many researchers in this field have developed sophisticated computational models for application to specific cell biological questions. However, it is often difficult to reconcile conflicting computational results that use different approaches to describe the same phenomenon. To address this issue systematically, we have defined a series of computational test cases ranging from very simple to moderately complex, varying key features of dimensionality, reaction type, reaction speed, crowding, and cell size. We then quantified how explicit spatial and/or stochastic implementations alter outcomes, even when all methods use the same reaction network, rates, and concentrations. For simple cases, we generally find minor differences in solutions of the same problem. However, we observe increasing discordance as the effects of localization, dimensionality reduction, and irreversible enzymatic reactions are combined. We discuss the strengths and limitations of commonly used computational approaches for exploring cell biological questions and provide a framework for decision making by researchers developing new models. As computational power and speed continue to increase at a remarkable rate, the dream of a fully comprehensive computational model of a living cell may be drawing closer to reality, but our analysis demonstrates that it will be crucial to evaluate the accuracy of such models critically and systematically.

A Monochromophoric Approach to Succinct Ratiometric Fluorescent Probes without Probe-Product Crosstalk

Ratiometric probes facilitate quantitative studies via self-calibration and are cherished for bioimaging. Often, a small probe-product spectral separation leads to crosstalk, but the rational devel...

Lysosomotropic Features and Autophagy Modulators among Medical Drugs: Evaluation of Their Role in Pathologies.

The concept of lysosomotropic agents significantly changed numerous aspects of cellular biochemistry, biochemical pharmacology, and clinical medicine. In the present review, we focused on numerous low-molecular and high-molecular lipophilic basic compounds and on the role of lipophagy and autophagy in experimental and clinical medicine. Attention was primarily focused on the most promising agents acting as autophagy inducers, which offer a new window for treatment and/or prophylaxis of various diseases, including type 2 diabetes mellitus, Parkinson’s disease, and atherosclerosis. The present review summarizes current knowledge on the lysosomotropic features of medical drugs, as well as autophagy inducers, and their role in pathological processes.

Drought response of European beech (Fagus sylvatica L.)—A review

European beech (Fagus sylvatica) is the dominant tree species of Central Europe’s natural forests and one of the continent’s most important timber species. This highly competitive species is known to be drought-sensitive and thus may increasingly be threatened by climate change-related heat waves and drought in part of its distribution range. Tree responses and tolerance to drought are complex processes that are best understood by adopting a multi-level analysis spanning from the molecular physiological to the tree and stand levels. Here I review recent progress in our understanding of beech drought responses in all relevant fields of research, notably adjustments in cellular biochemistry, responses of photosynthesis, stomatal regulation and leaf water status, the vulnerability of the hydraulic and phloem systems, adaptive responses of the fine root system, leaf area adjustments, long-term declines in radial growth, and drought-induced tree mortality. A special focus is on population differences in drought tolerance as the basis for the selection of drought-hardier provenances. The evidence from the different research fields is brought together with the aim to develop a multi-disciplinary perception of beech drought response, to identify weak components in the species’ drought response strategy, and to suggest future research efforts to close knowledge gaps.

Last Word on Viewpoint: The interaction between SARS-CoV-2 and ACE2 may have consequences for skeletal muscle viral susceptibility and myopathies.

Last Word on Viewpoint: The interaction between SARS-CoV-2 and ACE2 may have consequences for skeletal muscle viral susceptibility and myopathies.

Machine learning linked evolutionary biosensor array for highly sensitive and specific molecular identification

Bacteria initiate complicated signaling cascades from the detection of intracellular metabolites or exogenous substances by hundreds of transcription factors, which have been widely investigated as genetically-encoded biosensors for molecular recognition. However, the limited number of transcription factors and their broad substrate specificity result in ambiguity in small molecule identification. This study presents a new small molecule fingerprinting technique using evolutionary biosensor arrays with a machine learning technique that can capture highly specific substrate signals. Employing multiple mutant transcription factors derived from a single transcription factor has effectively circumvented the limited availability of transcription factors induced by a small molecule of our interest. This method achieved up to 95.3% true positive rate for identifying small molecules, and the high-resolution protein engineering technique improved the limit of detection 75-fold. The signal trade-offs with background noises caused by the complex cellular biochemistry of mutant transcription factors enable the biosensor arrays to be more informative in terms of statistical variance. The machine learning technology, coupled with the single transcription factor-driven evolutionary biosensor array, will open new avenues for molecular fingerprinting technologies.

Multiple Functions of Spectrin: Convergent Effects

Spectrin is a multifunctional, multi-domain protein most well known in the membrane skeleton of mature human erythrocytes. Here we review the literature on the crosstalk of the chaperone activity of spectrin with its other functionalities. We hypothesize that the chaperone activity is derived from the surface exposed hydrophobic patches present in individual "spectrin-repeat" domains and show a competition between the membrane phospholipid binding functionality and chaperone activity of spectrin. Moreover, we show that post-translational modifications such as glycation which shield these surface exposed hydrophobic patches, reduce the chaperone function. On the other hand, oligomerization which is linked to increase of hydrophobicity is seen to increase it. We note that spectrin seems to prefer haemoglobin as its chaperone client, binding with it preferentially over other denatured proteins. Spectrin is also known to interact with unstable haemoglobin variants with a higher affinity than in the case of normal haemoglobin. We propose that chaperone activity of spectrin could be important in the cellular biochemistry of haemoglobin, particularly in the context of diseases.

Attention and Capacity Limits in Perception: A Cellular Metabolism Account.

Limits on perceptual capacity result in various phenomena of inattentional blindness. Here we propose a neurophysiological account attributing these perceptual capacity limits directly to limits on cerebral cellular metabolism. We hypothesized that overall cerebral energy supply remains constant, regardless of overall mental processing demands; therefore, an attention mechanism is required to regulate limited cellular metabolism levels in line with attended task demands. Increased perceptual load in a task (imposing a greater demand on neural computations) should thus result in increased metabolism underlying attended processing, and reduced metabolism mediating unattended processing. We tested this prediction measuring oxidation states of cytochrome c oxidase (oxCCO), an intracellular marker of cellular metabolism. Broadband near-infrared spectroscopy was used to record oxCCO levels from human visual cortex while participants (both sexes) performed a rapid sequential visual search task under either high perceptual load (complex feature-conjunction search) or low load (feature pop-out search). A task-irrelevant, peripheral checkerboard was presented on a random half of trials. Our findings showed that oxCCO levels in visual cortex regions responsive to the attended-task stimuli were increased in high versus low perceptual load, whereas oxCCO levels related to unattended processing were significantly reduced. A negative temporal correlation of these load effects further supported our metabolism trade-off account. These results demonstrate an attentional compensation mechanism that regulates cellular metabolism levels according to processing demands. Moreover, they provide novel evidence for the widely held stipulation that overall cerebral metabolism levels remain constant regardless of mental task demand and establish a neurophysiological account for capacity limits in perception.SIGNIFICANCE STATEMENT We investigated whether capacity limits in perception can be explained by the effects of attention on the allocation of limited cellular metabolic energy for perceptual processing. We measured the oxidation state of cytochrome c oxidase, an intracellular measure of metabolism, in human visual cortex during task performance. The results showed increased levels of cellular metabolism associated with attended processing and reduced levels of metabolism underlying unattended processing when the task was more demanding. A temporal correlation between these effects supported an attention-directed metabolism trade-off. These findings support an account for inattentional blindness grounded in cellular biochemistry. They also provide novel evidence for the claim that cerebral processing is limited by a constant energy supply, which thus requires attentional regulation.

Human ARMT1 structure and substrate specificity indicates that it is a DUF89 family damage-control phosphatase

Metabolite damage control is a critical but poorly defined aspect of cellular biochemistry, which likely involves many of the so far functionally uncharacterized protein domain (domains of unknown function; DUFs). We have determined the crystal structure of the human DUF89 protein product of the C6ORF211 gene to 1.85 Å. The crystal structure shows that the protein contains a core α-β-α fold with an active site-bound metal ion and α-helical bundle N-terminal cap, which are both conserved features of subfamily III DUF89 domains. The biochemical activities of the human protein are conserved with those of a previously characterized budding yeast homolog, where an in vitro phosphatase activity is supported by divalent cations that include Co2+, Ni2+, Mn2+ or Mg2+. Full steady-state kinetics parameters of human DUF89 using a standard PNPP phosphatase assay revealed a six times higher catalytic efficiency in presence of Co2+ compared to Mg2+. The human enzyme targets a number of phosphate substrates similar to the budding yeast homolog, while it lacks a previously indicated methyltransferase activity. The highest activity on substrate was observed with fructose-1-phosphate, a potent glycating agent, and thus human DUF89 phosphatase activity may also play a role in limiting the buildup of phospho-glycan species and their related damaged metabolites.

Transient droplets are key to cellular biochemistry

Clifford P. Brangwynne smashed the single-celled embryo between a microscope slide and a thin glass cover slip. Then he peered into the microscope to get a look at the cell, taken from a worm commo...

PB-Net: Automatic peak integration by sequential deep learning for multiple reaction monitoring

Mass spectrometry (MS) based proteomics has become an indispensable component of modern molecular and cellular biochemistry analysis. Multiple reaction monitoring (MRM) is one of the most well-established MS techniques for molecule detection and quantification. Despite its wide usage, there lacks an accurate computational framework to analyze MRM data, and expert annotation is often required, especially to perform peak integration. Here we propose a deep learning method PB-Net (Peak Boundary Neural Network), built upon recent advances in sequential neural networks, for fully automatic chromatographic peak integration. To train PB-Net, we generated a large dataset of over 170,000 expert annotated peaks from MS transitions spanning a wide dynamic range, including both peptides and intact glycopeptides. Our model demonstrated outstanding performances on unseen test samples, reaching near-perfect agreement (Pearson's r 0.997) with human annotated ground truth. Systematic evaluations also show that PB-Net is substantially more robust and accurate compared to previous state-of-the-art peak integration software. PB-Net can benefit the wide community of mass spectrometry data analysis, especially in applications involving high-throughput MS experiments. Codes and test data used in this work are available at https://github.com/miaecle/PB-net. SignificanceHuman annotations serve an important role in accurate quantification of multiple reaction monitoring (MRM) experiments, though they are costly to collect and limit analysis throughput. In this work we proposed and developed a novel technique for the peak-integration step in MRM, based on recent innovations in sequential deep learning models. We collected in total 170,000 expert-annotated MRM peaks and trained a set of accurate and robust neural networks for the task. Results demonstrated a substantial improvement over the current state-of-the-art software for mass spectrometry analysis and comparable level of accuracy and precision as human annotators.

Combining optogenetics with sensitive FRET imaging to monitor local microtubule manipulations

Optogenetic methods for switching molecular states in cells are increasingly prominent tools in life sciences. Förster Resonance Energy Transfer (FRET)-based sensors can provide quantitative and sensitive readouts of altered cellular biochemistry, e.g. from optogenetics. However, most of the light-inducible domains respond to the same wavelength as is required for excitation of popular CFP/YFP-based FRET pairs, rendering the techniques incompatible with each other. In order to overcome this limitation, we red-shifted an existing CFP/YFP-based OP18 FRET sensor (COPY) by employing an sYFP2 donor and mScarlet-I acceptor. Their favorable quantum yield and brightness result in a red-shifted FRET pair with an optimized dynamic range, which could be further enhanced by an R125I point mutation that stimulates intramolecular interactions. The new sensor was named ROPY and it visualizes the interaction between the microtubule regulator stathmin/OP18 and free tubulin heterodimers. We show that through phosphorylation of the ROPY sensor, its tubulin sequestering ability can be locally regulated by photo-activatable Rac1 (PARac1), independent of the FRET readout. Together, ROPY and PARac1 provide spatiotemporal control over free tubulin levels. ROPY/PARac1-based optogenetic regulation of free tubulin levels allowed us to demonstrate that depletion of free tubulin prevents the formation of pioneer microtubules, while local upregulation of tubulin concentration allows localized microtubule extensions to support the lamellipodia.

Cellular Fractionation Identifies the N‐ and C‐ Termini Contributions Toward Membrane Association of the Rho GTPase TCL

TCL is a poorly characterized Rho‐family GTPase; however, our lab has performed detailed structure/function analysis of TCL to gain insight into its cellular biochemistry. We have found that its unique N‐terminal extension promotes GTP‐loading of TCL and plasma membrane localization. Deletion of this TCL‐specific sequence promotes localization of TCL to intracellular vesicles. To further explore the role of the N‐terminal and C‐terminal sequences on TCL localization, plasmids encoding Venus were manipulated to include amino acids 1–24 of TCL at the N‐terminus of Venus and amino acids 198–214 at the C‐terminus of Venus. Additional N‐ and C‐terminus deletion mutants of YFP‐tagged TCL were also produced. These plasmids have been transfected into HeLa cells, and subcellular localization of the expressed proteins assessed by fluorescence microscopy and a fractionation procedure. Our results show a clear role for the C‐ terminus of TCL in facilitating membrane localization, but no significant localization directly related to the N‐terminus. Defining how TCL localizes to different cellular membrane environments will help us to understand more clearly its role focal adhesion dynamics, as well as how these dynamics may relate to melanoma development and tumor‐associated angiogenesis.Support or Funding InformationThis work was supported by Bemidji State University Biology Department and the College of Business, Mathematics, and Science. Support was also provided through the Neilson Foundation, Bemidji, MN and Regenerative Medicine Minnesota (RMM‐2017‐EP‐04).

Perception of Pharmacology Education by Medical Sophomore students from 2000 to the present: impact of digital technology and educational policy

Pharmacology is one of the disciplines that medical students find difficult to study. Reasons are that there are numerous drug names to remember with a variety of side effects; even within a given class, each drug may have unique advantages or disadvantages with regard to drug‐drug interaction, pharmacokinetics, and contraindications. To make things worse, a deep anxiety about the medical licensing step 1 exam induces students to learn pharmacology through simple rote memorization by relying predominantly on pharm cards and online visual‐mnemonics interfaces, and too little time is spent trying to understand drug action in the context of physiology and cellular biochemistry. Thus, the goal of Pharmacology education seems not to be achieved during the basic science years. Our SIU School of Medicine medical curriculum has introduced unique curriculum methods. This includes problem‐based learning modules (PBLM) and group discussions as major curricular activities in Year 1 and 2 basic science teaching, with optional attendance at resource sessions. In this study, AAMC Graduation Questionnaires and students’ comments on Pharmacology curriculum at SIU School of Medicine are analyzed for the academic years 2001 to 2019. One question addressed if the basic science curriculum provides a sufficient foundation for clinical clerkship. The percentage of ‘Good’ and ‘Fair’ remained similar (35–38%) across 18 years. However, ‘Disagree’ was notably reduced from 16% to less than 10% around 2003, whereas the national average remained about the same. The nature of students’ perceptions on Pharmacology Education and their performance will be discussed in relation to the incorporation of digital technologies like online interfaces, cloud and social media into the curriculum, as well as the introduction of innovative teaching methods.Support or Funding InformationNone

Tracking mTORC1 Activity in the Nucleus: Distinct Regulation Revealed by Locationspecific Inhibition of Akt

The mechanistic target of rapamycin complex 1 (mTORC1) is a kinase complex that contains the protein kinase mTOR, the key scaffolding protein Raptor, and several other core components. It is known to sense diverse upstream signals, such as growth factors, nutrient and energy status. When activated, mTORC1 integrates these signals to critically regulate distinct cellular processes, including cell growth, metabolism, and autophagy. An intriguing question is how mTORC1 is spatially regulated to exert widespread controls on downstream effectors. Besides the well studied mTORC1 activation at the lysosome, the regulation of this complex within other subcellular compartments is less clear. Previously, we showed that growth factor induces nuclear mTORC1 activity by utilizing a genetically encoded mTORC1 activity reporter targeted to the nucleus, providing initial evidence for active mTORC1 in the nucleus. Here we probe the regulation of growth factor‐induced nuclear mTORC1 activity by combining location‐selective kinase inhibition, live‐cell imaging and cellular biochemistry. We developed Akt Substrate‐based Tandem Occupancy Peptide Sponge (Akt‐STOPS) that allows location‐specific perturbation of Akt, a critical upstream kinase, at distinct subcellular compartments. Expression of Akt‐STOPS in the nucleus largely suppressed the growth factor stimulated Akt activity without significant inhibition of cytosolic Akt activity. Using Akt‐STOPS, we showed that growth factor‐stimulated nuclear mTORC1 activity requires nuclear Akt activity. Furthermore, we showed that growth factor stimulation induces nuclear translocation of Raptor, which is sensitive to importin inhibition, and that Raptor forms a complex with mTOR in the nucleus . The nuclear translocation of Raptor specifically depends on the nuclear Akt activity, as expression of nuclear Akt‐STOPS significantly lowered the nuclear translocation of Raptor and reduced the phosphorylation of RanBP, a critical regulator of Ran GTPase. Lastly, localizing exogenous Raptor to the nucleus in Raptor knock‐down cells rescues nuclear mTORC1 activity. Taken together, these results reveal a new mode of dynamic regulation of mTORC1 by recruiting a critical scaffolding protein, distinct from the mode of lysosomal mTORC1 activation. Diversity in the regulation of mTORC1 could lead to specificity in functional controls and also provide opportunities for selective targeting.Support or Funding InformationThis work was supported by NIH grants, R35 CA197622 and R01 GM111665 to J.Z., and NS047101 to UCSD Microscopy Core.