Local Oxygen Levels Research Articles

Bioassembly of hemoglobin-loaded photopolymerizable spheroids alleviates hypoxia-induced cell death

The delivery of oxygen within tissue engineered constructs is essential for cell survivability; however, achieving this within larger biofabricated constructs poses a significant challenge. Efforts to overcome this limitation often involve the delivery of synthetic oxygen generating compounds. The application of some of these compounds is problematic for the biofabrication of living tissues due to inherent issues such as cytotoxicity, hyperoxia and limited structural stability due to oxygen inhibition of radical-based crosslinking processes. This study aims to develop an oxygen delivering system relying on natural-derived components which are cytocompatible, allow for photopolymerization and advanced biofabrication processes, and improve cell survivability under hypoxia (1% O2). We explore the binding of human hemoglobin (Hb) as a natural oxygen deposit within photopolymerizable allylated gelatin (GelAGE) hydrogels through the spontaneous complex formation of Hb with negatively charged biomolecules (heparin, hyaluronic acid, and bovine serum albumin). We systematically study the effect of biomolecule inclusion on cytotoxicity, hydrogel network properties, Hb incorporation efficiency, oxygen carrying capacity, cell viability, and compatibility with 3D-bioassembly processes within melt electrowritten (MEW) scaffolds. All biomolecules were successfully incorporated within GelAGE hydrogels, displaying controllable mechanical properties and cytocompatibility. Results demonstrated efficient and tailorable Hb incorporation within GelAGE-Heparin hydrogels. The developed system was compatible with microfluidics and photopolymerization processes, allowing for the production of GelAGE-Heparin-Hb spheres. Hb-loaded spheres were assembled into MEW polycaprolactone scaffolds, significantly increasing the local oxygen levels. Ultimately, cells within Hb-loaded constructs demonstrated good cell survivability under hypoxia. Taken together, we successfully developed a hydrogel system that retains Hb as a natural oxygen deposit post-photopolymerization, protecting Hb from free-radical oxidation while remaining compatible with biofabrication of large constructs. The developed GelAGE-Heparin-Hb system allows for physoxic oxygen delivery and thus possesses a vast potential for use across broad tissue engineering and biofabrication strategies to help eliminate cell death due to hypoxia.

Targeted Delivery of Catalase and Photosensitizer Ce6 by a Tumor-Specific Aptamer Is Effective against Bladder Cancer In Vivo.

Photodynamic therapy (PDT) is often applied in a clinical setting to treat bladder cancer. However, current photosensitizers report drawbacks such as low efficacy, low selectivity, and numerous side effects, which have limited the clinical values of PDT for bladder cancer. Previously, we developed the first bladder cancer-specific aptamer that can selectively bind to and be internalized by bladder tumor cells versus normal uroepithelium cells. Here, we use an aptamer-based drug delivery system to deliver photosensitizer chlorine e6 (Ce6) into bladder tumor cells. In addition to Ce6, we also incorporate catalase into the drug complex to increase local oxygen levels in the tumor tissue. Compared with free Ce6, an aptamer-guided DNA nanotrain (NT) loaded with Ce6 and catalase (NT-Catalase-Ce6) can specifically recognize bladder cancer cells, produce oxygen locally, induce ROS in tumor cells, and cause mitochondrial apoptosis. In an orthotopic mouse model of bladder cancer, the intravesical instillation of NT-Catalase-Ce6 exhibits faster drug internalization and a longer drug retention time in tumor tissue compared with that in normal urothelium. Moreover, our modified PDT significantly inhibits tumor growth with fewer side effects such as cystitis than free Ce6. This aptamer-based photosensitizer delivery system can therefore improve the selectivity and efficacy and reduce the side effects of PDT treatment in mouse models of bladder cancer, bearing a great translational value for bladder cancer intravesical therapy.

Caffeine exacerbates seizure-induced death via postictal hypoxia

Sudden unexpected death in epilepsy (SUDEP) is the leading epilepsy-related cause of premature mortality in people with intractable epilepsy, who are 27 times more likely to die than the general population. Impairment of the central control of breathing following a seizure has been identified as a putative cause of death, but the mechanisms underlying this seizure-induced breathing failure are largely unknown. Our laboratory has advanced a vascular theory of postictal behavioural dysfunction, including SUDEP. We have recently reported that seizure-induced death occurs after seizures invade brainstem breathing centres which then leads to local hypoxia causing breathing failure and death. Here we investigated the effects of caffeine and two adenosine receptors in two models of seizure-induced death. We recorded local oxygen levels in brainstem breathing centres as well as time to cessation of breathing and cardiac activity relative to seizure activity. The administration of the non-selective A1/A2A antagonist caffeine or the selective A1 agonist N6-cyclopentyladenosine reveals a detrimental effect on postictal hypoxia, providing support for caffeine modulating cerebral vasculature leading to brainstem hypoxia and cessation of breathing. Conversely, A2A activation with CGS-21680 was found to increase the lifespan of mice in both our models of seizure-induced death.

Remodeling Tumor Immune Microenvironment by Using Polymer-Lipid-Manganese Dioxide Nanoparticles with Radiation Therapy to Boost Immune Response of Castration-Resistant Prostate Cancer.

Despite substantial progress in the treatment of castration-resistant prostate cancer (CRPC), including radiation therapy and immunotherapy alone or in combination, the response to treatment remains poor due to the hypoxic and immunosuppressive nature of the tumor microenvironment. Herein, we exploited the bioreactivity of novel polymer-lipid manganese dioxide nanoparticles (PLMDs) to remodel the tumor immune microenvironment (TIME) by increasing the local oxygen levels and extracellular pH and enhancing radiation-induced immunogenic cell death. This study demonstrated that PLMD treatment sensitized hypoxic human and murine CRPC cells to radiation, significantly increasing radiation-induced DNA double-strand breaks and ultimately cell death, which enhanced the secretion of damage-associated molecular patterns, attributable to the induction of autophagy and endoplasmic reticulum stress. Reoxygenation via PLMDs also polarized hypoxic murine RAW264.7 macrophages toward the M1 phenotype, enhancing tumor necrosis factor alpha release, and thus reducing the viability of murine CRPC TRAMP-C2 cells. In a syngeneic TRAMP-C2 tumor model, intravenous injection of PLMDs suppressed, while radiation alone enhanced recruitment of regulatory T cells and myeloid-derived suppressor cells. Pretreatment with PLMDs followed by radiation down-regulated programmed death-ligand 1 and promoted the infiltration of antitumor CD8+ T cells and M1 macrophages to tumor sites. Taken together, TIME modulation by PLMDs plus radiation profoundly delayed tumor growth and prolonged median survival compared with radiation alone. These results suggest that PLMDs plus radiation is a promising treatment modality for improving therapeutic efficacy in radioresistant and immunosuppressive solid tumors.

Spatio-temporal modelling of phenotypic heterogeneity in tumour tissues and its impact on radiotherapy treatment

We present a mathematical model that describes how tumour heterogeneity evolves in a tissue slice that is oxygenated by a single blood vessel. Phenotype is identified with the stemness level of a cell and determines its proliferative capacity, apoptosis propensity and response to treatment. Our study is based on numerical bifurcation analysis and dynamical simulations of a system of coupled, non-local (in phenotypic “space”) partial differential equations that link the phenotypic evolution of the tumour cells to local tissue oxygen levels. In our formulation, we consider a 1D geometry where oxygen is supplied by a blood vessel located on the domain boundary and consumed by the tumour cells as it diffuses through the tissue. For biologically relevant parameter values, the system exhibits multiple steady states; in particular, depending on the initial conditions, the tumour is either eliminated (“tumour-extinction”) or it persists (“tumour-invasion”). We conclude by using the model to investigate tumour responses to radiotherapy, and focus on identifying radiotherapy strategies which can eliminate the tumour. Numerical simulations reveal how phenotypic heterogeneity evolves during treatment and highlight the critical role of tissue oxygen levels on the efficacy of radiation protocols that are commonly used in the clinic.

Oxygen gradients dictate angiogenesis but not barriergenesis in a 3D brain microvascular model.

A variety of biophysical properties are known to regulate angiogenic sprouting, and in vitro systems can parse the individual effects of these factors in a controlled setting. Here, a three-dimensional brain microvascular model interrogates how variables including extracellular matrix composition, fluid shear stress, and radius of curvature affect angiogenic sprouting of cerebral endothelial cells. Tracking endothelial migration over several days reveals that application of fluid shear stress and enlarged vessel radius of curvature both attenuate sprouting. Computational modeling informed by oxygen consumption assays suggests that sprouting correlates to reduced oxygen concentration: both fluid shear stress and vessel geometry alter the local oxygen levels dictated by both ambient conditions and cellular respiration. Moreover, increasing cell density and consequently lowering the local oxygen levels yields significantly more sprouting. Further analysis reveals that the magnitude of oxygen concentration is not as important as its spatial concentration gradient: decreasing ambient oxygen concentration causes significantly less sprouting than applying an external oxygen gradient to the vessels. In contrast, barriergenesis is dictated by shear stress independent of local oxygen concentrations, suggesting that different mechanisms mediate angiogenesis and barrier formation and that angiogenic sprouting can occur without compromising the barrier. Overall, these results improve our understanding of how specific biophysical variables regulate the function and activation of cerebral vasculature, and identify spatial oxygen gradients as the driving factor of angiogenesis in the brain.

Multiphoton Phosphorescence Quenching Microscopy Reveals Kinetics of Tumor Oxygenation during Antiangiogenesis and Angiotensin Signaling Inhibition.

The abnormal function of tumor blood vessels causes tissue hypoxia, promoting disease progression and treatment resistance. Although tumor microenvironment normalization strategies can alleviate hypoxia globally, how local oxygen levels change is not known because of the inability to longitudinally assess vascular and interstitial oxygen in tumors with sufficient resolution. Understanding the spatial and temporal heterogeneity should help improve the outcome of various normalization strategies. We developed a multiphoton phosphorescence quenching microscopy system using a low-molecular-weight palladium porphyrin probe to measure perfused vessels, oxygen tension, and their spatial correlations in vivo in mouse skin, bone marrow, and four different tumor models. Further, we measured the temporal and spatial changes in oxygen and vessel perfusion in tumors in response to an anti-VEGFR2 antibody (DC101) and an angiotensin-receptor blocker (losartan). We found that vessel function was highly dependent on tumor type. Although some tumors had vessels with greater oxygen-carrying ability than those of normal skin, most tumors had inefficient vessels. Further, intervessel heterogeneity in tumors is associated with heterogeneous response to DC101 and losartan. Using both vascular and stromal normalizing agents, we show that spatial heterogeneity in oxygen levels persists, even with reductions in mean extravascular hypoxia. High-resolution spatial and temporal responses of tumor vessels to two agents known to improve vascular perfusion globally reveal spatially heterogeneous changes in vessel structure and function. These dynamic vascular changes should be considered in optimizing the dose and schedule of vascular and stromal normalizing strategies to improve the therapeutic outcome.

Abstract LB557: Multiphoton phosphorescence quenching microscopy reveals kinetics of tumor oxygenation during anti-angiogenesis and angiotensin signaling inhibition

Abstract Purpose: The abnormal function of tumor blood vessels causes hypoxia fueling disease progression and conferring treatment resistance. The local level of oxygen experienced by a cell will determine its response, making it critical to understand tissue oxygen levels with a spatial resolution on the order of the size of a cell. While microenvironment normalization strategies alleviate global hypoxia, how local oxygen levels change are not known because there are no in vivo techniques to longitudinally assess tumor vessels and interstitial oxygen in tumors with sufficient resolution. Understanding the heterogeneity of oxygen levels after microenvironmental normalization will help improve the efficacy of various normalization strategies. Experimental Design: We developed a multiphoton phosphorescence quenching microscopy system using a low-molecular weight palladium porphyrin probe to measure perfused vessels, oxygen tension and their spatial correlations in vivo in mouse skin, bone marrow, and tumors. Further, we measured the temporal and spatial changes in oxygen and vessel perfusion in tumors in response to microenvironmental normalization. Results: We found that vessel function was highly dependent on tumor type. Although some tumors had vessels with greater oxygen carrying ability than normal skin, most tumors had inefficient vessels. Further, inter-vessel heterogeneity in tumors coincided with heterogeneous response to microenvironmental normalizing agents. Using both vascular and stromal normalizing agents, we show that spatial heterogeneity in oxygen levels persist, even with global reductions in hypoxia. Conclusions: We present the first study to examine the high-resolution spatial and temporal response of tumor vessels to two agents known to improve vascular perfusion globally. Our measurements demonstrate that the heterogeneities in the local imbalance of pro- and anti-angiogenic signaling lead to spatially heterogeneous changes in vessel structure and function. Microscale dynamic vascular changes should be considered in optimizing the dose and schedule of microenvironment normalizing therapies to improve function. Citation Format: John Martin, Ryan Lanning, Dai Fukumura, Timothy Padera, Rakesh Jain. Multiphoton phosphorescence quenching microscopy reveals kinetics of tumor oxygenation during anti-angiogenesis and angiotensin signaling inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB557.

Temporal Lobe Spikes Affect Distant Intrinsic Connectivity Networks.

Objective: To evaluate local and distant blood oxygen level dependent (BOLD) signal changes related to interictal epileptiform discharges (IED) in drug-resistant temporal lobe epilepsy (TLE).Methods: Thirty-three TLE patients undergoing EEG–functional Magnetic Resonance Imaging (fMRI) as part of the presurgical workup were consecutively enrolled. First, a single-subject spike-related analysis was performed: (a) to verify the BOLD concordance with the presumed Epileptogenic Zone (EZ); and (b) to investigate the Intrinsic Connectivity Networks (ICN) involvement. Then, a group analysis was performed to search for common BOLD changes in TLE.Results: Interictal epileptiform discharges were recorded in 25 patients and in 19 (58%), a BOLD response was obtained at the single-subject level. In 42% of the cases, BOLD changes were observed in the temporal lobe, although only one patient had a pure concordant finding, with a single fMRI cluster overlapping (and limited to) the EZ identified by anatomo-electro-clinical correlations. In the remaining 58% of the cases, BOLD responses were localized outside the temporal lobe and the presumed EZ. In every patient, with a spike-related fMRI map, at least one ICN appeared to be involved. Four main ICNs were preferentially involved, namely, motor, visual, auditory/motor speech, and the default mode network. At the single-subject level, EEG–fMRI proved to have high specificity (above 65%) in detecting engagement of an ICN and the corresponding ictal/postictal symptom, and good positive predictive value (above 67%) in all networks except the visual one. Finally, in the group analysis of BOLD changes related to IED revealed common activations at the right precentral gyrus, supplementary motor area, and middle cingulate gyrus.Significance: Interictal temporal spikes affect several distant extra-temporal areas, and specifically the motor/premotor cortex. EEG–fMRI in patients with TLE eligible for surgery is recommended not for strictly localizing purposes rather it might be useful to investigate ICNs alterations at the single-subject level.

Decoding Activity in Broca's Area Predicts the Occurrence of Auditory Hallucinations Across Subjects

BackgroundFunctional magnetic resonance imaging (fMRI) capture aims at detecting auditory-verbal hallucinations (AVHs) from continuously recorded brain activity. Establishing efficient capture methods with low computational cost that easily generalize between patients remains a key objective in precision psychiatry. To address this issue, we developed a novel automatized fMRI-capture procedure for AVHs in patients with schizophrenia (SCZ). MethodsWe used a previously validated but labor-intensive personalized fMRI-capture method to train a linear classifier using machine learning techniques. We benchmarked the performances of this classifier on 2320 AVH periods versus resting-state periods obtained from SCZ patients with frequent symptoms (n = 23). We characterized patterns of blood oxygen level–dependent activity that were predictive of AVH both within and between subjects. Generalizability was assessed with a second independent sample gathering 2000 AVH labels (n = 34 patients with SCZ), while specificity was tested with a nonclinical control sample performing an auditory imagery task (840 labels, n = 20). ResultsOur between-subject classifier achieved high decoding accuracy (area under the curve = 0.85) and discriminated AVH from rest and verbal imagery. Optimizing the parameters on the first schizophrenia dataset and testing its performance on the second dataset led to an out-of-sample area under the curve of 0.85 (0.88 for the converse test). We showed that AVH detection critically depends on local blood oxygen level–dependent activity patterns within Broca's area. ConclusionsOur results demonstrate that it is possible to reliably detect AVH states from fMRI blood oxygen level–dependent signals in patients with SCZ using a multivariate decoder without performing complex preprocessing steps. These findings constitute a crucial step toward brain-based treatments for severe drug-resistant hallucinations.

Conjugated Polymer-Ferrocence Nanoparticle as an NIR-II Light Powered Nanoamplifier to Enhance Chemodynamic Therapy.

Chemodynamic therapy (CDT) is a promising therapeutic modality with transition metal ions and endogenous H2O2 as reagents, but its efficiency is impaired by low endogenous H2O2 levels and nonregeneration of metal ions. Most intracellular H2O2 supplement strategies use oxidases and are intensively dependent on oxygen participation. The hypoxia microenvironments of solid tumors weaken their performance. Here, we develop a near-infrared II light powered nanoamplifier to improve the local oxygen level and to enhance CDT. The nanoamplifier CPNP-Fc/Pt consists of ferrocene (Fc)- and cisplatin prodrug (Pt(IV))-modified conjugated polymer nanoparticles (CPNPs). CPNP has a donor-acceptor structure and demonstrates a good photothermal effect under 1064 nm light irradiation, which accelerates blood flow and efficiently elevates the local oxygen content. In response to intracellular glutathione, Pt(II) is released from CPNP-Fc/Pt and triggers enzymatic cascade reactions with nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) and superoxide dismutase to convert oxygen into H2O2. The enhanced oxygen level results in efficient intracellular H2O2 supply. Fc is reacted with H2O2 and converted to Fc+ via the Fenton reaction, with the generation of hydroxyl radicals for CDT. Unlike free metal ions, the Fe(III) in Fc+ is reduced to Fe(II) by intracellular NAD(P)H, which achieves the regeneration of Fc. The sufficient intracellular H2O2 supply and efficient Fc regeneration effectively enhance the Fenton reaction and demonstrate good in vivo CDT results with tumor growth suppression. This design offers a promising strategy to enhance CDT efficiency in the hypoxia microenvironment of solid tumors.

The ketogenic diet raises brain oxygen levels, attenuates postictal hypoxia, and protects against learning impairments

ObjectivesA prolonged vasoconstriction/hypoperfusion/hypoxic event follows self-terminating focal seizures. The ketogenic diet (KD) has demonstrated efficacy as a metabolic treatment for intractable epilepsy and other disorders but its effect on local brain oxygen levels is completely unknown. This study investigated the effects of the KD on tissue oxygenation in the hippocampus before and after electrically elicited (kindled) seizures and whether it could protect against a seizure-induced learning impairment. We also examined the effects of the ketone β-hydroxybutyrate (BHB) as a potential underlying mechanism. MethodsMale and female rats were given access to one of three diet protocols 2 weeks prior to the initiation of seizures: KD, caloric restricted standard chow, and ad libitum standard chow. Dorsal hippocampal oxygen levels were measured prior to initiation of diets as well as before and after a 10-day kindling paradigm. Male rats were then tested on a novel object recognition task to assess postictal learning impairments. In a separate cohort, BHB was administered 30 min prior to seizure elicitation to determine whether it influenced oxygen dynamics. ResultsThe KD increased dorsal hippocampal oxygen levels, ameliorated postictal hypoxia, and prevented postictal learning impairments. Acute BHB administration did not alter oxygen levels before or after seizures. InterpretationThe ketogenic diet raised brain oxygen levels and attenuated severe postictal hypoxia likely through a mechanism independent of ketosis and shows promise as a non-pharmacological treatment to prevent the postictal state.

Biomimetic CoO@AuPt nanozyme responsive to multiple tumor microenvironmental clues for augmenting chemodynamic therapy

Chemodynamic therapy (CDT), an emerging therapeutic strategy, has been recently exploited for in situ treatment through Fenton or Fenton-like reactions to generate cytotoxic reactive oxygen species (ROS). However, current systems rely significantly on the high local oxygen levels and strongly acidic conditions (pH = 3.0–5.0). Simultaneously, the produced ROS can be rapidly consumed by intracellular glutathione (GSH) in the electron transport chain. Herein, an original and biomimetic CoO@AuPt nanocatalyst was prepared based on the assembly of Au and Pt nanoparticles (NPs) on the surface of hollow CoO nanocapsules. The as-synthesized nanozyme exhibits extremely high stability under physiological conditions, whereas it undergoes spontaneous disintegration in the unique tumor microenvironment (TME). Subsequently, the decomposition products can catalyze a cascade of biochemical reactions to produce abundant ROS without any external stimuli. Thus, the present nanoplatform can increase intracellular ROS levels through continuous supply of H2O2, relief of local hypoxia and depletion of GSH, which result in remarkable and specific tumor damage both in vitro and in vivo. The findings of this study highlight the promising potential of CoO@AuPt nanocatalyst as a TME-responsive CDT nanomagnet for highly efficient tumor therapy.

Scalable surrogate deconvolution for identification of partially-observable systems and brain modeling

Objective. For many biophysical systems, direct measurement of all state-variables, in − vivo is not feasible. Thus, a key challenge in biological modeling and signal processing is to reconstruct the activity and structure of interesting biological systems from indirect measurements. These measurements are often generated by approximately linear time-invariant dynamical interactions with the hidden system and may therefore be described as a convolution of hidden state-variables with an unknown kernel. Approach. In the current work, we present an approach termed surrogate deconvolution, to directly identify such coupled systems (i.e. parameterize models). Surrogate deconvolution reframes certain non linear partially-observable identification problems, which are common in neuroscience/biology, as analytical objectives that are compatible with almost any user-chosen optimization procedure. Main results. We show that the proposed technique is highly scalable, low in computational complexity, and performs competitively with the current gold-standard in partially-observable system estimation: the joint Kalman Filters (Unscented and Extended). We show the benefits of surrogate deconvolution for model identification when applied to simulations of the Local Field Potential and blood oxygen level dependent (BOLD) signal. Lastly, we demonstrate the empirical stability of Hemodynamic Response Function (HRF) kernel estimates for Mesoscale Individualized NeuroDynamic (MINDy) models of individual human brains. The recovered HRF parameters demonstrate reliable individual variation as well as a stereotyped spatial distribution, on average. Significance. These results demonstrate that surrogate deconvolution promises to enhance brain-modeling approaches by simultaneously and rapidly fitting large-scale models of brain networks and the physiological processes which generate neuroscientific measurements (e.g. hemodynamics for BOLD fMRI).

Electrochemical Evaluation of a Multi-Site Clinical Depth Recording Electrode for Monitoring Cerebral Tissue Oxygen.

The intracranial measurement of local cerebral tissue oxygen levels—PbtO2—has become a useful tool for the critical care unit to investigate severe trauma and ischemia injury in patients. Our preliminary work in animal models supports the hypothesis that multi-site depth electrode recording of PbtO2 may give surgeons and critical care providers needed information about brain viability and the capacity for better recovery. Here, we present a surface morphology characterization and an electrochemical evaluation of the analytical properties toward oxygen detection of an FDA-approved, commercially available, clinical grade depth recording electrode comprising 12 Pt recording contacts. We found that the surface of the recording sites is composed of a thin film of smooth Pt and that the electrochemical behavior evaluated by cyclic voltammetry in acidic and neutral electrolyte is typical of polycrystalline Pt surface. The smoothness of the Pt surface was further corroborated by determination of the electrochemical active surface, confirming a roughness factor of 0.9. At an optimal working potential of −0.6 V vs. Ag/AgCl, the sensor displayed suitable values of sensitivity and limit of detection for in vivo PbtO2 measurements. Based on the reported catalytical properties of Pt toward the electroreduction reaction of O2, we propose that these probes could be repurposed for multisite monitoring of PbtO2 in vivo in the human brain.

Engineered algae: A novel oxygen-generating system for effective treatment of hypoxic cancer.

Microalgae, a naturally present unicellular microorganism, can undergo light photosynthesis and have been used in biofuels, nutrition, etc. Here, we report that engineered live microalgae can be delivered to hypoxic tumor regions to increase local oxygen levels and resensitize resistant cancer cells to both radio- and phototherapies. We demonstrate that the hypoxic environment in tumors is markedly improved by in situ-generated oxygen through microalgae-mediated photosynthesis, resulting in notably radiotherapeutic efficacy. Furthermore, the chlorophyll from microalgae produces reactive oxygen species during laser irradiation, further augmenting the photosensitizing effect and enhancing tumor cell apoptosis. Thus, the sequential combination of oxygen-generating algae system with radio- and phototherapies has the potential to create an innovative treatment strategy to improve the outcome of cancer management. Together, our findings demonstrate a novel approach that leverages the products of photosynthesis for treatment of tumors and provide proof-of-concept evidence for future development of algae-enhanced radio- and photodynamic therapy.

Caffeine Exacerbates Postictal Hypoxia

A stroke-like event follows seizures which may be responsible for the postictal state and a contributing factor to the development of seizure-induced brain abnormalities and behavioral dysfunction associated with epilepsy. Caffeine is the world’s most popular drug with ∼85% of people in the USA consuming it daily. Thus, persons with epilepsy are likely to have caffeine in their body and brain during seizures. This preclinical study investigated the effects of acute caffeine on local hippocampal tissue oxygenation pre and post seizure. We continuously measured local oxygen levels in the CA1 region of the hippocampus and utilized the electrical kindling model in rats. Rats were acutely administered either caffeine, or one of its metabolites, or agonists and antagonists at adenosine sub-receptor types or ryanodine receptors prior to the elicitation of seizures. Acute caffeine administration caused a significant drop in pre-seizure hippocampal pO2. Following a seizure, caffeine, as well as two of its metabolites paraxanthine, and theophylline, increased the time below the severe hypoxic threshold (10 mmHg). Likewise, the specific A2A receptor antagonist, SCH-58261, mimicked caffeine by causing a significant drop in pre-seizure pO2 and the area and time below the severe hypoxic threshold. Moreover, the A2A receptor agonist, CGS-21680 was able to prevent the effect of both caffeine and SCH-58261 adding further evidence that caffeine is likely acting through the A2A receptor. Clinical tracking and investigations are needed to determine the effect of caffeine on postictal symptomology and blood flow in persons with epilepsy.

Kinetic analysis of a globin-coupled diguanylate cyclase, YddV: Effects of heme iron redox state, axial ligands, and heme distal mutations on catalysis

Heme-based oxygen sensors allow bacteria to regulate their activity based on local oxygen levels. YddV, a globin-coupled oxygen sensor with diguanylate cyclase activity from Escherichia coli, regulates cyclic-di-GMP synthesis based on oxygen availability. Stable and active samples of the full-length YddV protein were prepared by attaching it to maltose binding protein (MBP). To better understand the full-length protein's structure, the interactions between its domains were examined by performing a kinetic analysis. The diguanylate cyclase reaction catalyzed by YddV-MBP exhibited Michaelis-Menten kinetics. Its pH optimum was 8.5–9.0, and catalysis required either Mg2+ or Mn2+; other divalent metal ions gave no activity. The most active form of YddV-MBP had a 5-coordinate Fe(III) heme complex; its kinetic parameters were KmGTP 84 ± 21 μM and kcat 1.2 min−1. YddV-MBP with heme Fe(II), heme Fe(II)-O2, and heme Fe(II)-CO complexes had kcat values of 0.3 min−1, 0.95 min−1, and 0.3 min−1, respectively, suggesting that catalysis is regulated by the heme iron's redox state and axial ligand binding. The kcat values for heme Fe(III) complexes of L65G, L65Q, and Y43A YddV-MBP mutants bearing heme distal amino acid replacements were 0.15 min−1, 0.26 min−1 and 0.54 min−1, respectively, implying that heme distal residues play key regulatory roles by mediating signal transduction between the sensing and functional domains. Ultracentrifugation and size exclusion chromatography experiments showed that YddV-MBP is primarily dimeric in solution, with a sedimentation coefficient around 8. The inactive heme-free H93A mutant is primarily octameric, suggesting that catalytically active dimer formation requires heme binding.

Oxygen tension-mediated erythrocyte membrane interactions regulate cerebral capillary hyperemia.

The tight coupling between cerebral blood flow and neural activity is a key feature of normal brain function and forms the basis of functional hyperemia. The mechanisms coupling neural activity to vascular responses, however, remain elusive despite decades of research. Recent studies have shown that cerebral functional hyperemia begins in capillaries, and red blood cells (RBCs) act as autonomous regulators of brain capillary perfusion. RBCs then respond to local changes of oxygen tension (PO2) and regulate their capillary velocity. Using ex vivo microfluidics and in vivo two-photon microscopy, we examined RBC capillary velocity as a function of PO2 and showed that deoxygenated hemoglobin and band 3 interactions on RBC membrane are the molecular switch that responds to local PO2 changes and controls RBC capillary velocity. Capillary hyperemia can be controlled by manipulating RBC properties independent of the neurovascular unit, providing an effective strategy to treat or prevent impaired functional hyperemia.

Microbial eukaryotes in the suboxic chemosynthetic ecosystem of Movile Cave, Romania.

Movile Cave is a small system of partially inundated galleries in limestone settings close to the Black Sea in Southeast Romania. Isolated from the surface for 6 million years, its sulfidic, methane and ammonia-rich waters harbour unique chemosynthetic prokaryotic communities that include sulphur and ammonium-metabolizing chemolithotrophs, methanogens, methanotrophs and methylotrophs. The cave also harbours cave-dwelling invertebrates and fungi, but the diversity of other microbial eukaryotes remained completely unknown. Here, we apply an 18S rRNA gene-based metabarcoding approach to study the composition of protist communities in floating microbial mats and plankton from a well-preserved oxygen-depleted cave chamber. Our results reveal a wide protist diversity with, as dominant groups, ciliates (Alveolata), Stramenopiles, especially bicosoecids, and jakobids (Excavata). Ciliate sequences dominated both, microbial mats and plankton, followed by either Stramenopiles or excavates. Stramenopiles were more prominent in microbial mats, whereas jakobids dominated the plankton fraction of the oxygen-depleted water column. Mats cultured in the laboratory were enriched in Cercozoa. Consistent with local low oxygen levels, Movile Cave protists are most likely anaerobic or microaerophilic. Several newly detected OTU clades were very divergent from cultured species or environmental sequences in databases and represent phylogenetic novelty, notably within jakobids. Movile Cave protists likely cover a variety of ecological roles in this ecosystem including predation, parasitism, saprotrophy and possibly diverse prokaryote-protist syntrophies.