Computational approaches for evaluating morphological changes in the corneal stroma associated with decellularization.

Decellularized corneas offer a promising and sustainable source of replacement grafts, mimicking native tissue and reducing the risk of immune rejection post-transplantation. Despite great success in achieving acellular scaffolds, little consensus exists regarding the quality of the decellularized extracellular matrix. Metrics used to evaluate extracellular matrix performance are study-specific, subjective, and semi-quantitative. Thus, this work focused on developing a computational method to examine the effectiveness of corneal decellularization. We combined conventional semi-quantitative histological assessments and automated scaffold evaluations based on textual image analyses to assess decellularization efficiency. Our study highlights that it is possible to develop contemporary machine learning (ML) models based on random forests and support vector machine algorithms, which can identify regions of interest in acellularized corneal stromal tissue with relatively high accuracy. These results provide a platform for developing machine learning biosensing systems for evaluating subtle morphological changes in decellularized scaffolds, which are crucial for assessing their functionality.

Theoretical Study of Hydroxylation of α- and ß-Pinene by a Cytochrome P450 Monooxygenase Model.

Previous studies on biocatalytic transformations of pinenes by cytochrome P450 (CYP) enzymes reveal the formation of different oxygenated products from a single substrate due to the multistate reactivity of CYP and the many reactive sites in the pinene scaffold. Up until now, the detailed mechanism of these biocatalytic transformations of pinenes have not been reported. Hereby, we report a systematic theoretical study of the plausible hydrogen abstraction and hydroxylation reactions of α- and ß-pinenes by CYP using the density functional theory (DFT) method. All DFT calculations in this study were based on B3LYP/LAN computational methodology using the Gaussian09 software. We used the B3LYP functional with corrections for dispersive forces, BSSE, and anharmonicity to study the mechanism and thermodynamic properties of these reactions using a bare model (without CYP) and a pinene-CYP model. According to the potential energy surface and Boltzmann distribution for radical conformers, the major reaction products of CYP-catalyzed hydrogen abstraction from ß-pinene are the doublet trans (53.4%) and doublet cis (46.1%) radical conformer at delta site. The formation of doublet cis/trans hydroxylated products released a total Gibbs free energy of about 48 kcal/mol. As for alpha pinene, the most stable radicals were trans-doublet (86.4%) and cis-doublet (13.6%) at epsilon sites, and their hydroxylation products released a total of ~50 kcal/mol Gibbs free energy. Our results highlight the likely C-H abstraction and oxygen rebounding sites accounting for the multi-state of CYP (doublet, quartet, and sextet spin states) and the formation of different conformers due to the presence of cis/trans allylic hydrogen in α-pinene and ß-pinene molecules.

Gray level co-occurrence matrix and wavelet analyses reveal discrete changes in proximal tubule cell nuclei after mild acute kidney injury.

Acute kidney injury (AKI) relates to an abrupt reduction in renal function resulting from numerous conditions. Morbidity, mortality, and treatment costs related to AKI are relatively high. This condition is strongly associated with damage to proximal tubule cells (PTCs), generating distinct patterns of transcriptional and epigenetic alterations that result in structural changes in the nuclei of this epithelium. To this date, AKI-related nuclear chromatin redistribution in PTCs is poorly understood, and it is unclear whether changes in PTC chromatin patterns can be detected using conventional microscopy during mild AKI, which can progress to more debilitating forms of injury. In recent years, gray level co-occurrence matrix (GLCM) analysis and discrete wavelet transform (DWT) have emerged as potentially valuable methods for identifying discrete structural changes in nuclear chromatin architecture that are not visible during the conventional histopathological exam. Here we present findings indicating that GLCM and DWT methods can be successfully used in nephrology to detect subtle nuclear morphological alterations associated with mild tissue injury demonstrated in rodents by inducing a mild form of AKI through ischemia-reperfusion injury. Our results show that mild ischemic AKI is associated with the reduction of local textural homogeneity of PTC nuclei quantified by GLCM and the increase of nuclear structural heterogeneity indirectly assessed with DWT energy coefficients. This rodent model allowed us to show that mild ischemic AKI is associated with the significant reduction of textural homogeneity of PTC nuclei, indirectly assessed by GLCM indicators and DWT energy coefficients.

Methylene Blue Inhibits Cromakalim-Activated K+ Currents in Follicle-Enclosed Oocytes.

The effects of methylene blue (MB) on cromakalim-induced K+ currents were investigated in follicle-enclosed Xenopus oocytes. In concentrations ranging from 3-300 µM, MB inhibited K+ currents (IC50: 22.4 µM) activated by cromakalim, which activates KATP channels. MB inhibited cromakalim-activated K+ currents in a noncompetitive and voltage-independent manner. The respective EC50 and slope values for cromakalim-activation of K+ currents were 194 ± 21 µM and 0.91 for controls, and 206 ± 24 µM and 0.87 in the presence of 30 µM MB. The inhibition of cromakalim-induced K+ currents by MB was not altered by pretreatment with the Ca2+ chelator BAPTA, which suggests that MB does not influence Ca2+-activated second messenger pathways. K+ currents mediated through a C-terminally deleted form of Kir6.2 (KirΔC26), which does not contain the sulfonylurea receptor, were still inhibited by MB, indicating direct interaction of MB with the channel-forming Kir6.2 subunit. The binding characteristics of the KATP ligand [3H]glibenclamide are not altered by MB in a concentration range between 1 µM-1 mM, as suggested by radioligand binding assay. The presence of a membrane permeable cGMP analogue (8-Br-cGMP, 100 µM) and a guanylate cyclase activator (BAY 58-2667, 3 µM) did not affect the inhibitory effects of MB, suggesting that MB does not inhibit cromakalim-activated K+ currents through guanylate cyclase. Collectively, these results suggest that MB directly inhibits cromakalim-activated K+ currents in follicular cells of Xenopus oocytes.

Neuropathic pain: Mechanisms and therapeutic strategies.

The physiopathology and neurotransmission of pain are of an owe inspiring complexity. Our ability to satisfactorily suppress neuropathic or other forms of chronic pain is limited. The number of pharmacodynamically distinct and clinically available medications is low and the successes achieved modest. Pain Medicine practitioners are confronted with the ethical dichotomy imposed by Hippocrates: On one hand the mandate of primum non nocere, on the other hand, the promise of heavenly joys if successful divinum est opus sedare dolorem. We briefly summarize the concepts associated with nociceptive pain from nociceptive input (afferents from periphery), modulatory output [descending noradrenergic (NE) and serotoninergic (5-HT) fibers] to local control. The local control is comprised of the "inflammatory soup" at the site of pain origin and synaptic relay stations, with an ATP-rich environment promoting inflammation and nociception while an adenosine-rich environment having the opposite effect. Subsequently, we address the transition from nociceptor pain to neuropathic pain (independent of nociceptor activation) and the process of sensitization and pain chronification (transient pain progressing into persistent pain). Having sketched a model of pain perception and processing we attempt to identify the sites and modes of action of clinically available drugs used in chronic pain treatment, focusing on adjuvant (co-analgesic) medication.

Artificial neural networks in contemporary toxicology research.

Artificial neural networks (ANNs) have a huge potential in toxicology research. They may be used to predict toxicity of various chemical compounds or classify the compounds based on their toxic effects. Today, numerous ANN models have been developed, some of which may be used to detect and possibly explain complex chemico-biological interactions. Fully connected multilayer perceptrons may in some circumstances have high classification accuracy and discriminatory power in separating damaged from intact cells after exposure to a toxic substance. Regularized and not fully connected convolutional neural networks can detect and identify discrete changes in patterns of two-dimensional data associated with toxicity. Bayesian neural networks with weight marginalization sometimes may have better prediction performance when compared to traditional approaches. With the further development of artificial intelligence, it is expected that ANNs will in the future become important parts of various accurate and affordable biosensors for detection of various toxic substances and evaluation of their biochemical properties. In this concise review article, we discuss the recent research focused on the scientific value of ANNs in evaluation and prediction of toxicity of chemical compounds.

Advanced Glycation End Products and Diabetes Mellitus: Mechanisms and Perspectives.

Persistent hyperglycemic state in type 2 diabetes mellitus leads to the initiation and progression of non-enzymatic glycation reaction with proteins and lipids and nucleic acids. Glycation reaction leads to the generation of a heterogeneous group of chemical moieties known as advanced glycated end products (AGEs), which play a central role in the pathophysiology of diabetic complications. The engagement of AGEs with its chief cellular receptor, RAGE, activates a myriad of signaling pathways such as MAPK/ERK, TGF-ß, JNK, and NF-κB, leading to enhanced oxidative stress and inflammation. The downstream consequences of the AGEs/RAGE axis involve compromised insulin signaling, perturbation of metabolic homeostasis, RAGE-induced pancreatic beta cell toxicity, and epigenetic modifications. The AGEs/RAGE signaling instigated modulation of gene transcription is profoundly associated with the progression of type 2 diabetes mellitus and pathogenesis of diabetic complications. In this review, we will summarize the exogenous and endogenous sources of AGEs, their role in metabolic dysfunction, and current understandings of AGEs/RAGE signaling cascade. The focus of this review is to recapitulate the role of the AGEs/RAGE axis in the pathogenesis of type 2 diabetes mellitus and its associated complications. Furthermore, we present an overview of future perspectives to offer new therapeutic interventions to intervene with the AGEs/RAGE signaling pathway and to slow down the progression of diabetes-related complications.

Analysis of Vascular Architecture and Parenchymal Damage Generated by Reduced Blood Perfusion in Decellularized Porcine Kidneys Using a Gray Level Co-occurrence Matrix.

There is no cure for kidney failure, but a bioartificial kidney may help address this global problem. Decellularization provides a promising platform to generate transplantable organs. However, maintaining a viable vasculature is a significant challenge to this technology. Even though angiography offers a valuable way to assess scaffold structure/function, subtle changes are overlooked by specialists. In recent years, various image analysis methods in radiology have been suggested to detect and identify subtle changes in tissue architecture. The aim of our research was to apply one of these methods based on a gray level co-occurrence matrix (Topalovic et al.) computational algorithm in the analysis of vascular architecture and parenchymal damage generated by hypoperfusion in decellularized porcine. Perfusion decellularization of the whole porcine kidneys was performed using previously established protocols. We analyzed and compared angiograms of kidneys subjected to pathophysiological arterial perfusion of whole blood. For regions of interest Santos et al. covering kidney medulla and the main elements of the vascular network, five major GLCM features were calculated: angular second moment as an indicator of textural uniformity, inverse difference moment as an indicator of textural homogeneity, GLCM contrast, GLCM correlation, and sum variance of the co-occurrence matrix. In addition to GLCM, we also performed discrete wavelet transform analysis of angiogram ROIs by calculating the respective wavelet coefficient energies using high and low-pass filtering. We report statistically significant changes in GLCM and wavelet features, including the reduction of the angular second moment and inverse difference moment, indicating a substantial rise in angiogram textural heterogeneity. Our findings suggest that the GLCM method can be successfully used as an addition to conventional fluoroscopic angiography analyses of micro/macrovascular integrity following in vitro blood perfusion to investigate scaffold integrity. This approach is the first step toward developing an automated network that can detect changes in the decellularized vasculature.

Tocilizumab and COVID-19: Timing of Administration and Efficacy.

Elevated concentrations of interleukin-6 have been demonstrated to be an important key factor in COVID-19 host immune impairment. It represents an important prognostic factor of harm associated with COVID-19 infection by stimulating a vigorous proinflammatory response, leading to the so-called "cytokine storm". Therefore, immunomodulatory interventions targeting interleukin-6 receptor antagonism have been investigated as potential treatments to counterbalance the host immune dysregulation and to support the advantageous effects of corticosteroids. Tocilizumab is a recombinant humanized monoclonal antibody that has gained much interest during the COVID-19 pandemic as an interleukin-6 receptor antagonist. Various early observational studies have reported beneficial effects of tocilizumab. Moreover, consequent randomized controlled trials have subsequently shown significant positive results about tocilizumab efficacy and safety, focusing on outcomes like mortality, risk of intensive care unit admission, and the need for mechanical ventilation, while others presented conflicting findings. In this review, we first described the pathophysiology of COVID-19 infection while highlighting the role of interleukin-6. Furthermore, we also discussed the non-conclusive evidence about tocilizumab to be used as the standard of care therapy for all patients with COVID-19 pneumonia, as well as its beneficial effects in selected patients.

Hyperthermia and Serotonin: The Quest for a "Better Cyproheptadine".

Fine temperature control is essential in homeothermic animals. Both hyper- and hypothermia can have deleterious effects. Multiple, efficient and partly redundant mechanisms of adjusting the body temperature to the value set by the internal thermostat exist. The neural circuitry of temperature control and the neurotransmitters involved are reviewed. The GABAergic inhibitory output from the brain thermostat in the preoptic area POA to subaltern neural circuitry of temperature control (Nucleus Raphe Dorsalis and Nucleus Raphe Pallidus) is a function of the balance between the (opposite) effects mediated by the transient receptor potential receptor TRPM2 and EP3 prostaglandin receptors. Activation of TRPM2-expressing neurons in POA favors hypothermia, while inhibition has the opposite effect. Conversely, EP3 receptors induce elevation in body temperature. Activation of EP3-expressing neurons in POA results in hyperthermia, while inhibition has the opposite effect. Agonists at TRPM2 and/or antagonists at EP3 could be beneficial in hyperthermia control. Activity of the neural circuitry of temperature control is modulated by a variety of 5-HT receptors. Based on the theoretical model presented the "ideal" antidote against serotonin syndrome hyperthermia appears to be an antagonist at the 5-HT receptor subtypes 2, 4 and 6 and an agonist at the receptor subtypes 1, 3 and 7. Very broadly speaking, such a profile translates in a sympatholytic effect. While a compound with such an ideal profile is presently not available, better matches than the conventional antidote cyproheptadine (used off-label in severe serotonin syndrome cases) appear to be possible and need to be identified.

Cannabidiol Inhibits Multiple Ion Channels in Rabbit Ventricular Cardiomyocytes.

Cannabidiol (CBD), a major non-psychotropic cannabinoid found in the Cannabis plant, has been shown to exert anti-nociceptive, anti-psychotic, and anti-convulsant effects and to also influence the cardiovascular system. In this study, the effects of CBD on major ion currents were investigated using the patch-clamp technique in rabbit ventricular myocytes. CBD inhibited voltage-gated Na+ and Ca2+ channels with IC50 values of 5.4 and 4.8 µM, respectively. In addition, CBD, at lower concentrations, suppressed ion currents mediated by rapidly and slowly activated delayed rectifier K+ channels with IC50 of 2.4 and 2.1 µM, respectively. CBD, up to 10 µM, did not have any significant effect on inward rectifier I K1 and transient outward I to currents. The effects of CBD on these currents developed gradually, reaching steady-state levels within 5-8 min, and recoveries were usually slow and partial. Hill coefficients higher than unity in concentration-inhibition curves suggested multiple CBD binding sites on these channels. These findings indicate that CBD affects cardiac electrophysiology by acting on a diverse range of ion channels and suggest that caution should be exercised when CBD is administered to carriers of cardiac channelopathies or to individuals using drugs known to affect the rhythm or the contractility of the heart.

Gray-Level Co-occurrence Matrix Analysis for the Detection of Discrete, Ethanol-Induced, Structural Changes in Cell Nuclei: An Artificial Intelligence Approach.

Gray-level co-occurrence matrix (GLCM) analysis is a contemporary and innovative computational method for the assessment of textural patterns, applicable in almost any area of microscopy. The aim of our research was to perform the GLCM analysis of cell nuclei in Saccharomyces cerevisiae yeast cells after the induction of sublethal cell damage with ethyl alcohol, and to evaluate the performance of various machine learning (ML) models regarding their ability to separate damaged from intact cells. For each cell nucleus, five GLCM parameters were calculated: angular second moment, inverse difference moment, GLCM contrast, GLCM correlation, and textural variance. Based on the obtained GLCM data, we applied three ML approaches: neural network, random trees, and binomial logistic regression. Statistically significant differences in GLCM features were observed between treated and untreated cells. The multilayer perceptron neural network had the highest classification accuracy. The model also showed a relatively high level of sensitivity and specificity, as well as an excellent discriminatory power in the separation of treated from untreated cells. To the best of our knowledge, this is the first study to demonstrate that it is possible to create a relatively sensitive GLCM-based ML model for the detection of alcohol-induced damage in Saccharomyces cerevisiae cell nuclei.

On saline infusion, clonus, molecules and forgotten scientists: Who was Dr Julius Sander (1840-1909)?

Zitterbewegungen des Fusses bei Dorsalflexion (shaking movements of the foot upon dorsal flexion) were observed independently from each other and described in the same issue of a German peer reviewed journal by Carl Westphal (1833-1890) at the Charité in Berlin and by Wilhelm Erb (1840-1921) in Heidelberg. While Westphal used the term Fussphaenomen, Erb is credited with coining the term clonus for the phenomenon. Both scientists are immortalized by various eponyms acknowledging their respective contributions to science. Little is known however about Julius Sander (1840-1909), in those days resident at Charité, who noticed the phenomenon and presented it to his superiors, Wilhelm Griesinger (1817 -1868) and Westphal. In addition to such observations, Sander made original contributions in resuscitation physiology while working with Hugo Kronecker (1839-1914). With Kronecker, Sander published observations on life saving transfusions with inorganic salt solutions in dogs "Bemerkung über lebensrettende Transfusion mit anorganischer Salzlösung bei Hunden" a very early work on isovolemic fluid resuscitation. The purpose of this communication is to highlight Sander's scientific contributions and to shed some light on his life, of which a German Lexicon stated that after 1870 no information on him can be ascertained anymore.

Metabolic conversion of ß-pinene to ß-ionone in rats.

Exposure to or ingestion of turpentine can alter the scent of urine, conferring it a flowery, violet-like scent. Turpentine's effect on urine was initially noticed after its use either as medicine or as a preservative in winemaking. Regardless of the source of exposure, the phenomenon requires metabolic conversion of turpentine component(s) to ionone, the molecule mainly responsible for the scent of violets.The purpose of this study was to identify the presence of ionone in the urine of rats that received ß-pinene, and thus to demonstrate that the postulated conversion occurs.We treated rats intraperitoneally with normal saline (negative control), ß-ionone (positive control), low-dose ß-pinene (1/3 of LD50), and high-dose ß-pinene (1/2 of LD50). Urine samples were collected up to 72 h after administration of the compounds, followed by gas chromatography/mass spectrometry identification of the presence of ionone.ß-Ionone was found in the urine of rats exposed to both low and high doses of ß-pinene. In contrast, α-ionone appears unlikely to have been formed in rats exposed to either low or high doses of ß-pinene. ß-pinene was converted to ß-ionone, followed by partial excretion in the urine of rats. ß-Ionone is a minor metabolite of ß-pinene.

Lymph Nodes-On-Chip: Promising Immune Platforms for Pharmacological and Toxicological Applications.

Organs-on-chip are gaining increasing attention as promising platforms for drug screening and testing applications. However, lymph nodes-on-chip options remain limited although the lymph node is one of the main determinants of the immunotoxicity of newly developed pharmacological drugs. In this review, we describe existing biomimetic lymph nodes-on-chip, their design, and their physiological relevance to pharmacology and shed the light on future directions associated with lymph node-on-chip design and implementation in drug discovery and development.

Experimental and Established Oximes as Pretreatment before Acute Exposure to Azinphos-Methyl.

Poisoning with organophosphorus compounds (OPCs) represents an ongoing threat to civilians and rescue personal. We have previously shown that oximes, when administered prophylactically before exposure to the OPC paraoxon, are able to protect from its toxic effects. In the present study, we have assessed to what degree experimental (K-27; K-48; K-53; K-74; K-75) or established oximes (pralidoxime, obidoxime), when given as pretreatment at an equitoxic dosage of 25% of LD01, are able to reduce mortality induced by the OPC azinphos-methyl. Their efficacy was compared with that of pyridostigmine, the only FDA-approved substance for such prophylaxis. Efficacy was quantified in rats by Cox analysis, calculating the relative risk of death (RR), with RR=1 for the reference group given only azinphos-methyl, but no prophylaxis. All tested compounds significantly (p ≤ 0.05) reduced azinphos-methyl-induced mortality. In addition, the efficacy of all tested experimental and established oximes except K-53 was significantly superior to the FDA-approved compound pyridostigmine. Best protection was observed for the oximes K-48 (RR = 0.20), K-27 (RR = 0.23), and obidoxime (RR = 0.21), which were significantly more efficacious than pralidoxime and pyridostigmine. The second-best group of prophylactic compounds consisted of K-74 (RR = 0.26), K-75 (RR = 0.35) and pralidoxime (RR = 0.37), which were significantly more efficacious than pyridostigmine. Pretreatment with K-53 (RR = 0.37) and pyridostigmine (RR = 0.52) was the least efficacious. Our present data, together with previous results on other OPCs, indicate that the experimental oximes K-27 and K-48 are very promising pretreatment compounds. When penetration into the brain is undesirable, obidoxime is the most efficacious prophylactic agent already approved for clinical use.

Remedia Sternutatoria over the Centuries: TRP Mediation.

Sneezing (sternutatio) is a poorly understood polysynaptic physiologic reflex phenomenon. Sneezing has exerted a strange fascination on humans throughout history, and induced sneezing was widely used by physicians for therapeutic purposes, on the assumption that sneezing eliminates noxious factors from the body, mainly from the head. The present contribution examines the various mixtures used for inducing sneezes (remedia sternutatoria) over the centuries. The majority of the constituents of the sneeze-inducing remedies are modulators of transient receptor potential (TRP) channels. The TRP channel superfamily consists of large heterogeneous groups of channels that play numerous physiological roles such as thermosensation, chemosensation, osmosensation and mechanosensation. Sneezing is associated with the activation of the wasabi receptor, (TRPA1), typical ligand is allyl isothiocyanate and the hot chili pepper receptor, (TRPV1), typical agonist is capsaicin, in the vagal sensory nerve terminals, activated by noxious stimulants.

The action of aripiprazole and brexpiprazole at the receptor level in singultus.

The hiccup (Latin singultus) is an involuntary periodic contraction of the diaphragm followed by glottic closure, which can be a rare side effect of aripiprazole. In contrast to the structurally closely related aripiprazole, brexpiprazole was not associated with this particular adverse drug reaction. Having two very similar drugs that differ in their ability to induce hiccups represents a unique opportunity to gain insight into the receptors involved in the pathophysiology of the symptom and differences in clinical effects between aripiprazole and brexpiprazole. The overlap between maneuvers used to terminate paroxysmal supraventricular tachycardia and those employed to terminate bouts of hiccups suggests that activation of efferent vagal fibers can be therapeutic in both instances. Recent work seems to support a pivotal role for serotonin receptors in such vagal activation. It is unlikely that a unique receptor-drug interaction could explain the different effects of the examined drugs on hiccup. The different effect is most likely the consequence of several smaller effects at more than one receptor. Brexpiprazole is a highly affine (potent) α2C antagonist and, therefore, also an indirect 5-HT1A agonist. In contrast, aripiprazole is a partial 5-HT1A agonist (weak antagonist) and an HT3 antagonist. Activation of 5-HT1A receptors enhances vagal activity while HT3 blockade reduces it. Vagus nerve activation is therapeutic for hiccups. A definitive answer continues to be elusive.

Multi-Compartment Lymph-Node-on-a-Chip Enables Measurement of Immune Cell Motility in Response to Drugs.

Organs On-a-Chip represent novel platforms for modelling human physiology and disease. The lymph node (LN) is a relevant immune organ in which B and T lymphocytes are spatially organized in a complex architecture, and it is the place where the immune response initiates. The present study addresses the utility of a recently designed LN-on-a-chip to dissect and understand the effect of drugs delivered to cells in a fluidic multicellular 3D setting that mimics the human LN. To do so, we analyzed the motility and viability of human B and T cells exposed to hydroxychloroquine (HCQ). We show that the innovative LN platform, which operates at a microscale level, allows real-time monitoring of co-cultured B and T cells by imaging, and supports cellular random movement. HCQ delivered to cells through a constant and continuous flow induces a reduction in T cell velocity while promotes persistent rotational motion. We also find that HCQ increases the production of reactive oxygen species in T cells. Taken together, these results highlight the potential of the LN-on-a-chip to be applied in drug screening and development, and in cellular dynamics studies.