Evaluation of N4-hydroxycytidine incorporation into nucleic acids of SARS-CoV-2-infected host cells by direct measurement with liquid chromatography-mass spectrometry.

Molnupiravir, an orally administered prodrug of ß-d-N4-hydroxycytidine (NHC), is incorporated into newly synthesized RNA by viral RNA-dependent RNA polymerase (RdRp). It is used for treatment of SARS-CoV-2 infections. Incorporation of NHC triphosphate into viral RNA inhibits replication of the virus, at least in part by introducing deleterious mutations. However, there is limited information on NHC incorporation into host RNA and reports on the risk of mutagenicity that molnupiravir/NHC pose to the host are conflicting. We used two liquid chromatography-mass spectrometry (LC-MS) methods to evaluate the incorporation of NHC into RNA and DNA of host Vero E6 cells in a SARS-CoV-2 infection model. To test this, host and viral RNA were degraded to their ribonucleosides, while host DNA was degraded to deoxyribonucleosides. Subsequently, nucleic acid constituents were analyzed by LC-MS, which offers specific, direct, and quantitative determination of incorporation. Our findings revealed concentration dependent NHC incorporation into host cell RNA in both infected and uninfected cell cultures, reaching a maximum of 1 in 7,093 bases. Analysis of host DNA revealed no presence of deoxy-N4-hydroxycytidine down to a detection limit of 1 in 133,000 bases. Our findings therefore suggest minimal to no NHC incorporation into host DNA, indicating a low probability of significant host cell mutagenicity associated with its use.

The proton-sensing receptors TDAG8 and GPR4 are differentially expressed in human and mouse oligodendrocytes: Exploring their role in neuroinflammation and multiple sclerosis.

Acidosis is one of the hallmarks of demyelinating central nervous system (CNS) lesions in multiple sclerosis (MS). The response to acidic pH is primarily mediated by a family of G protein-coupled proton-sensing receptors: OGR1, GPR4 and TDAG8. These receptors are inactive at alkaline pH, reaching maximal activation at acidic pH. Genome-wide association studies have identified a locus within the TDAG8 gene associated with several autoimmune diseases, including MS. Accordingly, we here found that expression of TDAG8, as opposed to GPR4 or OGR1, is upregulated in MS plaques. This led us to investigate the expression of TDAG8 in oligodendrocytes using mouse and human in vitro and in vivo models. We observed significant upregulation of TDAG8 in human MO3.13 oligodendrocytes during maturation and in response to acidic conditions. However, its deficiency did not impact normal myelination in the mouse CNS, and its expression remained unaltered under demyelinating conditions in mouse organotypic cerebellar slices. Notably, our data revealed no expression of TDAG8 in primary mouse oligodendrocyte progenitor cells (OPCs), in contrast to its expression in primary human OPCs. Our investigations have revealed substantial species differences in the expression of proton-sensing receptors in oligodendrocytes, highlighting the limitations of the employed experimental models in fully elucidating the role of TDAG8 in myelination and oligodendrocyte biology. Consequently, the study does not furnish robust evidence for the role of TDAG8 in such processes. Nonetheless, our findings tentatively point towards a potential association between TDAG8 and myelination processes in humans, hinting at a potential link between TDAG8 and the pathophysiology of MS and warrants further research.

3D-printed extraction devices fabricated from silica particles suspended in acrylate resin.

In recent years, there has been an increasing worldwide interest in the use of alternative sample preparation methods. Digital light processing (DLP) is a 3D printing technique based on using UV light to form photo-curable resin layer upon layer, which results in a printed shape. This study explores the application of this technique for the development of novel drug extraction devices in analytical chemistry. A composite material consisting of a photocurable resin and C18-modified silica particles was employed as a sorbent device, demonstrating its effectiveness in pharmaceutical analysis. Apart from estimating optimal printing parameters, microscopic examination of the material surface, and sorbent powder to resin ratio, the extraction procedure was also optimised. Optimisation included the type and amount of sample matrix additives, desorption solvent, sorption and desorption times, and proper number of sorbent devices needed in extraction protocol. To demonstrate this method's applicability for sample analysis, the solid-phase extraction followed by gas chromatography coupled with mass spectrometry (SPE-GC-MS) method was validated for its ability to quantify benzodiazepine-type drugs. This evaluation confirmed good linearity in the concentration range of 50-1000 ng/mL, with R2 values being 0.9932 and 0.9952 for medazepam and diazepam, respectively. Validation parameters proved that the presented method is precise (with values ranging in-between 2.98 %-7.40 %), and accurate (88.81 % to 110.80 %). A negative control was also performed to investigate possible sorption properties of the resin itself, proving that the addition of C18-modified silica particles significantly increases the extraction efficiency and repeatability. The cost-effectiveness of this approach makes it particularly advantageous for single-use scenarios, eliminating the need for time-consuming sorbent-cleaning procedures, common in traditional solid-phase extraction techniques. Future optimisation opportunities include refining sorbent size, shape, and geometry to achieve lower limits of quantification. As a result of these findings, 3D-printed extraction devices can serve as a viable alternative to commercially available SPE or solid-phase microextraction (SPME) protocols for studying new sample preparation approaches.

The Role of Arginine-Vasopressin in Stroke and the Potential Use of Arginine-Vasopressin Type 1 Receptor Antagonists in Stroke Therapy: A Narrative Review.

Stroke is a life-threatening condition in which accurate diagnoses and timely treatment are critical for successful neurological recovery. The current acute treatment strategies, particularly non-invasive interventions, are limited, thus urging the need for novel therapeutical targets. Arginine vasopressin (AVP) receptor antagonists are emerging as potential targets to treat edema formation and subsequent elevation in intracranial pressure, both significant causes of mortality in acute stroke. Here, we summarize the current knowledge on the mechanisms leading to AVP hyperexcretion in acute stroke and the subsequent secondary neuropathological responses. Furthermore, we discuss the work supporting the predictive value of measuring copeptin, a surrogate marker of AVP in stroke patients, followed by a review of the experimental evidence suggesting AVP receptor antagonists in stroke therapy. As we highlight throughout the narrative, critical gaps in the literature exist and indicate the need for further research to understand better AVP mechanisms in stroke. Likewise, there are advantages and limitations in using copeptin as a prognostic tool, and the translation of findings from experimental animal models to clinical settings has its challenges. Still, monitoring AVP levels and using AVP receptor antagonists as an add-on therapeutic intervention are potential promises in clinical applications to alleviate stroke neurological consequences.

Recent Advances in Assessment and Treatment in Kienböck's Disease.

Kienböck's disease is a rare disease described as progressive avascular osteonecrosis of the lunate. The typical manifestations include a unilateral reduction in wrist motion with accompanying pain and swelling. Besides recent advances in treatment options, the etiology and pathophysiology of the disease remain poorly understood. Common risk factors include anatomical features including ulnar variance, differences in blood supply, increased intraosseous pressure along with direct trauma, and environmental influence. The staging of Kienböck's disease depends mainly on radiographic characteristics assessed according to the modified Lichtman scale. The selection of treatment options is often challenging, as radiographic features may not correspond directly to initial clinical symptoms and differ among age groups. At the earliest stages of Kienböck disease, the nonoperative, unloading management is generally preferred. Patients with negative ulnar variance are usually treated with radial shortening osteotomy. For patients with positive or neutral ulnar variance, a capitate shortening osteotomy is a recommended option. One of the most recent surgical techniques used in Stage III Kienböck cases is vascularized bone grafting. One of the most promising procedures is a vascularized, pedicled, scaphoid graft combined with partial radioscaphoid arthrodesis. This technique provides excellent pain management and prevents carpal collapse. In stage IV, salvage procedures including total wrist fusion or total wrist arthroplasty are often required.

Neuroprotective Effects of Guanosine in Ischemic Stroke-Small Steps towards Effective Therapy.

Guanosine (Guo) is a nucleotide metabolite that acts as a potent neuromodulator with neurotrophic and regenerative properties in neurological disorders. Under brain ischemia or trauma, Guo is released to the extracellular milieu and its concentration substantially raises. In vitro studies on brain tissue slices or cell lines subjected to ischemic conditions demonstrated that Guo counteracts destructive events that occur during ischemic conditions, e.g., glutaminergic excitotoxicity, reactive oxygen and nitrogen species production. Moreover, Guo mitigates neuroinflammation and regulates post-translational processing. Guo asserts its neuroprotective effects via interplay with adenosine receptors, potassium channels, and excitatory amino acid transporters. Subsequently, guanosine activates several prosurvival molecular pathways including PI3K/Akt (PI3K) and MEK/ERK. Due to systemic degradation, the half-life of exogenous Guo is relatively low, thus creating difficulty regarding adequate exogenous Guo distribution. Nevertheless, in vivo studies performed on ischemic stroke rodent models provide promising results presenting a sustained decrease in infarct volume, improved neurological outcome, decrease in proinflammatory events, and stimulation of neuroregeneration through the release of neurotrophic factors. In this comprehensive review, we discuss molecular signaling related to Guo protection against brain ischemia. We present recent advances, limitations, and prospects in exogenous guanosine therapy in the context of ischemic stroke.

Subacute transverse myelitis with optic symptoms in neuroborreliosis: a case report.

BACKGROUND: Subacute transverse myelitis is one of the late manifestations of neuroborreliosis with only a few cases described to the present day. CASE PRESENTATION: We present magnetic resonance imaging, cerebrospinal fluid, and electroneurography findings of a young female patient suffering from neuroborreliosis-associated transverse myelitis with a wide constellation of symptoms including papilloedema. Magnetic resonance imaging of the cervical spine has shown an enlargement of the spinal cord in the mid-cervical region. Cerebrospinal fluid findings included lymphocytic pleocytosis, increased levels of anti - Borrelia antibodies, and increased intrathecal anti -Borrelia antibody index. Following the 28-day course of intravenous ceftriaxone, the patient attained complete recovery. CONCLUSIONS: Subacute transverse myelitis in the course of neuroborreliosis should be considered in the differential diagnosis of patients with abnormal magnetic resonance scans of the spinal cord, lymphocytic pleocytosis, and intrathecal antibody production, especially in the tick-endemic areas, even if the tick bite was not reported. Infrequent accompanying symptoms such as papilloedema are diagnostically challenging and cannot be treated as clinching evidence.

The effect of trehalose on intracellular and extracellular nucleotide metabolism. A pilot study.

Trehalose is a stable, non-reducing disaccharide, which was found recently to stimulate autophagy, limit the inflammatory response and suppress the growth of specific types of cancer. Purinergic signaling and dysregulation of nucleotide metabolism are the key factors, which play a role in the pathophysiology of cancer development and inflammation. Therefore, this study took a novel approach and aimed to find the effect of trehalose on intracellular, and the extracellular metabolism of nucleotides and NAD + in endothelial and breast cancer cells. The results of this study indicated that in vitro concentrations of trehalose between 0.5 and 5 mM reduced the levels of intracellular NAD + in breast cancer cells. The decrease of intracellular guanosine, independent of GTP energy metabolism, was also observed in both endothelial and cancer cells. Trehalose decreased the activity of ecto-adenosine deaminase. Maximal 3-fold decrease in adenosine deamination was observed in both cell types. Trehalose causes changes in both intracellular and extracellular nucleotide metabolism that is more significant in cancer cells than in endothelium. This effect may have therapeutic potential in cancer and endothelial dysfunction, but its full clarification requires further studies.