Nanogels with covalently bound and releasable trehalose for autophagy stimulation in atherosclerosis.

Atherosclerosis, cholesterol-driven plaque formation in arteries, is a complex multicellular disease which is a leading cause of vascular diseases. During the progression of atherosclerosis, the autophagic function is impaired, resulting in lipid accumulation-mediated foam cell formation. The stimulation of autophagy is crucial for the recovery of cellular recycling process. One of the potential autophagy inducers is trehalose, a naturally occurring non-reducing disaccharide. However, trehalose has poor bioavailability due to its hydrophilic nature which results in poor penetration through cell membranes. To enhance its bioavailability, we developed trehalose-releasing nanogels (TNG) for the treatment of atherosclerosis. The nanogels were fabricated through copolymerization of 6-O-acryloyl-trehalose with the selected acrylamide-type monomers affording a high trehalose conjugation (~ 58%, w/w). TNG showed a relatively small hydrodynamic diameter (dH, 67 nm) and a uniform spherical shape and were characterized by negative ζ potential (-18 mV). Thanks to the trehalose-rich content, TNG demonstrated excellent colloidal stability in biological media containing serum and were non-hemolytic to red blood cells. In vitro study confirmed that TNG could stimulate autophagy in foam cells and enhance lipid efflux and in vivo study in ApoE-/- mice indicated a significant reduction in atherosclerotic plaques, while increasing autophagic markers. In conclusion, TNG hold great promise as a trehalose delivery system to restore impaired autophagy-mediated lipid efflux in atherosclerosis and subsequently reduce atherosclerotic plaques.

Trehalose-Bearing Carriers to Target Impaired Autophagy and Protein Aggregation Diseases.

In recent years, trehalose, a natural disaccharide, has attracted growing attention because of the discovery of its potential to induce autophagy. Trehalose has also been demonstrated to preserve the protein's structural integrity and to limit the aggregation of pathologically misfolded proteins. Both of these properties have made trehalose a promising therapeutic candidate to target autophagy-related disorders and protein aggregation diseases. Unfortunately, trehalose has poor bioavailability due to its hydrophilic nature and susceptibility to enzymatic degradation. Recently, trehalose-bearing carriers, in which trehalose is incorporated either by chemical conjugation or physical entrapment, have emerged as an alternative option to free trehalose to improve its efficacy, particularly for the treatment of neurodegenerative diseases, atherosclerosis, nonalcoholic fatty liver disease (NAFLD), and cancers. In the current Perspective, we discuss all existing literature in this emerging field and try to identify key challenges for researchers intending to develop trehalose-bearing carriers to stimulate autophagy or inhibit protein aggregation.

Predictive modelling of response to neoadjuvant therapy in HER2+ breast cancer.

HER2-positive (HER2+) breast cancer accounts for 20-25% of all breast cancers. Predictive biomarkers of neoadjuvant therapy response are needed to better identify patients with early stage disease who may benefit from tailored treatments in the adjuvant setting. As part of the TCHL phase-II clinical trial (ICORG10-05/NCT01485926) whole exome DNA sequencing was carried out on normal-tumour pairs collected from 22 patients. Here we report predictive modelling of neoadjuvant therapy response using clinicopathological and genomic features of pre-treatment tumour biopsies identified age, estrogen receptor (ER) status and level of immune cell infiltration may together be important for predicting response. Clonal evolution analysis of longitudinally collected tumour samples show subclonal diversity and dynamics are evident with potential therapy resistant subclones detected. The sources of greater pre-treatment immunogenicity associated with a pathological complete response is largely unexplored in HER2+ tumours. However, here we point to the possibility of APOBEC associated mutagenesis, specifically in the ER-neg/HER2+ subtype as a potential mediator of this immunogenic phenotype.

A Simple Synthesis of Reduction-Responsive Acrylamide-Type Nanogels for miRNA Delivery.

MicroRNAs (miRNAs) have great therapeutic potential; however, their delivery still faces huge challenges, especially given the short half-life of naked miRNAs due to rapid hydrolysis or inactivation by abundant nucleases in the systemic circulation. Therefore, the search for reliable miRNA delivery systems is crucial. Nanogels are one of the more effective nanocarriers because they are biocompatible and have a high drug-loading capacity. In this study, acrylamide-based nanogels containing cationic groups and redox-sensitive crosslinkers were developed for cellular delivery of anti-miR21 (a-miR21). To achieve this, post-polymerization loading of a-miR21 oligonucleotides into nanogels was performed by utilizing the electrostatic interaction between positively charged nanogels and negatively charged oligonucleotides. Different molar ratios of the amine groups (N) on the cationic nanogel and phosphate groups (P) on the miRNA were investigated. An N/P ratio of 2 allowed high miRNA loading capacity (MLC, 6.7% w/w) and miRNA loading efficiency (MLE, 99.7% w/w). Successful miRNA loading was confirmed by dynamic light scattering (DLS) and electrophoretic light scattering (ELS) measurements. miRNA-loaded nanogels (NG/miRNA) formed stable dispersions in biological media and showed an enhanced miRNA release profile in the presence of glutathione (GSH). Moreover, the addition of heparin to dissociate the miRNA from the cationic nanogels resulted in the complete release of miRNA. Lastly, a cell uptake study indicated that NG/miRNA could be easily taken up by cancer cells.

The tetraspanin transmembrane protein CD53 mediates dyslipidemia and integrates inflammatory and metabolic signaling in hepatocytes.

Tetraspanins are transmembrane signaling and proinflammatory proteins. Prior work demonstrates that the tetraspanin, CD53/TSPAN25/MOX44, mediates B-cell development and lymphocyte migration to lymph nodes and is implicated in various inflammatory diseases. However, CD53 is also expressed in highly metabolic tissues, including adipose and liver; yet its function outside the lymphoid compartment is not defined. Here, we show that CD53 demarcates the nutritional and inflammatory status of hepatocytes. High-fat exposure and inflammatory stimuli induced CD53 in vivo in liver and isolated primary hepatocytes. In contrast, restricting hepatocyte glucose flux through hepatocyte glucose transporter 8 deletion or through trehalose treatment blocked CD53 induction in fat- and fructose-exposed contexts. Furthermore, germline CD53 deletion in vivo blocked Western diet-induced dyslipidemia and hepatic inflammatory transcriptomic activation. Surprisingly, metabolic protection in CD53 KO mice was more pronounced in the presence of an inciting inflammatory event. CD53 deletion attenuated tumor necrosis factor alpha-induced and fatty acid + lipopolysaccharide-induced cytokine gene expression and hepatocyte triglyceride accumulation in isolated murine hepatocytes. In vivo, CD53 deletion in nonalcoholic steatohepatitis diet-fed mice blocked peripheral adipose accumulation and adipose inflammation, insulin tolerance, and liver lipid accumulation. We then defined a stabilized and trehalase-resistant trehalose polymer that blocks hepatocyte CD53 expression in basal and over-fed contexts. The data suggest that CD53 integrates inflammatory and metabolic signals in response to hepatocyte nutritional status and that CD53 blockade may provide a means by which to attenuate pathophysiology in diseases that integrate overnutrition and inflammation, such as nonalcoholic steatohepatitis and type 2 diabetes.

pH and Reduction Dual-Responsive Nanogels as Smart Nanocarriers to Resist Doxorubicin Aggregation.

The use of smart nanocarriers that can modulate therapeutic release aided by biological cues can prevent undesirable cytotoxicity caused by the premature release of cytotoxic drugs during nanocarrier circulation. In this report, degradable nanocarriers based on pH/reduction dual-responsive nanogels were synthesized to encapsulate doxorubicin hydrochloride (DOX) and specifically boost the release of DOX in conditions characteristic of the cancer microenvironment. Nanogels containing anionic monomer 2-carboxyethyl acrylate (CEA) and N,N'-bis(acryloyl)cystamine (CBA) as a degradable crosslinker have been successfully synthesized via photoinitiated free radical polymerization. The loading process was conducted after polymerization by taking advantage of the electrostatic interaction between the negatively charged nanogels and the positively charged DOX. In this case, a high drug loading capacity (DLC) of up to 27.89% was achieved. The entrapment of DOX into a nanogel network could prevent DOX from aggregating in biological media at DOX concentrations up to ~160 µg/mL. Anionic nanogels had an average hydrodynamic diameter (dH) of around 90 nm with a negative zeta (ζ) potential of around -25 mV, making them suitable for targeting cancer tissue via the enhanced permeation effect. DOX-loaded nanogels formed a stable dispersion in different biological media, including serum-enriched cell media. In the presence of glutathione (GSH) and reduced pH, drug release was enhanced, which proves dual responsivity. An in vitro study using the HCT 116 colon cancer cell line demonstrated the enhanced cytotoxic effect of the NG-CBA/DOX-1 nanogel compared to free DOX. Taken together, pH/reduction dual-responsive nanogels show promise as drug delivery systems for anticancer therapy.

CTCF shapes chromatin structure and gene expression in health and disease.

CCCTC-binding factor (CTCF) is an eleven zinc finger (ZF), multivalent transcriptional regulator, that recognizes numerous motifs thanks to the deployment of distinct combinations of its ZFs. The great majority of the ~50,000 genomic locations bound by the CTCF protein in a given cell type is intergenic, and a fraction of these sites overlaps with transcriptional enhancers. Furthermore, a proportion of the regions bound by CTCF intersect genes and promoters. This suggests multiple ways in which CTCF may impact gene expression. At promoters, CTCF can directly affect transcription. At more distal sites, CTCF may orchestrate interactions between regulatory elements and help separate eu- and heterochromatic areas in the genome, exerting a chromatin barrier function. In this review, we outline how CTCF contributes to the regulation of the three-dimensional structure of chromatin and the formation of chromatin domains. We discuss how CTCF binding and architectural functions are regulated. We examine the literature implicating CTCF in controlling gene expression in development and disease both by acting as an insulator and a factor facilitating regulatory elements to efficiently interact with each other in the nuclear space.

Trehalose-releasing nanogels: A step toward a trehalose delivery vehicle for autophagy stimulation.

Trehalose has been widely studied as a treatment for a variety of human disorders due to its ability to stimulate autophagy. Trehalose, however, is poorly adsorbed and is hydrolyzed in the intestinal mucosa, and oral delivery requires relatively high doses to induce autophagy. The parenteral injection of trehalose-releasing nanogels proposed in this study offers an alternative mode of delivery. This study aimed to develop stable colloidal dispersions of trehalose-rich nanogels that could sustainably release trehalose under physiologically relevant conditions. The nanogel design was based on the covalent incorporation of 6-O-acryloyl-trehalose within a polymer network. A series of nine trehalose-rich nanogels with highly conjugated trehalose (up to 59 % w/w) were synthesized and shown to sustainably release trehalose at a rate that is not dose dependent. The nanogels were optimized to keep colloidal stability in serum-enriched cell culture media. The stable nanogels were not cytotoxic to primary HUVECs. Two selected nanogels with opposite surface charges were subjected to extended in vitro characterization that included a cellular uptake study and a hemocompatibility assay. Both nanogels were efficiently taken up by HUVECs during a short incubation. They also proved not to be hemolytic to human RBCs in concentrations up to 2.0 mg/mL. Finally, an in vivo autophagy stimulation study employing transgenic zebrafish and Drosophila larvae demonstrated that prolonged exposure to a cationic trehalose-releasing nanogel can induce autophagic activity in in vivo systems without any detectable toxicity.

Ultrasound-guided posterior quadratus lumborum block for postoperative pain control after minimally invasive radical prostatectomy: a randomized, double-blind, placebo-controlled trial.

A minimally invasive approach to radical prostatectomy offers improved ambulation and discharge times. Postoperative pain control is one of the key factors that facilitates rapid recovery. With the aim to assure adequate analgesia and minimize the use of opioids, application of truncal nerve blocks has been proposed in a number of endoscopic procedures. The aim of this double-blind, placebo-controlled study was to evaluate the efficacy of bilateral posterior quadratus lumborum block (pQLB) in alleviating pain and reducing postoperative opioid demand in patients following endoscopic extraperitoneal and laparoscopic prostatectomy. We enrolled 50 patients who were diagnosed with prostate cancer and scheduled for prostatectomy. They were randomized to receive preoperative, ultrasound-guided pQLB with the use of either 30 ml of 0.375 % ropivacaine (ropivacaine group) or 30 ml of 0.9 % NaCl (placebo group). Our primary endpoint was opioid consumption in the first 24 hours after surgery. Secondary endpoints were pain intensity at predefined timepoints and the incidence of nausea and vomiting and pruritus. No differences were detected between the ropivacaine and placebo groups in intravenous oxycodone consumption during the first 24 hours after surgery. Similarly, there were no differences in pain intensity at any of the timepoints assessed. The rate of nausea and vomiting was equal in both groups and pruritus was not observed. Application of bilateral pQLB does not reduce opioid consumption after minimally invasive prostatectomy.

Analysis of Antidepressant, Benzodiazepine Anxiolytic, and Hypnotic Use When Treating Depression, Anxiety, and Aggression in Pain Clinic Patients Treated for Neuropathic Pain.

Depression, anxiety, and aggression accompany neuropathic pain. Effective treatment of these comorbidities enhances the outcomes of pain management. Therefore, the study was designed to analyze the relationship between the intensity of depression, anxiety, and aggression and the pharmacotherapy applied in the daily practice of treating neuropathic pain. The aim of the study was to evaluate the frequency of using antidepressants (ADs), benzodiazepine anxiolytics (BDAs), and hypnotics, and the influence of administering these on the intensity of depression, anxiety, and aggression in patients diagnosed with neuropathic pain. A multi-center survey was conducted among 421 patients. An evaluation of the severity of depression, anxiety, and aggression was made using the Hospital Anxiety and Depression Scale-Modified Version (HADS-M). Among the patients treated due to neuropathic pain, ADs are used much more often than BDAs and hypnotics. Depression was well controlled, while anxiety was identified as a possible uncontrolled therapeutic problem in these patients, despite the correlation between the frequency of AD and hypnotics usage and the severity of anxiety. We also found that women show a higher level of intensity in both anxiety and depression, but this does not influence the frequency of their being administered ADs, BDAs, and hypnotics.

Psychometric validation of the Polish version of the Central Sensitization Inventory in subjects with chronic spinal pain.

BACKGROUND: Central sensitization is an amplification of neuronal signaling within the central nervous system. The Central Sensitization Inventory was introduced in 2012. A Polish version of the CSI (CSI-Pol) was developed in 2019, but it was not psychometrically validated. The aim of this study was to validate the CSI-Pol in a sample of Polish-speaking patients with chronic spinal pain and compare them with a group of healthy control subjects. METHODS: The CSI-Pol was administered to 151 patients with chronic spinal pain recruited from two centers. It was re-administered 7 days later. The psychometric properties were then evaluated, including test-retest reliability, construct validity, factor structure and internal consistency. We correlated the CSI-Pol with functional scales, depression and social support scales and compared CSI-Pol scores in the clinical subjects with 30 healthy control subjects recruited from medical staff and their families. RESULTS: The CSI-Pol demonstrated excellent internal consistency (Cronbach's α =0,933) and test-retest reliability (Intraclass Correlation Coefficients - ICC =0.96), as well as significant positive associations with other patient-reported scales, including the Neck Disability Index (r = 0.593), Revised Oswestry Low Back Pain Disability Questionnaire (r = 0.422), and other measures of functional and depressive states. An exploratory factor analysis resulted in a 4-factor model. CSI-Pol scores in the clinical sample (35.27 ± 17.25) were significantly higher than the control sample (23.3 ± 8.9). CONCLUSION: The results of this study suggest that the CSI-Pol may be a useful clinical tool for assessing central sensitization related symptoms and guiding appropriate treatment in Polish-speaking patients with spinal pain.

The Core-Shell Structure, Not Sugar, Drives the Thermal Stabilization of Single-Enzyme Nanoparticles.

Trehalose is widely assumed to be the most effective sugar for protein stabilization, but exactly how unique the structure is and the mechanism by which it works are still debated. Herein, we use a polyion complex micelle approach to control the position of trehalose relative to the surface of glucose oxidase within cross-linked and non-cross-linked single-enzyme nanoparticles (SENs). The distribution and density of trehalose molecules in the shell can be tuned by changing the structure of the underlying polymer, poly(N-[3-(dimethylamino)propyl] acrylamide (PDMAPA). SENs in which the trehalose is replaced with sucrose and acrylamide are prepared as well for comparison. Isothermal titration calorimetry, dynamic light scattering, and asymmetric flow field-flow fraction in combination with multiangle light scattering reveal that two to six polymers bind to the enzyme. Binding either trehalose or sucrose close to the enzyme surface has very little effect on the thermal stability of the enzyme. By contrast, encapsulation of the enzyme within a cross-linked polymer shell significantly enhances its thermal stability and increases the unfolding temperature from 70.3 °C to 84.8 °C. Further improvements (up to 92.8 °C) can be seen when trehalose is built into this shell. Our data indicate that the structural confinement of the enzyme is a far more important driver in its thermal stability than the location of any sugar.

Noninvasive celiac plexus radiosurgery in palliative treatment for patients with symptomatic pancreatic cancer.

Advanced pancreatic cancer is commonly associated with significant visceral pain, radiating in a belt-like distribution to the upper abdomen, referring to the lower back, and significantly affecting patients' quality of life (QoL). The pain is often poorly controlled by pharmacotherapy, or the doses necessary to control the pain produce substantial adverse effects. Other available pain management options include invasive celiac plexus block or neurolysis, palliative radiotherapy, and systemic chemotherapy, all with limited efficacy. In this case report, we present the first non-invasive celiac plexus radiosurgery performed in Europe in a patient with pancreatic cancer, demonstrating that significant pain relief can be achieved through a non-invasive procedure performed within 2 outpatient visits.

Chromatin Alterations in Neurological Disorders and Strategies of (Epi)Genome Rescue.

Neurological disorders (NDs) comprise a heterogeneous group of conditions that affect the function of the nervous system. Often incurable, NDs have profound and detrimental consequences on the affected individuals' lives. NDs have complex etiologies but commonly feature altered gene expression and dysfunctions of the essential chromatin-modifying factors. Hence, compounds that target DNA and histone modification pathways, the so-called epidrugs, constitute promising tools to treat NDs. Yet, targeting the entire epigenome might reveal insufficient to modify a chosen gene expression or even unnecessary and detrimental to the patients' health. New technologies hold a promise to expand the clinical toolkit in the fight against NDs. (Epi)genome engineering using designer nucleases, including CRISPR-Cas9 and TALENs, can potentially help restore the correct gene expression patterns by targeting a defined gene or pathway, both genetically and epigenetically, with minimal off-target activity. Here, we review the implication of epigenetic machinery in NDs. We outline syndromes caused by mutations in chromatin-modifying enzymes and discuss the functional consequences of mutations in regulatory DNA in NDs. We review the approaches that allow modifying the (epi)genome, including tools based on TALENs and CRISPR-Cas9 technologies, and we highlight how these new strategies could potentially change clinical practices in the treatment of NDs.

Identification of Toxocara canis Antigen-Interacting Partners by Yeast Two-Hybrid Assay and a Putative Mechanism of These Host-Parasite Interactions.

Toxocara canis is a zoonotic roundworm that infects humans and dogs all over the world. Upon infection, larvae migrate to various tissues leading to different clinical syndromes. The host-parasite interactions underlying the process of infection remain poorly understood. Here, we describe the application of a yeast two-hybrid assay to screen a human cDNA library and analyse the interactome of T. canis larval molecules. Our data identifies 16 human proteins that putatively interact with the parasite. These molecules were associated with major biological processes, such as protein processing, transport, cellular component organisation, immune response and cell signalling. Some of these identified interactions are associated with the development of a Th2 response, neutrophil activity and signalling in immune cells. Other interactions may be linked to neurodegenerative processes observed during neurotoxocariasis, and some are associated with lung pathology found in infected hosts. Our results should open new areas of research and provide further data to enable a better understanding of this complex and underestimated disease.

Mirogabalin-A Novel Selective Ligand for the α2δ Calcium Channel Subunit.

The efficacy of neuropathic pain control remains unsatisfactory. Despite the availability of a variety of therapies, a significant proportion of patients suffer from poorly controlled pain of this kind. Consequently, new drugs and treatments are still being sought to remedy the situation. One such new drug is mirogabalin, a selective ligand for the α2δ subunits of voltage-gated calcium channels (VGCC) developed by Sankyo group for the management of neuropathic pain. In 2019 in Japan, mirogabalin was approved for peripheral neuropathic pain following the encouraging results of clinical trials conducted with diabetic peripheral neuropathic pain (DPNP) and postherpetic neuralgia (PHN) patients. The ligand selectivity of mirogabalin for α2δ-1 and α2δ-2 and its slower dissociation rate for α2δ-1 than for α2δ-2 subunits of VGCC may contribute to its strong analgesic effects, wide safety margin, and relatively lower incidence of adverse effects compared to pregabalin and gabapentin. This article discusses the mechanism of action of mirogabalin, presents data on its pharmacodynamics and pharmacokinetics, and reviews the available experimental and clinical studies that have assessed the efficacy and safety of the drug in the treatment of selected neuropathic pain syndromes.

Guidelines of the Polish Society of Anaesthesiology and Intensive Therapy regarding prevention of inadvertent intraoperative hypothermia.

Core body temperature is strictly regulated (± 0.2 °C) and coordinated at the level of central nervous system located in the hypothalamus via several protective effector mechanisms that prevent overcooling and overheating. The central regulation permits both circadian and monthly variations of even 1°C; under normal conditions, however, the activation of effective protective mechanisms prevents even the slightest overcooling and core temperature elevation at any moment of the day.

Microchamber microfluidics combined with thermogellable glycomicrogels - Platform for single cells study in an artificial cellular microenvironment.

Microfluidic technology is a powerful tool to precisely establish artificial microenvironments and has been used to generate numerous biomimetic devices. Here, we present a combined microenvironment platform, which consists of microchamber microfluidics filled with thermoresponsive glycomicrogels, and enables live-cell immobilization and continuous observation. Poly(N-isopropylacrylamide) microgels containing trehalose has been selected from our previous study to possess adequate physicochemical characteristics and provide potential multivalent interactions with cell surfaces. We show that the designed microplatform enables small population of cells to be trapped in individual parallel microchambers and further immobilized in an artificial extracellular matrix. We applied our platform to long-term imaging experiments and studied HeLa cell growth dynamics under continuous, diffusion-dominated medium exchange. The mathematical modeling revealed that regardless of the initial number of cells, the growth dynamic follows the exponential growth pattern over the analyzed timespan (one week). These results confirm that the presented microsystem facilitates the long-term cell culture in a cellular-mimicking microenvironment without reaching environmental constraints.

Fixed Dose versus Height-Adjusted Conventional Dose of Intrathecal Hyperbaric Bupivacaine for Caesarean Delivery: A Prospective, Double-Blinded Randomised Trial.

The optimal intrathecal dose of local anaesthetic for caesarean section (CS) anaesthesia is still being debated. We performed a study to compare the effectiveness and safety of spinal anaesthesia with 12.5 mg of hyperbaric bupivacaine and a dosing regimen of conventional doses adjusted to parturient height. One hundred and forty parturients scheduled for elective CS were enrolled. The fixed-dose group (FD) received a spinal block with 12.5 mg of hyperbaric bupivacaine with fentanyl, whereas the adjusted-dose group (AD) received a height-adjusted dose of bupivacaine (9-13 mg) with fentanyl. Sensory block ≥ T5 dermatome within 10 min and no need for supplementary analgesia were set as the composite primary outcome (success). Rates of successful blocks and complications were compared. Complete data were available for 134 cases. Spinal anaesthesia was successful in 58 out of 67 patients in the FD group and 57 out of 67 in the AD group (p > 0.05). Eight spinals in each group failed to produce a block ≥ T5 in 10 min, and one patient in the FD group and two in the AD group required i.v. analgesics despite sensory block ≥ T5. No differences were noted in terms of hypotension, bradycardia and nausea between the FD and AD groups. Compared to the height-adjusted dose regimen based on conventional doses of hyperbaric bupivacaine, the fixed dose regimen of 12.5 mg was equally effective and did not increase the risk of spinal block-related complications.