Upconversion Nanoparticles Intercalated in Large Polymer Micelles for Tumor Imaging and Chemo/Photothermal Therapy.

Frontiers in theranostics are driving the demand for multifunctional nanoagents. Upconversion nanoparticle (UCNP)-based systems activated by near-infrared (NIR) light deeply penetrating biotissue are a powerful tool for the simultaneous diagnosis and therapy of cancer. The intercalation into large polymer micelles of poly(maleic anhydride-alt-1-octadecene) provided the creation of biocompatible UCNPs. The intrinsic properties of UCNPs (core@shell structure NaYF4:Yb3+/Tm3+@NaYF4) embedded in micelles delivered NIR-to-NIR visualization, photothermal therapy, and high drug capacity. Further surface modification of micelles with a thermosensitive polymer (poly-N-vinylcaprolactam) exhibiting a conformation transition provided gradual drug (doxorubicin) release. In addition, the decoration of UCNP micelles with Ag nanoparticles (Ag NPs) synthesized in situ by silver ion reduction enhanced the cytotoxicity of micelles at cell growth temperature. Cell viability assessment on Sk-Br-3, MDA-MB-231, and WI-26 cell lines confirmed this effect. The efficiency of the prepared UCNP complex was evaluated in vivo by Sk-Br-3 xenograft regression in mice for 25 days after peritumoral injection and photoactivation of the lesions with NIR light. The designed polymer micelles hold promise as a photoactivated theranostic agent with quattro-functionalities (NIR absorption, photothermal effect, Ag NP cytotoxicity, and Dox loading) that provides imaging along with chemo- and photothermal therapy enhanced with Ag NPs.

New Cysteine-Containing PEG-Glycerolipid Increases the Bloodstream Circulation Time of Upconverting Nanoparticles.

Upconverting nanoparticles have unique spectral and photophysical properties that make them suitable for development of theranostics for imaging and treating large and deep-seated tumors. Nanoparticles based on NaYF4 crystals doped with lanthanides Yb3+ and Er3+ were obtained by the high-temperature decomposition of trifluoroacetates in oleic acid and 1-octadecene. Such particles have pronounced hydrophobic properties. Therefore, to obtain stable dispersions in aqueous media for the study of their properties in vivo and in vitro, the polyethylene glycol (PEG)-glycerolipids of various structures were obtained. To increase the circulation time of PEG-lipid coated nanoparticles in the bloodstream, long-chain substituents are needed to be attached to the glycerol backbone using ether bonds. To prevent nanoparticle aggregation, an L-cysteine-derived negatively charged carboxy group should be included in the lipid molecule.

Natural and Synthetic Polymer Scaffolds Comprising Upconversion Nanoparticles as a Bioimaging Platform for Tissue Engineering.

Modern biocompatible materials of both natural and synthetic origin, in combination with advanced techniques for their processing and functionalization, provide the basis for tissue engineering constructs (TECs) for the effective replacement of specific body defects and guided tissue regeneration. Here we describe TECs fabricated using electrospinning and 3D printing techniques on a base of synthetic (polylactic-co-glycolic acids, PLGA) and natural (collagen, COL, and hyaluronic acid, HA) polymers impregnated with core/shell ß-NaYF4:Yb3+,Er3+/NaYF4 upconversion nanoparticles (UCNPs) for in vitro control of the tissue/scaffold interaction. Polymeric structures impregnated with core/shell ß-NaYF4:Yb3+,Er3+/NaYF4 nanoparticles were visualized with high optical contrast using laser irradiation at 976 nm. We found that the photoluminescence spectra of impregnated scaffolds differ from the spectrum of free UCNPs that could be used to control the scaffold microenvironment, polymer biodegradation, and cargo release. We proved the absence of UCNP-impregnated scaffold cytotoxicity and demonstrated their high efficiency for cell attachment, proliferation, and colonization. We also modified the COL-based scaffold fabrication technology to increase their tensile strength and structural stability within the living body. The proposed approach is a technological platform for "smart scaffold" development and fabrication based on bioresorbable polymer structures impregnated with UCNPs, providing the desired photoluminescent, biochemical, and mechanical properties for intravital visualization and monitoring of their behavior and tissue/scaffold interaction in real time.

Prevalence of abnormal left ventricular global longitudinal strain by speckle tracking echocardiography and its prognostic value in patients with COVID-19.

IMPORTANCE: Although cardiac injury is a known complication of COVID-19 infection, there is no established tool to predict cardiac involvement and in-hospital mortality in this patient population. OBJECTIVE: To assess if left ventricular global longitudinal strain (LV-GLS) can detect cardiac involvement and be used as a risk-stratifying parameter for hospitalised patients with COVID-19. MAIN OUTCOMES AND MEASURES: In-hospital mortality. RESULTS: We found a statistically significant association between LV-GLS and in-hospital mortality (adjusted OR (aOR)=1.09; 95% CI 1.0 to 1.19, p=0.050). Furthermore, right ventricular fractional area change was significantly associated with in-hospital mortality (aOR=1.04; 95% CI 1.0 to 1.08, p=0.043). Troponin level had no statistically significant association with in-hospital mortality (aOR=3.43; 95% CI 0.78 to 15.03, p=0.101). CONCLUSION AND RELEVANCE: LV-GLS can be a useful parameter for cardiovascular risk assessment in hospitalised patients with COVID-19 infection.

Photoluminescent Scaffolds Based on Natural and Synthetic Biodegradable Polymers for Bioimaging and Tissue Engineering.

Non-invasive visualization and monitoring of tissue-engineered structures in a living organism is a challenge. One possible solution to this problem is to use upconversion nanoparticles (UCNPs) as photoluminescent nanomarkers in scaffolds. We synthesized and studied scaffolds based on natural (collagen-COL and hyaluronic acid-HA) and synthetic (polylactic-co-glycolic acids-PLGA) polymers loaded with ß-NaYF4:Yb3+, Er3+ nanocrystals (21 ± 6 nm). Histomorphological analysis of tissue response to subcutaneous implantation of the polymer scaffolds in BALB/c mice was performed. The inflammatory response of the surrounding tissues was found to be weak for scaffolds based on HA and PLGA and moderate for COL scaffolds. An epi-luminescent imaging system with 975 nm laser excitation was used for in vivo visualization and photoluminescent analysis of implanted scaffolds. We demonstrated that the UCNPs' photoluminescent signal monotonously decreased in all the examined scaffolds, indicating their gradual biodegradation followed by the release of photoluminescent nanoparticles into the surrounding tissues. In general, the data obtained from the photoluminescent analysis correlated satisfactorily with the histomorphological analysis.

Epigenetics of the far northern Yakutian population.

BACKGROUND: Yakuts are one of the indigenous populations of the subarctic and arctic territories of Siberia characterized by a continental subarctic climate with severe winters, with the regular January average temperature in the regional capital city of Yakutsk dipping below - 40 °C. The epigenetic mechanisms of adaptation to such ecologies and environments and, in particular, epigenetic age acceleration in the local population have not been studied before. RESULTS: This work reports the first epigenetic study of the Yakutian population using whole-blood DNA methylation data, supplemented with the comparison to the residents of Central Russia. Gene set enrichment analysis revealed, among others, geographic region-specific differentially methylated regions associated with adaptation to climatic conditions (water consumption, digestive system regulation), aging processes (actin filament activity, cell fate), and both of them (channel activity, regulation of steroid and corticosteroid hormone secretion). Further, it is demonstrated that the epigenetic age acceleration of the Yakutian representatives is significantly higher than that of Central Russia counterparts. For both geographic regions, we showed that epigenetically males age faster than females, whereas no significant sex differences were found between the regions. CONCLUSIONS: We performed the first study of the epigenetic data of the Yakutia cohort, paying special attention to region-specific features, aging processes, age acceleration, and sex specificity.

Assessment of select synthetic cannabinoid receptor agonist bias and selectivity between the type 1 and type 2 cannabinoid receptor.

The first synthetic cannabinoid receptor agonists (SCRAs) were designed as tool compounds to study the endocannabinoid system's two predominant cannabinoid receptors, CB1R and CB2R. Unfortunately, novel SCRAs now represent the most rapidly proliferating novel psychoactive substances (NPS) of abuse globally. Unlike ∆9-tetrahydrocannabinol, the CB1R and CB2R partial agonist and the intoxicating constituent of Cannabis, many SCRAs characterized to date are full agonists of CB1R. Gaining additional insight into the pharmacological activity of these SCRAs is critical to assess and regulate NPSs as they enter the marketplace. The purpose of this study was to assess select SCRAs recently identified by Canadian police, border service agency, private companies and the illicit market as potential CB1R and CB2R agonists. To this end, fifteen SCRAs were screened for in vitro activity and in silico interactions at CB1R and CB2R. Several SCRAs were identified as being highly biased for cAMP inhibition or ßarrestin2 recruitment and receptor subtype selectivity between CB1R and CB2R. The indazole ring and halogen-substituted butyl or pentyl moieties were identified as two structural features that may direct ßarrestin2 bias. Two highly-biased SCRAs-JWH-018 2'-napthyl-N-(3-methylbutyl) isomer (biased toward cAMP inhibition) and 4-fluoro MDMB-BINACA (biased toward ßarrestin2 recruitment) displayed unique and differential in vivo activity in mice. These data provide initial insight into the correlations between structure, signalling bias, and in vivo activity of the SCRAs.

Effects of the noradrenergic system in rat white matter exposed to oxygen-glucose deprivation in vitro.

Norepinephrine (NE) is released in excess into the extracellular space during oxygen-glucose deprivation (OGD) in brain, increasing neuronal metabolism and aggravating glutamate excitoxicity. We used isolated rat optic nerve and spinal cord dorsal columns to determine whether the noradrenergic system influences axonal damage in white matter. Tissue was studied electrophysiologically by recording the compound action potential (CAP) before and after exposure to 60 min of OGD at 36 degrees C. Depleting catecholamine stores with reserpine was protective and improved CAP recovery after 1 h of reperfusion from 17% (control) to 35%. Adding NE during OGD decreased CAP recovery to 8%, and adding NE to reserpine during OGD eliminated the protective effect of the latter. Selective inhibitors of Na(+)-dependent norepinephrine transport desipramine and nisoxetine improved recovery to 58% and 44%, respectively. alpha2 adrenergic receptor agonists UK14,304 and medetomidine improved CAP recovery to 41% and 46% after 1 h of OGD. Curiously, alpha2 antagonists alone were also highly protective (e.g., atipamezole: 86% CAP recovery), at concentrations that did not affect baseline excitability. The protective effect of alpha2 receptor modulation was corroborated by imaging fluorescent Ca(2+) and Na(+) indicators within axons during OGD. Both agonists and antagonists significantly reduced axonal Ca(2+) and Na(+) accumulation in injured axons. These data suggest that the noradrenergic system plays an active role in the pathophysiology of axonal ischemia and that alpha2 receptor modulation may be useful against white matter injury.

Depolarization-induced Ca2+ release in ischemic spinal cord white matter involves L-type Ca2+ channel activation of ryanodine receptors.

The mechanisms of Ca(2+) release from intracellular stores in CNS white matter remain undefined. In rat dorsal columns, electrophysiological recordings showed that in vitro ischemia caused severe injury, which persisted after removal of extracellular Ca(2+); Ca(2+) imaging confirmed that an axoplasmic Ca(2+) rise persisted in Ca(2+)-free perfusate. However, depletion of Ca(2+) stores or reduction of ischemic depolarization (low Na(+), TTX) were protective, but only in Ca(2+)-free bath. Ryanodine or blockers of L-type Ca(2+) channel voltage sensors (nimodipine, diltiazem, but not Cd(2+)) were also protective in zero Ca(2+), but their effects were not additive with ryanodine. Immunoprecipitation revealed an association between L-type Ca(2+) channels and RyRs, and immunohistochemistry confirmed colocalization of Ca(2+) channels and RyR clusters on axons. Similar to "excitation-contraction coupling" in skeletal muscle, these results indicate a functional coupling whereby depolarization sensed by L-type Ca(2+) channels activates RyRs, thus releasing damaging amounts of Ca(2+) under pathological conditions in white matter.

Na+-dependent sources of intra-axonal Ca2+ release in rat optic nerve during in vitro chemical ischemia.

The contribution of intracellular stores to axonal Ca2+ overload during chemical ischemia in vitro was examined by confocal microscopy. Ca2+ accumulation was measured by fluo-4 dextran (low-affinity dye, KD approximately 4 microM) or by Oregon Green 488 BAPTA-1 dextran (highaffinity dye, KD approximately 450 nM). Axonal Na+ was measured using CoroNa Green. Ischemia in CSF containing 2 mM Ca2+ caused an approximately 3.5-fold increase in fluo-4 emission after 30 min, indicating a large axonal Ca2+ rise well into the micromolar range. Axonal Na+ accumulation was enhanced by veratridine and reduced, but not abolished, by TTX. Ischemia in Ca2+-free (plus BAPTA) perfusate resulted in a smaller but consistent Ca2+ increase monitored by Oregon Green 488 BAPTA-1, indicating release from intracellular sources. This release was eliminated in large part when Na+ influx was reduced by replacement with N-methyl-D-glucamine (NMDG+; even in depolarizing high K+ perfusate), Li+, or by the application of TTX and significantly increased by veratridine. Intracellular release also was reduced significantly by neomycin or 1-(6-[(17beta-methoxyestra-1,3,5 [10]-trien-17-yl) amino] hexyl)-1H-pyrrole-2,5-dione (U73122 [GenBank]) (phospholipase C inhibitors), heparin [inositol trisphosphate (IP3) receptor blocker], or 7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one (CGP37157; mitochondrial Na+/Ca2+ exchange inhibitor) as well as ryanodine. Combining CGP37157 with U73122 [GenBank] or heparin decreased the response more than either agent alone and significantly improved electrophysiological recovery. Our conclusion is that intra-axonal Ca2+ release during ischemia in rat optic nerve is mainly dependent on Na+ influx. This Na+ accumulation stimulates three distinct intra-axonal sources of Ca2+: (1) the mitochondrial Na+/Ca2+ exchanger driven in the Na+ import/Ca2+ export mode, (2) positive modulation of ryanodine receptors, and (3) promotion of IP3 generation by phospholipase C.

Spatiotemporal distribution of spectrin breakdown products induced by anoxia in adult rat optic nerve in vitro.

Hypoxic/ischemic and traumatic injury to central nervous system myelinated axons is heavily dependent on accumulation of Ca ions in the axoplasm, itself promoted by Na influx from the extracellular space. Given the high density of nodal Na channels, we hypothesized that nodes of Ranvier might be particularly vulnerable to Ca overload and subsequent damage, as this is the expected locus of maximal Na influx. Adult rat optic nerves were exposed to in vitro anoxia and analyzed immunohistochemically for the presence of spectrin breakdown. Cleavage of spectrin became detectable between 15 and 30 mins of anoxia, and increased homogeneously along the lengths of fibers; localized breakdown was not observed at nodes of Ranvier at any time point analyzed. Spectrin breakdown was also found in glial processes surrounding axons. Confocal imaging of axoplasmic Ca also revealed a gradual and nonlocalized increase as anoxia progressed, without evidence of Ca 'hot-spots' anywhere along the axons at any time between 0 and 30 mins of anoxic exposure in vitro. Calculations of Ca diffusion rates indicated that even if Ca entered or was released focally in axons, this ion would diffuse rapidly into the internodes and likely produce diffuse injury by activating Ca-dependent proteases. Western blot analysis for voltage-gated Na channel protein revealed that key functional proteins such as these are also degraded by anoxia/ischemia. Thus, proteolysis of structural and functional proteins will conspire to irreversibly injure central axons and render them nonfunctional, eventually leading to transection, degradation, and Wallerian degeneration.

Caspase inhibitor z-DEVD-fmk attenuates calpain and necrotic cell death in vitro and after traumatic brain injury.

In studies designed to evaluate the therapeutic window for treatment of traumatic brain injury, the caspase 3 inhibitor z-DEVD-fmk improved neurologic function and reduced lesion volumes when administered at 1 but not at 4, 8, or 24 hours after injury. Moreover, neither caspase 3 nor PARP, a caspase 3 substrate, were cleaved in injured, untreated cortex from 1 to 72 hours after injury. Few cortical neurons expressed active caspase 3 or were TUNEL positive from 6 to 24 hours after injury, and TUNEL staining was primarily Type I (necrotic). Nissl staining revealed extensive neuronal necrosis in the injured cortex from 6 to 24 hours after impact. Considered together, these data suggested that z-DEVD-fmk may reduce neuronal necrosis, so we used an in vitro model of necrotic cell death induced by maitotoxin to test this further and explore the potential mechanism(s) involved. Z-DEVD-fmk (1 nM-100 microM) significantly attenuated maitotoxin induced neuronal cell death and markedly reduced expression of the 145 kD calpain-mediated alpha-spectrin breakdown product after maitotoxin injury. Neither the 120 kD caspase-mediated alpha-spectrin cleavage product nor cathepsin B were expressed after maitotoxin injury. In a cell free assay, z-DEVD-fmk reduced hydrolysis of casein by purified calpain I. Finally, z-DEVD-fmk reduced expression of the 145 kD calpain-mediated alpha-spectrin cleavage fragment after traumatic brain injury in vivo. These data suggest that neuroprotection by z-DEVD-fmk may, in part, reflect inhibition of calpain-related necrotic cell death.

Neuroprotective and nootropic actions of a novel cyclized dipeptide after controlled cortical impact injury in mice.

1-ARA-35b (35b) is a cyclized dipeptide that shows considerable neuroprotective activity in vitro and improves neurologic recovery after fluid percussion-induced traumatic brain injury in rats. The authors evaluated the effects of treatment with 35b in mice subjected to controlled cortical impact brain injury. Animals treated with intravenous 35b after traumatic injury showed significantly enhanced recovery of beam walking and place learning functions compared with vehicle-treated controls, in addition to reduced lesion volumes. Beneficial effects were dose related and showed an inverted U-shaped dose-response curve between 0.1 and 10 mg/kg. Protective actions were found when the drug was administered initially at 30 minutes or 1, 4, or 8 hours, but not at 24 hours, after trauma. In separate experiments, rats treated with 35b on days 7 through 10 after injury showed remarkably improved place learning in comparison with injured controls. These studies confirm and extend the neuroprotective effects of this diketopiperazine in traumatic brain injury. In addition, they show that 35b has a relatively wide therapeutic window and improves cognitive function after both acute and chronic injury.

Multiple caspases are activated after traumatic brain injury: evidence for involvement in functional outcome.

Caspase-3 is a cysteine protease that is strongly implicated in neuronal apoptosis. Activation of caspase-3 may be induced by at least two major initiator pathways: a caspase-8-mediated pathway activated through cell surface death receptors (extrinsic pathway), and a caspase-9-mediated pathway activated by signals from the mitochondria that lead to formation of an apoptosomal complex (intrinsic pathway). In the present studies, we compare the activation of caspases-3, -8, and -9 after lateral fluid-percussion traumatic brain injury (TBI) in rats. Immunoblot analysis identified cleaved forms of caspases-3 and -9, but not caspase-8, at 1, 12, and 48 h after injury. Immunocytochemistry specific for cleaved caspases-3 and -9 revealed their expression primarily in neurons. These caspases were also frequently localized in TUNEL-positive cells, some of which demonstrated morphological features of apoptosis. However, caspases-3 and -9 were also found in neurons that were not TUNEL-positive, and other TUNEL-positive cells did not show activated caspases. In contrast to caspases-3 or -9, caspase-8 expression was only minimally changed by injury. An increase in expression of this caspase was undetectable by immunoblotting methods, and appeared as positive immunostaining restricted to a few cells within the injured cortex. Treatment with the pan-caspase inhibitor z-VAD-fmk at 15 min after TBI improved performance on motor and spatial learning tests. These data suggest that several caspases may be involved in the pathophysiology of TBI and that pan-caspase inhibition strategies may improve neurological outcomes.