Chemical Targeting of Rhodol Voltage Sensitive Dyes to Dopaminergic Neurons

Optical imaging of changes in membrane potential of living cells can be achieved by the means of fluorescent voltage sensitive dyes (VSDs). A particularly challenging task is to efficiently deliver these highly lipophilic probes to specific neuronal subpopulations in brain tissue. We have tackled this task by designing a solubilizing, hydrophilic polymer platform that carries a high-affinity ligand for a membrane protein marker of interest and a fluorescent VSD. Here, we disclose an improved design of polymer supported probes for chemical, non-genetic targeting of voltage sensors to axons natively expressing the dopamine transporter in ex vivo mouse brain tissue. We first show that for negatively charged rhodol VSDs functioning on the photoinduced electron transfer principle, poly(ethylene glycol) (PEG) as a carrier enables targeting with higher selectivity than the polysaccharide dextran in HEK cell culture. In the same experimental setting, we also demonstrate that incorporation of an azetidine ring in the rhodol chromophore substantially increases the brightness and voltage sensitivity of the respective VSD. We show that the superior properties of the optimized sensor are transferable to recording of electrically evoked activity from dopaminergic axons in mouse striatal slices after averaging of multiple trials. Finally, we suggest the next milestones for the field to achieve single-scan recordings with non-genetically targeted VSDs in native brain tissue.

Hybrid-Integrated Biosensor for Express Determination of Protein Markers of Diseases based on Molecular Recognition and Direct Fluorimetric Detection

Ways of creating new generation biosensors for multiparametric express diagnostics based on molecular recognition and direct fluorimetric registration of a peptide aptamer — protein marker complex were considered. The biosensor platform comprises a microfluidic channel for delivery sample solutions, coupled with flow-through zones containing covalently attached arrays of peptide probes — aptamers. An outer glass window of the biochip assembly contains a layer of luminophore ZnS:Cu, bound on it via an acrylic lacquer and intended for the re-emitting native fluorescence of bound proteins into the longer wavelength range, more efficient in registering signals with CMOS sensors. The aptamers were designed using "Protein 3D" program for analysis of spatial complementarity of protein structures. The peptide, complementary to Troponin T, was modified by replacement of aromatic amino acid residue while maintaining the spatial configuration. The complementarity of peptide and Troponin T was confirmed using a capillary electrophoresis-on-a-chip. Biosensors are manufactured using thick-film technology and photolithography. The fluorescence of marker proteins was excited using UV-LED with a radiation wavelength of 275 nm. The limit of detection achieved for Troponin T was 6 ng/ml.

Gold nanoclusters conjugated berberine reduce inflammation and alleviate neuronal apoptosis by mediating M2 polarization for spinal cord injury repair

Spinal cord injury (SCI) leads to nerve cell apoptosis and loss of motor function. Herein, excessive activation of the M1 phenotype macrophages/microglia is found to be the main reason for the poor prognosis of SCI, but the selective activation phenotype (M2) macrophages/microglia facilitates the recovery of SCI. Thereafter, we used gold nanoclusters loaded berberine (BRB-AuNCs) to reduce inflammation by inhibiting the activation of M1 phenotype macrophages/microglia, which simultaneously inhibited neuronal apoptosis after SCI. In vitro and in vivo experiments showed that BRB-AuNCs reduced M1 protein marker CD86, increased M2 protein marker CD206, reduced inflammation and apoptotic cytokines (IL-1β, IL-6, TNF-α, Cleaved Caspase-3, Bax). These results indicate that BRB-AuNCs have excellent anti-inflammatory and anti-apoptotic effects by inducing the polarization of macrophages/microglia from M1 phenotype to M2 phenotype. Thereafter, the motor functions of SCI rats were significantly improved after treatment with BRB-AuNCs. This work not only provides a new way for the treatment of SCI but also broadens BRB utilization strategies.

Quantitative single-cell analysis of immunofluorescence protein multiplex images illustrates biomarker spatial heterogeneity within breast cancer subtypes

Background The extent of cellular heterogeneity in breast cancer could have potential impact on diagnosis and long-term outcome. However, pathology evaluation is limited to biomarker immunohistochemical staining and morphology of the bulk cancer. Inter-cellular heterogeneity of biomarkers is not usually assessed. As an initial evaluation of the extent of breast cancer cellular heterogeneity, we conducted quantitative and spatial imaging of Estrogen Receptor (ER), Progesterone Receptor (PR), Epidermal Growth Factor Receptor-2 (HER2), Ki67, TP53, CDKN1A (P21/WAF1), CDKN2A (P16INK4A), CD8 and CD20 of a tissue microarray (TMA) representing subtypes defined by St. Gallen surrogate classification. Methods Quantitative, single cell-based imaging was conducted using an Immunofluorescence protein multiplexing platform (MxIF) to study protein co-expression signatures and their spatial localization patterns. The range of MxIF intensity values of each protein marker was compared to the respective IHC score for the TMA core. Extent of heterogeneity in spatial neighborhoods was analyzed using co-occurrence matrix and Diversity Index measures. Results On the 101 cores from 59 cases studied, diverse expression levels and distributions were observed in MxIF measures of ER and PR among the hormonal receptor-positive tumor cores. As expected, Luminal A-like cancers exhibit higher proportions of cell groups that co-express ER and PR, while Luminal B-like (HER2-negative) cancers were composed of ER+, PR- groups. Proliferating cells defined by Ki67 positivity were mainly found in groups with PR-negative cells. Triple-Negative Breast Cancer (TNBC) exhibited the highest proliferative fraction and incidence of abnormal P53 and P16 expression. Among the tumors exhibiting P53 overexpression by immunohistochemistry, a group of TNBC was found with much higher MxIF-measured P53 signal intensity compared to HER2+, Luminal B-like and other TNBC cases. Densities of CD8 and CD20 cells were highest in HER2+ cancers. Spatial analysis demonstrated variability in heterogeneity in cellular neighborhoods in the cancer and the tumor microenvironment. Conclusions Protein marker multiplexing and quantitative image analysis demonstrated marked heterogeneity in protein co-expression signatures and cellular arrangement within each breast cancer subtype. These refined descriptors of biomarker expressions and spatial patterns could be valuable in the development of more informative tools to guide diagnosis and treatment.

Macrophages expressing uncoupling protein 1 increase in adipose tissue in response to cold in humans

Acute cold induces beige adipocyte protein marker expression in human subcutaneous white adipose tissue (SC WAT) from both the cold treated and contralateral leg, and the immune system regulates SC WAT beiging in mice. Cold treatment significantly increased the gene expression of the macrophage markers CD68 and 86 in SC WAT. Therefore, we comprehensively investigated the involvement of macrophages in SC WAT beiging in lean and obese humans by immunohistochemistry. Cold treatment significantly increased CD163/CD68 macrophages in SC WAT from the cold treated and contralateral legs of lean and obese subjects, and had similar effects on CD206/CD68 macrophages, whereas the effects on CD86/CD68 macrophages were inconsistent between lean and obese. However, linear regression analysis did not find significant relationships between the change in macrophage numbers and the change in UCP1 protein abundance. A high percentage of CD163 macrophages in SC WAT expressed UCP1, and these UCP1 expressing CD163 macrophages were significantly increased by cold treatment in SC WAT of lean subjects. In conclusion, our results suggest that CD163 macrophages are involved in some aspect of the tissue remodeling that occurs during SC WAT beiging in humans after cold treatment, but they are likely not direct mediators of the beiging process.

Following the SPARC: tracking Secreted Protein Acidic and Rich in Cysteine associated with LX-2´s extracellular vesicles as a non-destructive modality to evaluate lipid-based antifibrotic treatments

Uncovering the complex cellular mechanisms underlying hepatic fibrogenesis, a highly dynamic and active process ultimately responsible for liver failure if left untreated, could expedite the development of effective treatments and noninvasive diagnostic modalities for this often silent pathology. The biochemical complexity of extracellular vesicles (EVs) and their role in intercellular communication make them an attractive tool to look for biomarkers that might become a viable alternative to invasive liver biopsies. We developed a solid set of methods to isolate and characterize EVs from differently treated human hepatic stellate cell (HSC) line LX-2 in vitro, and we investigated the biological effect they exert onto naïve LX-2, proving that EVs do play an active role in fibrogenesis. Electrical/asymmetric flow field-flow fractionation (EAF4) revealed EV subpopulations with different physicochemical behaviors. Proteomic data from our samples was mined for EV-associated proteins whose expression correlated with HSC treatment. Consequently, we chose the secreted protein acidic and cysteine rich (SPARC), a matricellular protein previously reported to be upregulated in activated HSCs, as a proof-of-concept protein to explore the feasibility of using fluorescence nanoparticle tracking analysis as a non-destructive tool for the determination of HSCs’ fibrogenic phenotype based on EVs. We could thus use EVs to directly evaluate the efficacy of treatment with S80, a lipid rich (>75 %) in polyenylphosphatidylcholines (PPC). We found that PPC-rich S80 reduces the relative presence of SPARC-positive EVs. For the first time, we could correlate the cellular response to lipid-based antifibrotic treatment to the relative presence of a candidate protein marker associated with the released EVs. In addition to providing novel insights into PPC treatments, our findings pave the way for more precise and less invasive diagnostic analyses of hepatic fibrogenesis.

Necrotizing Gingivitis: Microbial Diversity and Quantification of Protein Secretion in Necrotizing Gingivitis

Necrotizing gingivitis (NG) is a necrotizing periodontal disease that differs from chronic gingivitis (CG). To date, both the microbiological causes and the involved host cytokine response of NG still remain unclear. Here, we investigated corresponding interdental plaque and serum samples from two groups of Chinese patients with CG (n = 21) or NG (n = 21). The microbiota were studied by 16S rRNA Illumina MiSeq sequencing of the microbial metagenome and by assessing quantitatively the abundance of the phylum Bacteroidetes, the genus Prevotella and the species T. forsythia, P. endodontalis, and P. gingivalis using fluorescence in situ hybridization (FISH). With respect to the associated host response, the levels of 30 inflammatory mediators were quantified by multiplex immunoassay analysis. Differential microbial abundance analysis of the two disease groups revealed at the phylum level that Proteobacteria accounted for 67% of the differentially abundant organisms, followed by organisms of Firmicutes (21%) and Actinobacteria (9%). At the species level, significant differences in abundance were seen for 75 species of which 58 species were significantly more abundant in CG patients. Notably, the FISH analysis revealed that Bacteroidetes was the most prevalent phylum in NG. The multiplex cytokine assay showed significant quantitative differences between the disease groups for eight analytes (GM–CSF, G–CSF, IFN–α, IL–4, IL–13, TNF–α, MIG, and HGF). The G–CSF was found to be the most significantly increased inflammatory protein marker in NG. The next-generation sequencing (NGS) data supported the understanding of NG as a multi-microbial infection with distinct differences to CG in regard to the microbial composition.

Voluntary oral methamphetamine increases memory deficits and contextual sensitization during abstinence associated with decreased PKMζ and increased κOR in the hippocampus of female mice

Background: Female populations exhibit vulnerabilities to psychostimulant addiction, as well as cognitive dysfunction following bouts of abuse. Aims: The goal for this study was to advance our understanding of the mechanisms that produce sex disparities in drug addiction. Methods: We used an animal model for voluntary oral methamphetamine administration (VOMA) and focused on male and female mice that consumed 7.6–8.2 mg/kg of methamphetamine (MA) per day during the last 18 days of the paradigm. Results: The VOMA-exposed female mice displayed increased locomotor activity in the drug-administration context compared to male mice, demonstrating sex-specific changes in contextual sensitization. During 2 weeks of forced abstinence, mice underwent further behavioral testing. We show that abstinence increased open-arm entries on the elevated plus maze in both sexes. There were no differences in immobility on the tail suspension test. In a hippocampal-dependent radial arm maze task, VOMA-treated female mice, but not male mice, showed working memory deficits. Hippocampal tissue was collected and analyzed using Western blotting. VOMA-exposed female mice exhibited increased kappa opioid receptor (κOR) expression in the hippocampus compared to male mice, suggesting a vulnerability toward abstinence-induced dysphoria. Female VOMA mice also exhibited a decrease in the memory protein marker, protein kinase M zeta (PKMζ), in the hippocampus. Conclusions: Our study reveals sex-specific effects following abstinence from chronic MA consumption on hippocampal κOR and PKMζ expression, suggesting that these neural changes in female mice may underlie spatial memory deficits and identify an increased susceptibility to dysregulated neural mechanisms. These data validate VOMA as a model sensitive to sex differences in behavior and hippocampal neurochemistry following chronic MA exposure.