The Neuronal Ischemic Tolerance Is Conditioned by the Tp53 Arg72Pro Polymorphism.

Cerebral preconditioning (PC) confers endogenous brain protection after stroke. Ischemic stroke patients with a prior transient ischemic attack (TIA) may potentially be in a preconditioned state. Although PC has been associated with the activation of pro-survival signals, the mechanism by which preconditioning confers neuroprotection is not yet fully clarified. Recently, we have described that PC-mediated neuroprotection against ischemic insult is promoted by p53 destabilization, which is mediated by its main regulator MDM2. Moreover, we have previously described that the human Tp53 Arg72Pro single nucleotide polymorphism (SNP) controls susceptibility to ischemia-induced neuronal apoptosis and governs the functional outcome of patients after stroke. Here, we studied the contribution of the human Tp53 Arg72Pro SNP on PC-induced neuroprotection after ischemia. Our results showed that cortical neurons expressing the Pro72-p53 variant exhibited higher PC-mediated neuroprotection as compared with Arg72-p53 neurons. PC prevented ischemia-induced nuclear and cytosolic p53 stabilization in Pro72-p53 neurons. However, PC failed to prevent mitochondrial p53 stabilization, which occurs in Arg72-p53 neurons after ischemia. Furthermore, PC promoted neuroprotection against ischemia by controlling the p53/active caspase-3 pathway in Pro72-p53, but not in Arg72-p53 neurons. Finally, we found that good prognosis associated to TIA within 1 month prior to ischemic stroke was restricted to patients harboring the Pro72 allele. Our findings demonstrate that the Tp53 Arg72Pro SNP controls PC-promoted neuroprotection against a subsequent ischemic insult by modulating mitochondrial p53 stabilization and then modulates TIA-induced ischemic tolerance.

Mesencephalic astrocyte-derived neurotrophic factor affords neuroprotection to early brain injury induced by subarachnoid hemorrhage via activating Akt-dependent prosurvival pathway and defending blood-brain barrier integrity.

This study aimed to explore the neuroprotective effect of mesencephalic astrocyte-derived neurotrophic factor (MANF) protein on early brain injury caused by subarachnoid hemorrhage (SAH) and the relevant mechanisms in experimental rats, expecting to understand whether MANF was a potential therapeutic target for SAH treatment. A perforation model of SAH was introduced into the study. Recombinant human MANF (rh-MANF) and protein kinase B (Akt) inhibitor (MK2206) were used to explore the effect and the mechanisms. Multiple approaches for systemic assessment were employed in the research, including the Garcia test, the SAH grade, Evans blue (EB) dye leakage, brain-water content (BWC), the rotarod test, and the Morris water-navigation task, as were biotechniques, such as immunohistochemistry, Western blot, transmission electron microscopy, and flow cytometry. MANF was mainly expressed in rat neurons, and its expression increased significantly at 3 h after SAH induction and peaked at 24 h. Stereotactic injection of rh-MANF into the cerebroventricle significantly increased the level of MANF, p-Akt, p-mouse double minute 2 homolog (p-MDM2), and B-cell lymphoma 2 (Bcl-2) in brain tissue, whereas it down-regulated the expression of P53, Bcl-2-associated X protein (Bax), and cleaved caspase-3, which indicated that neuronal apoptosis was remarkably suppressed. Expression of matrix metallopeptidase 9 (MMP-9) was also suppressed by the rh-MANF injection. Furthermore, neurologic deficits, EB dye leakage, and BWC were reduced, and long-lasting neuroprotection was noted with rh-MANF administration. The antiapoptotic and blood-brain barrier (BBB) protective effect could be offset by administering MK2206. MANF could alleviate neuronal apoptosis by activating Akt-dependent prosurvival pathway and abate BBB damage via MMP-9 suppression. MANF showed not only transient but also long-lasting neuroprotective properties. The rh-MANF as a potential drug for treating SAH might be of clinical use.-Li, T., Xu, W., Gao, L., Guan, G., Zhang, Z., He, P., Xu, H., Fan, L., Yan, F., Chen, G. Mesencephalic astrocyte-derived neurotrophic factor affords neuroprotection to early brain injury induced by subarachnoid hemorrhage via activating Akt-dependent prosurvival pathway and defending blood-brain barrier integrity.

A Complete Proteomic Workflow to Study Brain-Related Disorders via Postmortem Tissue.

Here we describe a mass spectrometry-based proteomics workflow to discovery proteins differentially regulated in brains collected postmortem from mental, neurological, or substance abuse disorders (MNS) patients. One way to maximize protein detection is to carry out enrichment of cellular compartments such as the nucleus, mitochondria and cytosol. Subcellular fractionation improves proteome coverage and may shed light on the role of these organelles in the pathophysiology of MNS.

Evidence of intraneuronal Aß accumulation preceding tau pathology in the entorhinal cortex.

Growing evidence gathered from transgenic animal models of Alzheimer's disease (AD) indicates that the intraneuronal accumulation of amyloid-ß (Aß) peptides is an early event in the AD pathogenesis, producing cognitive deficits before the deposition of insoluble plaques. Levels of soluble Aß are also a strong indicator of synaptic deficits and concurrent AD neuropathologies in post-mortem AD brain; however, it remains poorly understood how this soluble amyloid pool builds within the brain in the decades leading up to diagnosis, when a patient is likely most amenable to early therapeutic interventions. Indeed, characterizing early intracellular Aß accumulation in humans has been hampered by the lack of Aß-specific antibodies, variability in the quality of available human brain tissue and the limitations of conventional microscopy. We therefore sought to investigate the development of the intraneuronal Aß pathology using extremely high-quality post-mortem brain material obtained from a cohort of non-demented subjects with short post-mortem intervals and processed by perfusion-fixation. Using well-characterized monoclonal antibodies, we demonstrate that the age-dependent intraneuronal accumulation of soluble Aß is pervasive throughout the entorhinal cortex and hippocampus, and that this phase of the amyloid pathology becomes established within AD-vulnerable regions before the deposition of Aß plaques and the formation of tau neurofibrillary tangles. We also show for the first time in post-mortem human brain that Aß oligomers do in fact accumulate intraneuronally, before the formation of extracellular plaques. Finally, we validated the origin of the Aß-immunopositive pool by resolving Aß- and APP/CTF-immunoreactive sites using super resolution structured illumination microscopy. Together, these findings indicate that the lifelong accrual of intraneuronal Aß may be a potential trigger for downstream AD-related pathogenic events in early disease stages.

Novel antibodies reveal presynaptic localization of C9orf72 protein and reduced protein levels in C9orf72 mutation carriers.

Hexanucleotide repeat expansion in C9orf72 is the most common genetic cause of frontotemporal dementia and amyotrophic lateral sclerosis, but the pathogenic mechanism of this mutation remains unresolved. Haploinsufficiency has been proposed as one potential mechanism. However, insights if and how reduced C9orf72 proteins levels might contribute to disease pathogenesis are still limited because C9orf72 expression, localization and functions in the central nervous system (CNS) are uncertain, in part due to the poor specificity of currently available C9orf72 antibodies.Here, we generated and characterized novel knock-out validated monoclonal rat and mouse antibodies against C9orf72. We found that C9orf72 is a low abundant, cytoplasmic, highly soluble protein with the long 481 amino acid isoform being the predominant, if not exclusively, expressed protein isoform in mouse tissues and human brain. As consequence of the C9orf72 repeat expansion, C9orf72 protein levels in the cerebellum were reduced to 80% in our series of C9orf72 mutation carriers (n = 17) compared to controls (n = 26). However, no associations between cerebellar protein levels and clinical phenotypes were seen. Finally, by utilizing complementary immunohistochemical and biochemical approaches including analysis of human iPSC derived motor neurons, we identified C9orf72, in addition to its association to lysosomes, to be localized to the presynapses and able to interact with all members of the RAB3 protein family, suggestive of a role for C9orf72 in regulating synaptic vesicle functions by potentially acting as guanine nucleotide exchange factor for RAB3 proteins.In conclusion, our findings provide further evidence for haploinsufficiency as potential mechanism in C9orf72 pathogenesis by demonstrating reduced protein levels in C9orf72 mutation carriers and important novel insights into the physiological role of C9orf72 in the CNS. Moreover, the described novel monoclonal C9orf72 antibodies will be useful tools to further dissect the cellular and molecular functions of C9orf72.

Subcellular origin of mitochondrial DNA deletions in human skeletal muscle.

OBJECTIVE: In patients with mitochondrial DNA (mtDNA) maintenance disorders and with aging, mtDNA deletions sporadically form and clonally expand within individual muscle fibers, causing respiratory chain deficiency. This study aimed to identify the sub-cellular origin and potential mechanisms underlying this process. METHODS: Serial skeletal muscle cryosections from patients with multiple mtDNA deletions were subjected to subcellular immunofluorescent, histochemical, and genetic analysis. RESULTS: We report respiratory chain-deficient perinuclear foci containing mtDNA deletions, which show local elevations of both mitochondrial mass and mtDNA copy number. These subcellular foci of respiratory chain deficiency are associated with a local increase in mitochondrial biogenesis and unfolded protein response signaling pathways. We also find that the commonly reported segmental pattern of mitochondrial deficiency is consistent with the three-dimensional organization of the human skeletal muscle mitochondrial network. INTERPRETATION: We propose that mtDNA deletions first exceed the biochemical threshold causing biochemical deficiency in focal regions adjacent to the myonuclei, and induce mitochondrial biogenesis before spreading across the muscle fiber. These subcellular resolution data provide new insights into the possible origin of mitochondrial respiratory chain deficiency in mitochondrial myopathy. Ann Neurol 2018;84:289-301.

Cellular and Subcellular Localization of Endoplasmic Reticulum Chaperone GRP78 Following Transient Focal Cerebral Ischemia in Rats.

The 78-kDa glucose-regulated protein (GRP78), a chaperone protein located in the endoplasmic reticulum (ER), has been reported to have neuroprotective effects in the injured central nervous system. Our aim was to examine the expression profiles and subcellular distributions of GRP78 and its association with the neuroglial reaction in the rat striatum after transient, focal cerebral ischemia. In sham-operated rats, constitutive, specific immunoreactivity for GRP78 was almost exclusively localized to the rough ER of striatal neurons, with none in the resting, ramified microglia or astrocytes. At 1 day post reperfusion, increased expression was observed in ischemia-resistant cholinergic interneurons, when most striatal neurons had lost GRP78 expression (this occurred earlier than the loss of other neuronal markers). By 3 days post reperfusion, GRP78 expression had re-emerged in association with the activation of glial cells in both infarct and peri-infarct areas but showed different patterns in the two regions. Most of the expression induced in the infarct area could be attributed to brain macrophages, while expression in the peri-infarct area predominantly occurred in neurons and reactive astrocytes. A gradual, sustained induction of GRP78 immunoreactivity occurred in reactive astrocytes localized to the astroglial scar, lasting for at least 28 days post reperfusion. Using correlative light- and electron-microscopy, we found conspicuous GRP78 protein localized to abnormally prominent, dilated rough ER in both glial cell types. Thus, our data indicate a link between GRP78 expression and the activated functional status of neuroglial cells, predominantly microglia/macrophages and astrocytes, occurring in response to ischemia-induced ER stress.

Sodium Channel Remodeling in Subcellular Microdomains of Murine Failing Cardiomyocytes.

BACKGROUND: Cardiac sodium channel (NaV1.5) dysfunction contributes to arrhythmogenesis during pathophysiological conditions. Nav1.5 localizes to distinct subcellular microdomains within the cardiomyocyte, where it associates with region-specific proteins, yielding complexes whose function is location specific. We herein investigated sodium channel remodeling within distinct cardiomyocyte microdomains during heart failure. METHODS AND RESULTS: Mice were subjected to 6 weeks of transverse aortic constriction (TAC; n=32) to induce heart failure. Sham-operated on mice were used as controls (n=20). TAC led to reduced left ventricular ejection fraction, QRS prolongation, increased heart mass, and upregulation of prohypertrophic genes. Whole-cell sodium current (INa) density was decreased by 30% in TAC versus sham-operated on cardiomyocytes. On macropatch analysis, INa in TAC cardiomyocytes was reduced by 50% at the lateral membrane (LM) and by 40% at the intercalated disc. Electron microscopy and scanning ion conductance microscopy revealed remodeling of the intercalated disc (replacement of [inter-]plicate regions by large foldings) and LM (less identifiable T tubules and reduced Z-groove ratios). Using scanning ion conductance microscopy, cell-attached recordings in LM subdomains revealed decreased INa and increased late openings specifically at the crest of TAC cardiomyocytes, but not in groove/T tubules. Failing cardiomyocytes displayed a denser, but more stable, microtubule network (demonstrated by increased α-tubulin and Glu-tubulin expression). Superresolution microscopy showed reduced average NaV1.5 cluster size at the LM of TAC cells, in line with reduced INa. CONCLUSIONS: Heart failure induces structural remodeling of the intercalated disc, LM, and microtubule network in cardiomyocytes. These adaptations are accompanied by alterations in NaV1.5 clustering and INa within distinct subcellular microdomains of failing cardiomyocytes.

ALS-Related Mutant FUS Protein Is Mislocalized to Cytoplasm and Is Recruited into Stress Granules of Fibroblasts from Asymptomatic FUS P525L Mutation Carriers.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) shows a strong genetic basis, with SOD1, FUS, TARDBP, and C9ORF72 being the genes most frequently involved. This has allowed identification of asymptomatic mutation carriers, which may be of help in understanding the molecular changes preceding disease onset. OBJECTIVES: We studied the cellular expression of FUS protein and the effect of heat-shock- and dithiothreitol-induced stress in fibroblasts from FUS P525L mutation carriers, healthy controls, and patients with sporadic ALS. METHODS: Western blots and immunocytochemistry were performed to study the subcellular localization of FUS protein. Control and stressed cells were double stained with FUS and the stress marker TIA-R. RESULTS: Fibroblasts from healthy controls and sporadic ALS cases showed a prominent nuclear FUS expression. In the 2 FUS P525L mutation carriers, instead, most cells showed a protein localization in both nucleus and cytoplasm, or exclusively in the cytoplasm. Stress prompted the formation of cytoplasmic granules in all subjects and in sporadic ALS FUS mislocalization to the cytoplasm. Cytoplasmic FUS was recruited into stress granules, which showed a time-dependent decrease in all subjects. However, in the FUS P525L fibroblasts, the granules persisted longer, and they were more numerous than those detected in the cells from controls and sporadic ALS patients. CONCLUSIONS: We show that in fibroblasts of FUS P525L mutation carriers, FUS mislocalized to the cytoplasm where it redistributed into stress granules with likely a dose effect, i.e. a higher number of cells with granules, which persist longer, than in controls and ALS cases. These data represent an early molecular change occurring before ALS onset, suggesting a transient preaggregative state.

Differences in cisplatin distribution in sensitive and resistant ovarian cancer cells: a TEM/NanoSIMS study.

Cisplatin is a widely used anti-cancer drug, but its effect is often limited by acquired resistance to the compound during treatment. Here, we use a combination of transmission electron microscopy (TEM) and nanoscale-secondary ion mass spectrometry (NanoSIMS) to reveal differences between cisplatin uptake in human ovarian cancers cells, which are known to be susceptible to acquired resistance to cisplatin. Both cisplatin sensitive and resistant cell lines were studied, revealing markedly less cisplatin in the resistant cell line. In cisplatin sensitive cells, Pt was seen to distribute diffusely in the cells with hotspots in the nucleolus, mitochondria, and autophagosomes. Inductively coupled plasma mass spectrometry (ICP-MS) was used to validate the NanoSIMS results.

RAGE maintains high levels of NFκB and oncogenic Kras activity in pancreatic cancer.

Oncogenic KRas activity is central to several cancer types including pancreatic ductal adenocarcinoma (PDAC) but has been determined to be "undruggable". Recent studies have indicated that oncogenic KRas is not constitutively active but relies on a feed-forward stimulatory mechanism involving NFκB mediated inflammation. In the current study, we investigated the role of the receptor for advanced glycation end-products (RAGE) in maintaining oncogenic signaling in PDAC. We observed that there was a shift in the levels of specific RAGE isoforms and altered cellular localization in PDAC. Furthermore, inhibition of RAGE using a pharmacological antagonist, FPS-ZM1, or a blocking antibody, decreased phosphorylation of IKBα and inhibited Erk activity down-stream of Kras in PDAC cell lines. In vivo, inhibition of RAGE using FPS-ZM1 reduced the growth of PDAC syngeneic orthotopic xenografts and prolonged survival. These data indicate that RAGE plays a central role in maintaining inflammatory signaling in PDAC that benefits tumor growth. These observations support the development of approaches to inhibit the carcinogenic actions of Kras indirectly by blocking the mechanisms which maintain its activity.

Loss of M1 Receptor Dependent Cholinergic Excitation Contributes to mPFC Deactivation in Neuropathic Pain.

In chronic pain, the medial prefrontal cortex (mPFC) is deactivated and mPFC-dependent tasks such as attention and working memory are impaired. We investigated the mechanisms of mPFC deactivation in the rat spared nerve injury (SNI) model of neuropathic pain. Patch-clamp recordings in acute slices showed that, 1 week after the nerve injury, cholinergic modulation of layer 5 (L5) pyramidal neurons was severely impaired. In cells from sham-operated animals, focal application of acetylcholine induced a left shift of the input/output curve and persistent firing. Both of these effects were almost completely abolished in cells from SNI-operated rats. The cause of this impairment was an ∼60% reduction of an M1-coupled, pirenzepine-sensitive depolarizing current, which appeared to be, at least in part, the consequence of M1 receptor internalization. Although no changes were detected in total M1 protein or transcript, both the fraction of the M1 receptor in the synaptic plasma membrane and the biotinylated M1 protein associated with the total plasma membrane were decreased in L5 mPFC of SNI rats. The loss of excitatory cholinergic modulation may play a critical role in mPFC deactivation in neuropathic pain and underlie the mPFC-specific cognitive deficits that are comorbid with neuropathic pain.SIGNIFICANCE STATEMENT The medial prefrontal cortex (mPFC) undergoes major reorganization in chronic pain. Deactivation of mPFC output is causally correlated with both the cognitive and the sensory component of neuropathic pain. Here, we show that cholinergic excitation of commissural layer 5 mPFC pyramidal neurons is abolished in neuropathic pain rats due to a severe reduction of a muscarinic depolarizing current and M1 receptor internalization. Therefore, in neuropathic pain rats, the acetylcholine (ACh)-dependent increase in neuronal excitability is reduced dramatically and the ACh-induced persisting firing, which is critical for working memory, is abolished. We propose that the blunted cholinergic excitability contributes to the functional mPFC deactivation that is causal for the pain phenotype and represents a cellular mechanism for the attention and memory impairments comorbid with chronic pain.

Activation of ß-Glucocerebrosidase Reduces Pathological α-Synuclein and Restores Lysosomal Function in Parkinson's Patient Midbrain Neurons.

UNLABELLED: Parkinson's disease (PD) is characterized by the accumulation of α-synuclein (α-syn) within Lewy body inclusions in the nervous system. There are currently no disease-modifying therapies capable of reducing α-syn inclusions in PD. Recent data has indicated that loss-of-function mutations in the GBA1 gene that encodes lysosomal ß-glucocerebrosidase (GCase) represent an important risk factor for PD, and can lead to α-syn accumulation. Here we use a small-molecule modulator of GCase to determine whether GCase activation within lysosomes can reduce α-syn levels and ameliorate downstream toxicity. Using induced pluripotent stem cell (iPSC)-derived human midbrain dopamine (DA) neurons from synucleinopathy patients with different PD-linked mutations, we find that a non-inhibitory small molecule modulator of GCase specifically enhanced activity within lysosomal compartments. This resulted in reduction of GCase substrates and clearance of pathological α-syn, regardless of the disease causing mutations. Importantly, the reduction of α-syn was sufficient to reverse downstream cellular pathologies induced by α-syn, including perturbations in hydrolase maturation and lysosomal dysfunction. These results indicate that enhancement of a single lysosomal hydrolase, GCase, can effectively reduce α-syn and provide therapeutic benefit in human midbrain neurons. This suggests that GCase activators may prove beneficial as treatments for PD and related synucleinopathies. SIGNIFICANCE STATEMENT: The presence of Lewy body inclusions comprised of fibrillar α-syn within affected regions of PD brain has been firmly documented, however no treatments exist that are capable of clearing Lewy bodies. Here, we used a mechanistic-based approach to examine the effect of GCase activation on α-syn clearance in human midbrain DA models that naturally accumulate α-syn through genetic mutations. Small molecule-mediated activation of GCase was effective at reducing α-syn inclusions in neurons, as well as associated downstream toxicity, demonstrating a therapeutic effect. Our work provides an example of how human iPSC-derived midbrain models could be used for testing potential treatments for neurodegenerative disorders, and identifies GCase as a critical therapeutic convergence point for a wide range of synucleinopathies.

The atypical cadherin Dachsous1 localizes to the base of the ciliary apparatus in airway epithelia.

Mucociliary clearance requires the distinct orientation and coordinated movement of airway cilia, which is established through planar cell polarity signaling (PCP). The atypical cadherin Dachsous1 (Dchs1) is a transmembrane protein that regulates PCP in D. melanogaster. However, little is known about Dchs1 expression and its potential role in PCP in mammalian adult tissues. Here, we show that Dchs1 is ubiquitously expressed in mouse embryos, but exhibits a highly restricted expression to lung tissues in the adult stage. Strikingly, human Dchs1 localized exclusively to the base of the ciliary apparatus in cultured human respiratory epithelial cells with differentiated motile 9 + 2 cilia. This localization could be functionally important as we observed aberrant DCHS1 mRNA expression in human non-small cell lung cancer tissue. In sum, we establish Dchs1 as a component of the membrane domain surrounding the ciliary base. This suggests a specific role of Dchs1 in PCP-dependent organization of ciliary function and a possible role in lung disease.

Multiscale investigation of USPIO nanoparticles in atherosclerotic plaques and their catabolism and storage in vivo.

The storage and catabolism of Ultrasmall SuperParamagnetic Iron Oxide (USPIO) nanoparticles were analyzed through a multiscale approach combining Two Photon Laser Scanning Microscopy (TPLSM) and High-Resolution Transmission Electron Microscopy (HRTEM) at different times after intravenous injection in an atherosclerotic ApoE(-/-) mouse model. The atherosclerotic plaque features and the USPIO heterogeneous biodistribution were revealed down from organ's scale to subcellular level. The biotransformation of the nanoparticle iron oxide (maghemite) core into ferritin, the non-toxic form of iron storage, was demonstrated for the first time ex vivo in atherosclerotic plaques as well as in spleen, the iron storage organ. These results rely on an innovative spatial and structural investigation of USPIO's catabolism in cellular phagolysosomes. This study showed that these nanoparticles were stored as non-toxic iron compounds: maghemite oxide or ferritin, which is promising for MRI detection of atherosclerotic plaques in clinics using these USPIOs. From the Clinical Editor: Advance in nanotechnology has brought new contrast agents for clinical imaging. In this article, the authors investigated the use and biotransformation of Ultrasmall Super-paramagnetic Iron Oxide (USPIO) nanoparticles for analysis of atherosclerotic plagues in Two Photon Laser Scanning Microscopy (TPLSM) and High-Resolution Transmission Electron Microscopy (HRTEM). The biophysical data generated from this study could enable the possible use of these nanoparticles for the benefits of clinical patients.

Neuronal ceroid lipofuscinosis with DNAJC5/CSPα mutation has PPT1 pathology and exhibit aberrant protein palmitoylation.

Neuronal ceroid lipofuscinoses (NCL) are a group of inherited neurodegenerative disorders with lysosomal pathology (CLN1-14). Recently, mutations in the DNAJC5/CLN4 gene, which encodes the presynaptic co-chaperone CSPα were shown to cause autosomal-dominant NCL. Although 14 NCL genes have been identified, it is unknown if they act in common disease pathways. Here we show that two disease-associated proteins, CSPα and the depalmitoylating enzyme palmitoyl-protein thioesterase 1 (PPT1/CLN1) are biochemically linked. We find that in DNAJC5/CLN4 patient brains, PPT1 is massively increased and mis-localized. Surprisingly, the specific enzymatic activity of PPT1 is dramatically reduced. Notably, we demonstrate that CSPα is depalmitoylated by PPT1 and hence its substrate. To determine the consequences of PPT1 accumulation, we compared the palmitomes from control and DNAJC5/CLN4 patient brains by quantitative proteomics. We discovered global changes in protein palmitoylation, mainly involving lysosomal and synaptic proteins. Our findings establish a functional link between two forms of NCL and serve as a springboard for investigations of NCL disease pathways.

Anti-Mesothelin Nanobodies for Both Conventional and Nanoparticle-Based Biomedical Applications.

Mesothelin, a cancer biomarker overexpressed in tumors of epithelial origin, is a target for nanotechnology-based diagnostic, therapeutic, and prognostic applications. The currently available anti-mesothelin antibodies present limitations, including low penetration due to large size and/or lack of in vivo stability. Single domain antibodies (sdAbs) or nanobodies (Nbs) provide powerful solutions to these specific problems. We generated a phage-display library of Nbs that were amplified from B cells of a llama that was immunized with human recombinant mesothelin. Two nanobodies (Nb A1 and Nb C6) were selected on the basis of affinity (K(D) = 15 and 30 nM, respectively). Nb A1 was further modified by adding either a cysteine to permit maleimide-based bioconjugations or a sequence for the site-specific metabolic addition of a biotin in vivo. Both systems of conjugation (thiol-maleimide and streptavidin/biotin) were used to characterize and validate Nb A1 and to functionalize nanoparticles. We showed that anti-mesothelin Nb A1 could detect native and denatured mesothelin in various diagnostic applications, including flow cytometry, western blotting, immunofluorescence, and optical imaging. In conclusion, anti-mesothelin Nbs are novel, cost-effective, small, and single domain reagents with high affinity and specificity for the tumor-associated antigen mesothelin, which can be simply bioengineered for attachment to nanoparticles or modified surfaces using multiple bioconjugation strategies. These anti-mesothelin Nbs can be useful in both conventional and nanotechnology-based diagnostic, therapeutic and prognostic biomedical applications.

Functionalization of graphene oxide nanostructures improves photoluminescence and facilitates their use as optical probes in preclinical imaging.

Recently reported photoluminescent nanographene oxides (nGOs), i.e. nanographene oxidised with a sulfuric/nitric acid mixture (SNOx method), have tuneable photoluminescence and are scalable, simple and fast to produce optical probes. This material belongs to the vast class of photoluminescent carbon nanostructures, including carbon dots, nanodiamonds (NDs), graphene quantum dots (GQDs), all of which demonstrate a variety of properties that are attractive for biomedical imaging such as low toxicity and stable photoluminescence. In this study, the nGOs were organically surface-modified with poly(ethylene glycol)-poly(ethylene imine) (PEG-PEI) copolymers tagged with folic acid as the affinity ligand for cancer cells expressing folate receptors. The functionalization enhanced both the cellular uptake and quantum efficiency of the photoluminescence as compared to non-modified nGOs. The nGOs exhibited an excitation dependent photoluminescence that facilitated their detection with a wide range of microscope configurations. The functionalized nGOs were non-toxic, they were retained in the stained cell population over a period of 8 days and they were distributed equally between daughter cells. We have evaluated their applicability in in vitro and in vivo (chicken embryo CAM) models to visualize and track migratory cancer cells. The good biocompatibility and easy detection of the functionalized nGOs suggest that they could address the limitations faced with quantum dots and organic fluorophores in long-term in vivo biomedical imaging.

Simultaneous topographic and recognition imaging of epidermal growth factor receptor (EGFR) on single human breast cancer cells.

Epidermal growth factor receptor (EGFR) plays an important role in signaling pathway of the development of breast cancer cells. Since EGFR overexpresses in most breast cancer cells, it is regarded as a biomarker molecule of breast cancer cells. Here we demonstrated a new AFM technique-topography and recognition (TREC) imaging-to simultaneously obtain highly sensitive and specific molecular recognition images and high-resolution topographic images of EGFR on single breast cancer cells.

Labeling of HeLa cells using ZrO2:Yb(3+)-Er(3+) nanoparticles with upconversion emission.

This work reports the synthesis, structural characterization, and optical properties of ZrO2:Yb(3+)-Er(3+) (2­1 mol%) nanocrystals. The nanoparticles were coated with 3-aminopropyl triethoxysilane (APTES) and further modified with biomolecules, such as Biotin-Anti-rabbit (mouse IgG) and rabbit antibody-AntiKi-67, through a conjugation method. The conjugation was successfully confirmed by Fourier transform infrared, zeta potential, and dynamic light scattering. The internalization of the conjugated nanoparticles in human cervical cancer (HeLa) cells was followed by two-photon confocal microscopy. The ZrO2:Yb(3+)-Er(3+) nanocrystals exhibited strong red emission under 970-nm excitation. Moreover, the luminescence change due to the addition of APTES molecules and biomolecules on the nanocrystals was also studied. These results demonstrate that ZrO2:Yb(3+)-Er(3+) nanocrystals can be successfully functionalized with biomolecules to develop platforms for biolabeling and bioimaging.