White matter hyperintensity genetic risk factor TRIM47 regulates autophagy in brain endothelial cells.

White matter hyperintensity (WMH) is strongly correlated with age-related dementia and hypertension, but its pathogenesis remains obscure. GWAS identified TRIM47 at 17q25 locus as a top genetic risk factor for WMH formation. TRIM family is a class of E3 ubiquitin ligase with pivotal functions in autophagy, which is critical for brain endothelial cell (ECs) remodeling during hypertension. We hypothesize that TRIM47 regulates autophagy and its loss-of-function disturbs cerebrovasculature. Based on transcriptomics and immunohistochemistry, TRIM47 is found selectively expressed by brain ECs in human and mouse, and its transcription is upregulated by artificially-induced autophagy while downregulated in hypertension-like conditions. Using in silico simulation, immunocytochemistry and super-resolution microscopy, we identified the highly conserved binding site between TRIM47 and the LIR (LC3-interacting region) motif of LC3B. Importantly, pharmacological autophagy induction increased Trim47 expression on mouse ECs (b.End3) culture, while silencing Trim47 significantly increased autophagy with ULK1 phosphorylation induction, transcription and vacuole formation. Together, we confirm that TRIM47 is an endogenous inhibitor of autophagy in brain ECs, and such TRIM47-mediated regulation connects genetic and physiological risk factors for WMH formation but warrants further investigation. SUMMARY STATEMENT: TRIM47, top genetic risk factor for white matter hyperintensity formation, is a negative regulator of autophagy in brain endothelial cells and implicates a novel cellular mechanism for age-related cerebrovascular changes.

Cuprizone drives divergent neuropathological changes in different mouse models of Alzheimer's disease.

Myelin degradation is a normal feature of brain aging that accelerates in Alzheimer's disease (AD). To date, however, the underlying biological basis of this correlation remains elusive. The amyloid cascade hypothesis predicts that demyelination is caused by increased levels of the ß-amyloid (Aß) peptide. Here we report on work supporting the alternative hypothesis that early demyelination is upstream of amyloid. We challenged two different mouse models of AD (R1.40 and APP/PS1) using cuprizone-induced demyelination and tracked the responses with both neuroimaging and neuropathology. In oppose to amyloid cascade hypothesis, R1.40 mice, carrying only a single human mutant APP (Swedish; APP SWE ) transgene, showed a more abnormal changes of magnetization transfer ratio and diffusivity than in APP/PS1 mice, which carry both APP SWE and a second PSEN1 transgene (delta exon 9; PSEN1 dE9 ). Although cuprizone targets oligodendrocytes (OL), magnetic resonance spectroscopy and targeted RNA-seq data in R1.40 mice suggested a possible metabolic alternation in axons. In support of alternative hypotheses, cuprizone induced significant intraneuronal amyloid deposition in young APP/PS1, but not in R1.40 mice, and it suggested the presence of PSEN deficiencies, may accelerate Aß deposition upon demyelination. In APP/PS1, mature OL is highly vulnerable to cuprizone with significant DNA double strand breaks (53BP1 + ) formation. Despite these major changes in myelin, OLs, and Aß immunoreactivity, no cognitive impairment or hippocampal pathology was detected in APP/PS1 mice after cuprizone treatment. Together, our data supports the hypothesis that myelin loss can be the cause, but not the consequence, of AD pathology. SIGNIFICANCE STATEMENT: The causal relationship between early myelin loss and the progression of Alzheimer's disease remains unclear. Using two different AD mouse models, R1.40 and APP/PS1, our study supports the hypothesis that myelin abnormalities are upstream of amyloid production and deposition. We find that acute demyelination initiates intraneuronal amyloid deposition in the frontal cortex. Further, the loss of oligodendrocytes, coupled with the accelerated intraneuronal amyloid deposition, interferes with myelin tract diffusivity at a stage before any hippocampus pathology or cognitive impairments occur. We propose that myelin loss could be the cause, not the consequence, of amyloid pathology during the early stages of Alzheimer's disease.

Diagnostic Yield of Exome Sequencing in Fetuses with Sonographic Features of Skeletal Dysplasias but Normal Karyotype or Chromosomal Microarray Analysis: A Systematic Review.

Skeletal dysplasias are a group of diseases characterized by bone and joint abnormalities, which can be detected during prenatal ultrasound. Next-generation sequencing has rapidly revolutionized molecular diagnostic approaches in fetuses with structural anomalies. This review studies the additional diagnostic yield of prenatal exome sequencing in fetuses with prenatal sonographic features of skeletal dysplasias. This was a systematic review by searching PubMed for studies published between 2013 and July 2022 that identified the diagnostic yield of exome sequencing after normal karyotype or chromosomal microarray analysis (CMA) for cases with suspected fetal skeletal dysplasias based on prenatal ultrasound. We identified 10 out of 85 studies representing 226 fetuses. The pooled additional diagnostic yield was 69.0%. The majority of the molecular diagnoses involved de novo variants (72%), while 8.7% of cases were due to inherited variants. The incremental diagnostic yield of exome sequencing over CMA was 67.4% for isolated short long bones and 77.2% for non-isolated cases. Among phenotypic subgroup analyses, features with the highest additional diagnostic yield were an abnormal skull (83.3%) and a small chest (82.5%). Prenatal exome sequencing should be considered for cases with suspected fetal skeletal dysplasias with or without a negative karyotype or CMA results. Certain sonographic features, including an abnormal skull and small chest, may indicate a potentially higher diagnostic yield.

Chemical exchange saturation transfer MRI detects myelin changes in cuprizone mouse model at 3T.

Chemical exchange saturation transfer (CEST) sensitively detects molecular alterations in the brain, such as relayed nuclear Overhauser effect (rNOE) CEST contrast at -3.5 ppm representing aliphatic protons in both lipids and proteins, and CEST contrast at 3.5 ppm correlating with amide proton in proteins. Myelin is rich in lipids and proteins, and therefore CEST can be explored as a biomarker for myelin pathology, which could contribute to the diagnosis and prognosis of multiple sclerosis (MS). In the current study, we investigate the specificity of aliphatic rNOE and the amide pool in myelin detection using the cuprizone (CPZ) mouse model, which recapitulates the demyelination and remyelination of MS. In this study, preclinical 3T MRI was performed in 19 male C57BL/6 mice. Mice in the normal control (NC) group (n = 9) were fed a normal diet for the whole course, while mice in the CPZ group (n = 10) were fed with CPZ for 10 weeks, followed by 4 weeks with a normal diet. The CEST contrast of rNOE (-3.5 ppm) and amide (3.5 ppm) in brain regions of the corpus callosum (CC) and the caudate putamen were compared. Statistical differences between the groups were calculated using two-way ANOVA. We observed significantly decreased rNOE (NC: 4.85% ± 0.09%/s vs. CPZ: 3.88% ± 0.18%/s, p = 0.007) and amide pool (NC: 3.20% ± 0.10%/s vs. CPZ: 2.46% ± 0.16%/s, p = 0.02) in the CC after 8 weeks on CPZ diet (p < 0.05). Moreover, the rNOE in the CPZ group recovered to a level comparable with the NC group at week 14 (p = 0.39), while amide remained at a level as low as that for the NC group (p = 0.051). Significant rNOE and amide changes, validated by immunohistochemistry results for demyelination and remyelination, demonstrate the huge potential of CEST for revealing myelin pathology, which has implications for MS identification at the clinical field strength of 3T.

Multiple CEST contrast imaging of nose-to-brain drug delivery using iohexol liposomes at 3T MRI.

Image guided nose-to-brain drug delivery provides a non-invasive way to monitor drug delivered to the brain, and the intranasal administration could increase effective dose via bypassing Blood Brain Barrier (BBB). Here, we investigated the imaging of liposome-based drug delivery to the brain via intranasal administration, in which the liposome could penetrate mucus and could be detected by chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI) at 3T field strength. Liposomes were loaded with a computed tomography (CT) contrast agent, iohexol (Ioh-Lipo), which has specific amide protons exchanging at 4.3 ppm of Z-spectrum (or CEST spectrum). Ioh-Lipo generated CEST contrasts of 35.4% at 4.3 ppm, 1.8% at -3.4 ppm and 20.6% at 1.2 ppm in vitro. After intranasal administration, these specific CEST contrasts were observed in both olfactory bulb (OB) and frontal lobe (FL) in the case of 10% polyethylene glycol (PEG) Ioh-Lipo. We observed obvious increases in CEST contrast in OB half an hour after the injection of 10% PEG Ioh-Lipo, with a percentage increase of 62.0% at 4.3 ppm, 10.9% at -3.4 ppm and 25.7% at 1.2 ppm. Interestingly, the CEST map at 4.3 ppm was distinctive from that at -3.4 pm and 1.2 ppm. The highest contrast of 4.3 ppm was at the external plexiform layer (EPL) and the region between left and right OB (LROB), while the CEST contrast at -3.4 ppm had no significant difference among all investigated regions with slightly higher signal in olfactory limbus (OL, between OB and FL) and FL, as validated with histology. While no substantial increase of CEST contrast at 4.3 ppm, -3.4 ppm or 1.2 ppm was observed in OB and FL when 1% PEG Ioh-Lipo was administered. We demonstrated for the first time the feasibility of non-invasively detecting the nose-to-brain delivery of liposomes using CEST MRI. This multiple-contrast approach is necessary to image the specific distribution of iohexol and liposome simultaneously and independently, especially when designing drug carriers for nose-to-brain drug delivery.

Apolipoprotein E ε4 disrupts oligodendrocyte differentiation by interfering with astrocyte-derived lipid transport.

Carriers of the APOE4 (apolipoprotein E ε4) variant of the APOE gene are subject to several age-related health risks, including Alzheimer's disease (AD). The deficient lipid and cholesterol transport capabilities of the APOE4 protein are one reason for the altered risk profile. In particular, APOE4 carriers are at elevated risk for sporadic AD. While deposits o misfolded proteins are present in the AD brain, white matter (WM) myelin is also disturbed. As myelin is a lipid- and cholesterol-rich structure, the connection to APOE makes considerable biological sense. To explore the APOE-WM connection, we have analyzed the impact of human APOE4 on oligodendrocytes (OLs) of the mouse both in vivo and in vitro. We find that APOE proteins is enriched in astrocytes but sparse in OL. In human APOE4 (hAPOE4) knock-in mice, myelin lipid content is increased but the density of major myelin proteins (MBP, MAG, and PLP) is largely unchanged. We also find an unexpected but significant reduction of cell density of the OL lineage (Olig2+ ) and an abnormal accumulation of OL precursors (Nkx 2.2+ ), suggesting a disruption of OL differentiation. Gene ontology analysis of an existing RNA-seq dataset confirms a robust transcriptional response to the altered chemistry of the hAPOE4 mouse brain. In culture, the uptake of astrocyte-derived APOE during Lovastatin-mediated depletion of cholesterol synthesis is sufficient to sustain OL differentiation. While endogenous hAPOE protein isoforms have no effects on OL development, exogenous hAPOE4 abolishes the ability of very low-density lipoprotein to restore myelination in Apoe-deficient, cholesterol-depleted OL. Our data suggest that APOE4 impairs myelination in the aging brain by interrupting the delivery of astrocyte-derived lipids to the oligodendrocytes. We propose that high myelin turnover and OL exhaustion found in APOE4 carriers is a likely explanation for the APOE-dependent myelin phenotypes of the AD brain.

Apolipoprotein E ε4 Mediates Myelin Breakdown by Targeting Oligodendrocytes in Sporadic Alzheimer Disease.

White matter degradation in the frontal lobe is one of the earliest detectable changes in aging and Alzheimer disease. The ε4 allele of apolipoprotein E (APOE4) is strongly associated with such myelin pathology but the underlying cellular mechanisms remain obscure. We hypothesized that, as a lipid transporter, APOE4 directly triggers pathology in the cholesterol-rich myelin sheath independent of AD pathology. To test this, we performed immunohistochemistry on brain tissues from healthy controls, sporadic, and familial Alzheimer disease subjects. While myelin basic protein expression was largely unchanged, in frontal cortex the number of oligodendrocytes (OLs) was significantly reduced in APOE4 brains independent of their Braak stage or NIA-RI criteria. This high vulnerability of OLs was confirmed in humanized APOE3 or APOE4 transgenic mice. A gradual decline of OL numbers was found in the aging brain without associated neuronal loss. Importantly, the application of lipidated human APOE4, but not APOE3, proteins significantly reduced the formation of myelinating OL in primary cell culture derived from Apoe-knockout mice, especially in cholesterol-depleted conditions. Our findings suggest that the disruption of myelination in APOE4 carriers may represent a direct OL pathology, rather than an indirect consequence of amyloid plaque formation or neuronal loss.

Expanding the horizon of research into the pathogenesis of the white matter diseases: Proceedings of the 2021 Annual Workshop of the Albert Research Institute for White Matter and Cognition.

White matter pathologies are critically involved in the etiology of vascular cognitive impairment-dementia (VCID), Alzheimer's disease (AD), and Alzheimer's disease and related diseases (ADRD), and therefore need to be considered a treatable target ( Roseborough A, Hachinski V, Whitehead S. White matter degeneration - a treatable target? Roseborough et al. JAMA Neurol [Internet]. 2020 Apr 27;77(7):793-4, [1] . To help address this often-missed area of research, several workshops have been sponsored by the Leo and Anne Albert Charitable Trust since 2015, resulting in the incorporation of "The Albert Research Institute for White Matter and Cognition" in 2020. The first annual "Institute" meeting was held virtually on March 3-4, 2021. The Institute provides a forum and workspace for communication and support of the advancement of white matter science and research to better understand the evolution and prevention of dementia. It serves as a platform for young investigator development, to introduce new data and debate biology mechanisms and new ideas, and to encourage and support new research collaborations and directions to clarify how white matter changes, with other genetic and health risk factors, contribute to cognitive impairment. Similar to previous Albert Trust-sponsored workshops (Barone et al. in J Transl Med 14:1-14, [2]; Sorond et al. in GeroScience 42:81-96, [3]), established expert investigators were identified and invited to present. Opportunities to attend and present were also extended by invitation to talented research fellows and younger scientists. Also, updates on institute-funded research collaborations were provided and discussed. The summary that follows is a synopsis of topics and discussion covered in the workshop.

Deep neural network based CEST and AREX processing: Application in imaging a model of Alzheimer's disease at 3 T.

PURPOSE: To optimize and apply deep neural network based CEST (deepCEST) and apparent exchange dependent-relaxation (deepAREX) for imaging the mouse brain with Alzheimer's disease (AD) at 3T MRI. METHODS: CEST and T1 data of central and anterior brain slices of 10 AD mice and 10 age-matched wild type (WT) mice were acquired at a 3T animal MRI scanner. The networks of deepCEST/deepAREX were optimized and trained on the WT data. The CEST/AREX contrasts of AD and WT mice predicted by the networks were analyzed and further validated by immunohistochemistry. RESULTS: After optimization and training on CEST data of WT mice, deepCEST/deepAREX could rapidly (~1 s) generate precise CEST and AREX results for unseen CEST data of AD mice, indicating the accuracy and generalization of the networks. Significant lower amide weighted (3.5 ppm) signal related to amyloid ß-peptide (Aß) plaque depositions, which was validated by immunohistochemistry results, was detected in both central and anterior brain slices of AD mice compared to WT mice. Decreased magnetization transfer (MT) signal was also found in AD mice especially in the anterior slice. CONCLUSION: DeepCEST/deepAREX could rapidly generate accurate CEST/AREX contrasts in animal study. The well-optimized deepCEST/deepAREX have potential for AD differentiation at 3T MRI.

Transcriptome Profiling of Mouse Corpus Callosum After Cerebral Hypoperfusion.

White matter damage caused by cerebral hypoperfusion is a major hallmark of subcortical ischemic vascular dementia (SIVD), which is the most common subtype of vascular cognitive impairment and dementia (VCID) syndrome. In an aging society, the number of SIVD patients is expected to increase; however, effective therapies have yet to be developed. To understand the pathological mechanisms, we analyzed the profiles of the cells of the corpus callosum after cerebral hypoperfusion in a preclinical SIVD model. We prepared cerebral hypoperfused mice by subjecting 2-month old male C57BL/6J mice to bilateral carotid artery stenosis (BCAS) operation. BCAS-hypoperfusion mice exhibited cognitive deficits at 4 weeks after cerebral hypoperfusion, assessed by novel object recognition test. RNA samples from the corpus callosum region of sham- or BCAS-operated mice were then processed using RNA sequencing. A gene set enrichment analysis using differentially expressed genes between sham and BCAS-operated mice showed activation of oligodendrogenesis pathways along with angiogenic responses. This database of transcriptomic profiles of BCAS-hypoperfusion mice will be useful for future studies to find a therapeutic target for SIVD.

ATM loss disrupts the autophagy-lysosomal pathway.

ATM (ataxia telangiectasia mutated) protein is found associated with multiple organelles including synaptic vesicles, endosomes and lysosomes, often in cooperation with ATR (ataxia telangiectasia and Rad3 related). Mutation of the ATM gene results in ataxia-telangiectasia (A-T), an autosomal recessive disorder with defects in multiple organs including the nervous system. Precisely how ATM deficiency leads to the complex phenotypes of A-T, however, remains elusive. Here, we reported that part of the connection may lie in autophagy and lysosomal abnormalities. We found that ATM was degraded through the autophagy pathway, while ATR was processed by the proteasome. Autophagy and lysosomal trafficking were both abnormal in atm-/- neurons and the deficits impacted cellular functions such as synapse maintenance, neuronal survival and glucose uptake. Upregulated autophagic flux was observed in atm-/- lysosomes, associated with a more acidic pH. Significantly, we found that the ATP6V1A (ATPase, H+ transporting, lysosomal V1 subunit A) proton pump was an ATM kinase target. In atm-/- neurons, lysosomes showed enhanced retrograde transport and accumulated in the perinuclear regions. We attributed this change to an unexpected physical interaction between ATM and the retrograde transport motor protein, dynein. As a consequence, SLC2A4/GLUT4 (solute carrier family 4 [facilitated glucose transporter], member 4) translocation to the plasma membrane was inhibited and trafficking to the lysosomes was increased, leading to impaired glucose uptake capacity. Together, these data underscored the involvement of ATM in a variety of neuronal vesicular trafficking processes, offering new and therapeutically useful insights into the pathogenesis of A-T.Abbreviations: 3-MA: 3-methyladenine; A-T: ataxia-telangiectasia; ALG2: asparagine-linked glycosylation 2 (alpha-1,3-mannosyltransferase); AMPK: adenosine 5'-monophosphate (AMP)-activated protein kinase; ATG5: autophagy related 5; ATM: ataxia telangiectasia mutated; ATP6V1A: ATPase, H+ transporting, lysosomal V1 subunit A; ATR: ataxia-telangiectasia and Rad3 related; BFA1: bafilomycin A1; CC3: cleaved-CASP3; CGN: cerebellar granule neuron; CLQ: chloroquine; CN: neocortical neuron; CTSB: cathepsin B; CTSD: cathepsin D; DYNLL1: the light chain1 of dynein; EIF4EBP1/4E-BP1: eukaryotic translation initiation factor 4E binding protein 1; Etop: etoposide; FBS: fetal bovine serum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HBS: HEPES-buffered saline; HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HOMER1: homer protein homolog 1; KU: KU-60019; LAMP1: lysosomal-associated membrane protein 1; LC3B-II: LC3-phosphatidylethanolamine conjugate; Lyso: lysosome; LysopH-GFP: lysopHluorin-GFP; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MAP2: microtubule associated protein 2; MAPK14: mitogen-activated protein kinase 14; MAPK8/JNK1: mitogen-activated protein kinase 8; MCOLN1/TRPML1: mucolipin 1; OSBPL1A: oxysterol binding protein like 1A; PIKK: phosphatidylinositol 3 kinase related kinase; Rapa: rapamycin; RILP: rab interacting lysosomal protein; ROS: reactive oxygen species; SEM: standard error of mean; SLC2A4/GLUT4: solute carrier family 2 (facilitated glucose transporter), member 4; TSC2/tuberin: TSC complex subunit 2; ULK1: unc-51 like kinase 1; UPS: ubiquitin-proteasome system; VE: VE-822; WCL: whole-cell lysate; WT: wild type.

Clinical and contrast-enhanced image features in the prediction model for the detection of small hepatocellular carcinomas.

Purpose: To identify novel radiological features and clinical characteristics to improve diagnostic criteria for early detection of small hepatocellular carcinoma (HCC). Patients and Methods: We retrospectively recruited asymptomatic patients with no history of HCC but a high risk of HCC in whom a new, solitary, well-defined, solid nodule between 10 and 20 mm was detected through a screening ultrasound. We retrospectively collected all clinical data, and patients were examined using dynamic contrast-enhanced computed tomography or magnetic resonance imaging; subsequently, fine-needle biopsy was performed. A multivariate analysis of the predictors of small HCCs was performed by fitting a multiple logistic regression model with the stepwise variable selection method. Results: In total, 392 and 347 patients with a small liver nodule received a final pathologic confirmation of HCC and non-HCC, respectively. The estimated odds ratios and 95% confidence intervals of tumor size > 12.45 mm, age > 56.61 years, liver cirrhosis, hepatitis C virus (HCV) carrier status, ln alpha-fetoprotein (AFP) > 1.954, arterial phase enhancement, and portal or venous phase washout appearance without arterial phase enhancement were 2.0735 (1.4746-2.9155), 1.8878 (1.2949-2.7521), 1.6927 (1.1294-2.5369), 1.6186 (1.0347-2.5321), 2.0297 (1.3342-3.0876), 3.7451 (2.3845-5.8821), and 2.0327 (1.3500-3.0608), respectively. The area under the receiver operating characteristic curves for the diagnosis of small HCCs was 0.79 for arterial phase enhancement and 0.75 for portal or venous phase washout appearance without arterial phase enhancement. Conclusion: Clinical and contrast-enhanced image features are valuable in the prediction model for the detection and early diagnosis of small HCCs in patients with a high risk of HCC. In addition to negative portal or venous washout and negative arterial enhancement in images, age > 56.61 years, tumor size > 12.45 mm, HCV carrier status, and ln(AFP) > 1.954, are useful indicators for the early detection of small HCCs.

Synthesis of α-1,2- and α-1,3-linked di-rhamnolipids for biological studies.

For a detailed examination of the interaction of rhamnose containing derivatives with recombinant horseshoe crab plasma lectin (rHPL), two di-rhamno-di-lipids (an α-1,2- and an α-1,3-linked) were synthesized via a new simple method. The N-iodosuccinimide/triflic acid mediated glycosylation of the methyl (R)-3-hydroxydecanoate with phenyl-1-thio-rhamnobioside donors afforded the mono-lipid disaccharides. Removal of the methyl ester group followed by esterification of the mono-lipids with a second (R)-3-hydroxydecanoate unit resulted in fully protected di-lipid derivatives, transformation of which into the target compounds was accomplished in two steps. This method allows the synthesis of both regioisomers in only 6 steps starting from the corresponding free disaccharides. Both synthetic di-rhamnolipids were biologically active for lectin binding differential binding preference between two isomeric di-rhamno-di-lipids. The rHPL lectin favours the α-1,3-linked di-rhamno-di-lipids over its α-1,2-linked regioisomer.

Altered d-glucose in brain parenchyma and cerebrospinal fluid of early Alzheimer's disease detected by dynamic glucose-enhanced MRI.

Altered cerebral glucose uptake is one of the hallmarks of Alzheimer's disease (AD). A dynamic glucose-enhanced (DGE) magnetic resonance imaging (MRI) approach was developed to simultaneously monitor d-glucose uptake and clearance in both brain parenchyma and cerebrospinal fluid (CSF). We observed substantially higher uptake in parenchyma of young (6 months) transgenic AD mice compared to age-matched wild-type (WT) mice. Notably lower uptakes were observed in parenchyma and CSF of old (16 months) AD mice. Both young and old AD mice had an obviously slower CSF clearance than age-matched WT mice. This resembles recent reports of the hampered CSF clearance that leads to protein accumulation in the brain. These findings suggest that DGE MRI can identify altered glucose uptake and clearance in young AD mice upon the emergence of amyloid plaques. DGE MRI of brain parenchyma and CSF has potential for early AD stratification, especially at 3T clinical field strength MRI.

Activation of STAT3 is a key event in TLR4 signaling-mediated melanoma progression.

Malignant melanoma is aggressive and has a high mortality rate. Toll-like receptor 4 (TLR4) has been linked to melanoma growth, angiogenesis and metastasis. However, signal transduction mediated by TLR4 for driving melanoma progression is not fully understood. Signal transducer and activator of transcription 3 (STAT3) has been identified as a major oncogene in melanoma progression. We found: that TLR4 expression positively correlates with activation/phosphorylation of STAT3 in human melanoma samples; that TLR4 ligands activate STAT3 through MYD88 and TRIF in melanoma cells; and that intratumoral activation of TLR4 increases STAT3 activation in the tumor and promotes tumor growth, angiogenesis, epithelial-mesenchymal transition (EMT) and the formation of an immunosuppressive tumor microenvironment in mice. Further, we found that the effects mediated by activating TLR4 are weakened by suppressing STAT3 function with a dominant negative STAT3 variant in melanoma. Collectively, our work identifies STAT3 activation as a key event in TLR4 signaling-mediated melanoma progression, shedding new light on the pathophysiology of melanoma.

Investigation of Parameters that Affect Resin Swelling in Green Solvents.

The influence of various physical and chemical factors on the swelling of polystyrene and PEG based resins in greener organic solvents has been systematically investigated. In general, chemical factors: the nature of the functionality/linker and the degree of loading were found to have a far larger influence on the swelling of the resins than physical parameters such as bead size. The results are interpreted in terms of Hansen solubility parameters for the solvents and there is evidence that some solvents interact with the polymeric core of a resin whilst others interact with the functionality. The results are extended to a study of the changes in resin swelling observed during both deprotection and chain elongation reactions during solid phase peptide synthesis.

Cell Penetrating Peptide as a High Safety Anti-Inflammation Ingredient for Cosmetic Applications.

Cosmeceutical peptides have become an important topic in recent decades in both academic and industrial fields. Many natural or synthetic peptides with different biological functions including anti-ageing, anti-oxidation, anti-infection and anti-pigmentation have been developed and commercialized. Current cosmeceutical peptides have already satisfied most market demand, remaining: "cargos carrying skin penetrating peptide with high safety" still an un-met need. To this aim, a cell-penetrating peptide, CPPAIF, which efficiently transported cargos into epithelial cells was exanimated. CPPAIF was evaluated with cell model and 3D skin model following OECD guidelines without using animal models. As a highly stable peptide, CPPAIF neither irritated nor sensitized skin, also did not disrupt skin barrier. In addition, such high safety peptide had anti-inflammation activity without allergic effect. Moreover, cargo carrying activity of CPPAIF was assayed using HaCaT cell model and rapid CPPAIF penetration was observed within 30 min. Finally, CPPAIF possessed transepidermal activity in water in oil formulation without disruption of skin barrier. All evidences indicated that CPPAIF was an ideal choice for skin penetrating and its anti-inflammatory activity could improve skin condition, which made CPPAIF suitable and attractive for novel cosmeceutical product development.

Proceedings from the Albert Charitable Trust Inaugural Workshop on white matter and cognition in aging.

This third in a series of vascular cognitive impairment (VCI) workshops, supported by "The Leo and Anne Albert Charitable Trust," was held from February 8 to 12 at the Omni Resort in Carlsbad, CA. This workshop followed the information gathered from the earlier two workshops suggesting that we focus more specifically on brain white matter in age-related cognitive impairment. The Scientific Program Committee (Frank Barone, Shawn Whitehead, Eric Smith, and Rod Corriveau) assembled translational, clinical, and basic scientists with unique expertise in acute and chronic white matter injury at the intersection of cerebrovascular and neurodegenerative etiologies. As in previous Albert Trust workshops, invited participants addressed key topics related to mechanisms of white matter injury, biomarkers of white matter injury, and interventions to prevent white matter injury and age-related cognitive decline. This report provides a synopsis of the presentations and discussions by the participants, including the existing knowledge gaps and the delineation of the next steps towards advancing our understanding of white matter injury and age-related cognitive decline. Workshop discussions and consensus resulted in action by The Albert Trust to (1) increase support from biannual to annual "White Matter and Cognition" workshops; (2) provide funding for two collaborative, novel research grants annually submitted by meeting participants; and (3) coordinate the formation of the "Albert Research Institute for White Matter and Cognition." This institute will fill a gap in white matter science, providing white matter and cognition communications, including annual updates from workshops and the literature and interconnecting with other Albert Trust scientific endeavors in cognition and dementia, and providing support for newly established collaborations between seasoned investigators and to the development of talented young investigators in the VCI-dementia (VCID) and white matter cognition arena.

Rhamnose Binding Protein as an Anti-Bacterial Agent-Targeting Biofilm of Pseudomonas aeruginosa.

More than 80% of infectious bacteria form biofilm, which is a bacterial cell community surrounded by secreted polysaccharides, proteins and glycolipids. Such bacterial superstructure increases resistance to antimicrobials and host defenses. Thus, to control these biofilm-forming pathogenic bacteria requires antimicrobial agents with novel mechanisms or properties. Pseudomonas aeruginosa, a Gram-negative opportunistic nosocomial pathogen, is a model strain to study biofilm development and correlation between biofilm formation and infection. In this study, a recombinant hemolymph plasma lectin (rHPLOE) cloned from Taiwanese Tachypleus tridentatus was expressed in an Escherichia coli system. This rHPLOE was shown to have the following properties: (1) Binding to P. aeruginosa PA14 biofilm through a unique molecular interaction with rhamnose-containing moieties on bacteria, leading to reduction of extracellular di-rhamnolipid (a biofilm regulator); (2) decreasing downstream quorum sensing factors, and inhibiting biofilm formation; (3) dispersing the mature biofilm of P. aeruginosa PA14 to improve the efficacies of antibiotics; (4) reducing P. aeruginosa PA14 cytotoxicity to human lung epithelial cells in vitro and (5) inhibiting P. aeruginosa PA14 infection of zebrafish embryos in vivo. Taken together, rHPLOE serves as an anti-biofilm agent with a novel mechanism of recognizing rhamnose moieties in lipopolysaccharides, di-rhamnolipid and structural polysaccharides (Psl) in biofilms. Thus rHPLOE links glycan-recognition to novel anti-biofilm strategies against pathogenic bacteria.

Dingchuan tang essential oil inhibits the production of inflammatory mediators via suppressing the IRAK/NF-κB, IRAK/AP-1, and TBK1/IRF3 pathways in lipopolysaccharide-stimulated RAW264.7 cells.

BACKGROUND: Dingchuan tang (asthma-relieving decoction), a formula of nine herbs, has been used for treating respiratory inflammatory diseases for >400 years in the People's Republic of China. However, the mechanisms underlying the anti-inflammatory action of dingchuan tang is not fully understood. This study aims to investigate the effects of Dingchuan tang essential oil (DCEO) on inflammatory mediators and the underlying mechanism of action. MATERIALS AND METHODS: DCEO was extracted by steam distillation. Lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages were used as the cell model. Production of nitric oxide (NO) was determined by the Griess test. Protein secretion and mRNA levels of inflammatory mediators were measured by the enzyme-linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (qRT-PCR), respectively. Protein levels were examined by Western blot. Nuclear localization of nuclear factor-kappa B (NF-κB) was detected using immunofluorescence analyses. RESULTS: DCEO significantly reduced LPS-triggered production of NO and prostaglandin E2 (PGE2) and decreased protein and mRNA levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). LPS induced upregulation of protein and mRNA levels of cytokines (interleukin-1ß [IL-1ß], interleukin-6 [IL-6], tumor necrosis factor-α [TNF-α]), and chemokines (monocyte chemoattractant protein-1 [MCP-1], chemokine [C-C motif] ligand 5 [CCL-5], and macrophage inflammatory protein [MIP]-1α) were suppressed by DCEO treatment. Phosphorylation and nuclear protein levels of transcription factors (activator protein-1 [AP-1], NF-κB, interferon regulatory factor 3 [IRF3]) were decreased by DCEO. Protein levels of phosphorylated IκB-α, IκB kinase α/ß (IKKα/ß), phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt), TGF ß-activated kinase 1 (TAK1), TANK-binding kinase 1 (TBK1), extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (p38), and c-Jun N-terminal kinase (JNK) were lowered by DCEO. Moreover, degradation of interleukin-1 receptor-associated kinase 1 (IRAK1) and IRAK4 induced by LPS was inhibited by DCEO treatment. CONCLUSION: Suppression of the interleukin-1 receptor-associated kinase (IRAK)/NF-κB, IRAK/AP-1 and TBK1/IRF3 pathways was associated with the inhibitory effects of DCEO on inflammatory mediators in LPS-stimulated RAW264.7 macrophages. This study provides a pharmacological justification for the use of dingchuan tang in managing inflammatory disorders.