Molecular Weight Forms Research Articles

Individual evaluation of aging- and caloric restriction-related changes to distinct multimeric complexes of circulating adiponectin by immunoblotting

Adiponectin (APN), a major adipokine secreted from white adipose tissue, prevents inflammation and improves insulin sensitivity. APN exists as distinct multimeric complexes with different physiological activities, including low, middle and high molecular weight complexes (LMW, MMW and HMW, respectively) in peripheral blood. Caloric restriction (CR), an intervention that suppresses aging-related pathophysiological changes and extends lifespan, reportedly elevates the expression levels of Adipoq (encoding APN) and total circulating APN. Circulating APN levels have generally been measured using ELISA, but ELISA fails to directly and separately detect APN multimeric complexes other than HMW. Here, we aimed to evaluate the association of aging and CR with oligomerization of APN in rodent models, using immunoblotting to distinguish multimeric complexes based on molecular sizes. In mice, aging elevated plasma levels of HMW and MMW, while CR only elevated HMW. In contrast, LMW and monomeric APN levels were unchanged, suggesting that aging and CR can induce the assembly of APN oligomers in adipocytes. In rats, plasma levels of all multimeric complexes and monomeric APN were not significantly changed by aging or CR. Collectively, levels of circulating APN in mice were consistent with previous findings, whereas those of rats were partially inconsistent, probably because of experimental differences. Moreover, aging reduced Adipoq mRNA levels in mice and rats, while CR prevented this reduction only in rats. Such a discrepancy between Adipoq expression and circulating APN levels may be attributed to proteasomal regulation in adipocytes or tissue accumulation of APN. In conclusion, this study provides new findings of aging- and CR-related changes of each APN multimeric complex and underscores the importance of qualitative approaches for a greater understanding of physiological changes in APN.

PI3K\u03b3 stimulates a high molecular weight form of myosin light chain kinase to promote myeloid cell adhesion and tumor inflammation

Myeloid cells play key roles in cancer immune suppression and tumor progression. In response to tumor derived factors, circulating monocytes and granulocytes extravasate into the tumor parenchyma where they stimulate angiogenesis, immune suppression and tumor progression. Chemokines, cytokines and interleukins stimulate PI3Kγ-mediated Rap1 activation, leading to conformational changes in integrin α4β1 that promote myeloid cell extravasation and tumor inflammation Here we show that PI3Kγ activates a high molecular weight form of myosin light chain kinase, MLCK210, that promotes myosin-dependent Rap1 GTP loading, leading to integrin α4β1 activation. Genetic or pharmacological inhibition of MLCK210 suppresses integrin α4β1 activation, as well as tumor inflammation and progression. These results demonstrate a critical role for myeloid cell MLCK210 in tumor inflammation and serve as basis for the development of alternative approaches to develop immune oncology therapeutics.

An ELISA-based antigenicity test of rabies recombinant glycoprotein cannot predict its protective potency in vivo

The potency of human and veterinary rabies vaccines is measured based on the National Institute of Health (NIH) potency test that is laborious, time-consuming, variable, and requires sacrifice of large numbers of mice. ELISA-based methods quantifying rabies glycoprotein (rGP) are being developed as potential alternatives to the NIH potency test for release of rabies vaccines. The aim of the current study was focused on the evaluation of in vitro- and in vivo-based assays in order to assess their concurrence for adequate and reliable assessment of immunogenicity and protective potency of a plant-derived recombinant rGP. The recombinant rGP of strain ERA.KK was engineered, expressed and purified from Nicotiana benthamiana plants. The recombinant rGP excluded the transmembrane and intracytoplasmic domains. It was purified by chromatography (≥90%) from the plant biomass, characterized, and mainly presented as high molecular weight forms, most likely soluble aggregates, of the rGP ectodomain. It was well-recognized and quantified by an ELISA, which utilizes two mouse monoclonal antibodies, D1-25 and 1112-1, and which should only recognize the native trimeric form of the rGP. However, in mice, the recombinant rGP did not induce the production of anti-rabies virus neutralizing antibodies and did not confer protection after intracerebral viral challenge. Similar immunogenicity was observed in guinea pigs and rabbits. Our results demonstrate that use of the ELISA method described here is not predictive of performance in vivo. These data highlight the critical need to develop in vitro potency assays that reliably define the antigen content that can induce a protective response.

Construction and Evaluation of Recombinant Chimeric Fibrillin and Elastin Fragment in Human Mesenchymal Stem Cells.

Diverse extracellular matrix (ECM) proteins physically interact with stem cells and regulate stem cell function. However, the large molecular weight of the natural ECM renders large-scale fabrication of a similar functional structure challenging. The objective of this study was to construct a low molecular weight and multifunctional chimeric form of recombinant ECM to stimulate mesenchymal stem cell (MSC) for tissue repair. We engineered Fibrillin-1PF14 fused to an elastin-like polypeptide to develop a new biomimetic ECM for stem cell differentiation and investigated whether this recombinant chimeric Fibrillin-Elastin fragment (rcFE) was effective on human nasal inferior turbinate-derived mesenchymal stem cells (hTMSCs). hTMSCs were grown in the medium supplemented with rcFE, then the effect of the protein was confirmed through cell adhesion assay, proliferation assay, and real-time PCR. rcFE enhanced the adhesion activity of hTMSCs by 2.7-fold at the optimal concentration, and the proliferation activity was 2.6-fold higher than that of the control group (non-treatment rcFE). In addition, when smooth muscle cell differentiation markers were identified by real-time PCR, Calponin increased about 6-fold, α-actin about 9-fold, and MYH11 about 10-fold compared to the control group. Chimeric rcFE enhanced cellular functions such as cell adhesion, proliferation, and smooth muscle differentiation of hTMSCs, suggesting that the rcFE can facilitate the induction of tissue regeneration.

Vasculocentric Axonal NfH in Small Vessel Disease.

Cerebral small vessel disease (SVD) causes lacunar stroke and vascular cognitive impairment in older people. The pathogenic pathways from vessel pathology to parenchymal damage in SVD are unknown. Neurofilaments are axonal structural proteins. Neurofilament-light (NfL) is an emerging biomarker for neurological disease. Here, we examined the high molecular weight form neurofilament-heavy (NfH) and quantified a characteristic pattern of peri-arterial (vasculocentric) NfH labeling. Subcortical frontal and parietal white matter from young adult controls, aged controls, and older people with SVD or severe Alzheimer disease (n = 52) was immunohistochemically labeled for hyperphosphorylated NfH (pNfH). The extent of pNfH immunolabeling and the degree of vasculocentric axonal pNfH were quantified. Axonal pNfH immunolabeling was sparse in young adults but a common finding in older persons (controls, SVD, or AD). Axonal pNfH was often markedly concentrated around small penetrating arteries. This vasculocentric feature was more common in older people with SVD than in those with severe AD (p = 0.004). We conclude that axonal pNfH is a feature of subcortical white matter in aged brains. Vasculocentric axonal pNfH is a novel parenchymal lesion that is co-located with SVD arteriopathy and could be a consequence of vessel pathology.

The unique Akt inhibitor SC66 suppressed AMPK activity and abolished autophagy through the EGFR-p62 pathway.

Akt is usually considered to be a negative regulator of both autophagy and adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) signaling. In the present study, we found that SC66, a pyridine-based allosteric Akt inhibitor, suppressed basal and H2 O2 -induced autophagy concurrent with decreased phosphorylation and activity of AMPK. SC66 treatment led to the formation of a high molecular weight (HMW) form of SQSTM1/p62 (p62), which is an autophagic substrate and is essential for selective autophagy. Moreover, we observed that SC66 inhibited the binding of p62 and microtubule-associated protein light chain 3 (LC3). The immunoprecipitation results revealed the interaction between p62 and epidermal growth factor receptor (EGFR), and knockdown of EGFR reversed SC66-mediated autophagy inhibition without affecting the phosphorylation of acetyl-CoA carboxylase (ACC), a well-known substrate of AMPK. SC66 increased the interaction between EGFR and Beclin 1 and markedly decreased the association of EGFR with VPS34, a critical protein for autophagy induction. Collectively, the data presented here indicate that EGFR-p62 pathway plays a critical role in Akt-mediated positive regulation of autophagy.

Glycol Chitosan Functionalized with a Gd(III) Chelate as a Redox-responsive Magnetic Resonance Imaging Probe to Label Cell Embedding Alginate Capsules.

One possibility for the non-invasive imaging of encapsulated cell grafts is to label the lumen of cell embedding capsules with a redox-responsive probe, as an increased extracellular reducing potential can be considered as a marker of hypoxia-induced necrosis. A Gd(III)-HPDO3A-like chelate has been conjugated to glycol-chitosan through a redox-responsive disulphide bond to obtain a contrast agent for Magnetic Resonance Imaging (MRI). Such a compound can be interspersed with fibroblasts within the lumen of alginate-chitosan capsules. Increasing reducing conditions within the extracellular microenvironment lead to the reductive cleavage of the disulphide bond and to the release of gadolinium in the form of a low molecular weight, non-ionic chelate. The efflux of such chelate from capsules is readily detected by a decrease of contrast enhancement in T1 -weighted MR images.

Abstract NG09: Bioengineered tissue mimetic hydrogels to study brain tumor biology and screen therapeutics

Abstract NG09: Bioengineered tissue mimetic hydrogels to study brain tumor biology and screen therapeutics

Time dependent synthesis of gold nanoparticles using chitosan as reducing agent: A spectroscopic approach

The present study attempted synthesis of gold nanoparticle using chitosan from shrimp waste and evaluated the kinetics of gold nanoparticle (AuNPs) formation. Two different forms of chitosan (high molecular weight and medium molecular weight forms) were used for the preparation of AuNPs. The formation of nanoparticles was studied using UV-VIS spectra obtained at different time intervals during synthesis. The typical UV-VIS spectrum of non-aggregated spherical AuNPs at 520 nm was obtained for both chitosan forms and the maximum absorbance was higher for the sample prepared with medium molecular weight chitosan. The results clearly indicated that the characteristic surface plasmonic resonance (SPR) peak became recognisable at 5 min of heating for the sample with high molecular weight chitosan and 3 min of heating for medium molecular weight chitosan. This indicated that, the lower the molecular weight, the faster the formation of nanoparticles. The wavelength (λ) of absorption maxima was selected and normalised with respect to the absorption maxima at 15 min. The sigmoidal shape of kinetic curve implied the autocatalytic character of AuNP formation. The data from the samples were fitted with two step Finke-Watzky kinetics model and the kinetics constants were derived. The rate constant of the nucleation reaction was found higher for the medium molecular weight chitosan. Keywords: AuNPs, Chitosan, Gold nanoparticles, Molecular weight, Rate constant

Oral (−)-Epicatechin Inhibits Progressive Tau Pathology in rTg4510 Mice Independent of Direct Actions at GSK3β

Aggregation of the microtubule-associated protein tau into paired helical filaments (PHFs) and neurofibrillary tangles is a defining characteristic of Alzheimer’s Disease. Various plant polyphenols disrupt tau aggregation in vitro but display poor bioavailability and low potency, challenging their therapeutic translation. We previously reported that oral administration of the flavonoid (−)-epicatechin (EC) reduced Amyloid-β (Aβ) plaque pathology in APP/PS1 transgenic mice. Here, we investigated whether EC impacts on tau pathology, independent of actions on Aβ, using rTg4510 mice expressing P301L mutant tau. 4 and 6.5 months old rTg4510 mice received EC (∼18 mg/day) or vehicle (ethanol) via drinking water for 21 days and the levels of total and phosphorylated tau were assessed. At 4 months, tau appeared as two bands of ∼55 kDa, phosphorylated at Ser262 and Ser396 and was unaffected by exposure to EC. At 6.5 months an additional higher molecular weight form of tau was detected at ∼64 kDa which was phosphorylated at Ser262, Ser396 and additionally at the AT8 sites, indicative of the presence of PHFs. EC consumption reduced the levels of the ∼64 kDa tau species and inhibited phosphorylation at Ser262 and AT8 phosphoepitopes. Regulation of the key tau kinase glycogen synthase kinase 3β (GSK3β) by phosphorylation at Ser9 was not altered by exposure to EC in mice or primary neurons. Furthermore, EC did not significantly inhibit GSK3β activity at physiologically-relevant concentrations in a cell free assay. Therefore, a 21-day intervention with EC inhibits or reverses the development of tau pathology in rTg4510 mice independently of direct inhibition of GSK3β.

Cleaved PINK1 induces neuronal plasticity through PKA-mediated BDNF functional regulation.

Mutations in PTEN-induced kinase 1 (PINK1) lead to early onset autosomal recessive Parkinson's disease in humans. In healthy neurons, full-length PINK1 (fPINK1) is post-translationally cleaved into different lower molecular weight forms, and cleaved PINK1 (cPINK1) gets shuttled to the cytosolic compartments to support extra-mitochondrial functions. While numerous studies have exemplified the role of mitochondrially localized PINK1 in modulating mitophagy in oxidatively stressed neurons, little is known regarding the physiological role of cPINK1 in healthy neurons. We have previously shown that cPINK1, but not fPINK1, modulates the neurite outgrowth and the maintenance of dendritic arbors by activating downstream protein kinase A (PKA) signaling in healthy neurons. However, the molecular mechanisms by which cPINK1 promotes neurite outgrowth remain to be elucidated. In this report, we show that cPINK1 supports neuronal development by modulating the expression and extracellular release of brain-derived neurotrophic factor (BDNF). Consistent with this role, we observed a progressive increase in the level of endogenous cPINK1 but not fPINK1 during prenatal and postnatal development of mouse brains and during development in primary cortical neurons. In cultured primary neurons, the pharmacological activation of endogenous PINK1 leads to enhanced downstream PKA activity, subsequent activation of the PKA-modulated transcription factor cAMP response element-binding protein (CREB), increased intracellular production and extracellular release of BDNF, and enhanced activation of the BDNF receptor-TRKβ. Mechanistically, cPINK1-mediated increased dendrite complexity requires the binding of extracellular BDNF to TRKβ. In summary, our data support a physiological role of cPINK1 in stimulating neuronal development by activating the PKA-CREB-BDNF signaling axis in a feedforward loop.

LMW cyclin E and its novel catalytic partner CDK5 are therapeutic targets and prognostic biomarkers in salivary gland cancers

Salivary gland cancers (SGCs) are rare yet aggressive malignancies with significant histological heterogeneity, which has made prediction of prognosis and development of targeted therapies challenging. In majority of patients, local recurrence and/or distant metastasis are common and systemic treatments have minimal impact on survival. Therefore, identification of novel targets for treatment that can also be used as predictors of recurrence for multiple histological subtypes of SGCs is an area of unmet need. In this study, we developed a novel transgenic mouse model of SGC, efficiently recapitulating the major histological subtype (adenocarcinomas of the parotid gland) of human SGC. CDK2 knock out (KO) mice crossed with MMTV-low molecular weight forms of cyclin E (LMW-E) mice generated the transgenic mouse models of SGC, which arise in the parotid region of the salivary gland, similar to the common site of origin seen in human SGCs. To identify the CDK2 independent catalytic partner(s) of LMW-E, we used LMW-E expressing cell lines in mass spectrometric analysis and subsequent biochemical validation in pull down assays. These studies revealed that in the absence of CDK2, LMW-E preferentially binds to CDK5. Molecular targeting of CDK5, using siRNA, resulted in inhibition of cell proliferation of human SGCs overexpressing LMW-E. We also provide clinical evidence of significant association of LMW-E/CDK5 co-expression and decreased recurrence free survival in human SGC. Immunohistochemical analysis of LMW-E and CDK5 in 424 patients representing each of the four major histological subtypes of human salivary cancers (Aci, AdCC, MEC, and SDC) revealed that LMW-E and CDK5 are concordantly (positive/positive or negative/negative) expressed in 70% of these patients. The co-expression of LMW-E/CDK5 (both positive) robustly predicts the likelihood of recurrence, regardless of the histological classification of these tumors. Collectively, our results suggest that CDK5 is a novel and targetable biomarker for the treatment of patients with SGC presenting with LMW-E overexpressing tumors.

Expression of C9orf72 hexanucleotide repeat expansion leads to formation of RNA foci and dipeptide repeat proteins but does not influence autophagy or proteasomal function in neuronal cells

C9orf72 hexanucleotide repeat expansion (HRE) is the major genetic cause underpinning frontotemporal lobar degeneration (FLTD) and amyotrophic lateral sclerosis (ALS). C9orf72 HRE-associated pathogenesis involves both loss-of-function, through reduced C9orf72 levels, and gain-of-function mechanisms, including formation of RNA foci and generation of dipeptide repeat (DPR) proteins. In addition, dysfunctional protein degradation pathways, i.e. autophagy and ubiquitin-proteasome system (UPS), are suggested. Our aim was to study the gain-of-function mechanisms in the context of the function of protein degradation pathways as well as the regulation of the DPR proteins through these pathways. To this end, we expressed the pathological HRE in neuronal N2a cells and mouse primary cortical neurons. Protein degradation pathways were modulated to induce or block autophagy or to inhibit UPS. In addition, proteasomal activity was assessed. The C9orf72 HRE-expressing N2a cells and neurons were confirmed to produce RNA foci and DPR proteins, predominantly the Poly-GP proteins. However, the presence of these pathological hallmarks did not result in alterations in autophagy or proteasomal activity in either of the studied cell types. In N2a cells, Poly-GP proteins appeared in soluble forms and Lactacystin-mediated UPS inhibition increased their levels, indicating proteasomal regulation. Similar effects were not observed in cortical neurons, where the Poly-GP proteins formed also higher molecular weight forms. These results suggest a cell type-specific morphology and regulation of the DPR proteins. Further studies in other model systems may shed additional light onto the effects of the C9orf72 HRE on cellular protein degradation pathways and the regulation of the DPR protein levels.

Effects of Light Orientation and Mechanical Damage to Leaves on Isoflavone Accumulation in Soybean Seeds

Soybean is largely cultivated worldwide providing high amounts of proteins and oil for food and feed, and isoflavones for nutraceutical uses. The increasing interest in agroforestry practices for improving carbon sequestration and mitigating climate changes suggests the need to assess soybean response to variations of light availability and direction. A two-year pot trial was carried out at Legnaro (NE Italy) in order to mimic the response of the soybean var. Sponsor to contrasting light orientation (east or west) by artificial shading, associated or not with mechanical leaf damage, in terms of protein accumulation, total cotyledon isoflavone concentration (TCIC) and isoflavone profile. Here, we demonstrate that a different intensity of the isoflavone metabolism exists in response to lighting conditions, with higher TCIC and slightly increased seed crude proteins in plants lighted from the east (morning time) and after mechanical leaf damage. The isoflavone profile was not changed, but low temperatures and high rainfall during seed filling (1st year) were associated with increased accumulation of medium-high molecular weight (MW) forms (i.e., glycosyls and malonyls), while high temperatures and low rainfall (2nd year) with increased accumulation of medium-low MW forms (i.e., glycosyls and aglycones). It is concluded that within agroforestry systems, there is possibly a large scope for maximizing isoflavone accumulation by selecting the harvesting area in the neighboring of the east side of the tree alleys, with further improvements if a mild shoot stripping is applied before flowering.

Evaluation of monomeric prolactin level by TRACE method and precipitation with polyethylene glycol

Prolactin exists in various forms including the monomeric biologically active form (23kDa) and a higher molecular weight form, bound most commonly to IgG, known as macroprolactin (>100kDa). Macroprolactin lacks biological activity and is one of the causes of false-positive results. In Russian Federation the most common method for macroprolactin determination is PEG precipitation test. We had conducted a retrospective analysis of 37 samples of patients with hyperprolactinemia (3 of them were males). The mean age was 30 [25;35] years. Prolactin level was measured by the immunoenzyme method with manual PEG precipitation and TRACE. The mean values found by the immunoenzyme method with manual PEG precipitation were 461,6 [375,0;821,2] mU/l, by TRACE - 449,9 [357,2;749,2] mU/l. The number of patients with normal prolactin levels was 30% (11) confirmed by two methods, high prolactin level at 46% (17). The prevalence of clinical symptoms of hyperprolactinemia was not differ depend the groups. The phenomenon of macroprolactinemia was registered in 32% (12) of patients. In 8 persons of this group normal prolactin level was revealed and in 4 patients hyperprolactinemia was found by TRACE. Measurements of prolactin levels by the TRACE method is useful for correct diagnosis in patients with equivocal results received by traditional method with PEG precipitation.

Influence of unsaturated photoinitiators on acrylic pressure-sensitive adhesive properties

Solvent-based acrylic pressure-sensitive adhesives (PSAs) containing unsaturated copolymerizable photoinitiators were synthesized. This study describes novel photoreactive acrylic PSAs in which performance in the form of molecular weight, tack, peel adhesion and shear strength are dependent on photoinitiator kind and concentration, UV-dose, and UV-crosslinking time.

Molecular forms of neurogranin in cerebrospinal fluid.

Neurogranin (Ng) is a 78 amino acid neuronal protein and a biomarker candidate for Alzheimer's disease (AD). Ng has been suggested to bind to calmodulin and phosphatidic acid via its centrally located IQ domain. Ng is cleaved within this functionally important domain, yielding the majority of fragments identified in cerebrospinal fluid (CSF), suggesting that cleavage of Ng may be a mechanism to regulate its function. Up to now, Ng has been shown to be present in CSF as both C‐terminal fragments as well as full‐length protein. To obtain an overview of the different molecular forms of Ng present in CSF, we show by size exclusion chromatography (SEC), immunoblotting, immunoprecipitation, and MS that Ng is present in CSF as several molecular forms. Besides monomeric full‐length Ng, also higher molecular weight forms of Ng, and C‐terminal‐ and previously not identified N‐terminal fragments were observed. We found by immunodepletion that C‐terminal peptides contribute on average to ~50% of the total‐Ng ELISA signal in CSF samples. There were no differences in the overall C‐terminal fragment/total‐Ng ratios between samples from AD and control groups. In addition, we found that monomeric Ng and its C‐terminal fragments bind to heparin via a heparin‐binding motif, which might be of relevance for their export mechanism from neurons. Taken together, this study highlights the presence of several molecular forms of Ng in CSF, comprising monomeric full‐length Ng, and N‐ and C‐terminal truncations of Ng, as well as larger forms of still unknown composition.

Autophagy-mediated cytoplasmic accumulation of p53 leads to apoptosis through DRAM-BAX in cadmium-exposed human proximal tubular cells

The tumor suppressor p53 is involved in cadmium (Cd)-induced apoptosis and autophagy. However, the regulatory mechanisms of p53 in Cd-induced kidney injury are not well established. Here, we report the role of autophagy in Cd-induced p53 induction in human proximal tubular cells (HK-2). HK-2 cells treated with Cd induced the expression of p53, DNA damage autophagy modulator (DRAM), and Bcl-2-associated X protein (BAX), as well as caused poly [ADP-ribose] polymerase 1 (PARP-1) cleavage. Cd exposure also induced autophagy with the accumulation of monomeric p62 and multiple high molecular weight form (HMW)-p62. The expression levels of p53, p62, microtubule-associated protein 1A/1B-light chain 3 (LC3)-1, and LC3-II were similar in the sense that they increased up to 12 h and then gradually decreased. DRAM and BAX levels began to increase post autophagy induction and continued to increase, indicating that autophagy preceded apoptosis. While the genetic knockdown of p53 downregulated HWM-p62, DRAM, and BAX, the expression levels of these proteins were upregulated by p53 overexpression. The genetic knockdown of p62 downregulated p53, autophagy, DRAM, and BAX. The inhibition of autophagy through pharmacological and genetic knockdown reduced p53 and inhibited Cd-induced apoptosis. Collectively, Cd induces apoptosis through p53-mediated DRAM-BAX signaling, which can be regulated by autophagy.

Inhibition of the proteasome and proteaphagy enhances apoptosis in FLT3-ITD-driven acute myeloid leukemia.

Acute myeloid leukaemia (AML) is a clonal disorder that affects hematopoietic stem cells or myeloid progenitors. One of the most common mutations that results in AML occurs in the gene encoding fms-like tyrosine kinase 3 (FLT3). Previous studies have demonstrated that AML cells expressing FLT3-internal tandem duplication (ITD) are more sensitive to the proteasome inhibitor bortezomib (Bz) than FLT3 wild-type cells, with this cytotoxicity being mediated by autophagy (Atg). Here, we show that proteasome inhibition with Bz results in modest but consistent proteaphagy in MOLM-14 leukemic cells expressing the FLT3-ITD mutation, but not in OCI-AML3 leukemic cells with wild-type FLT3. Chemical inhibition of Atg with bafilomycin A simultaneously blocked proteaphagy and resulted in the accumulation of the p62 Atg receptor in Bz-treated MOLM-14 cells. The use of ubiquitin traps revealed that ubiquitin plays an important role in proteasome-Atg cross-talk. The p62 inhibitor verteporfin blocked proteaphagy and, importantly, resulted in accumulation of high molecular weight forms of p62 and FLT3-ITD in Bz-treated MOLM-14 cells. Both Atg inhibitors enhanced Bz-induced apoptosis in FLT3-ITD-driven leukemic cells, highlighting the therapeutic potential of these treatments.

Genetic markers and phosphoprotein forms of beta-catenin pβ-Cat552 and pβ-Cat675 are prognostic biomarkers of cervical cancer.

BACKGROUNDCervical cancer (CC) remains a leading cause of gynaecological cancer-related mortality world wide and constitutes the third most common malignancy in women. The RAIDs consortium (http://www.raids-fp7.eu/) conducted a prospective European study [BioRAIDs (NCT02428842)] with the objective to stratify CC patients for innovative treatments. A “metagene” of genomic markers in the PI3K pathway and epigenetic regulators had been previously associated with poor outcome [2].METHODSTo detect new, more specific, targets for treatment of patients who resist standard chemo-radiation, a high-dimensional Cox model was applied to define dominant molecular variants, copy number variations, and reverse phase protein arrays (RPPA).FINDINGSSurvival analysis on 89 patients with all omics data available, suggested loss-of-function (LOF) or activating molecular alterations in nine genes to be candidate biomarkers for worse prognosis in patients treated by chemo-radiation while LOF of ATRX, MED13 as well as CASP8 were associated with better prognosis. When protein expression data by RPPA were factored in, the supposedly low molecular weight and nuclear form, of beta-catenin, phosphorylated in Ser552 (pβ-Cat552), ranked highest for good prognosis, while pβ-Cat675 was associated with worse prognosis.INTERPRETATIONThese findings call for molecularly targeted treatments involving p53, Wnt pathway, PI3K pathway, and epigenetic regulator genes. Pβ-Cat552 and pβ-Cat675 may be useful biomarkers to predict outcome to chemo-radiation, which targets the DNA repair axis.FUNDINGEuropean Union's Seventh Program for research, technological development and demonstration (agreement N°304,810), the Fondation ARC pour la recherche contre le cancer.