Phosphorylation at the disordered N-end makes HuR accumulate and dimerize in the cytoplasm.

Human antigen R (HuR) is an RNA binding protein mainly involved in maintaining the stability and controlling the translation of mRNAs, critical for immune response, cell survival, proliferation and apoptosis. Although HuR is a nuclear protein, its mRNA translational-related function occurs at the cytoplasm, where the oligomeric form of HuR is more abundant. However, the regulation of nucleo-cytoplasmic transport of HuR and its connection with protein oligomerization remain unclear. In this work, we describe the phosphorylation of Tyr5 as a new hallmark for HuR activation. Our biophysical, structural and computational assays using phosphorylated and phosphomimetic HuR proteins demonstrate that phosphorylation of Tyr5 at the disordered N-end stretch induces global changes on HuR dynamics and conformation, modifying the solvent accessible surface of the HuR nucleo-cytoplasmic shuttling (HNS) sequence and releasing regions implicated in HuR dimerization. These findings explain the preferential cytoplasmic accumulation of phosphorylated HuR in HeLa cells, aiding to comprehend the mechanisms underlying HuR nucleus-cytoplasm shuttling and its later dimerization, both of which are relevant in HuR-related pathogenesis.

Molecular Insights of Cholestasis in MDR2 Knockout Murine Liver Organoids.

MDR3 (multidrug resistance 3) deficiency in humans (MDR2 in mice) causes progressive familial intrahepatic cholestasis type 3 (PFIC3). PFIC3 is a lethal disease characterized by an early onset of intrahepatic cholestasis progressing to liver cirrhosis, a preneoplastic condition, putting individuals at risk of hepatocellular carcinoma (HCC). Hepatocyte-like organoids from MDR2-deficient mice (MDR2KO) were used in this work to study the molecular alterations caused by the deficiency of this transporter. Proteomic analysis by mass spectrometry allowed characterization of 279 proteins that were differentially expressed in MDR2KO compared with wild-type organoids. Functional enrichment analysis indicated alterations in three main cellular functions: (1) interaction with the extracellular matrix, (2) remodeling intermediary metabolism, and (3) cell proliferation and differentiation. The affected cellular processes were validated by orthogonal molecular biology techniques. Our results point to molecular mechanisms associated with PFIC3 that may drive the progression to liver cirrhosis and HCC and suggest proteins and cellular processes that could be targeted for the development of early detection strategies for these severe liver diseases.

Distal hereditary motor neuronopathy as a new phenotype associated with variants in BAG3.

OBJECTIVE: To describe a new phenotype associated with a novel variant in BAG3: autosomal dominant adult-onset distal hereditary motor neuronopathy. METHODS: This study enrolled eight affected individuals from a single family and included a comprehensive evaluation of the clinical phenotype, neurophysiologic testing, muscle MRI, muscle biopsy and western blot of BAG3 protein in skeletal muscle. Genetic workup included whole exome sequencing and segregation analysis of the detected variant in BAG3. RESULTS: Seven patients developed slowly progressive and symmetric distal weakness and atrophy of lower limb muscles, along with absent Achilles reflexes. The mean age of onset was 46 years. The neurophysiological examination was consistent with the diagnosis of distal motor neuronopathy. One 57-year-old female patient was minimally symptomatic. The pattern of inheritance was autosomal dominant, with one caveat: one female patient who was an obligate carrier of the variant died at the age of 73 years without exhibiting any muscle weakness. The muscle biopsies revealed neurogenic changes. A novel heterozygous truncating variant c.1513_1514insGGAC (p.Val505GlyfsTer6) in the gene BAG3 was identified in all affected family members. CONCLUSIONS: We report an autosomal dominant adult-onset distal hereditary motor neuronopathy with incomplete penetrance in women as a new phenotype related to a truncating variant in the BAG3 gene. Our findings expand the phenotypic spectrum of BAG3-related disorders, which previously included dilated cardiomyopathy, myofibrillar myopathy and adult-onset Charcot-Marie-Tooth type 2 neuropathy. Variants in BAG3 should be considered in the differential diagnosis of distal hereditary motor neuronopathies.

Distal myopathy due to digenic inheritance of TIA1 and SQSTM1 variants in two unrelated Spanish patients.

Welander distal myopathy typically manifests in late adulthood and is caused by the founder TIA1 c.1150G>A (p.Glu384Lys) variant in families of Swedish and Finnish descent. Recently, a similar phenotype has been attributed to the digenic inheritance of TIA1 c.1070A>G (p.Asn357Ser) and SQSTM1 c.1175C>T (p.Pro392Leu) variants. We describe two unrelated Spanish patients presenting with slowly progressive gait disturbance, distal-predominant weakness, and mildly elevated creatine kinase (CK) levels since their 6th decade. Electromyography revealed abnormal spontaneous activity and a myopathic pattern. Muscle magnetic resonance imaging (MRI) showed marked fatty replacement in distal leg muscles. A muscle biopsy, performed on one patient, revealed myopathic changes with rimmed vacuoles. Both patients carried the TIA1 p.Asn357Ser and SQSTM1 p.Pro392Leu variants. Digenic inheritance is supported by evidence from unrelated pedigrees and a plausible biological interaction between both proteins in protein quality control processes. Recent functional studies and additional case descriptions further support this. Clinical suspicion is necessary to seek both variants.

Delayed Diagnosis of Congenital Myasthenic Syndromes Erroneously Interpreted as Mitochondrial Myopathies.

BACKGROUND: Congenital myasthenic syndromes (CMSs) and primary mitochondrial myopathies (PMMs) can present with ptosis, external ophthalmoplegia, and limb weakness. METHODS: Our method involved the description of three cases of CMS that were initially characterized as probable PMM. RESULTS: All patients were male and presented with ptosis and/or external ophthalmoplegia at birth, with proximal muscle weakness and fatigue on physical exertion. After normal repetitive nerve stimulation (RNS) studies performed on facial muscles, a muscle biopsy (at a median age of 9) was performed to rule out congenital myopathies. In all three cases, the biopsy findings (COX-negative fibers or respiratory chain defects) pointed to PMM. They were referred to our neuromuscular unit in adulthood to establish a genetic diagnosis. However, at this time, fatigability was evident in the physical exams and RNS in the spinal accessory nerve showed a decremental response in all cases. Targeted genetic studies revealed pathogenic variants in the MUSK, DOK7, and RAPSN genes. The median diagnostic delay was 29 years. Treatment resulted in functional improvement in all cases. CONCLUSIONS: Early identification of CMS is essential as medical treatment can provide clear benefits. Its diagnosis can be challenging due to phenotypic overlap with other debilitating disorders. Thus, a high index of suspicion is necessary to guide the diagnostic strategy.

Mapping early serum proteome signatures of liver regeneration in living donor liver transplant cases.

The liver is the only solid organ capable of regenerating itself to regain 100% of its mass and function after liver injury and/or partial hepatectomy (PH). This exceptional property represents a therapeutic opportunity for severe liver disease patients. However, liver regeneration (LR) might fail due to poorly understood causes. Here, we have investigated the regulation of liver proteome and phosphoproteome at a short time after PH (9 h), to depict a detailed mechanistic background of the early LR phase. Furthermore, we analyzed the dynamic changes of the serum proteome and metabolome of healthy living donor liver transplant (LDLT) donors at different time points after surgery. The molecular profiles from both analyses were then correlated. Insulin and FXR-FGF15/19 signaling were stimulated in mouse liver after PH, leading to the activation of the main intermediary kinases (AKT and ERK). Besides, inhibition of the hippo pathway led to an increased expression of its target genes and of one of its intermediary proteins (14-3-3 protein), contributing to cell proliferation. In association with these processes, metabolic reprogramming coupled to enhanced mitochondrial activity cope for the energy and biosynthetic requirements of LR. In human serum of LDLT donors, we identified 56 proteins and 13 metabolites statistically differential which recapitulate some of the main cellular processes orchestrating LR in its early phase. These results provide mechanisms and protein mediators of LR that might prove useful for the follow-up of the regenerative process in the liver after PH as well as preventing the occurrence of complications associated with liver resection.

Point-of-Care Peptide Hormone Production Enabled by Cell-Free Protein Synthesis.

In resource-limited settings, it can be difficult to safely deliver sensitive biologic medicines to patients due to cold chain and infrastructure constraints. Point-of-care drug manufacturing could circumvent these challenges since medicines could be produced locally and used on-demand. Toward this vision, we combine cell-free protein synthesis (CFPS) and a 2-in-1 affinity purification and enzymatic cleavage scheme to develop a platform for point-of-care drug manufacturing. As a model, we use this platform to synthesize a panel of peptide hormones, an important class of medications that can be used to treat a wide variety of diseases including diabetes, osteoporosis, and growth disorders. With this approach, temperature-stable lyophilized CFPS reaction components can be rehydrated with DNA encoding a SUMOylated peptide hormone of interest when needed. Strep-Tactin affinity purification and on-bead SUMO protease cleavage yield peptide hormones in their native form that are recognized by ELISA antibodies and that can bind their respective receptors. With further development to ensure proper biologic activity and patient safety, we envision that this platform could be used to manufacture valuable peptide hormone drugs in a decentralized way.

Targeted Proteomics for Monitoring One-Carbon Metabolism in Liver Diseases.

Liver diseases cause approximately 2 million deaths per year worldwide and had an increasing incidence during the last decade. Risk factors for liver diseases include alcohol consumption, obesity, diabetes, the intake of hepatotoxic substances like aflatoxin, viral infection, and genetic determinants. Liver cancer is the sixth most prevalent cancer and the third in mortality (second in males). The low survival rate (less than 20% in 5 years) is partially explained by the late diagnosis, which remarks the need for new early molecular biomarkers. One-carbon metabolism integrates folate and methionine cycles and participates in essential cell processes such as redox homeostasis maintenance and the regulation of methylation reactions through the production of intermediate metabolites such as cysteine and S-Adenosylmethionine. One-carbon metabolism has a tissue specific configuration, and in the liver, the participating enzymes are abundantly expressed-a requirement to maintain hepatocyte differentiation. Targeted proteomics studies have revealed significant differences in hepatocellular carcinoma and cirrhosis, suggesting that monitoring one-carbon metabolism enzymes can be useful for stratification of liver disease patients and to develop precision medicine strategies for their clinical management. Here, reprogramming of one-carbon metabolism in liver diseases is described and the role of mass spectrometry to follow-up these alterations is discussed.

[Comparison of the Pap smear with conventional technique versus modified technique]. / Comparación del Papanicolau con técnica convencional frente a técnica modificada.

Background: Despite the fact that the Papanicolaou technique is the most effective method of prevention and detection of cervical cancer, the precision of this tool remains controversial; Because of this, there are medical and scientific efforts to improve the quality of the procedure. Objective: Compare the quality of sampling between the conventional and modified technique. Material and methods: Descriptive and comparative observational study in 150 cervical cytology samples (75 conventional technique samples and 75 in modified technique) in women aged 25 to 64 years. Demographic variables, characteristics of the cervix and quality of the sample were analyzed. Descriptive statistics and association measures were performed. Study with risk greater than the minimum. All participants signed an informed consent. Results: The quality of the sample was satisfactory in 92.0% for the conventional technique vs 89.3% for the modified technique. The main cause of unsatisfactory samples was insufficient cellularity 6.7% in conventional technique vs 12% of the modified technique, with no significant difference between both techniques p = 0.575 (1.37; 0.45-4.1), findings that reject the working hypothesis. Conclusions: There was no significant difference when using both tests, the samples with satisfactory quality were similar between both techniques.

Introducción: a pesar de que la técnica de papanicolaou es el método más eficaz para la prevención y detección del cáncer cervicouterino, la precisión de esta herramienta sigue siendo controversial; debido a esto, existen esfuerzos médicos y científicos para mejorar la calidad del procedimiento. Objetivo: comparar la calidad en la toma de muestra entre la técnica convencional y la modificada. Material y métodos: estudio observacional descriptivo y comparativo en 150 muestras de citología cervical (75 muestras técnica convencional y 75 en técnica modificada) en mujeres de 25 a 64 años. Se analizaron variables demográficas, características del cérvix y calidad de la muestra. Se realizó estadística descriptiva y medidas de asociación. Estudio con riesgo mayor que el mínimo. Todas las participantes firmaron consentimiento informado. Resultados: la calidad de la muestra fue satisfactoria en el 92% para la técnica convencional frente al 89.3% para la técnica modificada. La causa principal de muestras insatisfactorias fue la celularidad insuficiente, la cual se presentó en el 6.7% de las muestras con técnica convencional frente al 12% de la técnica modificada, sin diferencia significativa entre ambas técnicas p = 0.575 (1.37; 0.45-4.1), hallazgos que rechazan la hipótesis de trabajo. Conclusiones: no hubo diferencia significativa al utilizar ambas pruebas, las muestras con calidad satisfactoria fueron similares entre ambas técnicas.

Comparación del Papanicolau con técnica convencional frente a técnica modificada / Comparison of the Pap smear with conventional technique versus modified technique

Introducción: a pesar de que la técnica de papanicolaou es el método más eficaz para la prevención y detección del cáncer cervicouterino, la precisión de esta herramienta sigue siendo controversial; debido a esto, existen esfuerzos médicos y científicos para mejorar la calidad del procedimiento. Objetivo: comparar la calidad en la toma de muestra entre la técnica convencional y la modificada. Material y métodos: estudio observacional descriptivo y comparativo en 150 muestras de citología cervical (75 muestras técnica convencional y 75 en técnica modificada) en mujeres de 25 a 64 años. Se analizaron variables emográficas, características del cérvix y calidad de la muestra. Se realizó estadística descriptiva y medidas de asociación. Estudio con riesgo mayor que el mínimo. Todas las participantes firmaron consentimiento informado. Resultados: la calidad de la muestra fue satisfactoria en el 92% para la técnica convencional frente al 89.3% para la técnica modificada. La causa principal de muestras insatisfactorias fue la celularidad insuficiente, la cual se presentó en el 6.7% de las muestras con técnica convencional frente al 12% de la técnica modificada, sin diferencia significativa entre ambas técnicas p = 0.575 (1.37; 0.45-4.1), hallazgos que rechazan la hipótesis de trabajo. Conclusiones: no hubo diferencia significativa al utilizar ambas pruebas, las muestras con calidad satisfactoria fueron similares entre ambas técnicas.

Background: Despite the fact that the Papanicolaou technique is the most effective method of prevention and detection of cervical cancer, the precision of this tool remains controversial; Because of this, there are medical and scientific efforts to improve the quality of the procedure. Objective: Compare the quality of sampling between the conventional and modified technique. Material and methods: Descriptive and comparative observational study in 150 cervical cytology samples (75 conventional technique samples and 75 in modified technique) in women aged 25 to 64 years. Demographic variables, characteristics of the cervix and quality of the sample were analyzed. Descriptive statistics and association measures were performed. Study with risk greater than the minimum. All participants signed an informed consent. Results: The quality of the sample was satisfactory in 92.0% for the conventional technique vs 89.3% for the modified technique. The main cause of unsatisfactory samples was insufficient cellularity 6.7% in conventional technique vs 12% of the modified technique, with no significant difference between both techniques p = 0.575 (1.37; 0.45-4.1), findings that reject the working hypothesis. Conclusions: There was no significant difference when using both tests, the samples with satisfactory quality were similar between both techniques.

Development of a Standardized MRM Method for the Quantification of One Carbon Metabolism Enzymes.

One-carbon metabolism (1CM) plays a central role in liver physiology, being the source of essential metabolites such as S-adenosylmethionine, the main alkylating agent in living cells, and glutathione, their most important nonenzymatic antioxidant defense. Impairment of 1CM in hepatocytes is a recognized factor associated to chronic liver disorders and hepatocellular carcinoma. With this in mind, we have proposed the concept of functional biomarker referring to a cellular pathway that can be systematically monitored as indicative of a particular physiological or pathological condition. Here we describe a targeted mass spectrometry (MRM) protocol to simultaneously quantify 13 1CM enzymes in liver tissue specimens.

Monitoring one-carbon metabolism by mass spectrometry to assess liver function and disease.

Precision medicine promises to overcome the constraints of the traditional "one-for-all" healthcare approach through a clear understanding of the molecular features of a disease, allowing for innovative and tailored treatments. State-of-the-art proteomics has the potential to accurately explore the human proteome to identify, quantify, and characterize proteins associated with disease progression. There is a pressing need for informative biomarkers to diagnose liver disease early in its course to prevent severe disease for which no efficient treatment is yet available. Here, we propose the concept of a cellular pathway as a functional biomarker, whose monitorization may inform normal and pathological status. We have developed a standardized targeted selected-reaction monitoring assay to detect and quantify 13 enzymes of one-carbon metabolism (1CM). The assay is compliant with Clinical Proteomics Tumor Analysis Consortium (CPTAC) guidelines and has been included in the protein quantification assays that can be accessed through the assay portal at the CPTAC web page. To test the feasibility of the assay, we conducted a retrospective, proof-of-concept study on a collection of liver samples from healthy controls and from patients with cirrhosis or hepatocellular carcinoma (HCC). Our results indicate a significant reconfiguration of 1CM upon HCC development resulting from a process that can already be identified in cirrhosis. Our findings indicate that the systematic and integrated quantification of 1CM enzymes is a promising cell function-based biomarker for patient stratification, although further experiments with larger cohorts are needed to confirm these findings.

Persistent asymptomatic or mild symptomatic hyperCKemia due to mutations in ANO5: the mildest end of the anoctaminopathies spectrum.

BACKGROUND: The ANO5 gene encodes for anoctamin-5, a chloride channel involved in muscle cell membrane repair. Recessive mutations in ANO5 are associated with muscular diseases termed anoctaminopathies, which are characterized by proximal or distal weakness, or isolated hyperCKemia. We present the largest series of patients with asymptomatic/paucisymptomatic anoctaminopathy reported so far, highlighting their clinical and radiological characteristics. METHODS: Twenty subjects were recruited retrospectively from the Neuromuscular Disorders Units database of two national reference centers. All had a confirmed genetic diagnosis (mean age of diagnosis was 48 years) established between 2015 and 2019. Clinical and complementary data were evaluated through clinical records. RESULTS: None of the patients complained about weakness or showed abnormal muscular balance. Among paucisymptomatic patients, the main complaints or findings were generalized myalgia, exercise intolerance and calf hypertrophy, occasionally associated with calf pain. All patients showed persistent hyperCKemia, ranging from mild-moderate to severe. Muscle biopsy revealed inflammatory changes in three cases. Muscle magnetic resonance imaging revealed typical signs (preferential involvement of adductor and gastrocnemius muscles) in all but one patient. In two cases, abnormal findings were detectable only in STIR sequences (not in T1). Three patients showed radiological progression despite remaining asymptomatic. Twelve different mutations in ANO5 were detected, of which seven are novel. CONCLUSIONS: Recessive mutations in ANO5 are a frequent cause of undiagnosed asymptomatic/paucisymptomatic hyperCKemia. Patients with an apparent indolent phenotype may show muscle involvement in complementary tests (muscle biopsy and imaging), which may progress over time. Awareness of anoctaminopathy as the cause of nonspecific muscular complaints or of isolated hyperCKemia is essential to correctly diagnose affected patients.

Molecular architecture of the DNA-binding sites of the P-loop ATPases MipZ and ParA from Caulobacter crescentus.

The spatiotemporal regulation of chromosome segregation and cell division in Caulobacter crescentus is mediated by two different P-loop ATPases, ParA and MipZ. Both of these proteins form dynamic concentration gradients that control the positioning of regulatory targets within the cell. Their proper localization depends on their nucleotide-dependent cycling between a monomeric and a dimeric state and on the ability of the dimeric species to associate with the nucleoid. In this study, we use a combination of genetic screening, biochemical analysis and hydrogen/deuterium exchange mass spectrometry to comprehensively map the residues mediating the interactions of MipZ and ParA with DNA. We show that MipZ has non-specific DNA-binding activity that relies on an array of positively charged and hydrophobic residues lining both sides of the dimer interface. Extending our analysis to ParA, we find that the MipZ and ParA DNA-binding sites differ markedly in composition, although their relative positions on the dimer surface and their mode of DNA binding are conserved. In line with previous experimental work, bioinformatic analysis suggests that the same principles may apply to other members of the P-loop ATPase family. P-loop ATPases thus share common mechanistic features, although their functions have diverged considerably during the course of evolution.

Blockade of the trans-sulfuration pathway in acute pancreatitis due to nitration of cystathionine ß-synthase.

Acute pancreatitis is an inflammatory process of the pancreatic gland that may lead to dysregulation of the trans-sulfuration pathway. The aims of this work were firstly to study the methionine cycle as well as the trans-sulfuration pathway using metabolomic and proteomic approaches identifying the causes of this dysregulation in an experimental model of acute pancreatitis; and secondly to reveal the effects of S-adenosylmethionine administration on these pathways. Acute pancreatitis was induced by cerulein in mice, and a group of animals received S-adenosylmethionine treatment. Cerulein-induced acute pancreatitis rapidly caused marked depletion of methionine, S-adenosylmethionine, 5'-methylthioadenosine, cystathionine, cysteine, and glutathione levels in pancreas, but S-adenosylhomocysteine and homocysteine remained unchanged. Protein steady-state levels of S-adenosylhomocysteine-hydrolase and cystathionine gamma-lyase diminished but methylthioadenosine phosphorylase levels increased in pancreas with acute pancreatitis. Although cystathionine ß-synthase protein levels did not change with acute pancreatitis, Nos2 mRNA and protein levels were markedly up-regulated and caused tyrosine nitration of cystathionine ß-synthase in pancreas. S-adenosylmethionine administration enhanced Nos2 mRNA expression and cystathionine ß-synthase nitration and triggered homocysteine accumulation in acute pancreatitis. Furthermore, S-adenosylmethionine administration promoted enrichment of the euchromatin marker H3K4me3 in the promoters of Tnf-α, Il-6, and Nos2 and enhanced the mRNA up-regulation of these genes. Accordingly, S-adenosylmethionine administration increased inflammatory infiltrate and edema in pancreas with acute pancreatitis. In conclusion, tyrosine-nitration of cystathionine ß-synthase blockades the trans-sulfuration pathway in acute pancreatitis promoting homocysteine accumulation upon S-adenosylmethionine treatment.

A gradient-forming MipZ protein mediating the control of cell division in the magnetotactic bacterium Magnetospirillum gryphiswaldense.

Cell division needs to be tightly regulated and closely coordinated with other cellular processes to ensure the generation of fully viable offspring. Here, we investigate division site placement by the cell division regulator MipZ in the alphaproteobacterium Magnetospirillum gryphiswaldense, a species that forms linear chains of magnetosomes to navigate within the geomagnetic field. We show that M. gryphiswaldense contains two MipZ homologs, termed MipZ1 and MipZ2. MipZ2 localizes to the division site, but its absence does not cause any obvious phenotype. MipZ1, by contrast, forms a dynamic bipolar gradient, and its deletion or overproduction cause cell filamentation, suggesting an important role in cell division. The monomeric form of MipZ1 interacts with the chromosome partitioning protein ParB, whereas its ATP-dependent dimeric form shows non-specific DNA-binding activity. Notably, both the dimeric and, to a lesser extent, the monomeric form inhibit FtsZ polymerization in vitro. MipZ1 thus represents a canonical gradient-forming MipZ homolog that critically contributes to the spatiotemporal control of FtsZ ring formation. Collectively, our findings add to the view that the regulatory role of MipZ proteins in cell division is conserved among many alphaproteobacteria. However, their number and biochemical properties may have adapted to the specific needs of the host organism.

Molecular iodine/doxorubicin neoadjuvant treatment impair invasive capacity and attenuate side effect in canine mammary cancer.

BACKGROUND: Mammary cancer has a high incidence in canines and is an excellent model of spontaneous carcinogenesis. Molecular iodine (I2) exerts antineoplastic effects on different cancer cells activating re-differentiation pathways. In co-administration with anthracyclines, I2 impairs chemoresistance installation and prevents the severity of side effects generated by these antineoplastic drugs. This study is a random and double-blind protocol that analyzes the impact of I2 (10 mg/day) in two administration schemes of Doxorubicin (DOX; 30 mg/m2) in 27 canine patients with cancer of the mammary gland. The standard scheme (sDOX) includes four cycles of DOX administered intravenously for 20 min every 21 days, while the modified scheme (mDOX) consists of more frequent chemotherapy (four cycles every 15 days) with slow infusion (60 min). In both schemes, I2 or placebo (colored water) was supplemented daily throughout the treatment. RESULTS: mDOX attenuated the severity of adverse events (VCOG-CTCAE) in comparison with the sDOX group. The overall tumor response rate (RECIST criteria) for all dogs was 18% (interval of reduction 48-125%), and no significant difference was found between groups. I2 supplementation enhances the antineoplastic effect in mDOX, exhibiting a significant decrease in the tumor epithelial fraction, diminished expression of chemoresistance (MDR1 and Survivin) and invasion (uPA) markers and enhanced expression of the differentiation factor known as peroxisome proliferator-activated receptors type gamma (PPARγ). Significant tumor lymphocytic infiltration was also observed in both I2-supplemented groups. The ten-month survival analysis showed that the entire I2 supplementation (before and after surgery) induced 67-73% of disease-free survival, whereas supplementation in the last period (only after surgery) produced 50% in both schemes. CONCLUSIONS: The mDOX+I2 scheme improves the therapeutic outcome, diminishes the invasive capacity, attenuates the adverse events and increases disease-free survival. These data led us to propose mDOX+I2 as an effective treatment for canine mammary cancer.

YTHDC1 mediates nuclear export of N6-methyladenosine methylated mRNAs.

N6-methyladenosine (m6A) is the most abundant internal modification of eukaryotic messenger RNA (mRNA) and plays critical roles in RNA biology. The function of this modification is mediated by m6A-selective 'reader' proteins of the YTH family, which incorporate m6A-modified mRNAs into pathways of RNA metabolism. Here, we show that the m6A-binding protein YTHDC1 mediates export of methylated mRNA from the nucleus to the cytoplasm in HeLa cells. Knockdown of YTHDC1 results in an extended residence time for nuclear m6A-containing mRNA, with an accumulation of transcripts in the nucleus and accompanying depletion within the cytoplasm. YTHDC1 interacts with the splicing factor and nuclear export adaptor protein SRSF3, and facilitates RNA binding to both SRSF3 and NXF1. This role for YTHDC1 expands the potential utility of chemical modification of mRNA, and supports an emerging paradigm of m6A as a distinct biochemical entity for selective processing and metabolism of mammalian mRNAs.

Phase I study of carfilzomib, lenalidomide, vorinostat, and dexamethasone in patients with relapsed and/or refractory multiple myeloma.

Research has shown that proteasome inhibitors (e.g., carfilzomib), immunomodulatory agents (e.g., lenalidomide), histone deacetylase inhibitors (e.g., vorinostat) and corticosteroids (e.g., dexamethasone) have synergistic anti-multiple myeloma (MM) activity. This phase I dose-escalation study evaluated a regimen combining carfilzomib, lenalidomide, vorinostat and dexamethasone (QUAD) in patients with relapsed and/or refractory MM. Seventeen patients received carfilzomib (15, 20, or 20/27 mg/m(2) ; 30-min infusion; days 1, 2, 8, 9, 15, 16), lenalidomide (15 or 25 mg; days 1-21), vorinostat (300 or 400 mg; days 1-7, 15-21), and dexamethasone (40 mg; days 1, 8, 15, 22) in 28-d cycles. No dose-limiting toxicities were observed; the maximum tolerated dose was not reached. The maximum administered dose was carfilzomib 20/27 mg/m(2) , lenalidomide 25 mg, vorinostat 400 mg, and dexamethasone 40 mg. Common grade ≥3 adverse events included neutropenia (53%), thrombocytopenia (53%) and anaemia (41%). The overall response rate was 53%: 12% of patients achieved a very good partial response (PR) and 41% of patients achieved a PR. At a median follow-up of 10 months, median progression-free survival was 12 months and median overall survival was not reached. Treatment with QUAD was feasible and had encouraging activity in patients with relapsed and/or refractory MM.