Senescence is a developmental mechanism that contributes to embryonic growth and patterning.

Senescence is a form of cell-cycle arrest linked to tumor suppression and aging. However, it remains controversial and has not been documented in nonpathologic states. Here we describe senescence as a normal developmental mechanism found throughout the embryo, including the apical ectodermal ridge (AER) and the neural roof plate, two signaling centers in embryonic patterning. Embryonic senescent cells are nonproliferative and share features with oncogene-induced senescence (OIS), including expression of p21, p15, and mediators of the senescence-associated secretory phenotype (SASP). Interestingly, mice deficient in p21 have defects in embryonic senescence, AER maintenance, and patterning. Surprisingly, the underlying mesenchyme was identified as a source for senescence instruction in the AER, whereas the ultimate fate of these senescent cells is apoptosis and macrophage-mediated clearance. We propose that senescence is a normal programmed mechanism that plays instructive roles in development, and that OIS is an evolutionarily adapted reactivation of a developmental process.

Alternative splicing of FKBP5 gene exerts control over T lymphocyte expansion.

FKBP51 is constitutively expressed by immune cells. As other FKBP family members, FKBP51 acts as a coreceptor for the natural products FK506 and rapamycin, which exhibit immunosuppressive effects. However, little is known about the intrinsic role of this large FKBP in the primary function of lymphocytes, that is, the adaptive immune response against foreign antigens, for example, pathogens. This paper aimed to investigate whether FKBP51 expression was modulated during lymphocyte activation. Moreover, as we recently identified a splicing isoform of FKBP51, namely FKBP51s, we also measured this splice protein, along with the canonical one, at different times of a peripheral blood mononuclear cell culture stimulated via T cell receptor. Our results show that the two FKBP51 isoforms were alternatively induced during the proliferative burst. Canonical FKBP51 increased in the time window between 48 and 96 h and its expression levels correlated with cyclin D levels. FKBP51s transiently increased earlier, at 24-36 h to reappearing later, at 120 h, when cyclin D expression returned at resting levels and proliferation ceased. Interestingly, within these two specific timeframes, FKBP51s accumulated in the nucleus. Here FKBP51s colocalized with the Foxp3 transcription factor at 36 h. Regulatory T cell (Treg) counts significantly decreased when FKBP51s was downmodulated. The coculture suppression assay suggested that FKBP51s supports the suppressive capability of Tregs. At 120 h, chromatin immunoprecipitation experiments found FKBP51s linked to CCND1 gene, suggesting a possible effect on gene transcription regulation, as previously demonstrated in melanoma. In conclusion, our study shows that FKBP5 isoforms are upregulated during lymphocyte activation, albeit on different timeframes. The activation of canonical FKBP51 coincides with proliferation hallmarks; FKBP5 splicing occurs early to sustain Treg development and late when proliferation ceases.

Gaucher disease prevalence in 600 patients affected by monoclonal gammopathy of undetermined significance.

BACKGROUND: Gaucher disease (GD) is a rare autosomal recessive inherited disorder caused by the lysosomal enzyme acid ß-glucosidase deficiency. Many patients experience a critical delay in the diagnosis of up to 8-10 years due to its rarity and variability in signs and symptoms, with the consultation of several specialists. PATIENTS AND METHODS: This prospective observational study analyzed the prevalence of GD in 600 patients with monoclonal gammopathy of uncertain significance (MGUS) from January 2018 until February 2022. RESULTS: The mean age of participants was 66 years, with a mean monoclonal component of 0.58 g/dL. In 433 MGUS patients with available data, anemia (hemoglobin level < 10 g/dL) was present in 31 patients (7%), and thrombocytopenia (platelet count <100.000/mm3 ) in 24 (5.5%). Of 600 MGUS patients tested for acid ß-glucosidase enzyme activity, 7 patients (1.2%) had activity below 2.5 nmol/h/mL. In comparison, GBA gene analysis was executed in 110 patients. It revealed 4 patients (0.7%) affected by GD (3 patients with compound heterozygous mutation and 1 with homozygous mutation), with a prevalence of 1 every 150 MGUS patients. Furthermore, 12 out of the remaining 106 evaluated patients (11%) were carriers of a single heterozygous mutation while having regular enzyme activity. CONCLUSIONS: The clinical heterogeneity of GD and frequent lack of awareness among physicians often lead to diagnostic delays and severe clinical manifestations. The role of MGUS in the presence of at least one clinical sign, such as low platelet count, organomegaly, bone pain, or bleeding tendency, could aid in initiating GD screening with DBS, thus reducing the period between symptom onset and the diagnosis of this rare disease.

Health-related quality of life and burden of fatigue in patients with primary immune thrombocytopenia by phase of disease.

The main objective of this study was to compare health-related quality of life (HRQOL) of primary immune thrombocytopenia (pITP) patients with that of general population, overall, and by patient group (i.e., newly diagnosed, persistent, and chronic patients). Fatigue was also investigated as a secondary objective. Overall, 424 adult patients were enrolled in a multicenter observational study and the control group consisted of a representative sample from the general population. Propensity score matching plus further multivariate linear regression adjustment was used to compare HRQOL outcomes between pITP patients and general population. Mean age of patients was 54 years. Of those with HRQOL assessment, 99 patients (23.6%) were newly diagnosed, 53 (12.6%) were persistent, and 268 (63.8%) were chronic pITP patients. Comparison by patient group versus their respective peers in the general population revealed greater impairments in persistent pITP patients. Persistent pITP patients reported clinically meaningful impairments in physical functioning (-15; 95% CI -24.1 to -5.8; P = 0.002), social functioning (-15.3; 95% CI -25.5 to -5.1; P = 0.004), role physical (-28.4; 95% CI -43.1 to -13.7; P < 0.001), role emotional (-23.9; 95% CI -40.1 to -7.7; P = 0.004), and mental health scales (-11.3; 95% CI -21.2 to -1.4; P = 0.026) of the SF-36 questionnaire. Higher fatigue severity was associated with lower physical and mental HRQOL outcomes. Our findings suggest that the burden of the disease and treatment might depend on the disease phase and that persistent pITP patients are the most vulnerable subgroup. Am. J. Hematol. 91:995-1001, 2016. © 2016 Wiley Periodicals, Inc.

Metabolic vulnerability of cancer stem cells and their niche.

Cancer stem cells (CSC) are the leading cause of the failure of anti-tumor treatments. These aggressive cancer cells are preserved and sustained by adjacent cells forming a specialized microenvironment, termed niche, among which tumor-associated macrophages (TAMs) are critical players. The cycle of tricarboxylic acids, fatty acid oxidation path, and electron transport chain have been proven to play central roles in the development and maintenance of CSCs and TAMs. By improving their oxidative metabolism, cancer cells are able to extract more energy from nutrients, which allows them to survive in nutritionally defective environments. Because mitochondria are crucial bioenergetic hubs and sites of these metabolic pathways, major hopes are posed for drugs targeting mitochondria. A wide range of medications targeting mitochondria, electron transport chain complexes, or oxidative enzymes are currently investigated in phase 1 and phase 2 clinical trials against hard-to-treat tumors. This review article aims to highlight recent literature on the metabolic adaptations of CSCs and their supporting macrophages. A focus is provided on the resistance and dormancy behaviors that give CSCs a selection advantage and quiescence capacity in particularly hostile microenvironments and the role of TAMs in supporting these attitudes. The article also describes medicaments that have demonstrated a robust ability to disrupt core oxidative metabolism in preclinical cancer studies and are currently being tested in clinical trials.

FKBP51 plays an essential role in Akt ubiquitination that requires Hsp90 and PHLPP.

FKBP51 plays a relevant role in sustaining cancer cells, particularly melanoma. This cochaperone participates in several signaling pathways. FKBP51 forms a complex with Akt and PHLPP, which is reported to dephosphorylate Akt. Given the recent discovery of a spliced FKBP51 isoform, in this paper, we interrogate the canonical and spliced isoforms in regulation of Akt activation. We show that the TPR domain of FKBP51 mediates Akt ubiquitination at K63, which is an essential step for Akt activation. The spliced FKBP51, lacking such domain, cannot link K63-Ub residues to Akt. Unexpectedly, PHLPP silencing does not foster phosphorylation of Akt, and its overexpression even induces phosphorylation of Akt. PHLPP stabilizes levels of E3-ubiquitin ligase TRAF6 and supports K63-ubiquitination of Akt. The interactome profile of FKBP51 from melanoma cells highlights a relevant role for PHLPP in improving oncogenic hallmarks, particularly, cell proliferation.

Response Assessment to Erythropoietin-Zeta (Epo-Alpha Biosimilar) Therapy in Low-Risk Myelodysplastic Syndromes.

BACKGROUND: This prospective observational study aimed to verify the efficacy of erythropoietin zeta in the treatment of patients with low-risk myelodysplastic syndrome. METHODS: Patients with low/int-1 IPSS risk and serum erythropoietin level below 500 U/L were enrolled. Treatment consisted of erythropoietin zeta 40,000 U subcutaneously once a week. The primary endpoint was the erythroid response. According to Simon's two-stage statistical design, 36 patients were recruited. The median age was 75 years (range 56-83 years), male/female ratio was 1.1/1, median baseline serum erythropoietin was 57.9 U/L (range 9.4-475 U/L). 53% of patients had low-risk disease, while the remaining had Int-1 risk. RESULTS: After 8 weeks, a significant response (rise in Hb levels of at least 1.5 g/dL) was achieved in 18 patients (50%) out of 36. However, 17 patients did not improve; 8/17 patients pursued the 40,000 U weekly schedule of erythropoietin zeta, and 4/8 (50%) of them reached the erythroid response after 16 weeks. Nine patients underwent dosage doubling (40,000 U twice per week), and 5/9 (55%) of them achieved the erythroid response. CONCLUSION: Compared with data from the literature, this prospective study revealed that EPO-zeta is a safe and effective therapeutic option in low-risk MDS patients.

ZeOncoTest: Refining and Automating the Zebrafish Xenograft Model for Drug Discovery in Cancer.

The xenograft of human cancer cells in model animals is a powerful tool for understanding tumor progression and metastatic potential. Mice represent a validated host, but their use is limited by the elevated experimental costs and low throughput. To overcome these restrictions, zebrafish larvae might represent a valuable alternative. Their small size and transparency allow the tracking of transplanted cells. Therefore, tumor growth and early steps of metastasis, which are difficult to evaluate in mice, can be addressed. In spite of its advantages, the use of this model has been hindered by lack of experimental homogeneity and validation. Considering these facts, the aim of our work was to standardize, automate, and validate a zebrafish larvae xenograft assay with increased translatability and higher drug screening throughput. The ZeOncoTest reliability is based on the optimization of different experimental parameters, such as cell labeling, injection site, automated individual sample image acquisition, and analysis. This workflow implementation finally allows a higher precision and experimental throughput increase, when compared to previous reports. The approach was validated with the breast cancer cell line MDA-MB-231, the colorectal cancer cells HCT116, and the prostate cancer cells PC3; and known drugs, respectively RKI-1447, Docetaxel, and Mitoxantrone. The results recapitulate growth and invasion for all tested tumor cells, along with expected efficacy of the compounds. Finally, the methodology has proven useful for understanding specific drugs mode of action. The insights gained bring a step further for zebrafish larvae xenografts to enter the regulated preclinical drug discovery path.

Enhancing Understanding of the Visual Cycle by Applying CRISPR/Cas9 Gene Editing in Zebrafish.

During the vertebrate visual cycle, all-trans-retinal is exported from photoreceptors to the adjacent RPE or Müller glia wherein 11-cis-retinal is regenerated. The 11-cis chromophore is returned to photoreceptors, forming light-sensitive visual pigments with opsin GPCRs. Dysfunction of this process perturbs phototransduction because functional visual pigment cannot be generated. Mutations in visual cycle genes can result in monogenic inherited forms of blindness. Though key enzymatic processes are well characterized, questions remain as to the physiological role of visual cycle proteins in different retinal cell types, functional domains of these proteins in retinoid biochemistry and in vivo pathogenesis of disease mutations. Significant progress is needed to develop effective and accessible treatments for inherited blindness arising from mutations in visual cycle genes. Here, we review opportunities to apply gene editing technology to two crucial visual cycle components, RPE65 and CRALBP. Expressed exclusively in the human RPE, RPE65 enzymatically converts retinyl esters into 11-cis retinal. CRALBP is an 11-cis-retinal binding protein expressed in human RPE and Muller glia. Loss-of-function mutations in either protein results in autosomal recessive forms of blindness. Modeling these human conditions using RPE65 or CRALBP murine knockout models have enhanced our understanding of their biochemical function, associated disease pathogenesis and development of therapeutics. However, rod-dominated murine retinae provide a challenge to assess cone function. The cone-rich zebrafish model is amenable to cost-effective maintenance of a variety of strains. Interestingly, gene duplication in zebrafish resulted in three Rpe65 and two Cralbp isoforms with differential temporal and spatial expression patterns. Functional investigations of zebrafish Rpe65 and Cralbp were restricted to gene knockdown with morpholino oligonucleotides. However, transient silencing, off-target effects and discrepancies between knockdown and knockout models, highlight a need for more comprehensive alternatives for functional genomics. CRISPR/Cas9 in zebrafish has emerged as a formidable technology enabling targeted gene knockout, knock-in, activation, or silencing to single base-pair resolution. Effective, targeted gene editing by CRISPR/Cas9 in zebrafish enables unprecedented opportunities to create genetic research models. This review will discuss existing knowledge gaps regarding RPE65 and CRALBP. We explore the benefits of CRISPR/Cas9 to establish innovative zebrafish models to enhance knowledge of the visual cycle.

[Lipid profile in hematologic neoplasms]. / Profilo lipidico nelle neoplasie ematologiche.

BACKGROUND AND OBJECTIVE: Abnormal blood lipid profiles have been reported in human malignancies. So, it is likely an overall involvement of tumoral cell metabolism. The aim of this study was to evaluate clinico-biological implications of altered lipid profiles in oncohaematologic patients. DESIGN AND METHODS: The plasma lipids, lipoproteins and apolipoproteins were determined at the time of diagnosis in 48 previously untreated patients (35M, 13F, median age 60 years), 11 with multiple myeloma (MM), 11 with non-Hodgkin's lymphoma (NHL), 11 with acute leukemia (AL), 10 with chronic myeloproliferative disorders (CMD) and 5 with B-chronic lymphocytic leukemia (B-CLL). The results were correlated with known prognostic serum markers, such as lactate dehydrogenase (LDH), beta-2-microglobulin (beta 2m), and soluble molecule ICAM1 (sICAM1). RESULTS: Altered blood lipid profiles were observed in all concohaematologic patients. Statistically significant values included reduced cholesterol (155 +/- 47.36 vs 205 +/- 35 mg/dl; p < 0.001), HDL-C (30.47 +/- 13.36 vs 45 +/- 10 mg/dl; p < 0.003) and apo A (118.86 +/- 49.98 vs 182.69 mg/dl; p < 0.0001) levels. No correlations were found between cholesterol levels and clinico-biological features representative of tumor mass (LDH, beta 2m, sICAM-1). A significant increase of cholesterol levels was observed in all patients responding to therapy. INTERPRETATION AND CONCLUSION: These results support the idea that the cholesterol, its fractions and the apolipoproteins determinations might be considered as useful biochemical and prognostic markers in hematologic neoplasms.

Mst4 and Ezrin induce brush borders downstream of the Lkb1/Strad/Mo25 polarization complex.

The human Lkb1 kinase, encoded by the ortholog of the invertebrate Par4 polarity gene, is mutated in Peutz-Jeghers cancer syndrome. Lkb1 activity requires complex formation with the pseudokinase Strad and the adaptor protein Mo25. The complex can induce complete polarization in a single isolated intestinal epithelial cell. We describe an interaction between Mo25alpha and a human serine/threonine kinase termed Mst4. A homologous interaction occurs in the yeast Schizosaccharomyces pombe in the control of polar tip growth. Human Mst4 translocates from the Golgi to the subapical membrane compartment upon activation of Lkb1. Inhibition of Mst4 activity inhibits Lkb1-induced brush border formation, whereas other aspects of polarity such as the formation of lateral junctions remain unaffected. As an essential event in brush border formation, Mst4 phosphorylates the regulatory T567 residue of Ezrin. These data define a brush border induction pathway downstream of the Lkb1/Strad/Mo25 polarization complex, yet separate from other polarity events.

Efeito de pasta com nanopartículas de hidroxiapatita e fluoreto sobre a desmineralizacão da dentina / The effect of paste containing hydroxyapatite nanoparticles and fluoride on dentin demineralization

Este estudo avaliou o potencial de uma formulação experimental à base de HAP e fluoreto de sódio, para reduzir a desmineralização da dentina bovina in vitro. Oitenta e quatro amostras de dentina radicular bovina foram divididas em 7 grupos de 12 amostras, sendo submetidas a um dos seguintes tratamentos: 1) pasta Nanop plus [HAP a 20% + 9.000 ppm F, NaF); 2) pasta Nanop [HAP 20%); 3) pasta F [9.000 ppm F, NaF); 4) pasta placebo [sem HAP e F); 5) MI Paste [caseína e fosfato de cálcio); 6) MI Paste plus [caseína e fosfato de cálcio, 900 ppm F, NaF); 7) sem tratamento [controle). Os tratamentos foram realizados duas vezes ao dia, intercalados por ciclagem de pH (des-rernineralização], por 7 dias. Na sequência, as amostras foram analisadas por microdureza superficial e longitudinal (10-220 um). Os dados foram tabulados e submetidos à análise estatística [p<0,05). Em geral, os únicos tratamentos que tiveram efeito significativo na redução da desmineralização da dentina foram as pastas Nanop plus e F. Portanto, os dados confirmaram que o fluoreto ainda continua sendo o melhor agente químico para controlar a desmineralização dentária. Já a pasta experimental contendo nanopartículas de HAP foi ineficaz em reduzir a desmineralização da dentina utilizando-se o modelo experimental proposto

This study evaluated the potential of an experimental formulation containing HAP and sodium fluoride to reduce bovine dentin demineralization in vitro. Eighty-four bovine rools dentin samples were divided into 7 groups of 12 samples and subjected to one of the following treatments: 1) Nanop plus paste (HAP 20% + 9000 ppm F, NaF); 2) Nanop paste (HAP 20010) 3) F paste (9000 ppm F, NaF); 4) placebo paste (without HAP and F); 5) MI paste (casein and calcium phosphate); 6) MI Paste plus (casein and calcium phosphate, 900 ppm F, NaF); 7) no treatment (controll. The treatments were performed twice a day, interspersed by pH cycling (de-remineralization) for 7 days. Thereafter, the samples were analyzed using surface and cross-sectional microhardness (10-220 J.1m). The data was plotted and statistically analyzed (p<0.05). The treatments that produced a significant effect on the reduction of dentin demi• neralization were Nanop plus and F pastes only. Therefore, the data confirmed that fluoride is still the best chemical agent to control dental demineralization. The experimental paste containing HAP nanoparticles was ineffective in reducing dentin demineralization when using this experimental model