Cordelli, E., Fresegna, A. M., Leter, G., Eleuteri, P., Spanò, M. and Villani, P. Evaluation of DNA Damage in Different Stages of Mouse Spermatogenesis after Testicular X Irradiation. Radiat. Res. ...160, 443–451 (2003). To evaluate whether DNA alterations in mature spermatozoa could stem from DNA damage induced in immature germ cells, testis cells and spermatozoa were analyzed by the comet assay and by the sperm chromatin structure assay 14, 45 and 100 days after in vivo X irradiation of the testes. These times were selected, according to the mouse seminiferous epithelium cycle, to follow the DNA damage induced in different germ cell compartments. The cytotoxic action was assessed by DNA flow cytometric analysis of testicular cells. A dose-dependent increase of DNA damage in testis cells was observed 14 days after irradiation, whereas mature sperm cells were not affected. On the other hand, an increase in DNA strand breaks was seen in spermatozoa 45 days after treatment. DNA damage returned to the control levels 100 days after irradiation. The methods used to evaluate DNA damage gave comparable results, emphasizing the correlation between DNA fragmentation and susceptibility of sperm chromatin to denaturation. Both techniques showed the high radiosensitivity of differentiating spermatogonia. The overall results showed that DNA damage induced in pre-meiotic germ cells is detectable in primary spermatocytes and is still present in mature spermatozoa.
High-flow nasal cannula (HFNC) and helmet noninvasive ventilation (NIV) are used for the management of acute hypoxemic respiratory failure.
Physiological comparison of HFNC and helmet NIV in patients ...with hypoxemia.
Fifteen patients with hypoxemia with Pa
/Fi
< 200 mm Hg received helmet NIV (positive end-expiratory pressure ≥ 10 cm H
O, pressure support = 10-15 cm H
O) and HFNC (50 L/min) in randomized crossover order. Arterial blood gases, dyspnea, and comfort were recorded. Inspiratory effort was estimated by esophageal pressure (Pes) swings. Pes-simplified pressure-time product and transpulmonary pressure swings were measured.
As compared with HFNC, helmet NIV increased Pa
/Fi
(median interquartile range: 255 mm Hg 140-299 vs. 138 101-172;
= 0.001) and lowered inspiratory effort (7 cm H
O 4-11 vs. 15 8-19;
= 0.001) in all patients. Inspiratory effort reduction by NIV was linearly related to inspiratory effort during HFNC (
= 0.84;
< 0.001). Helmet NIV reduced respiratory rate (24 breaths/min 23-31 vs. 29 26-32;
= 0.027), Pes-simplified pressure-time product (93 cm H
O ⋅ s ⋅ min
43-138 vs. 200 168-335;
= 0.001), and dyspnea (visual analog scale 3 2-5 vs. 8 6-9;
= 0.002), without affecting Pa
(
= 0.80) and comfort (
= 0.50). In the overall cohort, transpulmonary pressure swings were not different between treatments (NIV = 18 cm H
O 14-21 vs. HFNC = 15 8-19;
= 0.11), but patients exhibiting lower inspiratory effort on HFNC experienced increases in transpulmonary pressure swings with helmet NIV. Higher transpulmonary pressure swings during NIV were associated with subsequent need for intubation.
As compared with HFNC in hypoxemic respiratory failure, helmet NIV improves oxygenation, reduces dyspnea, inspiratory effort, and simplified pressure-time product, with similar transpulmonary pressure swings, Pa
, and comfort.
Electromagnetic fields are an assessed cause of prolonging free radicals lifespan. This study was carried out to investigate the influence of extremely low frequency electromagnetic fields on protein ...oxidation and on the 20S proteasome functionality, the complex responsible for the degradation of oxidized proteins. Caco 2 cells were exposed, for 24–72 hours, to 1 mT, 50 Hz electromagnetic fields. The treatment induced a time-dependent increase both in cell growth and in protein oxidation, more evident in the presence of TPA, while no changes in cell viability were detected. Exposing the cells to 50 Hz electromagnetic fields caused a global activation of the 20S proteasome catalytic components, particularly evident at 72 hours exposure and in the presence of TPA. The finding that EGCG, a natural antioxidant compound, counteracted the field-related pro-oxidant effects demonstrates that the increased proteasome activity was due to an enhancement in intracellular free radicals.
The G1/S cell cycle checkpoint is frequently dysregulated in cancer, leaving cancer cells reliant on a functional G2/M checkpoint to prevent excessive DNA damage. Wee1 regulates the G2/M checkpoint ...by phosphorylating CDK1 at Tyr15 to prevent mitotic entry. Previous drug development efforts targeting Wee1 resulted in the clinical-grade inhibitor, AZD1775. However, AZD1775 is burdened by dose-limiting adverse events, and has off-target PLK1 activity. In an attempt to overcome these limitations, we developed Wee1 degraders by conjugating AZD1775 to the cereblon (CRBN)-binding ligand, pomalidomide. The resulting lead compound, ZNL-02-096, degrades Wee1 while sparing PLK1, induces G2/M accumulation at 10-fold lower doses than AZD1775, and synergizes with Olaparib in ovarian cancer cells. We demonstrate that ZNL-02-096 has CRBN-dependent pharmacology that is distinct from AZD1775, which justifies further evaluation of selective Wee1 degraders.
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•A selective Wee1 degrader was generated by conjugating pomalidomide to AZD1775•Wee1 degradation induced G2/M accumulation at lower doses than Wee1 inhibition•Wee1 degradation synergized with Olaparib in ovarian cancer cells
Li, Pinch et al. develop and characterize the first selective Wee1 degrader, ZNL-02-096, by linking the clinical candidate inhibitor, AZD1775, to the cereblon-binding ligand, pomalidomide. They demonstrate that ZNL-02-096 exhibits cereblon-dependent pharmacology, induces G2/M phase accumulation and apoptosis at lower doses than AZD1775, and synergizes with Olaparib.
Adult neural stem, cell proliferation is dynamic and has the potential for massive self-renewal yet undergoes limited cell division in vivo. Here, we report an epigenetic mechanism regulating ...proliferation and self-renewal. The recruitment of the PI3K-related kinase signaling pathway and histone H2AX phosphorylation following GABA A receptor activation limits subventricular zone proliferation. As a result, NSC self-renewal and niche size is dynamic and can be directly modulated in both directions pharmacologically or by genetically targeting H2AX activation. Surprisingly, changes in proliferation have long-lasting consequences on stem cell numbers, niche size, and neuronal output. These results establish a mechanism that continuously limits proliferation and demonstrates its impact on adult neurogenesis. Such homeostatic suppression of NSC proliferation may contribute to the limited selfrepair capacity of the damaged brain.
Amino acid sequencing of subunits of the multicatalytic proteinase complex (MPC) isolated from bovine spleen showed an almost complete replacement of the X, Y, and Z subunits, constitutively ...expressed in most tissues, by the interferon-gamma-inducible LMP7, LMP2, and MECL1 subunits. A comparison with the pituitary MPC found a decreased chymotrypsin-like activity, a depressed peptidylglutamyl-peptide hydrolyzing activity, and a highly active component with properties similar to, but not identical with, that of the pituitary branched chain amino acid preferring (BrAAP) component. Unlike the pituitary BrAAP component, that of the spleen MPC exhibited a greatly decreased Km, a highly increased catalytic efficiency (kcat), and a 80-180 times greater specificity constant (kcat/Km) toward substrates with either branched chain or aromatic amino acid residues in the P1 position. Also, unlike the pituitary BrAAP component, that of the spleen was sensitive to inactivation by 3,4-dichloroisocoumarin and sensitive to inhibition by peptidyl-aldehydes with either phenylalaninal or leucinal residues. Several phenylalaninal peptidyl-aldehydes were identified which selectively inhibited components of the spleen but not of the pituitary MPC. Two of the inhibitors are dipeptidyl-aldehydes, two others are tetrapeptidyl-aldehydes with a Pro residue in the P3 position. The possibility is discussed that the properties and specificity of the spleen MPC are a consequence of the presence of the interferon-gamma-inducible subunits.
The clotting activity of human fibrinogen was fully inhibited in vitro by peroxynitrite. The decrease of activity followed an exponential function and the concentration of peroxynitrite needed to ...inhibit 50% of fibrinogen clotting was 22 μM at 25°C. The oxidative modification(s) induced by the peroxynitrite system (i.e. ONOO
−, ONOOH and ONOOH*) appeared specifically to affect fibrin clot formation (through the inhibition of fibrinogen polymerization) since the interaction of peroxynitrite-modified fibrinogen with thrombin appeared to be unaffected. The addition of NaHCO
3 decreased the peroxynitrite effect on fibrinogen clotting, suggesting that the reactive species formed by the reaction of CO
2 with peroxynitrite are less efficient oxidants of peroxynitrite itself. Similar effects were observed after addition of bilirubin, which also exerted a significant protection against peroxynitrite-mediated modification of fibrinogen.
Strong evidence indicates that regulated mRNA translation in neuronal dendrites underlies synaptic plasticity and brain development. The fragile X mental retardation protein (FMRP) is involved in ...this process; here, we show that it acts by inhibiting translation initiation. A binding partner of FMRP, CYFIP1/Sra1, directly binds the translation initiation factor eIF4E through a domain that is structurally related to those present in 4E-BP translational inhibitors. Brain cytoplasmic RNA 1 (
BC1), another FMRP binding partner, increases the affinity of FMRP for the CYFIP1-eIF4E complex in the brain. Levels of proteins encoded by known FMRP target mRNAs are increased upon reduction of CYFIP1 in neurons. Translational repression is regulated in an activity-dependent manner because BDNF or DHPG stimulation of neurons causes CYFIP1 to dissociate from eIF4E at synapses, thereby resulting in protein synthesis. Thus, the translational repression activity of FMRP in the brain is mediated, at least in part, by CYFIP1.
We estimated metastatic-death risk when the treatment of small choroidal melanomas is deferred until growth is observed.
In 24 patients with choroidal melanoma (median diameter 5.85 mm), the ...exponential growth rate estimated by a mixed-effects model was 4.3% per year. Using the Liverpool Uveal Melanoma Prognosticator Online v.3 (LUMPO3), we measured changes in 15-year metastatic and non-metastatic death risks according to whether the tumor is treated immediately or after observing growth 4 or 12 months later, considering age, sex, and metastasis predictors.
In 40-year-old females with 10 mm, disomy 3 and monosomy 3 choroidal melanomas (prevalence 16%), the 15-year absolute risks of metastatic death are 4.2% and 76.6%, respectively, increasing after a 4-month delay by 0.0% and 0.2% and by 3.0% and 2.3% with tumor growth rates of 5.0% and 20.0%, respectively. With 12-month delays, these risks increase by 0.0% and 0.5% and by 1.0% and 7.1%, respectively. Increases in metastatic-death risk are less with smaller tumors and with a higher risk of non-metastatic death.
Deferring treatment of choroidal melanomas until documentation of growth may delay iatrogenic visual loss by months or years and is associated with minimal increase in metastatic mortality, at least with small tumors with usual growth rates of up to 40% per year.
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