Ultrasound imaging of apoptosis: high-resolution non-invasive monitoring of programmed cell death in vitro, in situ and in vivo

A new non-invasive method for monitoring apoptosis has been developed using high frequency (40 MHz) ultrasound imaging. Conventional ultrasound backscatter imaging techniques were used to observe apoptosis occurring in response to anticancer agents in cells in vitro, in tissues ex vivo and in live animals. The mechanism behind this ultrasonic detection was identified experimentally to be the subcellular nuclear changes, condensation followed by fragmentation, that cells undergo during apoptosis. These changes dramatically increase the high frequency ultrasound scattering efficiency of apoptotic cells over normal cells (25- to 50-fold change in intensity). The result is that areas of tissue undergoing apoptosis become much brighter in comparison to surrounding viable tissues. The results provide a framework for the possibility of using high frequency ultrasound imaging in the future to non-invasively monitor the effects of chemotherapeutic agents and other anticancer treatments in experimental animal systems and in patients. © 1999 Cancer Research Campaig

The differences in thermal profiles between normal and leukemic cells exposed to anticancer drug evaluated by differential scanning calorimetry

Chronic lymphocytic leukemia (CLL) is a heterogenous disease with an imbalance between apoptosis and cell proliferation. Therefore, the main goal in CLL therapy is to induce apoptosis and effectively support this process in transformed B lymphocytes. In the current study, we have compared differential scanning calorimetry (DSC) profiles of nuclei isolated from CLL cells and normal mononuclear cells exposed to cladribine or fludarabine combined with mafosfamide (CM; FM), and additionally to CM combined with monoclonal antibody—rituximab (RCM) for 48 h, as well as in culture medium only (controls). Under current study, the mononuclear cells from peripheral blood (PBMCs) of healthy individuals have been included. The obtained results have shown the presence of thermal transition at 95 ± 5 °C in most of nuclear preparations (92.2 %) isolated from blood of CLL patients. This thermal characteristic parameter was changed after drug exposure, however, to a different extent. These thermal changes were accompanied by the decrease of cell viability, an elevation of apoptosis rate and the changes in expression/proteolysis of poly(ADP-ribose)polymerase-1—main marker of apoptosis. Importantly, in DSC profiles of nuclear preparations of PBMCs from blood of healthy donors exposed to investigated drug combinations and control CLL cells, the lack of such changes was observed. Our results confirmed that DSC technique complemented with other biological approaches could be helpful in tailoring therapy for CLL patients.Research was sponsored by Grant from the Polish National Science Centre (No. 2011/01/B/NZ/0102); Results of presented study were partially presented in oral presentation on 2nd Central and Eastern European Conference on Thermal Analysis and Calorimetry in Vilnius, Lithuania, 201

Drosophila DNA polymerase theta utilizes both helicase-like and polymerase domains during microhomology-mediated end joining and interstrand crosslink repair

Double strand breaks (DSBs) and interstrand crosslinks (ICLs) are toxic DNA lesions that can be repaired through multiple pathways, some of which involve shared proteins. One of these proteins, DNA Polymerase theta (Pol theta), coordinates a mutagenic DSB repair pathway named microhomology-mediated end joining (MMEJ) and is also a critical component for bypass or repair of ICLs in several organisms. Pol theta contains both polymerase and helicase-like domains that are tethered by an unstructured central region. While the role of the polymerase domain in promoting MMEJ has been studied extensively both in vitro and in vivo, a function for the helicase-like domain, which possesses DNA-dependent ATPase activity, remains unclear. Here, we utilize genetic and biochemical analyses to examine the roles of the helicase-like and polymerase domains of Drosophila Pol theta. We demonstrate an absolute requirement for both polymerase and ATPase activities during ICL repair in vivo. However, similar to mammalian systems, polymerase activity, but not ATPase activity, is required for ionizing radiation-induced DSB repair. Using a site-specific break repair assay, we show that overall end-joining efficiency is not affected in ATPase-dead mutants, but there is a significant decrease in templated insertion events. In vitro, Pol theta can efficiently bypass a model unhooked nitrogen mustard crosslink and promote DNA synthesis following microhomology annealing, although ATPase activity is not required for these functions. Together, our data illustrate the functional importance of the helicase-like domain of Pol theta and suggest that its tethering to the polymerase domain is important for its multiple functions in DNA repair and damage tolerance

Chromatin changes in cell transformation: progressive unfolding of the higher-order structure during the evolution of rat hepatocyte nodules. A differential scanning calorimetry study.

Using differential scanning calorimetry and complementary ultrastructural observations, we have characterized the status of chromatin during the transformation of rat hepatocytes in the resistant hepatocyte model of Solt and Farber (1976. Nature (Lond.). 263:701-703). Differential scanning calorimetry affords a measure of the degree of condensation of chromatin in situ and has therefore been used in this work for the purpose of establishing the nature of the structural changes associated with the emergence of successive cellular populations. Since the resistant hepatocyte model generates a series of synchronous phenotypic changes, it was possible to determine unambiguously the content of heterochromatin at each step of the process. The higher-order structure undergoes a partial relaxation in early developing nodules, isolated 16 weeks after initiation; the thermal transition at 90 degrees C, which is characteristic of noninteracting core particles, increases with respect to control hepatocytes. Dramatic changes occur in persistent (46-week) nodules. The 90 degrees C endotherm dominates the thermogram, while the transition at 107 degrees C, corresponding to the denaturation of the core particle packaged within the heterochromatic domains, disappears. The complete loss of the higher-order structure at this stage of transformation has been further verified by ultrastructural observations on thin nuclear sections. Ten-nm filaments, having a beaded appearance, are scattered throughout the nucleoplasm and clearly result from the decondensation of 30-nm-thick fibers. This catastrophic relaxation process cannot be related to an effective increase in gene activity. Rather, our observations suggest that during transformation chromatin is in a state of high transcriptional competence associated with the alert of general cellular programs. This view is consistent with the finding that in persistent nodules the DNA is extensively hypomethylated with respect to normal liver

Disturbo dell’equilibrio nel paziente con BPCO riacutizzata: efficacia del training cardiorespiratorio

Introduzione - Nella nostra esperienza clinica, confermata da diverse evidenze di letteratura il paziente con BPCO presenta spesso un deficit di equilibrio e del controllo posturale. Quest\u2019alterazione risulta correlata a un aumentato rischio di caduta e quindi all\u2019incremento delle comorbidit\ue0 associate e dei costi diretti e indiretti per questa patologia. Appare quindi di fondamentale importanza un\u2019accurata valutazione di questa funzione utilizzando strumenti il pi\uf9 possibile riproducibili e oggettivi. Attualmente quelli pi\uf9 comunemente utilizzati sono le scale di valutazione Berg Balance Scale (BBS) e la Balance Evaluation Systems Test (BESTest). Per quanto validate e attendibili, queste scale sono metodiche operatore-dipendente, pertanto oggetto di bassa riproducibilit\ue0 inter-operatore. Da questa osservazione deriva la necessit\ue0 di standardizzare questo tipo valutazione attraverso strumenti che garantiscano risultati riproducibili esenti da bias legati al singolo operatore. In quest\u2019ottica, il presente studio si propone d\u2019inserire nell\u2019iter di valutazione l\u2019esame di stabilit\ue0 posturale attraverso una pedana stabilometrica. Abbiamo inoltre voluto valutare se il training cardio-respiratorio, al quale sono sottoposti i pazienti affetti da BPCO nelle prime due settimane di trattamento, incidesse positivamente sul deficit di equilibrio, migliorando oltre la resistenza allo sforzo anche la stabilit\ue0 e quindi la sicurezza dei pazienti. Materiali e Metodi - Lo studio \ue8 stato eseguito presso l\u2019U. O. dell\u2019Ospedale San Carlo di Milano, tra febbraio e maggio 2019. Sono stati arruolati dieci pazienti, di et\ue0 compresa fra i 55 e gli 85 anni. I pazienti, durante la degenza, sono stati sottoposti unicamente al training cardio-respiratorio standard, oltre alla terapia medica adeguata a ciascun caso. Non sono stati eseguiti esercizi specifici per il miglioramento dell\u2019equilibrio. Sono stati rilevati i dati di emogasanalisi arteriosa, six minutes walking test, rilevazione della scala di GOLD e MRC, all\u2019inizio e alla fine del programma riabilitativo. Contestualmente \ue8 stata effettuata una valutazione in due tempi su pedana stabilometrica, oltre alla somministrazione della scala BBS. Le variabili considerate sono state: la frequenza di oscillazione del punto di proiezione del baricentro su pedana (misurata in Hz), l\u2019ampiezza totale di oscillazione (misurata in unit\ue0 pari alla minima variazione percettibile dallo strumento, pari a circa 0,5 mm) e l\u2019area dell\u2019ellissoide disegnato da tale proiezione (misurata in unit\ue0 quadratiche con intervallo di confidenza del 90%) Le valutazioni sono state eseguite al momento dell\u2019accesso presso l\u2019U.O. di Riabilitazione Intensiva Cardiopolmonare e al servizio di Fisioterapia e sono state ripetute a distanza di due settimane dalla prima valutazione. Come variabile di confronto \ue8 stata utilizzata la valutazione spirometrica per attestare il contestuale miglioramento della performance respiratoria, oltre all\u2019 emogasanalisi e alle scale di valutazione riportate. Risultati - Fino ad ora si \ue8 assistito a un miglioramento sia nei dati stabilometrici sopra riportati, sia in quelli rilevati attraverso la scala di valutazione BBS, parallelamente a un miglioramento della performance respiratoria misurati attraverso spirometria. Tutti i risultati sono provvisori e richiedono un parziale incremento di dimensioni del campione e una pi\uf9 accurata analisi statistica. Conclusioni I risultati preliminari sembrerebbero confermare l\u2019ipotesi secondo la quale la riacutizzazione della patologia BPCO, provochi nei pazienti, soprattutto se ipercapnici, un disturbo dell\u2019equilibrio, che migliora con la regolarizzazione degli scambi gassosi. Bibliografia - De Castro LA, Ribeiro LR, Mesquita R, et al. Static and functional balance in individuals With COPD: comparison with healthy controls and differences according to sex and disease severity. Respir Care 2016; 61: 1488\u20131496. Beuchamp MK, Balance assessment in people with COPD: An evidence-based guide. Chron Resp Dis 2018; 16: 1\u20138