Population dynamics of "null" and Thy1lo lymphocytes in mouse bone marrow: genesis of cells with natural killer cell lineage characteristics.

The population dynamics of "null" small lymphocytes lacking B and T lineage markers in mouse bone marrow have been examined using a combination of immunolabeling and hydroxyurea (HU) deletion techniques. The binding of the B lineage-associated mAb, 14.8, and anti-Thy1.2 to bone marrow cells has been detected radioautographically. Null cells lacking 14.8 and Thy1.2 determinants (14.8- Thy1-) formed a substantial subset (12-14%) of bone marrow small lymphocytes, representing 0.5 x 10(6) cells per femur (2-3% of nucleated cells). HU treatment revealed an exceptionally rapid turnover of the null small lymphocyte population (T1/2, 7.5 hr) compared with 14.8+ cells (T1/2, 20.5 hr) and Thy1+ cells (T1/2, 53 hr). Small lymphocytes bearing low intensities of Thy1 (Thy1lo) were also rapidly renewed (T1/2, 28 hr) whereas those with high intensities of Thy1 (Thy1hi) were renewed only slowly (T1/2, 123 hr). During ontogeny, null small lymphocytes first appeared in the fetal liver by Day 11 and the fetal spleen by Day 16, but increased rapidly in the bone marrow in early postnatal life. Double immunolabeling techniques demonstrated that 10% of null small lymphocytes in the bone marrow expressed NK1.1 antigen, while larger proportions bound to tumor (YAC.1) cells in vitro and displayed Fc receptors. The NK1.1-bearing fraction of null small lymphocytes in bone marrow was depleted by HU treatment only after an initial delay. NK1.1 was also expressed on subsets of Thy1lo cells and Thy1hi cells. The results have revealed the continuous production in mouse bone marrow of null and Thy1lo small lymphocytes, totaling 1-3 x 10(7) cells/day and 1.2 x 10(6) cells/day, respectively. The findings suggest that the large-scale production of null lymphocytes in mouse bone marrow includes the genesis of NK lineage cells which express NK1.1 and Thy1lo during a period of terminal maturation.     

Corticosteroid side-chain oxidations--I. Structural effects on the excreted isotope ratios of 4-14C- and 21-3H-labelled corticosteroid metabolites in rabbit urine

The metabolic fates of 4-14C- and 21-3H-labelled corticosteroids have been investigated in the rabbit by analysis of the normalized isotope ratios of neutral and acidic metabolites excreted in the urine. Isotope ratios of excreted radioactivity declined in the order cortisol (F) greater than corticosterone (B) greater than 11-desoxycortisol (S) greater than deoxycorticosterone (DOC). Steroid acids, isolated in alumina fraction C, represented 19.0, 15.0, 9.7 and 2.7% of the doses of DOC, B, S and F, respectively, and the isotope ratios declined in the order F greater than B greater than S greater than DOC. HPLC of steroid acid methyl ester derivatives indicated generally low isotope ratios for DOC and S steroid acids, consistent with complete side-chain oxidation to 20-oxo-21-oic acids and/or 17-carboxylic acids. Several B metabolite methyl esters peaks also exhibited low isotope ratios, but both B and F metabolites gave methyl esters that retained significant tritium consistent with the presence of 20-hydroxysteroid acids. The 21-hydroxy-steroid metabolite fractions had isotope ratios of F = S greater than B greater than DOC. HPLC showed that 20-oxo (tetrahydro) metabolites of B and F had reduced isotope ratios unlike the C-20 reduced (hexahydro) metabolites of DOC and S. It may be concluded that the metabolic fate of the corticoid side-chain in the rabbit is dependent on the steroid structure and may result in the excretion of both 20-oxo and 20-hydroxysteroid acids.     

Postnatal exposure to finasteride causes different effects on the prostate of male and female gerbils

The development and maintenance of prostate function depend on a fine balance between oestrogen and androgen levels. Finasteride inhibits 5α‐reductase, which is responsible for the conversion of testosterone into its most active form, dihydrotestosterone. Enzymes that metabolize these hormones have a highly relevant role in both the normal prostate metabolism and in the occurrence of pathological conditions. There are few studies on the impact of finasteride on male prostate development and fewer studies on the female prostate and possible intersexual differences. Therefore, we treated male and female gerbils from 7 to 14 days in postnatal life with a high dose of finasteride (500 μg/kg/day); the prostate complexes were then removed and submitted to immunohistochemistry, immunofluorescence and three‐dimensional reconstruction. In addition, hormonal serum dosages were administered. Treatment with finasteride resulted in an increased thickness of the periductal smooth musculature in the prostate of both male and female gerbils, such as well as a reduction in the thickness of developing prostate alveoli in both sexes. In addition, intersexual differences were observed as increased epithelial proliferation and decreases in the number of developing alveoli in females. Together, the data indicate that postnatal exposure to finasteride causes greater changes in the female gerbil prostate than in the male.     

Effect of curcumin-nanoemulsion associated with photodynamic therapy in breast adenocarcinoma cell line

Curcumin, a natural compound has several antineoplastic activities and is a promising natural photosensitizer used in photodynamic therapy. However, its low solubility in physiological medium limit the clinical use of curcumin. This study aimed to analyze the action of curcumin-nanoemulsion, a new and well-designed Drug Delivery System (DDS+) molecule, used as a photosensitizing agent in photodynamic therapy in an in vitro breast cancer model, MCF-7 cells. The empty nanoemulsion fulfils all necessary requirements to be an excellent DDS. Furthermore, the use of curcumin-nanoemulsion in photodynamic therapy resulted in a high phototoxic effect after activation at 440?nm, decreasing to <10% viable tumor cells after two irradiations and increasing the reactive oxygen species (ROS) production. The use of curcumin-nanoemulsion associated with photodynamic therapy resulted in an increase in the levels of caspase 3/7 activity for the studied MCF-7 cell model, indicating that this therapy triggers a cascade of events that lead to cell death, such as cellular apoptosis. In conclusion, curcumin-nanoemulsion proved to be efficient as a photosensitizing agent, had phototoxic effects, significantly decreased the proliferation of MCF-7 cells and stimulating the ROS production in combination with photodynamic therapy, so, this formulation has a great potential for use in treatment of breast cancer.     

Spectrofluorimetric determination of the anti-Covid 19 agent,remdesivir, in vials and spiked human plasma

Following the sudden widespread of the novel coronavirus (COVID-19) which first appeared in Wuhan city. Remdesivir (REM) was the first medicine licensed by the US Food and Drug Administration (FDA) for COVID-19 infected hospitalized patients. Hence, there was an urgent demand for the optimization of efficient selective and sensitive methods to be developed for the determination of REM in pharmaceuticals as well as biological samples. A sensitive and simple green spectrofluorimetric method has been developed to determine REM in pharmaceutical formulation, in addition to, spiked human plasma. The technique involves measuring the native fluorescence of REM in distilled water at 410 nm followed by excitation at 241 nm, giving a linear relationship over the range 50.00–500.00 ng/mL, and then improving the sensitivity of REM through micellar formation using 2.00% w/v sodium dodecyl sulfate (SDS). A linear relationship has been obtained over the range 10.00–350.00 ng/mL having detection and quantitation limits of 2.34 and 7.10 ng/mL, respectively. Different analytical parameters have been carefully studied. A validation study has been conducted successfully in accordance with the FDA and the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines. The developed methods' greenness was assessed utilizing a greenness profile and analytical eco-scale standards. Both methods were discovered to be environmentally friendly and could be successfully used for the determination of the studied drugs in pharmaceutical formulation and human plasma with good accuracy and high precision. As a result, the developed spectrofluorimetric methods could be ideally suited for determination of REM in quality control and medicinal laboratories.     

Chemoradiotherapy or chemotherapy as adjuvant treatment for resected gastric cancer: should we use selection criteria?

BackgroundThe management of gastric adenocarcinoma is essentially based on surgery followed by adjuvant treatment. Adjuvant chemotherapy (CT) as well as chemoradiotherapy (CTRT) have proven their effectiveness in survival outcomes compared to surgery alone. However, there is little data comparing the two adjuvant approaches. This study aimed to compare the prognosis and survival outcomes of patients with gastric adenocarcinoma operated and treated by adjuvant radio-chemotherapy or chemotherapyMaterials and methodsWe retrospectively evaluated 80 patients with locally advanced gastric cancer (LGC) who received adjuvant treatment. We compared survival outcomes and patterns of recurrence of 53 patients treated by CTRT and those of 27 patients treated by CT.ResultsAfter a median follow-up of 38.48 months, CTRT resulted in a significant improvement of the 5-year PFS (60.9% vs. 36%, p = 0.03) and the 5-year OS (55.9% vs. 33%, p = 0.015) compared to adjuvant CT. The 5-year OS was significantly increased by adjuvant CTRT (p = 0.046) in patients with lymph node metastasis, and particularly those with advanced pN stage (p = 0.0078) and high lymph node ratio (LNR) exceeding 25% (p = 0.012). Also, there was a significant improvement of the PFS of patients classified pN2–N3 (p = 0.022) with a high LNR (p = 0.018). CTRT was also associated with improved OS and PFS in patients with lymphovascular and perineural invasion (LVI and PNI) compared to chemotherapy.ConclusionThere is a particular survival benefit of adding radiotherapy to chemotherapy in patients with selected criteria such as lymph node involvement, high LNR LVI, and PNI.     

Evaluation of Canonical siRNA and Dicer Substrate RNA for Inhibition of Hepatitis C Virus Genome Replication – A Comparative Study

Hepatitis C virus (HCV) frequently establishes persistent infections in the liver, leading to the development of chronic hepatitis and, potentially, to liver cirrhosis and hepatocellular carcinoma at later stages. The objective of this study was to test the ability of five Dicer substrate siRNAs (DsiRNA) to inhibit HCV replication and to compare these molecules to canonical 21 nt siRNA. DsiRNA molecules were designed to target five distinct regions of the HCV genome – the 5’ UTR and the coding regions for NS3, NS4B, NS5A or NS5B. These molecules were transfected into Huh7.5 cells that stably harboured an HCV subgenomic replicon expressing a firefly luciferase/neoR reporter (SGR-Feo-JFH-1) and were also tested on HCVcc-infected cells. All of the DsiRNAs inhibited HCV replication in both the subgenomic system and HCVcc-infected cells. When DsiRNAs were transfected prior to infection with HCVcc, the inhibition levels reached 99.5%. When directly compared, canonical siRNA and DsiRNA exhibited similar potency of virus inhibition. Furthermore, both types of molecules exhibited similar dynamics of inhibition and frequencies of resistant mutants after 21 days of treatment. Thus, DsiRNA molecules are as potent as 21 nt siRNAs for the inhibition of HCV replication and may provide future approaches for HCV therapy if the emergence of resistant mutants can be addressed.     

ATM regulates Mre11-dependent DNA end-degradation and microhomology-mediated end joining

The human disorder ataxia telangiectasia (AT), which is characterized by genetic instability and neurodegeneration, results from mutation of the ataxia telangiectasia mutated (ATM) kinase. The loss of ATM leads to cell cycle checkpoint deficiencies and other DNA damage signaling defects that do not fully explain all pathologies associated with A-T including neuronal loss. In addressing this enigma, we find here that ATM suppresses DNA double-strand break (DSB) repair by microhomology-mediated end joining (MMEJ). We show that ATM repression of DNA end-degradation is dependent on its kinase activities and that Mre11 is the major nuclease behind increased DNA end-degradation and MMEJ repair in A-T. Assessment of MMEJ by an in vivo reporter assay system reveals decreased levels of MMEJ repair in Mre11-knockdown cells and in cells treated with Mre11-nuclease inhibitor mirin. Structure-based modeling of Mre11 dimer engaging DNA ends suggests the 5′ ends of a bridged DSB are juxtaposed such that DNA unwinding and 3′–5′ exonuclease activities may collaborate to facilitate simultaneous pairing of extended 5′ termini and exonucleolytic degradation of the 3′ ends in MMEJ. Together our results provide an integrated understanding of ATM and Mre11 in MMEJ: ATM has a critical regulatory function in controlling DNA end-stability and error-prone DSB repair and Mre11 nuclease plays a major role in initiating MMEJ in mammalian cells. These functions of ATM and Mre11 could be particularly important in neuronal cells, which are post-mitotic and therefore depend on mechanisms other than homologous recombination between sister chromatids to repair DSBs.Key words: ATM, Mre11, MRN complex, DNA degradation, double-strand break repair, microhomology-mediated end joining, PI-3-kinase-like kinases     

Bidirectional signaling of mammary epithelium and stroma: implications for breast cancer--preventive actions of dietary factors

The mammary gland is composed of two major cellular compartments: a highly dynamic epithelium that undergoes cycles of proliferation, differentiation and apoptosis in response to local and endocrine signals and the underlying stroma comprised of fibroblasts, endothelial cells and adipocytes, which collectively form the mammary fat pad. Breast cancer originates from subversions of normal growth regulatory pathways in mammary epithelial cells due to genetic mutations and epigenetic modifications in tumor suppressors, oncogenes and DNA repair genes. Diet is considered a highly modifiable determinant of breast cancer risk; thus, considerable efforts are focused on understanding how certain dietary factors may promote resistance of mammary epithelial cells to growth dysregulation. The recent indications that stromal cells contribute to the maintenance of the mammary epithelial ‘niche’ and the increasing appreciation for adipose tissue as an endocrine organ with a complex secretome have led to the novel paradigm that the mammary stromal compartment is itself a relevant target of bioactive dietary factors. In this review, we address the potential influence of dietary factors on mammary epithelial-stromal bidirectional signaling to provide mechanistic insights into how dietary factors may promote early mammary epithelial differentiation to decrease adult breast cancer risk.