Simultaneous Deletion of p21Cip1/Waf1 and Caspase-3 Accelerates Proliferation and Partially Rescues the Differentiation Defects of Caspase-3 Deficient Hematopoietic Stem Cells

Specialized blood cells are generated through the entire life of an organism by differentiation of a small number of hematopoietic stem cells (HSC). There are strictly regulated mechanisms assuring a constant and controlled production of mature blood cells. Although such mechanisms are not completely understood, some factors regulating cell cycle and differentiation have been identified. We have previously shown that Caspase-3 is an important regulator of HSC homeostasis and cytokine responsiveness. p21^cip1/waf1 is a known cell cycle regulator, however its role in stem cell homeostasis seems to be limited. Several reports indicate interactions between p21^cip1/waf1 and Caspase-3 in a cell type dependent manner. Here we studied the impact of simultaneous depletion of both factors on HSC homeostasis. Depletion of both Caspase-3 and p21^cip1/waf1 resulted in an even more pronounced increase in the frequency of hematopoietic stem and progenitor cells. In addition, simultaneous deletion of both genes revealed a further increase of cell proliferation compared to single knock-outs and WT control mice, while apoptosis or self-renewal ability were not affected in any of the genotypes. Upon transplantation, p21^cip1/waf1-/- bone marrow did not reveal significant alterations in engraftment of lethally irradiated mice, while Caspase-3 deficient HSPC displayed a significant reduction of blood cell production. However, when both p21^cip1/waf1 and Caspase-3 were eliminated this differentiation defect caused by Caspase-3 deficiency was abrogated.     

Quantitative Apparent Diffusion Coefficient Measurements Obtained by 3-Tesla MRI Are Correlated with Biomarkers of Bladder Cancer Proliferative Activity

Purpose

To investigate the association between Apparent Diffusion Coefficient (ADC) values and cell cycle and proliferative biomarkers (p53, p21, Ki67,) in order to establish its potential role as a noninvasive biomarker for prediction of cell cycle, proliferative activity and biological aggressiveness in bladder cancer.

Materials and Methods

Patients with bladder cancer who underwent 3,0 Tesla DW-MRI of the bladder before TUR-B or radical cystectomy were eligible for this prospective IRB-approved study. Histological specimen were immunohistochemically stained for the following markers: p53, p21 and ki67. Two board-certified uropathologists reviewed the specimens blinded to DW-MRI results. Histological grade and T-stage were classified according to the WHO 2004 and the 2009 TNM classification, respectively. Nonparametric univariate and multivariate statistics including correlation, logistic regression and ROC analysis were applied.

Results

Muscle invasive bladder cancer was histologically confirmed in 10 out of 41 patients. All examined tissue biomarkers were significantly correlated with ADC values (p<0.05, respectively). Based on multivariate analysis, p53 and ADC are both independent prognostic factors for muscle invasiveness of bladder cancer (>/ = T2). (p = 0.013 and p = 0.018).

Conclusion

ADC values are associated with cell cycle and proliferative biomarkers and do thereby reflect invasive and proliferative potential in bladder cancer. ADC and p53 are both independent prognostic factors for muscle invasiveness in bladder cancer.     

Where Do the Rural Poor Deliver When High Coverage of Health Facility Delivery Is Achieved? Findings from a Community and Hospital Survey in Tanzania

Introduction

As part of maternal mortality reducing strategies, coverage of delivery care among sub-Saharan African rural poor will improve, with a range of facilities providing services. Whether high coverage will benefit all socio-economic groups is unknown. Iringa rural District, Southern Tanzania, with high facility delivery coverage, offers a paradigm to address this question. Delivery services are available in first-line facilities (dispensaries, health centres) and one hospital. We assessed whether all socio-economic groups access the only comprehensive emergency obstetric care facility equally, and surveyed existing delivery services.

Methods

District population characteristics were obtained from a household community survey (n = 463). A Hospital survey collected data on women who delivered in this facility (n = 1072). Principal component analysis on household assets was used to assess socio-economic status. Hospital population socio-demographic characteristics were compared to District population using multivariable logistic regression. Deliveries'' distribution in District facilities and staffing were analysed using routine data.

Results

Women from the hospital compared to the District population were more likely to be wealthier. Adjusted odds ratio of hospital delivery increased progressively across socio-economic groups, from 1.73 for the poorer (p = 0.0031) to 4.53 (p<0.0001) for the richest. Remarkable dispersion of deliveries and poor staffing were found. In 2012, 5505/7645 (72%) institutional deliveries took place in 68 first-line facilities, the remaining in the hospital. 56/68 (67.6%) first-line facilities reported ≤100 deliveries/year, attending 33% of deliveries. Insufficient numbers of skilled birth attendants were found in 42.9% of facilities.

Discussion

Poorer women remain disadvantaged in high coverage, as they access lower level facilities and are under-represented where life-saving transfusions and caesarean sections are available. Tackling the challenges posed by low caseloads and staffing on first-line rural care requires confronting a dilemma between coverage and quality. Reducing number of delivery sites is recommended to improve quality and equity of care.     

Cigarette Smoke Toxins Deposited on Surfaces: Implications for Human Health

Cigarette smoking remains a significant health threat for smokers and nonsmokers alike. Secondhand smoke (SHS) is intrinsically more toxic than directly inhaled smoke. Recently, a new threat has been discovered – Thirdhand smoke (THS) – the accumulation of SHS on surfaces that ages with time, becoming progressively more toxic. THS is a potential health threat to children, spouses of smokers and workers in environments where smoking is or has been allowed. The goal of this study is to investigate the effects of THS on liver, lung, skin healing, and behavior, using an animal model exposed to THS under conditions that mimic exposure of humans. THS-exposed mice show alterations in multiple organ systems and excrete levels of NNAL (a tobacco-specific carcinogen biomarker) similar to those found in children exposed to SHS (and consequently to THS). In liver, THS leads to increased lipid levels and non-alcoholic fatty liver disease, a precursor to cirrhosis and cancer and a potential contributor to cardiovascular disease. In lung, THS stimulates excess collagen production and high levels of inflammatory cytokines, suggesting propensity for fibrosis with implications for inflammation-induced diseases such as chronic obstructive pulmonary disease and asthma. In wounded skin, healing in THS-exposed mice has many characteristics of the poor healing of surgical incisions observed in human smokers. Lastly, behavioral tests show that THS-exposed mice become hyperactive. The latter data, combined with emerging associated behavioral problems in children exposed to SHS/THS, suggest that, with prolonged exposure, they may be at significant risk for developing more severe neurological disorders. These results provide a basis for studies on the toxic effects of THS in humans and inform potential regulatory policies to prevent involuntary exposure to THS.     

Effect of Transcranial Direct-Current Stimulation Combined with Treadmill Training on Balance and Functional Performance in Children with Cerebral Palsy: A Double-Blind Randomized Controlled Trial

Background

Cerebral palsy refers to permanent, mutable motor development disorders stemming from a primary brain lesion, causing secondary musculoskeletal problems and limitations in activities of daily living. The aim of the present study was to determine the effects of gait training combined with transcranial direct-current stimulation over the primary motor cortex on balance and functional performance in children with cerebral palsy.

Methods

A double-blind randomized controlled study was carried out with 24 children aged five to 12 years with cerebral palsy randomly allocated to two intervention groups (blocks of six and stratified based on GMFCS level (levels I-II or level III).The experimental group (12 children) was submitted to treadmill training and anodal stimulation of the primary motor cortex. The control group (12 children) was submitted to treadmill training and placebo transcranial direct-current stimulation. Training was performed in five weekly sessions for 2 weeks. Evaluations consisted of stabilometric analysis as well as the administration of the Pediatric Balance Scale and Pediatric Evaluation of Disability Inventory one week before the intervention, one week after the completion of the intervention and one month after the completion of the intervention. All patients and two examiners were blinded to the allocation of the children to the different groups.

Results

The experimental group exhibited better results in comparison to the control group with regard to anteroposterior sway (eyes open and closed; p<0.05), mediolateral sway (eyes closed; p<0.05) and the Pediatric Balance Scale both one week and one month after the completion of the protocol.

Conclusion

Gait training on a treadmill combined with anodal stimulation of the primary motor cortex led to improvements in static balance and functional performance in children with cerebral palsy.

Trial Registration

Ensaiosclinicos.gov.br/RBR-9B5DH7     

Combined analysis of chromosomal instabilities and gene expression for colon cancer progression inference

Background

The human body plays host to a vast array of bacteria, found in oral cavities, skin, gastrointestinal tract and the vagina. Some bacteria are harmful while others are beneficial to the host. Despite the availability of many methods to identify bacteria, most of them are only applicable to specific and cultivable bacteria and are also tedious. Based on high throughput sequencing technology, this work derives 16S rRNA sequences of bacteria and analyzes probiotics and pathogens species.

Results

We constructed a database that recorded the species of probiotics and pathogens from literature, along with a modified Smith-Waterman algorithm for assigning the taxonomy of the sequenced 16S rRNA sequences. We also constructed a bacteria disease risk model for seven diseases based on 98 samples. Applicability of the proposed platform is demonstrated by collecting the microbiome in human gut of 13 samples.

Conclusions

The proposed platform provides a relatively easy means of identifying a certain amount of bacteria and their species (including uncultivable pathogens) for clinical microbiology applications. That is, detecting how probiotics and pathogens inhabit humans and how affect their health can significantly contribute to develop a diagnosis and treatment method.     

Resistance of p53 knockout cells to doxorubicin is related to reduced formation of DNA strand breaks rather than impaired apoptotic signaling

The anthracycline doxorubicin (adriamycin) is an important chemotherapeutic agent used in the treatment of solid epithelial and mesenchymal tumors as well as leukemias. A variety of mechanisms has been proposed to be involved in doxorubicin-induced cytotoxicity such as DNA intercalation, oxidative stress, DNA strand breakage by inhibition of topoisomerase II, activation of death receptors, and altered p53 expression. Concerning doxorubicin resistance and p53 status data reported are contradictory. Here, we show that mouse fibroblasts deficient in p53 (p53(-/-)) are more resistant to doxorubicin than p53 wild-type (p53 wt) cells. This is in contrast to other genotoxic agents (UV-light, alkylating drugs) for which p53(-/-) fibroblasts proved to be more sensitive. Resistance of p53(-/-) cells to doxorubicin is related to reduced induction of apoptosis. This is not likely to be due to altered apoptotic signaling since the expression of Bax and Bcl-2 was unchanged and the induction of Fas/CD95/APO-1 receptor and caspase-8 was the same in p53(-/-) and p53 wt cells on treatment with doxorubicin. However, we observed a clearly lower level of doxorubicin-induced DNA strand breaks in p53(-/-) cells compared to the wt. P170 glycoprotein was equally expressed and the accumulation and elimination of the drug occurred with identical kinetics in both cell types. p53 deficient cells were cross-resistant to another topoisomerase II inhibitor etoposide, which also provoked increased DNA strand breakage in p53 wt cells. Based on the data we conclude that the p53 status significantly impacts the generation of DNA strand breaks because of drug-induced topoisomerase inhibition rather than death receptor signaling. Since human tumors are frequently mutated in p53 the findings bear clinical implications.     

Pro-oxidant effect of dehydroepiandrosterone in rats is mediated by PPAR activation

DHEA-treatment exerts a dual effect, prooxidant or antioxidant, depending on the dosage and, therefore, on the tissue concentration reached. In agreement with previous studies showing a prooxidant effect of DHEA, here we show that pharmacological doses of DHEA produce increased H(2)O(2) levels and a marked reduction of GSH content in rat liver. DHEA, also increases both catalase (by 30%) and cytochrome-C-reductase (by 30%) activities in the liver cytosol. The effectiveness of the state of increased oxidative stress is also documented by changes in fatty acid pattern of the microsomal membranes. Moreover, DHEA, at high doses, enhances beta-oxidation, as demonstrated by an increase of acyl-CoA-oxidase activity and of cytochrome P450 4A content, confirming that it acts as a PPARs inducer. Both PPARs induction and proxidant effects completely disappear when DHEA is administered at lower doses. Seven days treatment (4 or 10 mg) is unable to affect either levels of proxidant species and of antioxidant molecules, or cytochrome P450 4A content and beta-oxidation. Prolonged DHEA treatment (4 mg/day) for three weeks not only is unable to affect PPARs activation and beta-oxidation, but it also exerts a protective effect against ADP/Fe(2+) induced lipid peroxidation. This latter result confirms the antioxidant effects of DHEA at low doses, as already previously documented.     

Conformationally constrained ethylenediamines: synthesis and receptor binding of 6,8-diazabicyclo[3.2.2]nonanes

The synthesis and receptor affinity of 6,8-diazabicyclo[3.2.2]nonanes representing conformationally constrained ethylenediamines are described. The Dieckmann analogous cyclization of the (piperazin-2-yl)propionate 9 provided the bicyclononane 10 only, when the first cyclization product was trapped with chlorotrimethylsilane. 10 was stereoselectively transformed into the bicyclic amines 19a,b and amides 22a,b, which were investigated in competition experiments with radioligands for their sigma(1)-, sigma(2)-, kappa-, and mu-receptor affinities. The (2R)-configured dimethylamine 19a showed promising sigma(1)-receptor affinity (K(i)=23.8 nM) and selectivity, whereas the (2S)-configured (dichlorophenyl)acetamide 22b displayed a sigma-receptor binding profile (sigma(1): K(i)=184 nM; sigma(2): K(i)=263 nM) very similar to the binding profile of the atypical antipsychotic BMY-14802 (26).