Restoration of Silenced Peutz-Jeghers Syndrome Gene, LKB1, Induces Apoptosis in Pancreatic Carcinoma Cells

Germ line mutations of the LKB1 tumor suppressor gene lead to Peutz-Jeghers syndrome (PJS) with a predisposition to cancer. Previous reports suggest that inactivation of this tumor-suppressor gene plays a role in the pathogenesis of gastrointestinal hamartomas as well as several cancers, including adenocarcinoma of the pancreas. Here, we have shown that LKB1 gene is silenced in the pancreatic cancer cell line AsPC-1, but can be recovered by treatment with the methylation inhibitor, 5-aza-2′-deoxycytidine (5aza2dC). Restoring the level of LKB1 through gene transfer initiated mitochondria-mediated apoptosis in AsPC-1cells, as evidenced by the release of cytochrome c from the mitochondria. By confocal microscopy as well as biochemical fractionation, we demonstrate that LKB1 is present in the nuclear and mitochondrial compartments of pancreatic cancer cells. Our observations also indicate that although functional p53 is absent, the p53 kin, p73, is inducible by doxorubicin in AsPC-1 cells. This suggests that LKB1-induced apoptosis is p53 independent but might be p73-mediated in the pancreatic tumor cell line, AsPC-1

Proteasomal Degradation of Human Peptidyl Prolyl Isomerase Pin1-pointing Phospho Bcl2 toward Dephosphorylation

Microtubule inhibitor-induced Bcl2 phosphorylation is detrimental to its antiapoptotic function. Phosphorylation of Bcl2 predominantly occurs on two serine residues (70 and 87) in cells arrested at G2-M phase by microtubule disarraying agents. Phospho Bcl2 can associate with a cis-trans peptidyl prolyl isomerase, Pin1. Pin1 and its homologues are known to target the proline residue carboxyl terminal to the phosphorylated threonine or serine residue of mitotic phosphoproteins, such as Bcl2. However, it was not clear how an extranuclear protein could associate with nuclear Pin1. The confocal images of the immunofluorescence studies employing phospho Bcl2-specific antibody developed in the laboratory demonstrated the translocation of phospho Bcl2 inside the nucleus. Interestingly, proteasomal degradation of Pin1 facilitates dephosphorylation of phospho Bcl2 due to longer exposure of Taxol. Here we show for the first time that proteasomal degradation of Pin1 is the key factor to determine the fate of phosphoforms of Bcl2. When Pin1 is degraded by proteasomes, phospho Bcl2 is converted to its native form. Thus, transient conformational change of Bcl2 due to association with peptidyl prolyl isomerase can contribute to irreversible apoptotic signaling

Teleconsent: A novel approach to obtain informed consent for research

Lack of recruitment of qualified research participants continues to be a significant bottleneck in clinical trials, often resulting in costly time extensions, underpowered results, and in some cases early termination. Some of the reasons for suboptimal recruitment include laborious consent processes and access to participants at remote locations. While new electronic consents technologies (eConsent) help overcome challenges related to readability and consent management, they do not adequately address challenges related to remote access. To address this, we have developed an innovative solution called “teleconsent”, which embeds the informed consent process into a telemedicine session. Teleconsent allows a researcher to remotely video conference with a prospective research participant, display and interactively guide participants in real-time through a consent form. When finished, the researcher and participant can electronically sign the consent form and print or download the signed document for archiving. This process can eliminate challenges related to travel and management of personnel at remote sites. Teleconsent has been successfully implemented in several clinical trials. Teleconsent can improve research recruitment by reducing the barriers related to informed consent, while preserving human interaction

Comparison of home-based palliative care delivered by community health workers versus usual care: research protocol for a pilot randomized controlled trial

Abstract Background Research studies demonstrate that palliative care can improve patient outcomes such as quality of life, symptom burden and patient satisfaction with care (Gomes B, et al. Effectiveness and cost-effectiveness of home palliative care services for adults with advanced illness and their caregivers. Cochrane Database Syst Rev. 2013(6):CD00776) (World Health Organization. Palliative Care. Published 2020.). While 76% of patients who need palliative care live in limited-resource countries, access to high quality palliative services in these countries is minimal (Worldwide Hospice and Palliative Care Association and World Health Organization. Global Atlas of Palliative Care (2nd ed). 2020.). In 2014 the Worldwide Hospice Palliative Care Alliance, with strong endorsement by the WHO, released the Palliative Care Toolkit to provide a training and implementation toolkit for empowering community members to deliver palliative care in resource poor settings (Worldwide Hospice and Palliative Care Association and World Health Organization. Global Atlas of Palliative Care at the End of Life. Geneva, Switzerland 2014.). They encouraged researchers and public health practitioners to conduct rigorous evaluation of the toolkit in diverse settings and contexts. To address this need, we will conduct a pilot randomized controlled trial (RCT) to examine implementation and explore potential effect of an intervention based upon the Palliative Care Toolkit, as adapted and used by community health workers (CHWs) working with a cancer center in Kolkata, India to deliver home-based palliative care for rural patients. Methods Utilizing a randomized controlled trial design, intervention patients (n = 45) receive home-based palliative services (Pal-Care) delivered by community health workers (CHWs), with comparison against a control group of patients (n = 45) who receive usual cancer-center-based palliative services. Primary outcome measures include evaluation of CHW training outcomes, roles and responsibilities of the CHWS and how they assist patients, trial recruitment, stakeholder perceptions of the intervention, and fidelity to study protocol. Secondary outcomes measure patient self-report of health-related quality of life, symptom burden, palliative needs and patient care experience, outcomes The RE-AIM framework guides our evaluation plan to measure the reach, effectiveness, adoption, implementation and maintenance of the Pal-Care intervention (Gaglio B, et al. The RE-AIM framework: a systematic review of use over time. Am J Public Health. 2013;103(6):e38?46.). Data will be analyzed in SAS. All measures will be evaluated overall and by patient age, gender and cancer type and by CHW caseload. Discussion Pal-Care is a RCT funded by the NCI to explore utilization of CHWs to deliver a home-based palliative care intervention built upon the WHO Palliative Care toolkit (PCT), as compared to a usual care control group. The long-term goal of this research is to develop an effective and sustainable model for delivering home-based palliative care for cancer patients in underserved areas. Trial registration (TRN) ClinicalTrials.gov ID# NCT04972630

Changes in urine microalbumin-to-creatinine ratio in children with sickle cell disease over time

Background: Approximately 20% of children with sickle cell disease (SCD) have microalbuminuria (MA). Very little is known about the progression of MA in children and young adults with SCD. Methods: In this study, we analyzed 5 year EMR data of 373 children (with ≥2 Microalbumin to creatinine (MA/Cr) ratio measurements) followed at the Medical University of South Carolina to determine the rate, direction, magnitude and predictors of MA/Cr change over time. Results: Age range was 1 – 22 years; mean 10.2 ± 5.2 years, 49.5% were male. Median follow up duration was 3.12 ±1.16 years. At baseline, 328 children had normal (<20 mg/L) MA level. Forty-five (12.1%) of children had MA (≥ 20 mg/L), of which 91% were ≥ 8 years and 21 (47%) continued to have MA at the end of the study period. On the other hand, during the study period, 24 new patients developed MA and 24 normalized their MA to levels <20 mg/L. In multivariate logistic regression model, age and bilirubin levels were predictive of MA/Cr increase in patients who received at least one blood transfusion during the study period. Baseline MA level was not predictive of the change in MA/Cr. Conclusions: In children and young adults, microalbuminuria is considered a marker of early renal injury. Over time, MA/Cr levels may increase or decrease. Further studies are needed to confirm our findings, assess the reliability of MA as marker of long-term renal injury and identify high risk patients likely to have worsening of MA over time

Barriers, Facilitators and Recommended Strategies for Implementing a Home-Based Palliative Care Intervention in Kolkata, India

Background:40 million people in the world are in need of palliative care, but only one-seventh of that population receive services. Underuse of palliative care in low resource countries exacerbates suffering in patients with life limiting illnesses such as cancer.Objectives:The current study was conducted to identify barriers, facilitators and recommended strategies for informing development of a home-based palliative care intervention for poor and medically underserved rural patients in Kolkata, India.Methods:Semi-structured interviews were conducted with 20 clinical and patient stakeholders in Kolkata, India. Questions queried current practices for delivering palliative care, along with barriers, facilitators and optimal strategies for implementing homebased palliative care.Results:We identified some key barriers to palliative care delivery in rural areas: lack of access to palliative care till late stages; patients unaware of their cancer stage; lack of affordability of medication and treatment costs; transportation challenges to access care; strict morphine distribution regulations making it challenging for patients to obtain morphine; cultural factors discouraging patients from seeking palliative care; resistance from medical community to use “rural medical practitioners (RMPs)” to deliver care. We also identified important facilitators, including availability of existing palliative care infrastructure at the cancer center, network of RMPs to serve as CHWs to facilitate palliative care delivery, low morphine cost and family support system for patients.Conclusion:Our findings provide evidence that a palliative care intervention which leverages an existing CHW infrastructure may be a feasible model for expanding the reach of palliative care to rural underserved patients

Aging-dependent changes in rat heart mitochondrial glutaredoxins—Implications for redox regulation

Clinical and animal studies have documented that hearts of the elderly are more susceptible to ischemia/reperfusion damage compared to young adults. Recently we found that aging-dependent increase in susceptibility of cardiomyocytes to apoptosis was attributable to decrease in cytosolic glutaredoxin 1 (Grx1) and concomitant decrease in NF-κB-mediated expression of anti-apoptotic proteins. Besides primary localization in the cytosol, Grx1 also exists in the mitochondrial intermembrane space (IMS). In contrast, Grx2 is confined to the mitochondrial matrix. Here we report that Grx1 is decreased by 50–60% in the IMS, but Grx2 is increased by 1.4–2.6 fold in the matrix of heart mitochondria from elderly rats. Determination of in situ activities of the Grx isozymes from both subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria revealed that Grx1 was fully active in the IMS. However, Grx2 was mostly in an inactive form in the matrix, consistent with reversible sequestration of the active-site cysteines of two Grx2 molecules in complex with an iron–sulfur cluster. Our quantitative evaluations of the active/inactive ratio for Grx2 suggest that levels of dimeric Grx2 complex with iron–sulfur clusters are increased in SSM and IFM in the hearts of elderly rats. We found that the inactive Grx2 can be fully reactivated by sodium dithionite or exogenous superoxide production mediated by xanthine oxidase. However, treatment with rotenone, which generates intramitochondrial superoxide through inhibition of mitochondrial respiratory chain Complex I, did not lead to Grx2 activation. These findings suggest that insufficient ROS accumulates in the vicinity of dimeric Grx2 to activate it in situ

Glutaredoxin Regulates Apoptosis in Cardiomyocytes via NFκB Targets Bcl-2 and Bcl-xL: Implications for Cardiac Aging

Cardiomyocyte apoptosis is a well-established contributor to irreversible injury following myocardial infarction (MI). Increased cardiomyocyte apoptosis is associated also with aging in animal models, exacerbated by MI; however, mechanisms for this increased sensitivity to oxidative stress are unknown. Protein mixed-disulfide formation with glutathione (protein glutathionylation) is known to change the function of intermediates that regulate apoptosis. Since glutaredoxin (Grx) specifically catalyzes protein deglutathionylation, we examined its status with aging and its influence on regulation of apoptosis. Grx1 content and activity are decreased by ∼40% in elderly (24-mo) Fischer 344 rat hearts compared to adult (6-mo) controls. A similar extent of Grx1 knockdown in H9c2 cardiomyocytes led to increased apoptosis, decreased NFκB-dependent transcriptional activity, and decreased production (mRNA and protein) of anti-apoptotic NFκB target genes, Bcl-2 and Bcl-xL. Knockdown of Bcl-2 and/or Bcl-xL in wild-type H9c2 cells to the same extent (∼50%) as observed in Grx1-knockdown cells increased baseline apoptosis; and knockdown of Bcl-xL, but not Bcl-2, also increased oxidant-induced apoptosis analogous to Grx1-knockdown cells. Natural Grx1-deficient cardiomyocytes isolated from elderly rats also displayed diminished NFκB activity and Bcl-xL content. Taken together, these data indicate diminution of Grx1 in elderly animals contributes to increased apoptotic susceptibility via regulation of NFκB function. Antioxid. Redox Signal. 12, 1339–1353