Yeast Assay Highlights the Intrinsic Genomic Instability of Human PML Intron 6 over Intron 3 and the Role of Replication Fork Proteins

Human acute promyelocytic leukemia (APL) is characterized by a specific balanced translocation t(15;17)(q22;q21) involving the PML and RARA genes. In both de novo and therapy-related APL, the most frequent PML breakpoints are located within intron 6, and less frequently in intron 3; the precise mechanisms by which these breakpoints arise and preferentially in PML intron 6 remain unsolved. To investigate the intrinsic properties of the PML intron sequences in vivo, we designed Saccharomyces cerevisiae strains containing human PML intron 6 or intron 3 sequences inserted in yeast chromosome V and measured gross chromosomal rearrangements (GCR). This approach provided evidence that intron 6 had a superior instability over intron 3 due to an intrinsic property of the sequence and identified the 3’ end of intron 6 as the most susceptible to break. Using yeast strains invalidated for genes that control DNA replication, we show that this differential instability depended at least upon Rrm3, a DNA helicase, and Mrc1, the human claspin homolog. GCR induction by hydrogen peroxide, a general genotoxic agent, was also dependent on genetic context. We conclude that: 1) this yeast system provides an alternative approach to study in detail the properties of human sequences in a genetically controlled situation and 2) the different susceptibility to produce DNA breaks in intron 6 versus intron 3 of the human PML gene is likely due to an intrinsic property of the sequence and is under replication fork genetic control.     

A Quantitative Diffuse Reflectance Imaging (QDRI) System for Comprehensive Surveillance of the Morphological Landscape in Breast Tumor Margins

In an ongoing effort to address the clear clinical unmet needs surrounding breast conserving surgery (BCS), our group has developed a next-generation multiplexed optical-fiber-based tool to assess breast tumor margin status during initial surgeries. Specifically detailed in this work is the performance and clinical validation of a research-grade intra-operative tool for margin assessment based on diffuse optical spectroscopy. Previous work published by our group has illustrated the proof-of-concept generations of this device; here we incorporate a highly optimized quantitative diffuse reflectance imaging (QDRI) system utilizing a wide-field (imaging area = 17cm²) 49-channel multiplexed fiber optic probe, a custom raster-scanning imaging platform, a custom dual-channel white LED source, and an astronomy grade imaging CCD and spectrograph. The system signal to noise ratio (SNR) was found to be greater than 40dB for all channels. Optical property estimation error was found to be less than 10%, on average, over a wide range of absorption (μ_a = 0–8.9cm^-1) and scattering (μ_s’ = 7.0–9.7cm^-1) coefficients. Very low inter-channel and CCD crosstalk was observed (2% max) when used on turbid media (including breast tissue). A raster-scanning mechanism was developed to achieve sub-pixel resolution and was found to be optimally performed at an upsample factor of 8, affording 0.75mm spatially resolved diffuse reflectance images (λ = 450–600nm) of an entire margin (area = 17cm²) in 13.8 minutes (1.23cm²/min). Moreover, controlled pressure application at the probe-tissue interface afforded by the imaging platform reduces repeated scan variability, providing <1% variation across repeated scans of clinical specimens. We demonstrate the clinical utility of this device through a pilot 20-patient study of high-resolution optical parameter maps of the ratio of the β-carotene concentration to the reduced scattering coefficient. An empirical cumulative distribution function (eCDF) analysis is used to reduce optical property maps to quantitative distributions representing the morphological landscape of breast tumor margins. The optimizations presented in this work provide an avenue to rapidly survey large tissue areas on intra-operative time scales with improved sensitivity to regions of focal disease that may otherwise be overlooked.     

Sleep Disruption and Daytime Sleepiness Correlating with Disease Severity and Insulin Resistance in Non-Alcoholic Fatty Liver Disease: A Comparison with Healthy Controls

Background & Aims

Sleep disturbance is associated with the development of obesity, diabetes and hepatic steatosis in murine models. Hepatic triglyceride accumulation oscillates in a circadian rhythm regulated by clock genes, light-dark cycle and feeding time in mice. The role of the sleep-wake cycle in the pathogenesis of human non-alcoholic fatty liver disease (NAFLD) is indeterminate. We sought to detail sleep characteristics, daytime sleepiness and meal times in relation to disease severity in patients with NAFLD.

Methods

Basic Sleep duration and latency, daytime sleepiness (Epworth sleepiness scale), Pittsburgh sleep quality index, positive and negative affect scale, Munich Chronotype Questionnaire and an eating habit questionnaire were assessed in 46 patients with biopsy-proven NAFLD and 22 healthy controls, and correlated with biochemical and histological parameters.

Results

In NAFLD compared to healthy controls, time to fall asleep was vastly prolonged (26.9 vs. 9.8 min., p = 0.0176) and sleep duration was shortened (6.3 vs. 7.2 hours, p = 0.0149). Sleep quality was poor (Pittsburgh sleep quality index 8.2 vs. 4.7, p = 0.0074) and correlated with changes in affect. Meal frequency was shifted towards night-times (p = 0.001). In NAFLD but not controls, daytime sleepiness significantly correlated with liver enzymes (ALAT [r = 0.44, p = 0.0029], ASAT [r = 0.46, p = 0.0017]) and insulin resistance (HOMA-IR [r = 0.5, p = 0.0009]) independent of cirrhosis. In patients with fibrosis, daytime sleepiness correlated with the degree of fibrosis (r = 0.364, p = 0.019).

Conclusions

In NAFLD sleep duration was shortened, sleep onset was delayed and sleep quality poor. Food-intake was shifted towards the night. Daytime sleepiness was positively linked to biochemical and histologic surrogates of disease severity. The data may indicate a role for sleep-wake cycle regulation and timing of food-intake in the pathogenesis of human NAFLD as suggested from murine models.     

Impact of Malaria Control on Mortality and Anemia among Tanzanian Children Less than Five Years of Age, 1999–2010

Background

Mainland Tanzania scaled up multiple malaria control interventions between 1999 and 2010. We evaluated whether, and to what extent, reductions in all-cause under-five child mortality (U5CM) tracked with malaria control intensification during this period.

Methods

Four nationally representative household surveys permitted trend analysis for malaria intervention coverage, severe anemia (hemoglobin <8 g/dL) prevalence (SAP) among children 6–59 months, and U5CM rates stratified by background characteristics, age, and malaria endemicity. Prevalence of contextual factors (e.g., vaccination, nutrition) likely to influence U5CM were also assessed. Population attributable risk percentage (PAR%) estimates for malaria interventions and contextual factors that changed over time were used to estimate magnitude of impact on U5CM.

Results

Household ownership of insecticide-treated nets (ITNs) rose from near zero in 1999 to 64% (95% CI, 61.7–65.2) in 2010. Intermittent preventive treatment of malaria in pregnancy reached 26% (95% CI, 23.6–28.0) by 2010. Sulfadoxine-pyrimethamine replaced chloroquine in 2002 and artemisinin-based combination therapy was introduced in 2007. SAP among children 6–59 months declined 50% between 2005 (11.1%; 95% CI, 10.0–12.3%) and 2010 (5.5%; 95% CI, 4.7–6.4%) and U5CM declined by 45% between baseline (1995–9) and endpoint (2005–9), from 148 to 81 deaths/1000 live births, respectively. Mortality declined 55% among children 1–23 months of age in higher malaria endemicity areas. A large reduction in U5CM was attributable to ITNs (PAR% = 11) with other malaria interventions adding further gains. Multiple contextual factors also contributed to survival gains.

Conclusion

Marked declines in U5CM occurred in Tanzania between 1999 and 2010 with high impact from ITNs and ACTs. High-risk children (1–24 months of age in high malaria endemicity) experienced the greatest declines in mortality and SAP. Malaria control should remain a policy priority to sustain and further accelerate progress in child survival.     

What Do Eye Gaze Metrics Tell Us about Motor Imagery?

Many of the brain structures involved in performing real movements also have increased activity during imagined movements or during motor observation, and this could be the neural substrate underlying the effects of motor imagery in motor learning or motor rehabilitation. In the absence of any objective physiological method of measurement, it is currently impossible to be sure that the patient is indeed performing the task as instructed. Eye gaze recording during a motor imagery task could be a possible way to “spy” on the activity an individual is really engaged in. The aim of the present study was to compare the pattern of eye movement metrics during motor observation, visual and kinesthetic motor imagery (VI, KI), target fixation, and mental calculation. Twenty-two healthy subjects (16 females and 6 males), were required to perform tests in five conditions using imagery in the Box and Block Test tasks following the procedure described by Liepert et al. Eye movements were analysed by a non-invasive oculometric measure (SMI RED250 system). Two parameters describing gaze pattern were calculated: the index of ocular mobility (saccade duration over saccade + fixation duration) and the number of midline crossings (i.e. the number of times the subjects gaze crossed the midline of the screen when performing the different tasks). Both parameters were significantly different between visual imagery and kinesthesic imagery, visual imagery and mental calculation, and visual imagery and target fixation. For the first time we were able to show that eye movement patterns are different during VI and KI tasks. Our results suggest gaze metric parameters could be used as an objective unobtrusive approach to assess engagement in a motor imagery task. Further studies should define how oculomotor parameters could be used as an indicator of the rehabilitation task a patient is engaged in.     

Revealing Different Roles of the mTOR-Targets S6K1 and S6K2 in Breast Cancer by Expression Profiling and Structural Analysis

Background

The AKT/mTORC1/S6K pathway is frequently overstimulated in breast cancer, constituting a promising therapeutic target. The benefit from mTOR inhibitors varies, likely as a consequence of tumour heterogeneity, and upregulation of several compensatory feed-back mechanisms. The mTORC1 downstream effectors S6K1, S6K2, and 4EBP1 are amplified and overexpressed in breast cancer, associated with a poor outcome and divergent endocrine treatment benefit. S6K1 and S6K2 share high sequence homology, but evidence of partly distinct biological functions is emerging. The aim of this work was to explore possible different roles and treatment target potentials of S6K1 and S6K2 in breast cancer.

Materials and methods

Whole-genome expression profiles were compared for breast tumours expressing high levels of S6K1, S6K2 or 4EBP1, using public datasets, as well as after in vitro siRNA downregulation of S6K1 and/or S6K2 in ZR751 breast cancer cells. In silico homology modelling of the S6K2 kinase domain was used to evaluate its possible structural divergences to S6K1.

Results

Genome expression profiles were highly different in S6K1 and S6K2 high tumours, whereas S6K2 and 4EBP1 profiles showed significant overlaps, both correlated to genes involved in cell cycle progression, among these the master regulator E2F1. S6K2 and 4EBP1 were inversely associated with IGF1 levels, and their prognostic value was shown to be restricted to tumours positive for IGFR and/or HER2. In vitro, S6K1 and S6K2 silencing resulted in upregulation of genes in the mTORC1 and mTORC2 complexes. Isoform-specific silencing also showed distinct patterns, e.g. S6K2 downregulation lead to upregulation of several cell cycle associated genes. Structural analyses of the S6K2 kinase domain showed unique structure patterns, deviating from those of S6K1, facilitating the development of isoform-specific inhibitors. Our data support emerging proposals of distinct biological features of S6K1 and S6K2, suggesting their importance as separate oncogenes and clinical markers, where specific targeting in different breast cancer subtypes could facilitate further individualised therapies.     

A Peptide to Reduce Pulmonary Edema in a Rat Model of Lung Transplantation

Background

Despite significant advances in organ preservation, surgical techniques and perioperative care, primary graft dysfunction is a serious medical problem in transplantation medicine in general and a specific problem in patients undergoing lung transplantation. As a result, patients develop lung edema, causing reduced tissue oxygenation capacity, reduced lung compliance and increased requirements for mechanical ventilatory support. Yet, there is no effective strategy available to protect the grafted organ from stress reactions induced by ischemia/reperfusion and by the surgical procedure itself.

Methods

We assessed the effect of a cingulin-derived peptide, XIB13 or a random peptide in an established rat model of allogeneic lung transplantation. Donor lungs and recipients received therapeutic peptide at the time of transplantation and outcome was analyzed 100min and 28 days post grafting.

Results

XIB13 improved blood oxygenation and reduced vascular leak 100min post grafting. Even after 28 days, lung edema was significantly reduced by XIB13 and lungs had reduced fibrotic or necrotic zones. Moreover, the induction of an allogeneic T cell response was delayed indicating a reduced antigen exchange between the donor and the host.

Conclusions

In summary, we provide a new tool to strengthen endothelial barrier function thereby improving outcomes in lung transplantation.     

Plasma HIV-2 RNA According to CD4 Count Strata among HIV-2-Infected Adults in the IeDEA West Africa Collaboration

Background

Plasma HIV-1 RNA monitoring is one of the standard tests for the management of HIV-1 infection. While HIV-1 RNA can be quantified using several commercial tests, no test has been commercialized for HIV-2 RNA quantification. We studied the relationship between plasma HIV-2 viral load (VL) and CD4 count in West African patients who were either receiving antiretroviral therapy (ART) or treatment-naïve.

Method

A cross sectional survey was conducted among HIV-2-infected individuals followed in three countries in West Africa from March to December 2012. All HIV-2 infected-patients who attended one of the participating clinics were proposed a plasma HIV-2 viral load measurement. HIV-2 RNA was quantified using the new ultrasensitive in-house real-time PCR assay with a detection threshold of 10 copies/ mL (cps/mL).

Results

A total of 351 HIV-2-infected individuals participated in this study, of whom 131 (37.3%) were treatment naïve and 220 (62.7%) had initiated ART. Among treatment-naïve patients, 60 (46.5%) had undetectable plasma HIV-2 viral load (<10 cps/mL), it was detectable between 10-100 cps/mL in 35.8%, between 100-1000 cps/mL in 11.7% and >1000 cps/mL in 6.0% of the patients. Most of the treatment-naïve patients (70.2%) had CD4-T cell count ≥500 cells/mm³ and 43 (46.7%) of these patients had a detectable VL (≥10 cps/mL). Among the 220 patients receiving ART, the median CD4-T cell count rose from 231 to 393 cells/mm³ (IQR [259-561]) after a median follow-up duration of 38 months and 145 (66.0%) patients had CD4-T cell count ≤ 500 cells/mm³ with a median viral load of 10 cps/mL (IQR [10-33]). Seventy five (34.0%) patients had CD4-T cell count ≥ 500 cells/mm³, among them 14 (18.7%) had a VL between 10-100 cps/mL and 2 (2.6%) had VL >100 cps/mL.

Conclusion

This study suggests that the combination of CD4-T cell count and ultrasensitive HIV-2 viral load quantification with a threshold of 10 cps/mL, could improve ART initiation among treatment naïve HIV-2-infected patients and the monitoring of ART response among patients receiving treatment.     

A Zebrafish Drug-Repurposing Screen Reveals sGC-Dependent and sGC-Independent Pro-Inflammatory Activities of Nitric Oxide

Tissue injury and infection trigger innate immune responses. However, dysregulation may result in chronic inflammation and is commonly treated with corticosteroids and non-steroidal anti-inflammatory drugs. Unfortunately, long-term administration of both therapeutic classes can cause unwanted side effects. To identify alternative immune-modulatory compounds we have previously established a novel screening method using zebrafish larvae. Using this method we here present results of an in vivo high-content drug-repurposing screen, identifying 63 potent anti-inflammatory drugs that are in clinical use for other indications. Our approach reveals a novel pro-inflammatory role of nitric oxide. Nitric oxide affects leukocyte recruitment upon peripheral sensory nervous system or epithelial injury in zebrafish larvae both via soluble guanylate cyclase and in a soluble guanylate cyclase -independent manner through protein S-nitrosylation. Together, we show that our screening method can help to identify novel immune-modulatory activities and provide new mechanistic insights into the regulation of inflammatory processes.