Hot off the presses! Nov 25 The Lancet

The Nov 25 issue of the The Lancet is now up on Pubget (About The Lancet): if you're at a subscribing institution, just click the link in the latest link at the home page. (Note you'll only be able to get all the PDFs in the issue if your institution subscribes to Pubget.)

Latest Articles Include:

• November 19â€"25, 2011
  - The Lancet 378(9805):i (2011)
  
• Politics and patient care
  - The Lancet 378(9805):1757 (2011)
  
• Breast cancer: old and new debates
  - The Lancet 378(9805):1758 (2011)
  
• CRASH-2 goes viral
  - The Lancet 378(9805):1758 (2011)
  
• Multiple sclerosisâ€"quenching the flames of inflammation
  - The Lancet 378(9805):1759-1760 (2011)
  
• Tackling domestic violence: is increasing referral enough?
  - The Lancet 378(9805):1760-1762 (2011)
  
• Community management of severe pneumonia in children
  - The Lancet 378(9805):1762-1764 (2011)
  
• The Global Fund: getting the reforms right
  - The Lancet 378(9805):1764-1765 (2011)
  
• The European Respiratory Roadmap
  - The Lancet 378(9805):1765-1767 (2011)
  
• Offline: Nursing, but not as you know it
  - The Lancet 378(9805):1768 (2011)
  
• Drug withdrawal sends critical care specialists back to basics
  - The Lancet 378(9805):1769 (2011)
  
• Health 2020: WHO's missing link?
  - The Lancet 378(9805):1770 (2011)
  
• Saving Chile's angelitos
  - The Lancet 378(9805):1771-1772 (2011)
  
• Book Lost words
  - The Lancet 378(9805):1772 (2011)
  
• Moving pictures of Parkinson's disease
  - The Lancet 378(9805):1773-1774 (2011)
  
• Effect of population-based screening on breast cancer mortality
  - The Lancet 378(9805):1775-1776 (2011)
  
• Leadership and public health
  - The Lancet 378(9805):1776 (2011)
  
• Leadership and public health
  - The Lancet 378(9805):1776 (2011)
  
• Leadership and public health
  - The Lancet 378(9805):1776-1777 (2011)
  
• Hydroxycarbamide use in young children with sickle-cell anaemia
  - The Lancet 378(9805):1777 (2011)
  
• Hydroxycarbamide use in young children with sickle-cell anaemia
  - The Lancet 378(9805):1777 (2011)
  
• Need to realign patient-oriented and commercial and academic research
  - The Lancet 378(9805):1777-1778 (2011)
  
• The forgotten psychosocial dimension of the obesity epidemic
  - The Lancet 378(9805):e8 (2011)
  
• Non-communicable diseases and surgery at the UN? “Fugetaboutit!”
  - The Lancet 378(9805):e8 (2011)
  
• Department of Error
  - The Lancet 378(9805):1778 (2011)
  
• Department of Error
  - The Lancet 378(9805):1778 (2011)
  
• Department of Error
  - The Lancet 378(9805):1778 (2011)
  
• Department of Error
  - The Lancet 378(9805):1778 (2011)
  
• Department of Error
  - The Lancet 378(9805):1778 (2011)
  
• Ocrelizumab in relapsing-remitting multiple sclerosis: a phase 2, randomised, placebo-controlled, multicentre trial
  - The Lancet 378(9805):1779-1787 (2011)
  Background B lymphocytes are implicated in the pathogenesis of multiple sclerosis. We aimed to assess efficacy and safety of two dose regimens of the humanised anti-CD20 monoclonal antibody ocrelizumab in patients with relapsing-remitting multiple sclerosis. Methods We did a multicentre, randomised, parallel, double-blind, placebo-controlled study involving 79 centres in 20 countries. Patients aged 18–55 years with relapsing-remitting multiple sclerosis were randomly assigned (1:1:1:1) via an interactive voice response system to receive either placebo, low-dose (600 mg) or high-dose (2000 mg) ocrelizumab in two doses on days 1 and 15, or intramuscular interferon beta-1a (30 亮g) once a week. The randomisation list was not disclosed to the study centres, monitors, project statisticians or to the project team at Roche. All groups were double blinded to group assignment, except the interferon beta-1a group who were rater masked. At week 24, patients in the initial placebo, 600 mg ocrelizumab, and interferon beta-1a groups received ocrelizumab 600 mg; the 2000 mg group received 1000 mg. Our primary endpoint was the total number of gadolinium-enhancing lesions (GEL) and T1-weighted MRI at weeks 12, 16, 20, and 24. Analyses were done on an! intention-to-treat basis. This trial is registered with ClinicalTrials.gov, number NCT00676715. Findings 218 (99%) of the 220 randomised patients received at least one dose of ocrelizumab, 204 (93%) completed 24 weeks of the study and 196 (89%) completed 48 weeks. In the intention-to-treat population of 218 patients, at week 24, the number of gadolinium-enhancing lesions was 89% (95% CI 68–97; p<0·0001) lower in the 600 mg ocrelizumab group than in the placebo group, and 96% (89–99; p<0·0001) lower in the 2000 mg group. In exploratory analyses, both 600 mg and 2000 mg ocrelizumab groups were better than interferon beta-1a for GEL reduction. We noted serious adverse events in two of 54 (4%; 95% CI 3·0–4·4) patients in the placebo group, one of 55 (2%; 1·3–2·3) in the 600 mg ocrelizumab group, three of 55 (5%; 4·6–6·3) in the 2000 mg group, and two of 54 (4%; 3·0–4·4) in the interferon beta-1a group. Interpretation The similarly pronounced effects of B-cell depletion with both ocrelizumab doses on MRI and relapse-related outcomes support a role for B-cells in disease pathogenesis and warrant further assessment in large, long-term trials. Funding F Hoffmann-La Roche Ltd, Biogen Idec Inc.
• Identification and Referral to Improve Safety (IRIS) of women experiencing domestic violence with a primary care training and support programme: a cluster randomised controlled trial
  - The Lancet 378(9805):1788-1795 (2011)
  Background Most clinicians have no training about domestic violence, fail to identify patients experiencing abuse, and are uncertain about management after disclosure. We tested the effectiveness of a programme of training and support in primary health-care practices to increase identification of women experiencing domestic violence and their referral to specialist advocacy services. Methods In this cluster randomised controlled trial, we selected general practices in two urban primary care trusts, Hackney (London) and Bristol, UK. Practices in which investigators from this trial were employed or those who did not use electronic records were excluded. Practices were stratified by proportion of female doctors, postgraduate training status, number of patients registered, and percentage of practice population on low incomes. Within every primary care trust area, we randomised practices with a computer-minimisation programme with a random component to intervention or control groups. The intervention programme included practice-based training sessions, a prompt within the medical record to ask about abuse, and a referral pathway to a named domestic violence advocate, who also delivered the training and further consultancy. The primary outcome was recorded referral of patients to domestic violence advocacy services. The prespecified secondary outcome was recorded iden! tification of domestic violence in the electronic medical records of the general practice. Poisson regression analyses accounting for clustering were done for all practices receiving the intervention. Practice staff and research associates were not masked and patients were not aware they were part of a study. This study is registered at Current Controlled Trials, ISRCTN74012786. Findings We randomised 51 (61%) of 84 eligible general practices in Hackney and Bristol. Of these, 24 received a training and support programme, 24 did not receive the programme, and three dropped out before the trial started. 1 year after the second training session, the 24 intervention practices recorded 223 referrals of patients to advocacy and the 24 control practices recorded 12 referrals (adjusted intervention rate ratio 22·1 [95% CI 11·5–42·4]). Intervention practices recorded 641 disclosures of domestic violence and control practices recorded 236 (adjusted intervention rate ratio 3·1 [95% CI 2·2–4·3). No adverse events were recorded. Interpretation A training and support programme targeted at primary care clinicians and administrative staff improved referral to specialist domestic violence agencies and recorded identification of women experiencing domestic violence. Our findings reduce the uncertainty about the benefit of training and support interventions in primary care settings for domestic violence and show that screening of women patients for domestic violence is not a necessary condition for improved identification and referral to advocacy services. Funding Health Foundation.
• Community case management of severe pneumonia with oral amoxicillin in children aged 2â€"59 months in Haripur district, Pakistan: a cluster randomised trial
  - The Lancet 378(9805):1796-1803 (2011)
  Background First dose oral co-trimoxazole and referral are recommended for WHO-defined severe pneumonia. Difficulties with referral compliance are reported in many low-resource settings, resulting in low access to appropriate treatment. The objective in this study was to assess whether community case management by lady health workers (LHWs) with oral amoxicillin in children with severe pneumonia was equivalent to current standard of care. Methods In Haripur district, Pakistan, 28 clusters were randomly assigned with stratification in a 1:1 ratio to intervention and control clusters by use of a computer-generated randomisation sequence. Children were included in the study if they were aged 2–59 months with WHO-defined severe pneumonia and living in the study area. In the intervention clusters, community-based LHWs provided mothers with oral amoxicillin (80–90 mg/kg per day or 375 mg twice a day for infants aged 2–11 months and 625 mg twice a day for those aged 12–59 months) with specific guidance on its use. In control clusters, LHWs gave the first dose of oral co-trimoxazole (age 2–11 months, sulfamethoxazole 200 mg plus trimethoprim 40 mg; age 12 months to 5 years, sulfamethoxazole 300 mg plus trimethoprim 60 mg) and referred the children to a health facility for standard of care. Participants, carers, and assessors were not masked to treatment assignment. The primary outcome was treatment failure by day 6! . Analysis was per protocol with adjustment for clustering within groups by use of generalised estimating equations. This study is registered, number ISRCTN10618300. Findings We assigned 1995 children to treatment in 14 intervention clusters and 1477 in 14 control clusters, and we analysed 1857 and 1354 children, respectively. Cluster-adjusted treatment failure rates by day 6 were significantly reduced in the intervention clusters (165 [9%] vs 241 [18%], risk difference −8·9%, 95% CI −12·4 to −5·4). Further adjustment for baseline covariates made little difference (−7·3%, −10·1 to −4·5). Two deaths were reported in the control clusters and one in the intervention cluster. Most of the risk reduction was in the occurrence of fever and lower chest indrawing on day 3 (−6·7%, −10·0 to −3·3). Adverse events were diarrhoea (n=4) and skin rash (n=1) in the intervention clusters and diarrhoea (n=3) in the control clusters. Interpretation Community case management could result in a standardised treatment for children with severe pneumonia, reduce delay in treatment initiation, and reduce the costs for families and health-care systems. Funding United States Agency for International Development (USAID).
• MRI for breast cancer screening, diagnosis, and treatment
  - The Lancet 378(9805):1804-1811 (2011)
  MRI is used widely both for screening women who are at increased risk of breast cancer and for treatment selection. Prospective studies confirm that MRI screening of women with known or suspected genetic mutation results in a higher sensitivity for cancer detection than does mammography. However, survival data are not available. In women with breast cancer, MRI detects cancer not identified with other types of screening. In two randomised trials, this increased sensitivity did not translate into improved selection of surgical treatment or a reduction in the number of operations. Data for longer-term outcomes such as ipsilateral breast tumour recurrence rates and contralateral breast cancer incidence are scarce, but to date do not show clear benefit for MRI. MRI is better than other methods of assessing the response to neoadjuvant chemotherapy, and is helpful in identifying the primary tumour in patients who present with axillary adenopathy.
• Gene expression profiling in breast cancer: classification, prognostication, and prediction
  - The Lancet 378(9805):1812-1823 (2011)
  Microarray-based gene expression profiling has had a major effect on our understanding of breast cancer. Breast cancer is now perceived as a heterogeneous group of different diseases characterised by distinct molecular aberrations, rather than one disease with varying histological features and clinical behaviour. Gene expression profiling studies have shown that oestrogen-receptor (ER)-positive and ER-negative breast cancers are distinct diseases at the transcriptomic level, that additional molecular subtypes might exist within these groups, and that the prognosis of patients with ER-positive disease is largely determined by the expression of proliferation-related genes. On the basis of these principles, a molecular classification system and prognostic multigene classifiers based on microarrays or derivative technologies have been developed and are being tested in randomised clinical trials and incorporated into clinical practice. In this review, we focus on the concep! tual effect and potential clinical use of the molecular classification of breast cancer, and discuss prognostic and predictive multigene predictors.
• A strange case of waitress headache
  - The Lancet 378(9805):1824 (2011)
  

Hot off the presses! Feb 01 Biomaterials

The Feb 01 issue of the Biomaterials is now up on Pubget (About Biomaterials): if you're at a subscribing institution, just click the link in the latest link at the home page. (Note you'll only be able to get all the PDFs in the issue if your institution subscribes to Pubget.)

Latest Articles Include:

• Editorial board
  - Biomaterials 33(4):IFC (2012)
  
• Microfluidic 3D bone tissue model for high-throughput evaluation of wound-healing and infection-preventing biomaterials
  - Biomaterials 33(4):999-1006 (2012)
  We report the use of a microfluidic 3D bone tissue model, as a high-throughput means of evaluating the efficacy of biomaterials aimed at accelerating orthopaedic implant-related wound-healing while preventing bacterial infection. As an example of such biomaterials, inkjet-printed micropatterns were prepared to contain antibiotic and biphasic calcium phosphate (BCP) nanoparticles dispersed in a poly(d,l-lactic-co-glycolic) acid matrix. The micropatterns were integrated with a microfluidic device consisting of eight culture chambers. The micropatterns immediately and completely killed Staphylococcus epidermidis upon inoculation, and enhanced the calcified extracellular matrix production of osteoblasts. Without antibiotic elution, bacteria rapidly proliferated to result in an acidic microenvironment which was detrimental to osteoblasts. These results were used to demonstrate the tissue model's potential in: (i) significantly reducing the number of biomaterial samples an! d culture experiments required to assess in vitro efficacy for wound-healing and infection prevention and (ii) in situ monitoring of dynamic interactions of biomaterials with bacteria as wells as with tissue cells simultaneously.
• Glycosaminoglycan (GAG) binding surfaces for characterizing GAG-protein interactions
  - Biomaterials 33(4):1007-1016 (2012)
  Glycosaminoglycans play an important role in tissue organisation through interactions with a diverse range of proteins, growth factors and other chemokines. In this report, we demonstrate the GAG-binding 'fingerprint' of two important GAG-binding proteins – osteoprotogerin and TIMP-3. The technique uses a straightforward method for attaching GAGs to assay surfaces in a non-covalent manner using plasma polymerization that leaves the adsorbed GAG able to participate in subsequent ligand binding. We show that OPG and TIMP-3 bind preferentially to different GAGs in a simple ELISA and that this binding does not correlate directly with simple GAG properties such as degree of sulfation. The methods outlined in this report can be easily applied to tissue engineering scaffolds in order to exploit the potential of surface-bound GAGs in influencing the structure of engineered tissues.
• Impact of processing parameters on the haemocompatibility of Bombyx mori silk films
  - Biomaterials 33(4):1017-1023 (2012)
  Silk has traditionally been used for surgical sutures due to its lasting strength and durability; however, the use of purified silk proteins as a scaffold material for vascular tissue engineering goes beyond traditional use and requires application-orientated biocompatibility testing. For this study, a library of Bombyx mori silk films was generated and exposed to various solvents and treatment conditions to reflect current silk processing techniques. The films, along with clinically relevant reference materials, were exposed to human whole blood to determine silk blood compatibility. All substrates showed an initial inflammatory response comparable to polylactide-co-glycolide (PLGA), and a low to moderate haemostasis response similar to polytetrafluoroethylene (PTFE) substrates. In particular, samples that were water annealed at 25 °C for 6 h demonstrated the best blood compatibility based on haemostasis parameters (e.g. platelet decay, thrombin-antithrombin complex,! platelet factor 4, granulocytes-platelet conjugates) and inflammatory parameters (e.g. C3b, C5a, CD11b, surface-associated leukocytes). Multiple factors such as treatment temperature and solvent influenced the biological response, though no single physical parameter such as β-sheet content, isoelectric point or contact angle accurately predicted blood compatibility. These findings, when combined with prior in vivo data on silk, support a viable future for silk-based vascular grafts.
• Three-dimensional paper-based electrochemiluminescence immunodevice for multiplexed measurement of biomarkers and point-of-care testing
  - Biomaterials 33(4):1024-1031 (2012)
  In this work, electrochemiluminescence (ECL) immunoassay was introduced into the recently proposed microfluidic paper-based analytical device (ﾎｼPADs) based on directly screen-printed electrodes on paper for the very first time. The screen-printed paper-electrodes will be more important for further development of this paper-based ECL device in simple, low-cost and disposable application than commercialized ones. To further perform high-performance, high-throughput, simple and inexpensive ECL immunoassay on ﾎｼPAD for point-of-care testing, a wax-patterned three-dimensional (3D) paper-based ECL device was demonstrated for the very first time. In this 3D paper-based ECL device, eight carbon working electrodes including their conductive pads were screen-printed on a piece of square paper and shared the same Ag/AgCl reference and carbon counter electrodes on another piece of square paper after stacking. Using typical tris-(bipyridine)-ruthenium (竇｡) - tri-n-prop! ylamine ECL system, the application test of this 3D paper-based ECL device was performed through the diagnosis of four tumor markers in real clinical serum samples. With the aid of a facile device-holder and a section-switch assembled on the analyzer, eight working electrodes were sequentially placed into the circuit to trigger the ECL reaction in the sweeping range from 0.5 to 1.1 V at room temperature. In addition, this 3D paper-based ECL device can be easily integrated and combined with the recently emerging paper electronics to further develop simple, sensitive, low-cost, disposable and portable ﾎｼPAD for point-of-care testing, public health and environmental monitoring in remote regions, developing or developed countries.
• Modulation of the migration and differentiation potential of adult bone marrow stromal stem cells by nitric oxide
  - Biomaterials 33(4):1032-1043 (2012)
  Nitric oxide (NO) is a diffusible free radical, which serves as a pluripotent intracellular messenger in numerous cell systems. NO has been demonstrated to regulate actin dependent cellular functions and functions as a putative inductive agent in directing stem cells differentiation. In this study, we investigated the effect of exogenous NO on the kinetics of movement and morphological changes in adult bone marrow stromal cells (BMSCs) in a wound healing model of cellular migration. Cellular migration and morphological changes were determined by measurement of changes in the area and fractal dimension of BMSCs monolayer as a function of time in the presence of an NO donor (S-Nitroso-N-Acetyl-D,l-Penicillamine, SNAP) compared to untreated BMSCs. Response of the BMSCs' actin cytoskeleton and desmin to NO was assessed by determining changes in their integrated optical density (IOD) and fractal dimension at 24 h and 7 days. NO suppressed BMSCs' migration accompanied by! a reduction in cell size, with maintenance of their stellate to polygonal morphology. In response to NO, the actin cytoskeleton expressed an increase in randomness but maintained a constant amount of F-actin relative to the cell size. The presence of NO also induced an increase in randomly organized cytoplasmic desmin. These data suggest that NO has an apparent inductive effect on adult BMSCs and is capable of initiating phenotypic change at the gross cellular, cytoskeletal and molecular levels. It is apparent, however, that additional factors or conditions are required to further drive the differentiation of adult BMSCs into specific phenotypes, such as cardiomyocytes.
• The mechanical stimulation of cells in 3D culture within a self-assembling peptide hydrogel
  - Biomaterials 33(4):1044-1051 (2012)
  The aim of this present study was to provide a scaffold as a tool for the investigation of the effect of mechanical stimulation on three-dimensionally cultured cells. For this purpose, we developed an artificial self-assembling peptide (SPG-178) hydrogel scaffold. The structural properties of the SPG-178 peptide were confirmed by attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) and transmission electron microscopy (TEM). The mechanical properties of the SPG-178 hydrogel were studied using rheology measurements. The SPG-178 peptide was able to form a stable, transparent hydrogel in a neutral pH environment. In the SPG-178 hydrogel, mouse skeletal muscle cells proliferated successfully (increased by 12.4 ± 1.5 times during 8 days of incubation; mean ± SEM). When the scaffold was statically stretched, a rapid phosphorylation of ERK was observed (increased by 2.8 ± 0.2 times; mean ± SEM). These results demonstrated that the developed self-! assembling peptide gel is non-cytotoxic and is a suitable tool for the investigation of the effect of mechanical stimulation on three-dimensional cell culture.
• Influence of perfusion and compression on the proliferation and differentiation of bone mesenchymal stromal cells seeded on polyurethane scaffolds
  - Biomaterials 33(4):1052-1064 (2012)
  In the present study, a porous meniscal-shaped scaffold consisting of polyurethane (PU)-based 1, 4-butanediisocyanate (BDI), which provided a 3-D culture condition for human bone mesenchymal stromal cells (hBMSC) was employed. A bioreactor was utilized to produce perfusion and mechanical stimulations. The viability, proliferation and fibro-cartilaginous differentiation of the hBMSC cultured on the PU-based meniscal scaffold were investigated during the perfusion and mechanical stimulation process. In addition, the mechanical properties of the cell-laden scaffolds were examined as well. Our finding indicated that the perfusion (10 ml/min) and on-off cyclic compressions mechanical stimulation (10% strain, 0.5 Hz, 4 times/day, 2 h/time with 4 h of rest thereafter) maintained the viability and promoted the proliferation of hBMSC over 2 weeks. The on-off cyclic compression caused a 1.85 fold increase in equilibrium modulus. Meanwhile, type I procollagen produced by hBMSC wa! s increased for 3.02-fold after 2 weeks culture. On the other hand, the irrigating medium enhanced the synthesis of type III procollagen for 2.24-fold after 2 weeks. Tensile modulus was elevated for 2.02-fold in perfusion group after 1 week, which was decreased after 2 weeks unexpectedly. Our study suggests that the perfusion and on-off compression are promising to enhance the functional properties of the hBMSC-laden PU-based meniscal scaffold.
• Three-dimensional cancer-bone metastasis model using ex-vivo co-cultures of live calvarial bones and cancer cells
  - Biomaterials 33(4):1065-1078 (2012)
  One of the major limitations of studying cancer-bone metastasis has been the lack of an appropriate ex-vivo model which can be used under defined conditions that simulates closely the in vivo live bone microenvironment in response to cancer-bone interactions. We have developed and utilized a three-dimensional (3D) cancer-bone metastasis model using free-floating live mouse calvarial bone organs in the presence of cancer cells in a roller tube system. In such co-cultures under hypoxia and a specifically defined bone remodeling stage, viz., resorption system, cancer cells showed a remarkable affinity and specificity for the "endosteal side" of the bone where they colonize and proliferate. This was concurrent with differentiation of resident stem/progenitor cells to osteoclasts and bone resorption. In contrast, under bone formation conditions this model revealed different pathophysiology where the breast cancer cells continued to induce osteoclastic bone resorption wh! ereas prostate cancer cells led to osteoblastic bone formation. The current 3D model was used to demonstrate its application to studies involving chemical and biochemical perturbations in the absence and presence of cancer cells and cellular responses. We describe proof-of-principle with examples of the broad versatility and multi-faceted application of this model that adds another dimension to the ongoing studies in the cancer-bone metastasis arena.
• Multifunctional nanoprobes for upconversion fluorescence, MR and CT trimodal imaging
  - Biomaterials 33(4):1079-1089 (2012)
  Early diagnosis probes that combine fluorescence, X-ray computed tomography (CT) and magnetic resonance (MR) imagings are anticipated to give three dimensional (3D) details of tissues and cells of high resolution and sensitivity. However, how to combine these three modalities together within a sub-50 nm sized structure is technically challenging. Here we report a trimodal imaging probe of PEGylated NaY/GdF4: Yb, Er, Tm @SiO2-Au@PEG5000 nanopaticles of uniform size of less than 50 nm. The as-designed nanoprobes showed (1) strong emissions ranging from the visible (Vis) to near infrared (NIR) for fluorescent imaging, (2) T1-weighted MRI by shorting T1 relaxation time and (3) enhanced HU value as a CT contrast agent. The structure was optimized based on a comprehensive investigation on the influence of the distance between the NaY/GdF4: Yb, Er, Tm core and Au nanoparticles (NPs) at the surface. The potential of trimodal imaging for cancerous cells and lesions was further ! demonstrated both in vitro and in vivo.
• Simultaneous electrochemical detection of multiple analytes based on dual signal amplification of single-walled carbon nanotubes and multi-labeled graphene sheets
  - Biomaterials 33(4):1090-1096 (2012)
  In this work, a sandwich-type electrochemical aptasensor for simultaneous sensitive detection of platelet-derived growth factor (PDGF) and thrombin is fabricated. Reduced graphene oxide sheets (rGS) are used as matrices to immobilize the redox probes, which are subsequently coated with platinum nanoparticles (PtNPs) to form the PtNPs-redox probes-rGS nanocomposites. With the employment of the as prepared nanocomposites, a signal amplification strategy was described based on bienzyme (glucose oxidase and horseradish peroxidase) modified PtNPs-redox probes-rGS nanocomposites as the tracer labels for secondary aptamers (Apt II) through sandwiched assay. Gold nanoparticles functionalized single-walled carbon nanotubes (AuNPs@SWCNTs) as the biosensor platform enhance the surface area to capture a large amount of primary aptamers (Apt I), thus amplifying the detection response. The experiment results show that the multi-labeled PtNPs-redox probes-rGS nanocomposites display s! atisfying electrochemical redox activity and highly electrocatalytic activity of PtNPs and bienzyme, which exhibit high sensitivity for detection of proteins. The linear range of PDGF is 0.01–35 nM with a detection limit of 8 pM, while the linear ranges from 0.02 to 45 nM and a detection limit of 11 pM for thrombin are obtained.
• Decorated graphene sheets for label-free DNA impedance biosensing
  - Biomaterials 33(4):1097-1106 (2012)
  An efficient DNA impedance biosensing platform is constructed, in which positively charged N,N-bis-(1-aminopropyl-3-propylimidazol salt)-3,4,9,10-perylene tetracarboxylic acid diimide (PDI) is anchored to graphene sheets. The π–π stacking and electronic interactions are elucidated by the distinct absorption features in UV–vis spectra and by quenching perylene fluorescence in contact with graphene. The rational design and tailoring of graphene surface invest it with desired properties (dispersive, structural, photoelectrical and conductive, etc.) and boost its application. Electrostatic interaction between PDI's positively charged imidazole rings and negatively charged phosphate backbones of single-stranded DNA (ssDNA) facilitates ssDNA immobilization. This manner is different from these mainly based on the attraction between the rings in DNA bases and the hexagonal cells of graphene, which is disturbed after hybridization and causes the leaving of formed double! -stranded DNA from graphene surface. The electrostatic ssDNA grafting occupies phosphate backbones and particularly leaves the bases available for efficient hybridization. DNA immobilization and hybridization lead to PDI/graphene interfacial property changes, which are monitored by electrochemical impedance spectroscopy and adopted as the analytical signal. The conserved sequence of the pol gene of human immunodeficiency virus 1 is satisfactorily detected via this PDI/graphene platform and shows high reproducibility, selectivity.
• PEGylated dendrimer-entrapped gold nanoparticles for in vivo blood pool and tumor imaging by computed tomography
  - Biomaterials 33(4):1107-1119 (2012)
  We report the synthesis and characterization of dendrimer-entrapped gold nanoparticles (Au DENPs) modified by polyethylene glycol (PEG) with enhanced biocompatibility for computed tomography (CT) imaging applications. In this study, amine-terminated poly(amidoamine) dendrimers of generation 5 (G5.NH2) modified by PEG monomethyl ether (G5.NH2-mPEG20) were used as templates to synthesize Au DENPs, followed by acetylation of the remaining dendrimer terminal amines to generate PEGylated Au DENPs. The partial PEGylation modification of dendrimer terminal amines allows high loading of Au within the dendrimer interior, and consequently by simply varying the Au salt/dendrimer molar ratio, the size of the PEGylated Au DENPs can be controlled at a range of 2–4 nm with a narrow size distribution. The formed PEGylated Au DENPs are water-dispersible, stable in a pH range of 5–8 and a temperature range of 0–50 °C, and non-cytotoxic at a concentration as high as 100 μm. X-ray! absorption coefficient measurements show that the attenuation intensity of the PEGylated Au DENPs is much higher than that of Omnipaque with iodine concentration similar to Au. With the sufficiently long half-decay time demonstrated by pharmacokinetics studies, the PEGylated Au DENPs enabled not only X-ray CT blood pool imaging of mice and rats after intravenous injection of the particles, but also effective CT imaging of a xenograft tumor model in nude mice. These findings suggest that the designed PEGylated Au DENPs can be used as a promising contrast agent with enhanced biocompatibility for CT imaging of various biological systems, especially in cancer diagnosis.
• The CD44/integrins interplay and the significance of receptor binding and re-presentation in the uptake of RGD-functionalized hyaluronic acid
  - Biomaterials 33(4):1120-1134 (2012)
  We have studied the interplay between two endocytic receptors for a carrier structure bearing two complementary ligands. Hyaluronic acid (HA; three different molecular weights) was functionalized with an RGD-containing peptide; this ancillary ligand allows the macromolecule to bind to 留v integrins in addition to the classical HA internalization receptor (CD44). The uptake of HA-RGD and of native HA was assessed in a phagocytic cell model (J774.2 murine macrophages), studying the kinetics of internalization and its mechanistic details. Indications of a synergic binding to integrins and CD44 emerged for HA-RGD; possibly, a first binding to integrins allows for a pre-concentration of the macromolecule on the cell surface, which is then followed by its binding to CD44. The endocytic mechanism and kinetics appeared then dominated by CD44, which has a much slower turnover than integrins. In this study we have demonstrated that the knowledge of the rate-determining steps of! the internalization of a carrier is necessary for assessing its performance. In this case, the presence of multiple ligands on a carrier was beneficial in some respect (e.g. in improved binding/targeting), but may not be sufficient to overcome penetration barriers that arise from slow receptor re-presentation.
• Intracellular pathways and nuclear localization signal peptide-mediated gene transfection by cationic polymeric nanovectors
  - Biomaterials 33(4):1135-1145 (2012)
  Polyethylenimine (PEI) - based polymers are promising cationic nanovectors. A good understanding of the mechanism by which cationic polymers/DNA complexes are internalized and delivered to nuclei helps to identify which transport steps may be manipulated in order to improve the transfection efficiency. In this work, cell internalization and trafficking of PEI-CyD (PC) composed of 硫-cyclodextrin (硫-CyD) and polyethylenimine (PEI, Mw 600) are studied. The results show that the PC transfected DNA is internalized by binding membrane-associated proteoglycans. The endocytic pathway of the PC particles is caveolae- and clathrin-dependent with both pathways converging to the lysosome. The intracellular fate of the PC provides visual evidence that it can escape from the lysosome. Lysosomal inhibition with chloroquine has no effect on PC mediated transfection implying that blocking the lysosomal traffic does not improve transfection. To improve the nuclear delivery of PC tra! nsfected DNA, nuclear localization signal (NLS) peptides are chosen to conjugate and combine with the PC. Compared to PC/pDNA, PC-NLS/pDNA, and PC/pDNA/NLS can effectively improve gene transfection in dividing and non-dividing cells.
• Highly dynamic biodegradable micelles capable of lysing Gram-positive and Gram-negative bacterial membrane
  - Biomaterials 33(4):1146-1153 (2012)
  The development of biodegradable antimicrobial polymers adds to the toolbox of attractive antimicrobial agents against antibiotic-resistant microbes. To this end, the potential of polycarbonate polymers as such materials were explored. A series of random polycarbonate polymers consisting of monomers MTC-OEt and MTC-CH2CH3Cl were designed and synthesized using metal-free organocatalytic ring-opening polymerization. Random polycarbonate polymers self-assembled in solution but appeared highly dynamic; such behaviors are desirable as ready disassembly of polymers at the microbial membrane facilitates membrane disruption. Their activities against clinically relevant Gram-positive (Staphylococcus aureus) and Gram-negative bacteria (E.coli and Pseudomonas aeruginosa) revealed that the hydrophobic-hydrophilic composition balance in polymers are important to render antimicrobial potency. Scanning electron microscopy (SEM) studies indicated microbial cell surface damage after tr! eatment with polymers, and confocal microscopy studies also showed entry of FITC-dextran dye in Escherichia coli as a result of membrane disruption. On the other hand, the polymers exhibited minimal toxicity against red blood cells in hemolysis tests. Therefore, these random polycarbonate polymers are promising antimicrobial agents against both Gram-positive and Gram-negative bacteria for various biomedical applications.
• Sustained local delivery of siRNA from an injectable scaffold
  - Biomaterials 33(4):1154-1161 (2012)
  Controlled gene silencing technologies have significant, unrealized potential for use in tissue regeneration applications. The design described herein provides a means to package and protect siRNA within pH-responsive, endosomolytic micellar nanoparticles (si-NPs) that can be incorporated into nontoxic, biodegradable, and injectable polyurethane (PUR) tissue scaffolds. The si-NPs were homogeneously incorporated throughout the porous PUR scaffolds, and they were shown to be released via a diffusion-based mechanism for over three weeks. The siRNA-loaded micelles were larger but retained nanoparticulate morphology of approximately 100 nm diameter following incorporation into and release from the scaffolds. PUR scaffold releasate collected in vitro in PBS at 37 °C for 1–4 days was able to achieve dose-dependent siRNA-mediated silencing with approximately 50% silencing achieved of the model gene GAPDH in NIH3T3 mouse fibroblasts. This promising platform technology provid! es both a research tool capable of probing the effects of local gene silencing and a potentially high-impact therapeutic approach for sustained, local silencing of deleterious genes within tissue defects.
• The therapeutic efficacy of camptothecin-encapsulated supramolecular nanoparticles
  - Biomaterials 33(4):1162-1169 (2012)
  Nanomaterials have been increasingly employed as drug(s)-incorporated vectors for drug delivery due to their potential of maximizing therapeutic efficacy while minimizing systemic side effects. However, there have been two main challenges for these vectors: (i) the existing synthetic approaches are cumbersome and incapable of achieving precise control of their structural properties, which will affect their biodistribution and therapeutic efficacies, and (ii) lack of an early checkpoint to quickly predict which drug(s)-incorporated vectors exhibit optimal therapeutic outcomes. In this work, we utilized a new rational developmental approach to rapidly screen nanoparticle (NP)-based cancer therapeutic agents containing a built-in companion diagnostic utility for optimal therapeutic efficacy. The approach leverages the advantages of a self-assembly synthetic method for preparation of two different sizes of drug-incorporated supramolecular nanoparticles (SNPs), and a positr! on emission tomography (PET) imaging-based biodistribution study to quickly evaluate the accumulation of SNPs at a tumor site in vivo and select the favorable SNPs for in vivo therapeutic study. Finally, the enhanced in vivo anti-tumor efficacy of the selected SNPs was validated by tumor reduction/inhibition studies. We foresee our rational developmental approach providing a general strategy in the search of optimal therapeutic agents among the diversity of NP-based therapeutic agents.
• Multifunctional nanocarrier mediated co-delivery of doxorubicin and siRNA for synergistic enhancement of glioma apoptosis in rat
  - Biomaterials 33(4):1170-1179 (2012)
  As the most fatal malignancy in brain, glioma cannot be effectively treated with the conventional chemotherapy and thus techniques which may improve the chemotherapeutic effect are of great importance in clinical glioma treatment. Based on the folate-targeted multifunctional nanocarrier developed in our lab, effective co-delivery of DOX and siRNA into rat C6 glioma cells over-expressing folate receptors was achieved. Although cell apoptosis was initiated even at low DOX doses such as 0.5 μg/mL in the DOX-alone treatment mediated by the folate-targeted nanocarrier, anti-apoptotic response in C6 cells was activated as well, as revealed by molecular biological investigations. Delivery of BCL-2 siRNA using the folate-targeted nanocarrier can effectively suppress the anti-apoptotic response and sensitized C6 cells to DOX treatment both in vitro and in vivo. In particular, animal studies using the in situ rat C6 glioma model showed that the folate-targeted co-delivery of ! BCL-2 siRNA and DOX caused not only an obvious down-regulation of the anti-apoptotic BCL-2 gene but also a remarkable up-regulation of the pro-apoptotic Bax gene, resulting in the significantly elevated level of caspase-3 activation and remarkable cell apoptosis in tumor tissues. Our results strongly demonstrated the synergistic effect of siRNA and DOX in inducing glioma C6 cell apoptosis, upon which an excellent therapeutic effect was achieved using the folate-targeted co-delivery strategy as indicated by the effective tumor growth inhibition and prolonged rat survival time in the animal test.
• Gold nanoparticles functionalized with therapeutic and targeted peptides for cancer treatment
  - Biomaterials 33(4):1180-1189 (2012)
  Functionalization of nanostructures such as gold nanoparticles (AuNPs) with different biological molecules has many applications in biomedical imaging, clinical diagnosis and therapy. Researchers mostly employed AuNPs larger than 10 nm for different biological and medicinal applications in previous studies. Herein, we synthesized a novel small (2 nm) AuNPs, which were functionalized with the therapeutic peptide, PMI (p12), and a targeted peptide, CRGDK for selective binding to neuropilin-1(Nrp-1) receptors which overexpressed on the cancer cells and regulated the process of membrane receptor-mediated internalization. It was found that CRGDK peptides increased intracellular uptake of AuNPs compared to other surface conjugations quantified by ICP-MS. Interestingly, CRGDK functionalized AuNPs resulted in maximal binding interaction between the CRGDK peptide and targeted Nrp-1 receptor overexpressed on MDA-MB-321 cell surface, which improved the delivery of therapeutic P12! peptide inside targeted cells. Au@p12 + CRGDK nanoparticles indicated with highly effective cancer treatment by increasing p53 expression upregulated with intracellular enhanced p12 therapeutic peptide. These results have implications to design and functionalize different molecules onto AuNPs surfaces to make hybrid model system for selective target binding as well as therapeutic effects for cancer treatment.
• Polyethylene glycol-conjugated hyaluronic acid-ceramide self-assembled nanoparticles for targeted delivery of doxorubicin
  - Biomaterials 33(4):1190-1200 (2012)
  Polyethylene glycol (PEG)-conjugated hyaluronic acid-ceramide (HACE) was synthesized for the preparation of doxorubicin (DOX)-loaded HACE-PEG-based nanoparticles, 160 nm in mean diameter with a negative surface charge. Greater uptake of DOX from these HACE-PEG-based nanoparticles was observed in the CD44 receptor highly expressed SCC7 cell line, compared to results from the CD44-negative cell line, NIH3T3. A strong fluorescent signal was detected in the tumor region upon intravenous injection of cyanine 5.5-labeled nanoparticles into the SCC7 tumor xenograft mice; the extended circulation time of the HACE-PEG-based nanoparticle was also observed. Pharmacokinetic study in rats showed a 73.0% reduction of the in vivo clearance of DOX compared to the control group. The antitumor efficacy of the DOX-loaded HACE-PEG-based nanoparticles was also verified in a tumor xenograft mouse model. DOX was efficiently delivered to the tumor site by active targeting via HA and CD44 rece! ptor interaction and by passive targeting due to its small mean diameter (<200 nm). Moreover, PEGylation resulted in prolonged nanoparticle circulation and reduced DOX clearance rate in an in vivo model. These results therefore indicate that PEGylated HACE nanoparticles represent a promising anticancer drug delivery system for cancer diagnosis and therapy.

Hot off the presses! Nov 18 Mol Cell

The Nov 18 issue of the Mol Cell is now up on Pubget (About Mol Cell): if you're at a subscribing institution, just click the link in the latest link at the home page. (Note you'll only be able to get all the PDFs in the issue if your institution subscribes to Pubget.)

Latest Articles Include:

• Staying Alive: Defensive Strategies in the BCL-2 Family Playbook
  - Mol Cell 44(4):509-510 (2011)
  Much debate surrounds how prosurvival members of the BCL-2 family repress opening of the BAX/BAK channel to block apoptosis; in this issue Llambi et al. (2011) identify two modes of apoptosis inhibition that exhibit surprisingly different behavior upon repeat proapoptotic challenges by BH3-only proteins.
• A20: More Than One Way to Skin a Cat
  - Mol Cell 44(4):511-512 (2011)
  In this issue of Molecular Cell, Skaug et al. (2011) propose a polyubiquitin-dependent, noncatalytic mechanism by which the deubiquitinase A20 inhibits IκB kinase and NF-κB activation.
• Mitosis Hit with an ATM Transaction Fee: Aurora B-Mediated Activation of ATM during Mitosis
  - Mol Cell 44(4):513-514 (2011)
  In this issue of Molecular Cell, Yang et al. (2011) demonstrate that Aurora B phosphorylates ATM, leading to its mitotic activation and ability to phosphorylate Bub1 and regulate the spindle checkpoint, thus maintaining genomic integrity.
• PGC1α Confers Specificityâ€"Metabolic Stress and p53-Dependent Transcription
  - Mol Cell 44(4):515-516 (2011)
  In this issue of Molecular Cell, Sen et al. (2011) report the involvement of PGC1α in modulating the transcriptional activity of p53 in metabolically challenged cells. They provide important insights into the mechanisms linking length and strength of the metabolic stress stimuli to the specific activation of p53 target genes.
• A Unified Model of Mammalian BCL-2 Protein Family Interactions at the Mitochondria
  - Mol Cell 44(4):517-531 (2011)
  During apoptosis, the BCL-2 protein family controls mitochondrial outer membrane permeabilization (MOMP), but the dynamics of this regulation remain controversial. We employed chimeric proteins composed of exogenous BH3 domains inserted into a tBID backbone that can activate the proapoptotic effectors BAX and BAK to permeabilize membranes without being universally sequestered by all antiapoptotic BCL-2 proteins. We thus identified two "modes" whereby prosurvival BCL-2 proteins can block MOMP, by sequestering direct-activator BH3-only proteins ("MODE 1") or by binding active BAX and BAK ("MODE 2"). Notably, we found that MODE 1 sequestration is less efficient and more easily derepressed to promote MOMP than MODE 2. Further, MODE 2 sequestration prevents mitochondrial fusion. We provide a unified model of BCL-2 family function that helps to explain otherwise paradoxical observations relating to MOMP, apoptosis, and mitochondrial dynamics.
• Fine-Tuning of Drp1/Fis1 Availability by AKAP121/Siah2 Regulates Mitochondrial Adaptation to Hypoxia
  - Mol Cell 44(4):532-544 (2011)
  Defining the mechanisms underlying the control of mitochondrial fusion and fission is critical to understanding cellular adaptation to diverse physiological conditions. Here we demonstrate that hypoxia induces fission of mitochondrial membranes, dependent on availability of the mitochondrial scaffolding protein AKAP121. AKAP121 controls mitochondria dynamics through PKA-dependent inhibitory phosphorylation of Drp1 and PKA-independent inhibition of Drp1-Fis1 interaction. Reduced availability of AKAP121 by the ubiquitin ligase Siah2 relieves Drp1 inhibition by PKA and increases its interaction with Fis1, resulting in mitochondrial fission. High AKAP121 levels, seen in cells lacking Siah2, attenuate fission and reduce apoptosis of cardiomyocytes under simulated ischemia. Infarct size and degree of cell death were reduced in Siah2−/− mice subjected to myocardial infarction. Inhibition of Siah2 or Drp1 in hatching C. elegans reduces their life span. Through modulating F! is1/Drp1 complex availability, our studies identify Siah2 as a key regulator of hypoxia-induced mitochondrial fission and its physiological significance in ischemic injury and nematode life span.
• Peroxiredoxin II Is an Essential Antioxidant Enzyme that Prevents the Oxidative Inactivation of VEGF Receptor-2 in Vascular Endothelial Cells
  - Mol Cell 44(4):545-558 (2011)
  Cellular antioxidant enzymes play crucial roles in aerobic organisms by eliminating detrimental oxidants and maintaining the intracellular redox homeostasis. Therefore, the function of antioxidant enzymes is inextricably linked to the redox-dependent activities of multiple proteins and signaling pathways. Here, we report that the VEGFR2 RTK has an oxidation-sensitive cysteine residue whose reduced state is preserved specifically by peroxiredoxin II (PrxII) in vascular endothelial cells. In the absence of PrxII, the cellular H2O2 level is markedly increased and the VEGFR2 becomes inactive, no longer responding to VEGF stimulation. Such VEGFR2 inactivation is due to the formation of intramolecular disulfide linkage between Cys1199 and Cys1206 in the C-terminal tail. Interestingly, the PrxII-mediated VEGFR2 protection is achieved by association of two proteins in the caveolae. Furthermore, PrxII deficiency suppresses tumor angiogenesis in vivo. This study thus demonstrate! s a physiological function of PrxII as the residential antioxidant safeguard specific to the redox-sensitive VEGFR2.
• Direct, Noncatalytic Mechanism of IKK Inhibition by A20
  - Mol Cell 44(4):559-571 (2011)
  A20 is a potent anti-inflammatory protein that inhibits NF-κB, and A20 dysfunction is associated with autoimmunity and B cell lymphoma. A20 harbors a deubiquitination enzyme domain and can employ multiple mechanisms to antagonize ubiquitination upstream of NEMO, a regulatory subunit of the IκB kinase complex (IKK). However, direct evidence of IKK inhibition by A20 is lacking, and the inhibitory mechanism remains poorly understood. Here we show that A20 can directly impair IKK activation without deubiquitination or impairment of ubiquitination enzymes. We find that polyubiquitin binding by A20, which is largely dependent on A20's seventh zinc-finger motif (ZnF7), induces specific binding to NEMO. Remarkably, this ubiquitin-induced recruitment of A20 to NEMO is sufficient to block IKK phosphorylation by its upstream kinase TAK1. Our results suggest a noncatalytic mechanism of IKK inhibition by A20 and a means by which polyubiquitin chains can specify a signaling ou! tcome.
• Heterotypic piRNA Ping-Pong Requires Qin, a Protein with Both E3 Ligase and Tudor Domains
  - Mol Cell 44(4):572-584 (2011)
  piRNAs guide PIWI proteins to silence transposons in animal germ cells. Reciprocal cycles of piRNA-directed RNA cleavage—catalyzed by the PIWI proteins Aubergine (Aub) and Argonaute3 (Ago3) in Drosophila melanogaster—expand the population of antisense piRNAs in response to transposon expression, a process called the Ping-Pong cycle. Heterotypic Ping-Pong between Aub and Ago3 ensures that antisense piRNAs predominate. We show that qin, a piRNA pathway gene whose protein product contains both E3 ligase and Tudor domains, colocalizes with Aub and Ago3 in nuage, a perinuclear structure implicated in transposon silencing. In qin mutants, less Ago3 binds Aub, futile Aub:Aub homotypic Ping-Pong prevails, antisense piRNAs decrease, many families of mobile genetic elements are reactivated, and DNA damage accumulates in nurse cells and oocytes. We propose that Qin enforces heterotypic Ping-Pong between Aub and Ago3, ensuring that transposons are silenced and maintaining the ! integrity of the germline genome.
• RAM/Fam103a1 Is Required for mRNA Cap Methylation
  - Mol Cell 44(4):585-596 (2011)
  The 7-methylguanosine cap added to the 5′ end of mRNA is required for efficient gene expression in eukaryotes. In mammals, methylation of the guanosine cap is catalyzed by RNMT (RNA guanine-7 methyltransferase), an enzyme previously thought to function as a monomer. We have identified an obligate component of the mammalian cap methyltransferase, RAM (RNMT-Activating Mini protein)/Fam103a1, a previously uncharacterized protein. RAM consists of an N-terminal RNMT-activating domain and a C-terminal RNA-binding domain. As monomers RNMT and RAM have a relatively weak affinity for RNA; however, together their RNA affinity is significantly increased. RAM is required for efficient cap methylation in vitro and in vivo, and is indirectly required to maintain mRNA expression levels, for mRNA translation and for cell viability. Our findings demonstrate that RAM is an essential component of the core gene expression machinery.
• Aurora-B Mediated ATM Serine 1403 Phosphorylation Is Required for Mitotic ATM Activation and the Spindle Checkpoint
  - Mol Cell 44(4):597-608 (2011)
  The ATM kinase plays a critical role in the maintenance of genetic stability. ATM is activated in response to DNA damage and is essential for cell-cycle checkpoints. Here, we report that ATM is activated in mitosis in the absence of DNA damage. We demonstrate that mitotic ATM activation is dependent on the Aurora-B kinase and that Aurora-B phosphorylates ATM on serine 1403. This phosphorylation event is required for mitotic ATM activation. Further, we show that loss of ATM function results in shortened mitotic timing and a defective spindle checkpoint, and that abrogation of ATM Ser1403 phosphorylation leads to this spindle checkpoint defect. We also demonstrate that mitotically activated ATM phosphorylates Bub1, a critical kinetochore protein, on Ser314. ATM-mediated Bub1 Ser314 phosphorylation is required for Bub1 activity and is essential for the activation of the spindle checkpoint. Collectively, our data highlight mechanisms of a critical function of ATM in mitosi! s.
• NSD2 Links Dimethylation of Histone H3 at Lysine 36 to Oncogenic Programming
  - Mol Cell 44(4):609-620 (2011)
  The histone lysine methyltransferase NSD2 (MMSET/WHSC1) is implicated in diverse diseases and commonly overexpressed in multiple myeloma due to a recurrent t(4;14) chromosomal translocation. However, the precise catalytic activity of NSD2 is obscure, preventing progress in understanding how this enzyme influences chromatin biology and myeloma pathogenesis. Here, we show that dimethylation of histone H3 at lysine 36 (H3K36me2) is the principal chromatin-regulatory activity of NSD2. Catalysis of H3K36me2 by NSD2 is sufficient for gene activation. In t(4;14)-positive myeloma cells, the normal genome-wide and gene-specific distribution of H3K36me2 is obliterated, creating a chromatin landscape that selects for a transcription profile favorable for myelomagenesis. Catalytically active NSD2 confers xenograft tumor formation upon t(4;14)-negative cells and promotes oncogenic transformation of primary cells in an H3K36me2-dependent manner. Together, our findings establish H3K3! 6me2 as the primary product generated by NSD2 and demonstrate that genomic disorganization of this canonical chromatin mark by NSD2 initiates oncogenic programming.
• PGC-1α, a Key Modulator of p53, Promotes Cell Survival upon Metabolic Stress
  - Mol Cell 44(4):621-634 (2011)
  Metabolic stress results in p53 activation, which can trigger cell-cycle arrest, ROS clearance, or apoptosis. However, what determines the p53-mediated cell fate decision upon metabolic stress is not very well understood. We show here that PGC-1α binds to p53 and modulates its transactivation function, resulting in preferential transactivation of proarrest and metabolic target genes. Thus glucose starvation results in p53-dependent cell-cycle arrest and ROS clearance, but abrogation of PGC-1α expression results in extensive apoptosis. Additionally, prolonged starvation results in PGC-1α degradation concomitant with induction of apoptosis. We have also identified RNF2, a Polycomb group (PcG) protein, as the cognate E3 ubiquitin ligase. Starvation of mice where PGC-1α expression is abrogated results in loss of p53-mediated ROS clearance, enhanced p53-dependent apoptosis, and consequent severe liver atrophy. These findings provide key insights into the role of! PGC-1α in regulating p53-mediated cell fate decisions in response to metabolic stress.
• Applied Force Provides Insight into Transcriptional Pausing and Its Modulation by Transcription Factor NusA
  - Mol Cell 44(4):635-646 (2011)
  Transcriptional pausing by RNA polymerase (RNAP) plays an essential role in gene regulation. Pausing is modified by various elongation factors, including prokaryotic NusA, but the mechanisms underlying pausing and NusA function remain unclear. Alternative models for pausing invoke blockade events that precede translocation (on-pathway), enzyme backtracking (off-pathway), or isomerization to a nonbacktracked, elemental pause state (off-pathway). We employed an optical trapping assay to probe the motions of individual RNAP molecules transcribing a DNA template carrying tandem repeats encoding the his pause, subjecting these enzymes to controlled forces. NusA significantly decreased the pause-free elongation rate of RNAP while increasing the probability of entry into short- and long-lifetime pauses, in a manner equivalent to exerting a ∼19 pN force opposing transcription. The effects of force and NusA on pause probabilities and lifetimes support a reaction scheme where ! nonbacktracked, elemental pauses branch off the elongation pathway from the pretranslocated state of RNAP.
• Telomere Protection by TPP1/POT1 Requires Tethering to TIN2
  - Mol Cell 44(4):647-659 (2011)
  To prevent ATR activation, telomeres deploy the single-stranded DNA binding activity of TPP1/POT1a. POT1a blocks the binding of RPA to telomeres, suggesting that ATR is repressed through RPA exclusion. However, comparison of the DNA binding affinities and abundance of TPP1/POT1a and RPA indicates that TPP1/POT1a by itself is unlikely to exclude RPA. We therefore analyzed the central shelterin protein TIN2, which links TPP1/POT1a (and POT1b) to TRF1 and TRF2 on the double-stranded telomeric DNA. Upon TIN2 deletion, telomeres lost TPP1/POT1a, accumulated RPA, elicited an ATR signal, and showed all other phenotypes of POT1a/b deletion. TIN2 also affected the TRF2-dependent repression of ATM kinase signaling but not to TRF2-mediated inhibition of telomere fusions. Thus, while TIN2 has a minor contribution to the repression of ATM by TRF2, its major role is to stabilize TPP1/POT1a on the ss telomeric DNA, thereby allowing effective exclusion of RPA and repression of ATR sig! naling.
• rRNA Pseudouridylation Defects Affect Ribosomal Ligand Binding and Translational Fidelity from Yeast to Human Cells
  - Mol Cell 44(4):660-666 (2011)
  How pseudouridylation (Ψ), the most common and evolutionarily conserved modification of rRNA, regulates ribosome activity is poorly understood. Medically, Ψ is important because the rRNA Ψ synthase, DKC1, is mutated in X-linked dyskeratosis congenita (X-DC) and Hoyeraal-Hreidarsson (HH) syndrome. Here, we characterize ribosomes isolated from a yeast strain in which Cbf5p, the yeast homolog of DKC1, is catalytically impaired through a D95A mutation (cbf5-D95A). Ribosomes from cbf5-D95A cells display decreased affinities for tRNA binding to the A and P sites as well as the cricket paralysis virus internal ribosome entry site (IRES), which interacts with both the P and the E sites of the ribosome. This biochemical impairment in ribosome activity manifests as decreased translational fidelity and IRES-dependent translational initiation, which are also evident in mouse and human cells deficient for DKC1 activity. These findings uncover specific roles for Ψ modification i! n ribosome-ligand interactions that are conserved in yeast, mouse, and humans.
• Genomic Maps of Long Noncoding RNA Occupancy Reveal Principles of RNA-Chromatin Interactions
  - Mol Cell 44(4):667-678 (2011)
  Long noncoding RNAs (lncRNAs) are key regulators of chromatin state, yet the nature and sites of RNA-chromatin interaction are mostly unknown. Here we introduce Chromatin Isolation by RNA Purification (ChIRP), where tiling oligonucleotides retrieve specific lncRNAs with bound protein and DNA sequences, which are enumerated by deep sequencing. ChIRP-seq of three lncRNAs reveal that RNA occupancy sites in the genome are focal, sequence-specific, and numerous. Drosophila roX2 RNA occupies male X-linked gene bodies with increasing tendency toward the 3′ end, peaking at CES sites. Human telomerase RNA TERC occupies telomeres and Wnt pathway genes. HOTAIR lncRNA preferentially occupies a GA-rich DNA motif to nucleate broad domains of Polycomb occupancy and histone H3 lysine 27 trimethylation. HOTAIR occupancy occurs independently of EZH2, suggesting the order of RNA guidance of Polycomb occupancy. ChIRP-seq is generally applicable to illuminate the intersection of RNA and ! chromatin with newfound precision genome wide.