Derailed protein turnover in the aging mammalian brain.

Efficient protein turnover is essential for cellular homeostasis and organ function. Loss of proteostasis is a hallmark of aging culminating in severe dysfunction of protein turnover. To investigate protein turnover dynamics as a function of age, we performed continuous in vivo metabolic stable isotope labeling in mice along the aging continuum. First, we discovered that the brain proteome uniquely undergoes dynamic turnover fluctuations during aging compared to heart and liver tissue. Second, trends in protein turnover in the brain proteome during aging showed sex-specific differences that were tightly tied to cellular compartments. Next, parallel analyses of the insoluble proteome revealed that several cellular compartments experience hampered turnover, in part due to misfolding. Finally, we found that age-associated fluctuations in proteasome activity were associated with the turnover of core proteolytic subunits, which was recapitulated by pharmacological suppression of proteasome activity. Taken together, our study provides a proteome-wide atlas of protein turnover across the aging continuum and reveals a link between the turnover of individual proteasome subunits and the age-associated decline in proteasome activity.

Amyloid fibril proteomics of AD brains reveals modifiers of aggregation and toxicity.

BACKGROUND: The accumulation of amyloid beta (Aß) peptides in fibrils is prerequisite for Alzheimer's disease (AD). Our understanding of the proteins that promote Aß fibril formation and mediate neurotoxicity has been limited due to technical challenges in isolating pure amyloid fibrils from brain extracts. METHODS: To investigate how amyloid fibrils form and cause neurotoxicity in AD brain, we developed a robust biochemical strategy. We benchmarked the success of our purifications using electron microscopy, amyloid dyes, and a large panel of Aß immunoassays. Tandem mass-spectrometry based proteomic analysis workflows provided quantitative measures of the amyloid fibril proteome. These methods allowed us to compare amyloid fibril composition from human AD brains, three amyloid mouse models, transgenic Aß42 flies, and Aß42 seeded cultured neurons. RESULTS: Amyloid fibrils are primarily composed by Aß42 and unexpectedly harbor Aß38 but generally lack Aß40 peptides. Multidimensional quantitative proteomics allowed us to redefine the fibril proteome by identifying 20 new amyloid-associated proteins. Notably, we confirmed 57 previously reported plaque-associated proteins. We validated a panel of these proteins as bona fide amyloid-interacting proteins using antibodies and orthogonal proteomic analysis. One metal-binding chaperone metallothionein-3 is tightly associated with amyloid fibrils and modulates fibril formation in vitro. Lastly, we used a transgenic Aß42 fly model to test if knock down or over-expression of fibril-interacting gene homologues modifies neurotoxicity. Here, we could functionally validate 20 genes as modifiers of Aß42 toxicity in vivo. CONCLUSIONS: These discoveries and subsequent confirmation indicate that fibril-associated proteins play a key role in amyloid formation and AD pathology.

Hydrological models for climate-based assessments at the watershed scale: A critical review of existing hydrologic and water quality models.

Global changes, including climate and land use changes, can result in significant impact to water resources. Planning for these changes requires making projections, even in the face of considerable uncertainties, to make informed management and policy decisions. A number of climate change scenarios and projections at global and regional levels are available that can be used to predict the likely range of outcomes. However, there is a need to translate these projections into potential implications for hydrology and water quality. Since there are dozens of hydrologic models, there is a need to evaluate them critically and to develop guidance regarding selecting the appropriate model for a given objective. We conducted a review of 21 different models commonly used for modeling hydrology (8), water quality (6) or both (7) at the watershed scale. Six of the models are strictly water quality models that depend on a separate model or observed data for hydrology. Seven additional models are useful for estimating hydrology and water quality simultaneously. The models were then evaluated based on ten different criteria, including functionality, scope, ability to model extreme events, data requirements, availability, and technical support, among others. The models were ranked Low, Medium or High in each of the criteria. The results indicate that three hydrologic models, MIKE-SHE, HEC-HMS, and MODHMS, as well as two full hydrology and water quality models, SWAT and WARMF, stand out in terms of functionality, availability, applicability to a wide range of watersheds and scales, ease of implementation, and availability of support. Modelers should carefully select the best model for their application, in part guided by the criteria discussed herein.

Electron Tomographic Methods for Studying Organelles of the Murine Chemical Synapse.

Electron tomography of the chemical synapse provides important architectural information regarding the organization of synaptic organelles including synaptic vesicles, Nissl bodies, and early endosomes. Here, we describe methods for the preparation of select murine brain regions for high-pressure freezing, freeze substitution, and EM tomographic analysis of synaptic structures. The method uses fresh brain slices prepared using a vibratome and biopsy punches to collect specific brain regions of interest suitable for subsequent preservation and EM tomographic imaging.

Levetiracetam Treatment Normalizes Levels of Presynaptic Endocytosis Machinery and Restores Nonamyloidogenic APP Processing in App Knock-in Mice.

Toxic amyloid-beta (Aß) peptides, produced by sequential proteolytic cleavage of the amyloid precursor protein (APP), play a key role in the initial stage of Alzheimer's disease (AD). Increasing evidence indicates that Aß42 induces neuronal circuit hyperexcitability in the early stages of AD pathology. As a result, researchers have investigated treatments that modulate the excitatory/inhibitory imbalance as potential AD therapies. For example, levetiracetam, an atypical antiepileptic drug used to quell hyperexcitability, has garnered recent interest in the AD field, even though its exact mechanism(s) of action remains elusive. Here, we show that in APP knock-in mouse models of amyloid pathology, chronic levetiracetam administration decreases cortical Aß42 levels and lowers the amyloid plaque burden. In addition, using multiplexed tandem mass tag-quantitative mass spectrometry-based proteomic analysis, we determined that chronic levetiracetam administration selectively normalizes levels of presynaptic endocytic proteins. Finally, we found that levetiracetam treatment selectively lowers beta carboxyl-terminal fragment levels, while the abundance of full-length APP remains unchanged. In summary, this work reports that chronic treatment with levetiracetam serves as a useful therapeutic in AD by normalizing levels of presynaptic endocytic proteins and altering APP cleavage preference, leading to a decrease in both Aß42 levels and the amyloid plaque burden. These novel findings provide novel evidence for the previously documented therapeutic value of levetiracetam to mitigate AD pathology.

Landscape of clinically actionable mutations in breast cancer 'A cohort study'.

Breast cancer (BC) is a heterogeneous disease. Numerous chemotherapeutic agents are available for early stage or advanced/metastatic breast cancer to provide maximum benefit with minimum side effects. However, the clinical outcome of patients with the same clinical and pathological characteristics and treated with similar treatments may show major differences and a vast majority of patients still develop treatment resistance and eventually succumb to disease. It remains an unmet need to identify specific molecular defects, new biomarkers to enable clinicians to adopt individualized treatment for every patient in terms of endocrine, chemotherapy or targeted therapy which will improve clinical outcomes in BC. Our study aimed to identify frequent hotspot mutation profile in BC by targeted deep sequencing in cancer-related genes using Illumina Truseq amplicon/Swift Accel-Amplicon panel and MiSeq technology in an IRB-approved prospective study in a CLIA compliant laboratory. All the cases had pathology review for stage, histological type, hormonal status and Ki-67. Data was processed using Strand NGS™. Mutations identified in the tumor were assessed for 'actionability' i.e. response to therapy and impact on prognosis.

Pulse-Chase Proteomics of the App Knockin Mouse Models of Alzheimer's Disease Reveals that Synaptic Dysfunction Originates in Presynaptic Terminals.

Compromised protein homeostasis underlies accumulation of plaques and tangles in Alzheimer's disease (AD). To observe protein turnover at early stages of amyloid beta (Aß) proteotoxicity, we performed pulse-chase proteomics on mouse brains in three genetic models of AD that knock in alleles of amyloid precursor protein (APP) prior to the accumulation of plaques and during disease progression. At initial stages of Aß accumulation, the turnover of proteins associated with presynaptic terminals is selectively impaired. Presynaptic proteins with impaired turnover, particularly synaptic vesicle (SV)-associated proteins, have elevated levels, misfold in both a plaque-dependent and -independent manner, and interact with APP and Aß. Concurrent with elevated levels of SV-associated proteins, we found an enlargement of the SV pool as well as enhancement of presynaptic potentiation. Together, our findings reveal that the presynaptic terminal is particularly vulnerable and represents a critical site for manifestation of initial AD etiology. A record of this paper's transparent peer review process is included in the Supplemental Information.

A Rare De Novo RAI1 Gene Mutation Affecting BDNF-Enhancer-Driven Transcription Activity Associated with Autism and Atypical Smith-Magenis Syndrome Presentation.

Deletions and mutations involving the Retinoic Acid Induced 1 (RAI1) gene at 17p11.2 cause Smith-Magenis syndrome (SMS). Here we report a patient with autism as the main clinical presentation, with some SMS-like features and a rare de novo RAI1 gene mutation, c.3440G > A (p.R1147Q). We functionally characterized the RAI1 p.R1147Q mutant protein. The mutation, located near the nuclear localization signal, had no effect on the subcellular localization of the mutant protein. However, similar to previously reported RAI1 missense mutations in SMS patients, the RAI1 p.R1147Q mutant protein showed a significant deficiency in activating in vivo transcription of a reporter gene driven by a BDNF (brain-derived neurotrophic factor) intronic enhancer. In addition, expression of other genes associated with neurobehavioral abnormalities and/or neurodevelopmental disorders were found to be altered in this patient. These results suggest a likely contribution of RAI1, either alone or in combination of other factors, to social behavior and reinforce the RAI1 gene as a candidate gene in patients with autistic manifestations or social behavioral abnormalities.

Effects of a Yoga Program on Mood States, Quality of Life, and Toxicity in Breast Cancer Patients Receiving Conventional Treatment: A Randomized Controlled Trial.

AIMS: The aim of this study is to compare the effects of yoga program with supportive therapy counseling on mood states, treatment-related symptoms, toxicity, and quality of life in Stage II and III breast cancer patients on conventional treatment. METHODS: Ninety-eight Stage II and III breast cancer patients underwent surgery followed by adjuvant radiotherapy (RT) or chemotherapy (CT) or both at a cancer center were randomly assigned to receive yoga (n = 45) and supportive therapy counseling (n = 53) over a 24-week period. Intervention consisted of 60-min yoga sessions, daily while the control group was imparted supportive therapy during their hospital visits. Assessments included state-trait anxiety inventory, Beck's depression inventory, symptom checklist, common toxicity criteria, and functional living index-cancer. Assessments were done at baseline, after surgery, before, during, and after RT and six cycles of CT. RESULTS: Both groups had similar baseline scores. There were 29 dropouts 12 (yoga) and 17 (controls) following surgery. Sixty-nine participants contributed data to the current analysis (33 in yoga, and 36 in controls). An ANCOVA, adjusting for baseline differences, showed a significant decrease for the yoga intervention as compared to the control group during RT (first result) and CT (second result), in (i) anxiety state by 4.72 and 7.7 points, (ii) depression by 5.74 and 7.25 points, (iii) treatment-related symptoms by 2.34 and 2.97 points, (iv) severity of symptoms by 6.43 and 8.83 points, (v) distress by 7.19 and 13.11 points, and (vi) and improved overall quality of life by 23.9 and 31.2 points as compared to controls. Toxicity was significantly less in the yoga group (P = 0.01) during CT. CONCLUSION: The results suggest a possible use for yoga as a psychotherapeutic intervention in breast cancer patients undergoing conventional treatment.

Rai1 Haploinsufficiency Is Associated with Social Abnormalities in Mice.

Background: Autism is characterized by difficulties in social interaction, communication, and repetitive behaviors; with different degrees of severity in each of the core areas. Haploinsufficiency and point mutations of RAI1 are associated with Smith-Magenis syndrome (SMS), a genetic condition that scores within the autism spectrum range for social responsiveness and communication, and is characterized by neurobehavioral abnormalities, intellectual disability, developmental delay, sleep disturbance, and self-injurious behaviors. Methods: To investigate the relationship between Rai1 and social impairment, we evaluated the Rai1+/- mice with a battery of tests to address social behavior in mice. Results: We found that the mutant mice showed diminished interest in social odors, abnormal submissive tendencies, and increased repetitive behaviors when compared to wild type littermates. Conclusions: These findings suggest that Rai1 contributes to social behavior in mice, and prompt it as a candidate gene for the social behaviors observed in Smith-Magenis Syndrome patients.

Effects of an integrated Yoga Program on Self-reported Depression Scores in Breast Cancer Patients Undergoing Conventional Treatment: A Randomized Controlled Trial.

AIM: To compare the effects of yoga program with supportive therapy on self-reported symptoms of depression in breast cancer patients undergoing conventional treatment. PATIENTS AND METHODS: Ninety-eight breast cancer patients with stage II and III disease from a cancer center were randomly assigned to receive yoga (n = 45) and supportive therapy (n = 53) over a 24-week period during which they underwent surgery followed by adjuvant radiotherapy (RT) or chemotherapy (CT) or both. The study stoppage criteria was progressive disease rendering the patient bedridden or any physical musculoskeletal injury resulting from intervention or less than 60% attendance to yoga intervention. Subjects underwent yoga intervention for 60 min daily with control group undergoing supportive therapy during their hospital visits. Beck's Depression Inventory (BDI) and symptom checklist were assessed at baseline, after surgery, before, during, and after RT and six cycles of CT. We used analysis of covariance (intent-to-treat) to study the effects of intervention on depression scores and Pearson correlation analyses to evaluate the bivariate relationships. RESULTS: A total of 69 participants contributed data to the current analysis (yoga, n = 33, and controls, n = 36). There was 29% attrition in this study. The results suggest an overall decrease in self-reported depression with time in both the groups. There was a significant decrease in depression scores in the yoga group as compared to controls following surgery, RT, and CT (P < 0.01). There was a positive correlation (P < 0.001) between depression scores with symptom severity and distress during surgery, RT, and CT. CONCLUSION: The results suggest possible antidepressant effects with yoga intervention in breast cancer patients undergoing conventional treatment.

Cilengitide combined with standard treatment for patients with newly diagnosed glioblastoma with methylated MGMT promoter (CENTRIC EORTC 26071-22072 study): a multicentre, randomised, open-label, phase 3 trial.

BACKGROUND: Cilengitide is a selective αvß3 and αvß5 integrin inhibitor. Data from phase 2 trials suggest that it has antitumour activity as a single agent in recurrent glioblastoma and in combination with standard temozolomide chemoradiotherapy in newly diagnosed glioblastoma (particularly in tumours with methylated MGMT promoter). We aimed to assess cilengitide combined with temozolomide chemoradiotherapy in patients with newly diagnosed glioblastoma with methylated MGMT promoter. METHODS: In this multicentre, open-label, phase 3 study, we investigated the efficacy of cilengitide in patients from 146 study sites in 25 countries. Eligible patients (newly diagnosed, histologically proven supratentorial glioblastoma, methylated MGMT promoter, and age ≥18 years) were stratified for prognostic Radiation Therapy Oncology Group recursive partitioning analysis class and geographic region and centrally randomised in a 1:1 ratio with interactive voice response system to receive temozolomide chemoradiotherapy with cilengitide 2000 mg intravenously twice weekly (cilengitide group) or temozolomide chemoradiotherapy alone (control group). Patients and investigators were unmasked to treatment allocation. Maintenance temozolomide was given for up to six cycles, and cilengitide was given for up to 18 months or until disease progression or unacceptable toxic effects. The primary endpoint was overall survival. We analysed survival outcomes by intention to treat. This study is registered with ClinicalTrials.gov, number NCT00689221. FINDINGS: Overall, 3471 patients were screened. Of these patients, 3060 had tumour MGMT status tested; 926 patients had a methylated MGMT promoter, and 545 were randomly assigned to the cilengitide (n=272) or control groups (n=273) between Oct 31, 2008, and May 12, 2011. Median overall survival was 26·3 months (95% CI 23·8-28·8) in the cilengitide group and 26·3 months (23·9-34·7) in the control group (hazard ratio 1·02, 95% CI 0·81-1·29, p=0·86). None of the predefined clinical subgroups showed a benefit from cilengitide. We noted no overall additional toxic effects with cilengitide treatment. The most commonly reported adverse events of grade 3 or worse in the safety population were lymphopenia (31 [12%] in the cilengitide group vs 26 [10%] in the control group), thrombocytopenia (28 [11%] vs 46 [18%]), neutropenia (19 [7%] vs 24 [9%]), leucopenia (18 [7%] vs 20 [8%]), and convulsion (14 [5%] vs 15 [6%]). INTERPRETATION: The addition of cilengitide to temozolomide chemoradiotherapy did not improve outcomes; cilengitide will not be further developed as an anticancer drug. Nevertheless, integrins remain a potential treatment target for glioblastoma. FUNDING: Merck KGaA, Darmstadt, Germany.

The influence of culture on couples coping with breast cancer: a comparative analysis of couples from China, India, and the United States.

This study examines the process of dyadic coping among couples who are managing stress related to a partner's breast cancer diagnosis and identifies cultural factors that affect how couples cope together. Utilizing a qualitative method based on relational psychologies, the "Listening Guide," the authors analyzed the narratives of 28 couples who where coping with early-stage breast cancer and lived in Hong Kong-China, India, and the United States. Analysis revealed four cultural factors influencing the process of coping with breast cancer. These factors included (1) family boundaries, (2) gender roles, (3) personal control, and (4) interdependence. Some couples were able to transcend prevailing cultural norms to re-establish balance in their lives and adapt to the cancer. Implications for using couple-based interventions with cancer patients in differing cultural contexts are discussed.

Extreme vulnerability of smallholder farmers to agricultural risks and climate change in Madagascar.

Across the tropics, smallholder farmers already face numerous risks to agricultural production. Climate change is expected to disproportionately affect smallholder farmers and make their livelihoods even more precarious; however, there is limited information on their overall vulnerability and adaptation needs. We conducted surveys of 600 households in Madagascar to characterize the vulnerability of smallholder farmers, identify how farmers cope with risks and explore what strategies are needed to help them adapt to climate change. Malagasy farmers are particularly vulnerable to any shocks to their agricultural system owing to their high dependence on agriculture for their livelihoods, chronic food insecurity, physical isolation and lack of access to formal safety nets. Farmers are frequently exposed to pest and disease outbreaks and extreme weather events (particularly cyclones), which cause significant crop and income losses and exacerbate food insecurity. Although farmers use a variety of risk-coping strategies, these are insufficient to prevent them from remaining food insecure. Few farmers have adjusted their farming strategies in response to climate change, owing to limited resources and capacity. Urgent technical, financial and institutional support is needed to improve the agricultural production and food security of Malagasy farmers and make their livelihoods resilient to climate change.

Preoperative decolonization effective at reducing staphylococcal colonization in total joint arthroplasty patients.

Staphylococcus decolonization prior to surgery is used to prevent surgical site infections (SSIs) after total joint arthroplasty (TJA). To determine if current treatment protocols result in successful decolonization of methicillin-sensitive S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA), 106 consecutive patients were screened for nasal MSSA/MRSA colonization pre-operatively and on the day of surgery. Colonized patients used intranasal mupirocin twice a day and chlorhexidine showers daily 5 days prior to surgery. Pre-operatively, 24 joints (22.0%) were positive for MSSA colonization and 5 joints (4.6%) were positive for MRSA colonization. On the day of surgery, 3 joints (2.8%) who underwent decolonization were positive for MSSA colonization and 0 joints were positive for MRSA colonization. The reduction in MSSA colonization was significant (P<0.001), while the eradication of MRSA colonization approached statistical significance (P=0.063). Current decolonization protocols using intranasal mupirocin and chlorhexidine washes are effective for reducing MRSA/MSSA colonization.

What is the rate of methicillin-resistant Staphylococcus aureus and Gram-negative infections in open fractures?

BACKGROUND: There have been increasing reports of methicillin-resistant Staphylococcus aureus (MRSA) infections in the community, but it is unclear whether infectious organisms in open fracture infections have changed and if our current regimen of antibiotic prophylaxis is therefore obsolete. QUESTIONS/PURPOSES: We determined the recent incidence of MRSA and Gram-negative organism infections after open fractures. METHODS: We performed a retrospective cohort study on 189 patients with 202 open fractures treated from 2009 to 2010. During the followup, patients were evaluated for signs of infection using the Centers for Disease Control and Prevention criteria. We determined the organisms using routine microbiology culture. The minimum followup was 3 months (median, 47 months; range, 3-108 months). RESULTS: Of the 202 open fractures, 20 (10%) developed infections. The most common organism was Staphylococcus, whereas five (25%) of those infected were positive for MRSA, and 11 (55%) of those with infection were cultured for at least one Gram-negative organism. Six (30%) open fractures had infections that grew out multiple organisms. The incidence of MRSA infections in our open fracture population was 2.5%. CONCLUSIONS: There is a high incidence of MRSA and Gram-negative infections after open fractures, which may indicate that current antibiotic regimens need to be changed. LEVEL OF EVIDENCE: Level IV, retrospective case-series. See the Guidelines for Authors for a complete description of levels of evidence.

Staphylococcus aureus screening and decolonization in orthopaedic surgery and reduction of surgical site infections.

BACKGROUND: Staphylococcus aureus is the most common organism responsible for orthopaedic surgical site infections (SSIs). Patients who are carriers for methicillin-sensitive S. aureus or methicillin-resistant S. aureus (MRSA) have a higher likelihood of having invasive S. aureus infections. Although some have advocated screening for S. aureus and decolonizing it is unclear whether these efforts reduce SSIs. QUESTIONS/PURPOSES: The purposes of this study were to determine (1) whether S. aureus screening and decolonization reduce SSIs in orthopaedic patients and (2) if implementing this protocol is cost-effective. METHODS: Studies for this systematic review were identified by searching PubMed, which includes MEDLINE (1946-present), EMBASE.com (1974-present), and the Cochrane Library's (John Wiley & Sons) Cochrane Database of Systematic Reviews (CDSR), Cochrane Central Register of Controlled Trials (CENTRAL), Database of Abstracts of Reviews of Effects (DARE), Health Technology Assessment Database (HTAD), and the NHS Economic Evaluation Database (NHSEED). Comprehensive literature searches were developed using EMTREE, MeSH, and keywords for each of the search concepts of decolonization, MRSA, and orthopedics/orthopedic surgery. Studies published before 1968 were excluded. We analyzed 19 studies examining the ability of the decolonization protocol to reduce SSIs and 10 studies detailing the cost-effectiveness of S. aureus screening and decolonization. RESULTS: All 19 studies showed a reduction in SSIs or wound complications by instituting a S. aureus screening and decolonization protocol in elective orthopaedic (total joints, spine, and sports) and trauma patients. The S. aureus screening and decolonization protocol also saved costs in orthopaedic patients when comparing the costs of screening and decolonization with the reduction of SSIs. CONCLUSIONS: Preoperative screening and decolonization of S. aureus in orthopaedic patients is a cost-effective means to reduce SSIs. LEVEL OF EVIDENCE: Level IV, systematic review of Level I-IV studies. See the Guidelines for Authors for a complete description of levels of evidence.