REAL-TIME OBSERVATION OF CELLULOSE BIODEGRADATION BY ATOMIC FORCE MICROSCOPY

Cellulose, the major structural component of plant cell walls, is a homopolymer of β-1,4-linked glucose residues. As cellulose is the most abundant biopolymer on Earth comprising approx. 50% of the bioshpere, it has attracted renewed interest as a potential source of energy through its biodegradation and fermentation to biofuels. The biodegradation of cellulose involves the concerted action of three types of enzymes, cellulases (EC 3.2.1.4, endo-β-1,4-glucanases), cellobiohydrolases (EC 3.2.1.91; cellulose 1,4-β-cellobiosidase), and β-glucosidases (EC 3.2.1.21; β-d-glucoside glucohydrolase). The former two classes of enzymes function to hydrolyze insoluble cellulose into soluble oligosaccharides which then serve as substrates for β-glucosidases to release free glucose. In many cases, these enzymes are multi-modular, being comprised of distinct catalytic and carbohydrate-binding modules (CBMs). The CBMS appear to aid in both the adsorption of the enzymes to the insoluble cellulose substrate and the destabilization of the hydrogen-bonding network within the crystalline substrate. An understanding of this latter process is extremely important because it has been demonstrated that binding of the enzymes to the insoluble cellulose represents the rate-limiting step in its hydrolysis. To this end, we have developed a protocol for the direct and real-time observation of cellulose biodegradation by atomic force microscopy (AFM). Working electrodes for AFM experiments consisted of a 200 nm thick gold film vapor deposited onto a glass slide pre-treated with a deposition of a 2 nm thick layer of chromium. After annealing in a muffle furnace at 700°C for 60 s, the slides were treated with thioglucose to provide a highly-ordered monolayer of hydrophilic glucose for the stable adsorption of cellulose. Thin films of bacterial microcrystalline cellulose on these electrodes were prepared using the Langmuir-Blodgett technique. Optimized conditions were established to involve a dispersion of a 2 mg/ml suspension of cellulose in methanol/chloroform (1:5) on aqueous phosphate buffer using a compression of 5 mN/m. With this protocol, drying of the cellulose film thereby precluding any associated structural alterations. AFM images were captured using a Pico SPM Microscope with AFMS 182 scanner and Pico-scan 5.2 software system using silicon nitride tips which had a nominal spring constant of 0.06 N m-1 for contact mode, and magnetically coated silicon tips for MAC mode. Under these conditions, the diameters of the microfibrils in a 50 nm fiber were observed to be 3 - 4 nm, which is smaller than the 7.5 nm previously reported by others. Homogeneous samples of the cellulase CenA from the bacterium Cellulomonas fimi were introduced into the liquid cell through capillary ports for the in situ imaging of cellulose disruption and hydrolysis. This activity was monitored over the course of 19 hours and initial evidence of degradation of the fibers was observed within three minutes of enzyme addition. In addition, details of the process of fiber fraying could be readily discerned. Genetic engineering was used to provide a mutant form of CenA involving a replacement of its catalytic aspartate nucleophile with alanine. Studies with this catalytically inactive enzyme derivative permit the analysis of cellulose fibril destabilization prior to hydrolysis

Cancer outcomes among Parkinson's disease patients with leucine rich repeat kinase 2 mutations, idiopathic Parkinson's disease patients, and nonaffected controls

BACKGROUND: Increased cancer risk has been reported in Parkinson's disease (PD) patients carrying the leucine rich repeat kinase 2 (LRRK2) G2019S mutation (LRRK2-PD) in comparison with idiopathic PD (IPD). It is unclear whether the elevated risk would be maintained when compared with unaffected controls. METHODS: Cancer outcomes were compared among 257 LRRK2-PD patients, 712 IPD patients, and 218 controls recruited from 7 LRRK2 consortium centers using mixed-effects logistic regression. Data were then pooled with a previous study to examine cancer risk between 401 LRRK2-PD and 1946 IPD patients. RESULTS: Although cancer prevalence was similar among LRRK2-PD patients (32.3%), IPD patients (27.5%), and controls (27.5%; P = 0.33), LRRK2-PD had increased risks of leukemia (odds ratio [OR] = 4.55; 95% confidence interval [CI], 1.46-10.61) and skin cancer (OR = 1.61; 95% CI, 1.09-2.37). In the pooled analysis, LRRK2-PD patients had also elevated risks of leukemia (OR = 9.84; 95% CI, 2.15-44.94) and colon cancer (OR = 2.34; 95% CI, 1.15-4.74) when compared with IPD patients. CONCLUSIONS: The increased risks of leukemia as well as skin and colon cancers among LRRK2-PD patients suggest that LRRK2 mutations heighten risks of certain cancers. © 2019 International Parkinson and Movement Disorder Society

Gene Expression Profiles of Sporadic Canine Hemangiosarcoma Are Uniquely Associated with Breed

The role an individual's genetic background plays on phenotype and biological behavior of sporadic tumors remains incompletely understood. We showed previously that lymphomas from Golden Retrievers harbor defined, recurrent chromosomal aberrations that occur less frequently in lymphomas from other dog breeds, suggesting spontaneous canine tumors provide suitable models to define how heritable traits influence cancer genotypes. Here, we report a complementary approach using gene expression profiling in a naturally occurring endothelial sarcoma of dogs (hemangiosarcoma). Naturally occurring hemangiosarcomas of Golden Retrievers clustered separately from those of non-Golden Retrievers, with contributions from transcription factors, survival factors, and from pro-inflammatory and angiogenic genes, and which were exclusively present in hemangiosarcoma and not in other tumors or normal cells (i.e., they were not due simply to variation in these genes among breeds). Vascular Endothelial Growth Factor Receptor 1 (VEGFR1) was among genes preferentially enriched within known pathways derived from gene set enrichment analysis when characterizing tumors from Golden Retrievers versus other breeds. Heightened VEGFR1 expression in these tumors also was apparent at the protein level and targeted inhibition of VEGFR1 increased proliferation of hemangiosarcoma cells derived from tumors of Golden Retrievers, but not from other breeds. Our results suggest heritable factors mold gene expression phenotypes, and consequently biological behavior in sporadic, naturally occurring tumors

The UBA-UIM Domains of the USP25 Regulate the Enzyme Ubiquitination State and Modulate Substrate Recognition

USP25m is the muscle isoform of the deubiquitinating (DUB) enzyme USP25. Similarly to most DUBs, data on USP25 regulation and substrate recognition is scarce. In silico analysis predicted three ubiquitin binding domains (UBDs) at the N-terminus: one ubiquitin-associated domain (UBA) and two ubiquitin-interacting motifs (UIMs), whereas no clear structural homology at the extended C-terminal region outside the catalytic domains were detected. In order to asses the contribution of the UBDs and the C-terminus to the regulation of USP25m catalytic activity, ubiquitination state and substrate interaction, serial and combinatorial deletions were generated. Our results showed that USP25m catalytic activity did not strictly depend on the UBDs, but required a coiled-coil stretch between amino acids 679 to 769. USP25 oligomerized but this interaction did not require either the UBDs or the C-terminus. Besides, USP25 was monoubiquitinated and able to autodeubiquitinate in a possible loop of autoregulation. UBDs favored the monoubiquitination of USP25m at the preferential site lysine 99 (K99). This residue had been previously shown to be a target for SUMO and this modification inhibited USP25 activity. We showed that mutation of K99 clearly diminished USP25-dependent rescue of the specific substrate MyBPC1 from proteasome degradation, thereby supporting a new mechanistic model, in which USP25m is regulated through alternative conjugation of ubiquitin (activating) or SUMO (inhibiting) to the same lysine residue (K99), which may promote the interaction with distinct intramolecular regulatory domains

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Developing Signs of Safety: A Deaf-accessible counselling toolkit for trauma and addiction

The U.S. Deaf community-more than half a million Americans who communicate using American Sign Language (ASL)-experiences higher rates of trauma exposure and substance use disorder (SUD) than the general population. Yet there are no evidence-based treatments for any behavioural health condition that have been evaluated for use with Deaf people. The driving aim of our work, therefore, has been to develop and formally evaluate a Deaf-accessible trauma/SUD counselling approach. Here we describe our initial intervention development work and a single-arm pilot that evaluated the feasibility, acceptability, and preliminary clinical efficacy of Signs of Safety-a Deaf-accessible toolkit to be used with an existing, widely adopted protocol for trauma and addiction (Seeking Safety). Preliminary efficacy results indicated clinically significant reductions in PTSD symptoms and frequency of alcohol use for the Seeking Safety/Signs of Safety model. Frequency of drug use did not change significantly-likely attributable to the mid-study legalization of recreational marijuana in our state. Next steps include the redesign and refilming of Signs of Safety based on pilot participant feedback, again using a Deaf-engaged development and production process. This new toolkit will be tested via a pilot randomized controlled trial designed based on present methodological lessons learned

REM sleep behavior disorder, as assessed by questionnaire, in G2019S LRRK2 mutation PD and carriers

BackgroundRapid eye movement sleep behavior disorder occurs with idiopathic Parkinson's disease (PD) and often precedes PD. Its frequency in LRRK2-PD and utility as a preclinical marker has not been established.MethodsOne hundred forty-four idiopathic PD, 142 LRRK2 G2019S mutation PD, 117 non-manifesting carriers, 93 related noncarriers, and 40 healthy controls completed the Rapid eye movement sleep Behavior Disorder Screening Questionnaire.ResultsCut scores were met by 30.6% idiopathic PD, 19.7% LRRK2-PD, 6% nonmanifesting carriers, 20.4% related noncarriers, and 15% controls. The likelihood of abnormal scores was decreased in LRRK2-PD versus idiopathic PD (odds ratio = 0.55, P = 0.03), nonmanifesting carriers versus related noncarriers (OR = 0.25, P &lt; 0.01), and PD of less than 3 years' duration, 1 of 19 LRRK2-PD versus 14 of 41 idiopathic PD (P &lt; 0.05).ConclusionsA lower frequency of abnormal questionnaire scores is seen in LRRK2-PD, especially in early LRRK2-PD, and in nonmanifesting carriers. Therefore, the Rapid eye movement sleep Behavior Disorder Questionnaire is unlikely to serve as a preclinical marker for phenoconversion to PD

Cancer outcomes among Parkinson's disease patients with leucine rich repeat kinase 2 mutations, idiopathic Parkinson's disease patients, and nonaffected controls

BackgroundIncreased cancer risk has been reported in Parkinson's disease (PD) patients carrying the leucine rich repeat kinase 2 (LRRK2) G2019S mutation (LRRK2-PD) in comparison with idiopathic PD (IPD). It is unclear whether the elevated risk would be maintained when compared with unaffected controls.MethodsCancer outcomes were compared among 257 LRRK2-PD patients, 712 IPD patients, and 218 controls recruited from 7 LRRK2 consortium centers using mixed-effects logistic regression. Data were then pooled with a previous study to examine cancer risk between 401 LRRK2-PD and 1946 IPD patients.ResultsAlthough cancer prevalence was similar among LRRK2-PD patients (32.3%), IPD patients (27.5%), and controls (27.5%; P = 0.33), LRRK2-PD had increased risks of leukemia (odds ratio [OR] = 4.55; 95% confidence interval [CI], 1.46-10.61) and skin cancer (OR = 1.61; 95% CI, 1.09-2.37). In the pooled analysis, LRRK2-PD patients had also elevated risks of leukemia (OR = 9.84; 95% CI, 2.15-44.94) and colon cancer (OR = 2.34; 95% CI, 1.15-4.74) when compared with IPD patients.ConclusionsThe increased risks of leukemia as well as skin and colon cancers among LRRK2-PD patients suggest that LRRK2 mutations heighten risks of certain cancers. © 2019 International Parkinson and Movement Disorder Society