Validation of Articulatory Rate and Imprecision Judgments in Speech of Individuals With Amyotrophic Lateral Sclerosis.

Purpose Perceptual judgments of articulatory function are commonly used by speech-language pathologists to evaluate articulatory performance in individuals with amyotrophic lateral sclerosis (ALS). The goal of this study was to evaluate the psychometric properties (e.g., reliability, validity) of these perceptual measures to inform their application as part of a comprehensive bulbar assessment tool in ALS. Method Preexisting data from 51 individuals with ALS were obtained from a larger longitudinal study. Five independent raters provided perceptual judgments of articulatory rate and imprecision in a sentence task. Inter- and intrarater reliability of these judgments were assessed. Perceptual ratings were correlated with an acoustic measure of articulatory rate, in syllables per second, obtained from passage-reading recordings. Both perceptual and acoustic measures were correlated with gold-standard kinematic tongue and jaw movement measures, recorded from sentences using electromagnetic articulography. Results The results revealed good inter- and intrarater reliability of perceptual judgments of articulatory function. Strong correlations were observed between perceptual ratings of articulatory rate and imprecision and acoustic measures of articulatory rate and kinematic measures of tongue speed. Conclusions These findings support the clinical application of perceptual judgments of articulatory function as valid and reliable measures of underlying articulatory changes in bulbar ALS. Additional research is needed to understand the responsiveness of these measures to clinical changes in articulatory function in ALS.

Using ddPCR to assess the DNA yield of FFPE samples.

OBJECTIVES: Detection of genomic alterations in diseases can be achieved with current molecular technologies. However, the molecules extracted from formalin-fixed, paraffin-embedded (FFPE) bio-samples are often limited possibly due to DNA fragmentation and crosslinking caused by the sample fixation and processing. The study objective was to design a droplet digital PCR (ddPCR) assay to assess the quality and quantity of DNA derived from various DNA extraction conditions on FFPE samples. METHODS: We used 10 µm-thick sections from 5 FFPE oral tumoral blocks, each consisting of 10-15 sections. The protocol variables tested included: 1) tissue staining; 2) duration and 3) temperature of post-digestion heat treatment; and 4) DNA extraction method. DNA quantity was assessed using the NanoDrop 2000 (Thermo Fisher Scientific, USA), the Qubit fluorometer (Thermo Fisher Scientific, USA), and a ddPCR-based assay. DNA quality was assessed using a ddPCR assay for the degree of fragmentation and the effectiveness of removing crosslinks with varying guanine-cytosine (GC)-content. RESULTS: Deparaffinization with xylene helped to increase the DNA yield. Tissue staining (methyl green staining, pH 6) prior to microdissection, comparing to no staining, caused additional DNA fragmentation. Compared to column-based method, DNA extracted with phenol chloroform and ethanol precipitation increased the degree of fragmentation and lowered the yield of amplifiable DNA. The cross-linking derived from GC-contents may not be the only factor impacting on the DNA quality. CONCLUSIONS: Samples undergoing different pre-treatment conditions prior to extraction can impact the yield of amplifiable DNA. Our ddPCR assay can be used to assess for both DNA quantity and quality.

Involvement of ALAD-20S Proteasome Complexes in Ubiquitination and Acetylation of Proteasomal α2 Subunits.

The ubiquitin-proteasome pathway has gained attention as a potential chemotherapeutic target, owing to its importance in the maintenance of protein homeostasis and the observation that cancer cells are more dependent on this pathway than normal cells. Additionally, inhibition of histone deacetylases (HDACs) by their inhibitors like Vorinostat (SAHA) has also proven a useful strategy in cancer therapy and the concomitant use of proteasome and HDAC inhibitors has been shown to be superior to either treatment alone. It has also been reported that delta-aminolevulinic acid dehydratase (ALAD) is a proteasome-associated protein, and may function as an endogenous proteasome inhibitor. While the role of ALAD in the heme biosynthetic pathway is well characterized, little is known about its interaction with, and the mechanism by which it inhibits, the proteasome. In the present study, this ALAD-proteasome complex was further characterized in cultured prostate cancer cells and the effects of SAHA treatment on the regulation of ALAD were investigated. ALAD interacts with the 20S proteasomal core, but not the 19S regulatory cap. Some ubiquitinated species were detected in ALAD immunoprecipitates that have similar molecular weights to ubiquitinated proteasomal α2 subunits, suggesting preferred binding of ALAD to ubiquitinated α2. Additionally, SAHA treatment increases levels of ALAD protein and an acetylated protein with a molecular weight similar to the ubiquitinated α2 subunit. Thus, the results of this study suggest that ALAD may play a regulatory role in a previously unreported post-translational modification of proteasomal α subunits.

Withaferin A inhibits the proteasome activity in mesothelioma in vitro and in vivo.

The medicinal plant Withania somnifera has been used for over centuries in Indian Ayurvedic Medicine to treat a wide spectrum of disorders. Withaferin A (WA), a bioactive compound that is isolated from this plant, has anti-inflammatory, immuno-modulatory, anti-angiogenic, and anti-cancer properties. Here we investigated malignant pleural mesothelioma (MPM) suppressive effects of WA and the molecular mechanisms involved. WA inhibited growth of the murine as well as patient-derived MPM cells in part by decreasing the chymotryptic activity of the proteasome that resulted in increased levels of ubiquitinated proteins and pro-apoptotic proteasome target proteins (p21, Bax, IκBα). WA suppression of MPM growth also involved elevated apoptosis as evidenced by activation of pro-apoptotic p38 stress activated protein kinase (SAPK) and caspase-3, elevated levels of pro-apoptotic Bax protein and cleavage of poly-(ADP-ribose)-polymerase (PARP). Our studies including gene-array based analyses further revealed that WA suppressed a number of cell growth and metastasis-promoting genes including c-myc. WA treatments also stimulated expression of the cell cycle and apoptosis regulatory protein (CARP)-1/CCAR1, a novel transducer of cell growth signaling. Knock-down of CARP-1, on the other hand, interfered with MPM growth inhibitory effects of WA. Intra-peritoneal administration of 5 mg/kg WA daily inhibited growth of murine MPM cell-derived tumors in vivo in part by inhibiting proteasome activity and stimulating apoptosis. Together our in vitro and in vivo studies suggest that WA suppresses MPM growth by targeting multiple pathways that include blockage of proteasome activity and stimulation of apoptosis, and thus holds promise as an anti-MPM agent.

L-glutamine Schiff base copper complex as a proteasome inhibitor and an apoptosis inducer in human cancer cells.

Interest in the use of metallic compounds for cancer treatment has been increasing since the discovery of cisplatin. Clinical studies suggest the use of proteasome inhibitors as potential novel anticancer agents. L-glutamine is the most abundant free amino acid in the body, and has been shown to play a regulatory role in several cellular processes, including metabolism, degradation, redox potential and cellular integrity. Although glutamine is reported to play a role in the regulation of apoptosis, the effect of glutamine copper complex on tumor cells and the involved molecular mechanism have not been investigated. Here, for the first time, we report that a newly synthesized L-glutamine-containing copper complex has proteasome-inhibitory activity in human breast cancer and leukemia cells. The inhibition of the tumor proteasomal activity results in the accumulation of ubiquitinated proteins and ubiquitinated form of IkappaB-alpha, a natural proteasome substrate, followed by induction of apoptosis. Furthermore, this glutamine Schiff base copper complex selectively inhibits the proteasomal activity and induces cell death in cultured breast cancer cells, but not normal, immortalized breast cells. Our data suggest that glutamine Schiff base copper complexes have a potential use for to be used in cancer treatment and prevention.

Induction of tumor cell apoptosis by a novel class of N-thiolated beta-lactam antibiotics with structural modifications at N1 and C3 of the lactam ring.

The investigation of novel anti-tumor agents that preferentially select for malignant cells with a tolerable toxicity level has been the focus of anti-cancer drug discovery. Our laboratories have previously reported that certain N-alkylthiolated beta-lactams had DNA-damaging and apoptosis-inducing activity in various tumor lines but not in nontransformed cells. In the current study, we further delineated the effects of substitutions at C3 or N1 of the lactam ring for cell death-inducing capability with close attention paid to a discernible structure-activity relationship (SAR). We found that two beta-lactam analogs (JG-5 and JG-19), both containing a branched-chain moiety at C3 of the lactam ring, exhibit potent apoptosis-inducing activity. Additionally, JG-5 exhibited superior in vitro biological activity over JG-19 owing to structural modifications made to substituents at the N1 and C3 positions of the lactam ring. Furthermore, the branched beta-lactams were able to inhibit growth of mice bearing breast cancer xenografts, associated with induction of DNA damage and apoptosis in tumor tissues. Our results strongly warrant further investigation into these novel beta-lactams as potential anti-cancer therapeutics.

Disulfiram promotes the conversion of carcinogenic cadmium to a proteasome inhibitor with pro-apoptotic activity in human cancer cells.

The ubiquitin-proteasome system is involved in various cellular processes, including transcription, apoptosis, and cell cycle. In vitro, in vivo, and clinical studies suggest the potential use of proteasome inhibitors as anticancer drugs. Cadmium (Cd) is a widespread environmental pollutant that has been classified as a human carcinogen. Recent study in our laboratory suggested that the clinically used anti-alcoholism drug disulfiram (DSF) could form a complex with tumor cellular copper, resulting in inhibition of the proteasomal chymotrypsin-like activity and induction of cancer cell apoptosis. In the current study, we report, for the first time, that DSF is able to convert the carcinogen Cd to a proteasome-inhibitor and cancer cell apoptosis inducer. Although the DSF-Cd complex inhibited the chymotrypsin-like activity of a purified 20S proteasome with an IC(50) value of 32 micromol/L, this complex was much more potent in inhibiting the chymotrypsin-like activity of prostate cancer cellular 26S proteasome. Inhibition of cellular proteasome activity by the DSF-Cd complex resulted in the accumulation of ubiquitinated proteins and the natural proteasome substrate p27, which was followed by activation of calpain and induction of apoptosis. Importantly, human breast cancer MCF10DCIS cells were much more sensitive to the DSF-Cd treatment than immortalized but non-tumorigenic human breast MCF-10A cells, demonstrating that the DSF-Cd complex could selectively induce proteasome inhibition and apoptosis in human tumor cells. Our work suggests the potential use of DSF for treatment of cells with accumulated levels of carcinogen Cd.